Inverse agonist activity at recombinant CXCR2 (unknown origin) measured after 20 mins by fluorescence based assayInverse agonist activity at recombinant CXCR2 (unknown origin) measured after 20 mins by fluorescence based assay
Inverse agonist activity at recombinant CXCR2 (unknown origin) measured after 20 mins by fluorescence based assayInverse agonist activity at recombinant CXCR2 (unknown origin) measured after 20 mins by fluorescence based assay
Antagonist activity at CXCR2 assessed as inhibition of CXCL8-induced neutrophil chemotaxisAntagonist activity at CXCR2 assessed as inhibition of CXCL8-induced neutrophil chemotaxis
Antagonist activity at CXCR2 assessed as inhibition of CXCL8-induced neutrophil chemotaxisAntagonist activity at CXCR2 assessed as inhibition of CXCL8-induced neutrophil chemotaxis
Antagonist activity at CXCR2 assessed as inhibition of CXCL8-induced neutrophil chemotaxisAntagonist activity at CXCR2 assessed as inhibition of CXCL8-induced neutrophil chemotaxis
Antagonist activity at CXCR2 assessed as inhibition of CXCL8-induced neutrophil chemotaxisAntagonist activity at CXCR2 assessed as inhibition of CXCL8-induced neutrophil chemotaxis
Antagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assayAntagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assay
Antagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assayAntagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity at CXCR2 expressed in CHO cells assessed as inhibition of IL-8-induced chemotaxisAntagonist activity at CXCR2 expressed in CHO cells assessed as inhibition of IL-8-induced chemotaxis
Antagonist activity at CXCR2 expressed in CHO cells assessed as inhibition of IL-8-induced chemotaxisAntagonist activity at CXCR2 expressed in CHO cells assessed as inhibition of IL-8-induced chemotaxis
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Calcium Fluorescence Assay: The assay is based on the detection of intracellular calcium changes detected by the selective, calcium-chelating dye, Fluo-4 (Molecular Probes). A large fluorescence intensity increase is observed upon calcium association with Fluo-4. The dye is delivered to the cell interior using an acetoxymethylester form of Fluo-4, where the intracellular esterase activity results in the charged species being released and trapped within the cytoplasm of the cell. hence, influx of calcium to this cytoplasmic pocket, via release from intracellular pools and the phospholipase C cascade can be detected. By co-expressing the CXCR2 receptor and the promiscuous Galpha 16 protein, activation of this chemokine receptor is directed into this phospholipase C cascade resulting in intracellular calcium mobilization.The CHOK1 cells stably transfected with human CXCR2 and the promiscuous Galpha 16 protein were maintained in a log phase of growth at 37 C. and 5% CO2 in the following media: Iscove's.Calcium Fluorescence Assay: The assay is based on the detection of intracellular calcium changes detected by the selective, calcium-chelating dye, Fluo-4 (Molecular Probes). A large fluorescence intensity increase is observed upon calcium association with Fluo-4. The dye is delivered to the cell interior using an acetoxymethylester form of Fluo-4, where the intracellular esterase activity results in the charged species being released and trapped within the cytoplasm of the cell. hence, influx of calcium to this cytoplasmic pocket, via release from intracellular pools and the phospholipase C cascade can be detected. By co-expressing the CXCR2 receptor and the promiscuous Galpha 16 protein, activation of this chemokine receptor is directed into this phospholipase C cascade resulting in intracellular calcium mobilization.The CHOK1 cells stably transfected with human CXCR2 and the promiscuous Galpha 16 protein were maintained in a log phase of growth at 37 C. and 5% CO2 in the following media: Iscove's.
Calcium Fluorescence Assay: The assay is based on the detection of intracellular calcium changes detected by the selective, calcium-chelating dye, Fluo-4 (Molecular Probes). A large fluorescence intensity increase is observed upon calcium association with Fluo-4. The dye is delivered to the cell interior using an acetoxymethylester form of Fluo-4, where the intracellular esterase activity results in the charged species being released and trapped within the cytoplasm of the cell. hence, influx of calcium to this cytoplasmic pocket, via release from intracellular pools and the phospholipase C cascade can be detected. By co-expressing the CXCR2 receptor and the promiscuous Galpha 16 protein, activation of this chemokine receptor is directed into this phospholipase C cascade resulting in intracellular calcium mobilization.The CHOK1 cells stably transfected with human CXCR2 and the promiscuous Galpha 16 protein were maintained in a log phase of growth at 37 C. and 5% CO2 in the following media: Iscove's.Calcium Fluorescence Assay: The assay is based on the detection of intracellular calcium changes detected by the selective, calcium-chelating dye, Fluo-4 (Molecular Probes). A large fluorescence intensity increase is observed upon calcium association with Fluo-4. The dye is delivered to the cell interior using an acetoxymethylester form of Fluo-4, where the intracellular esterase activity results in the charged species being released and trapped within the cytoplasm of the cell. hence, influx of calcium to this cytoplasmic pocket, via release from intracellular pools and the phospholipase C cascade can be detected. By co-expressing the CXCR2 receptor and the promiscuous Galpha 16 protein, activation of this chemokine receptor is directed into this phospholipase C cascade resulting in intracellular calcium mobilization.The CHOK1 cells stably transfected with human CXCR2 and the promiscuous Galpha 16 protein were maintained in a log phase of growth at 37 C. and 5% CO2 in the following media: Iscove's.
Antagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assayAntagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assay
Antagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assayAntagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assay
Antagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assayAntagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assay
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Antagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assayAntagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assay
Antagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assayAntagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assay
Antagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assayAntagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assay
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Inhibition of CXCR2 (unknown origin) expressed in HEK293 coexpressing snap-CXCR2-LGBit and beta-arrestin2-SMBit using furimazine as substrate preincubated for 1 hr followed by substrate addition and measured after 5 mins by bioluminescence based NanoBiT assayInhibition of CXCR2 (unknown origin) expressed in HEK293 coexpressing snap-CXCR2-LGBit and beta-arrestin2-SMBit using furimazine as substrate preincubated for 1 hr followed by substrate addition and measured after 5 mins by bioluminescence based NanoBiT assay
Inhibition of CXCR2 (unknown origin) expressed in HEK293 coexpressing snap-CXCR2-LGBit and beta-arrestin2-SMBit using furimazine as substrate preincubated for 1 hr followed by substrate addition and measured after 5 mins by bioluminescence based NanoBiT assayInhibition of CXCR2 (unknown origin) expressed in HEK293 coexpressing snap-CXCR2-LGBit and beta-arrestin2-SMBit using furimazine as substrate preincubated for 1 hr followed by substrate addition and measured after 5 mins by bioluminescence based NanoBiT assay
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysisAntagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysis
Antagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysisAntagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysis
Antagonist activity at CXCR2 receptor expressed in CHOK1 cells assessed as human IL8-induced calcium mobilization by FLIPRAntagonist activity at CXCR2 receptor expressed in CHOK1 cells assessed as human IL8-induced calcium mobilization by FLIPR
Antagonist activity at CXCR2 receptor expressed in CHOK1 cells assessed as human IL8-induced calcium mobilization by FLIPRAntagonist activity at CXCR2 receptor expressed in CHOK1 cells assessed as human IL8-induced calcium mobilization by FLIPR
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Inhibition of CXCR2 (unknown origin) expressed in HEK293 coexpressing snap-CXCR2-LGBit and beta-arrestin2-SMBit using furimazine as substrate preincubated for 1 hr followed by substrate addition and measured after 5 mins by bioluminescence based NanoBiT assayInhibition of CXCR2 (unknown origin) expressed in HEK293 coexpressing snap-CXCR2-LGBit and beta-arrestin2-SMBit using furimazine as substrate preincubated for 1 hr followed by substrate addition and measured after 5 mins by bioluminescence based NanoBiT assay
Inhibition of CXCR2 (unknown origin) expressed in HEK293 coexpressing snap-CXCR2-LGBit and beta-arrestin2-SMBit using furimazine as substrate preincubated for 1 hr followed by substrate addition and measured after 5 mins by bioluminescence based NanoBiT assayInhibition of CXCR2 (unknown origin) expressed in HEK293 coexpressing snap-CXCR2-LGBit and beta-arrestin2-SMBit using furimazine as substrate preincubated for 1 hr followed by substrate addition and measured after 5 mins by bioluminescence based NanoBiT assay
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysisAntagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysis
Antagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysisAntagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysis
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at CXCR2 expressed in CHO cells assessed as inhibition of IL-8-induced chemotaxisAntagonist activity at CXCR2 expressed in CHO cells assessed as inhibition of IL-8-induced chemotaxis
Antagonist activity at CXCR2 expressed in CHO cells assessed as inhibition of IL-8-induced chemotaxisAntagonist activity at CXCR2 expressed in CHO cells assessed as inhibition of IL-8-induced chemotaxis
Antagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assayAntagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assay
Antagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assayAntagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assay
Antagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assayAntagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assay
Antagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assayAntagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assay
Antagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assayAntagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assay
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Antagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assayAntagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assay
Antagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assayAntagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assay
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Antagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assayAntagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assay
Antagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assayAntagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assay
Antagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assayAntagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assayAntagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assay
Antagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assayAntagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assay
Antagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assayAntagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assay
Antagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assayAntagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assay
Calcium Fluorescence Assay: The assay is based on the detection of intracellular calcium changes detected by the selective, calcium-chelating dye, Fluo-4 (Molecular Probes). A large fluorescence intensity increase is observed upon calcium association with Fluo-4. The dye is delivered to the cell interior using an acetoxymethylester form of Fluo-4, where the intracellular esterase activity results in the charged species being released and trapped within the cytoplasm of the cell. hence, influx of calcium to this cytoplasmic pocket, via release from intracellular pools and the phospholipase C cascade can be detected. By co-expressing the CXCR2 receptor and the promiscuous Galpha 16 protein, activation of this chemokine receptor is directed into this phospholipase C cascade resulting in intracellular calcium mobilization.The CHOK1 cells stably transfected with human CXCR2 and the promiscuous Galpha 16 protein were maintained in a log phase of growth at 37 C. and 5% CO2 in the following media: Iscove's.Calcium Fluorescence Assay: The assay is based on the detection of intracellular calcium changes detected by the selective, calcium-chelating dye, Fluo-4 (Molecular Probes). A large fluorescence intensity increase is observed upon calcium association with Fluo-4. The dye is delivered to the cell interior using an acetoxymethylester form of Fluo-4, where the intracellular esterase activity results in the charged species being released and trapped within the cytoplasm of the cell. hence, influx of calcium to this cytoplasmic pocket, via release from intracellular pools and the phospholipase C cascade can be detected. By co-expressing the CXCR2 receptor and the promiscuous Galpha 16 protein, activation of this chemokine receptor is directed into this phospholipase C cascade resulting in intracellular calcium mobilization.The CHOK1 cells stably transfected with human CXCR2 and the promiscuous Galpha 16 protein were maintained in a log phase of growth at 37 C. and 5% CO2 in the following media: Iscove's.
Calcium Fluorescence Assay: The assay is based on the detection of intracellular calcium changes detected by the selective, calcium-chelating dye, Fluo-4 (Molecular Probes). A large fluorescence intensity increase is observed upon calcium association with Fluo-4. The dye is delivered to the cell interior using an acetoxymethylester form of Fluo-4, where the intracellular esterase activity results in the charged species being released and trapped within the cytoplasm of the cell. hence, influx of calcium to this cytoplasmic pocket, via release from intracellular pools and the phospholipase C cascade can be detected. By co-expressing the CXCR2 receptor and the promiscuous Galpha 16 protein, activation of this chemokine receptor is directed into this phospholipase C cascade resulting in intracellular calcium mobilization.The CHOK1 cells stably transfected with human CXCR2 and the promiscuous Galpha 16 protein were maintained in a log phase of growth at 37 C. and 5% CO2 in the following media: Iscove's.Calcium Fluorescence Assay: The assay is based on the detection of intracellular calcium changes detected by the selective, calcium-chelating dye, Fluo-4 (Molecular Probes). A large fluorescence intensity increase is observed upon calcium association with Fluo-4. The dye is delivered to the cell interior using an acetoxymethylester form of Fluo-4, where the intracellular esterase activity results in the charged species being released and trapped within the cytoplasm of the cell. hence, influx of calcium to this cytoplasmic pocket, via release from intracellular pools and the phospholipase C cascade can be detected. By co-expressing the CXCR2 receptor and the promiscuous Galpha 16 protein, activation of this chemokine receptor is directed into this phospholipase C cascade resulting in intracellular calcium mobilization.The CHOK1 cells stably transfected with human CXCR2 and the promiscuous Galpha 16 protein were maintained in a log phase of growth at 37 C. and 5% CO2 in the following media: Iscove's.
Antagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assayAntagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assay
Antagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assayAntagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assay
Antagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assayAntagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assay
Antagonist activity at CXCR2 (unknown origin) stably expressed in HEK293 cells co-expressing Galpha16 assessed as reduction in IL-8-induced intracellular calcium change incubated for 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity at CXCR2 (unknown origin) stably expressed in HEK293 cells co-expressing Galpha16 assessed as reduction in IL-8-induced intracellular calcium change incubated for 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity at CXCR2 (unknown origin) stably expressed in HEK293 cells co-expressing Galpha16 assessed as reduction in IL-8-induced intracellular calcium change incubated for 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity at CXCR2 (unknown origin) stably expressed in HEK293 cells co-expressing Galpha16 assessed as reduction in IL-8-induced intracellular calcium change incubated for 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonistic activity against human CXCR2 in neutrophils assessed as blockade of GROalpha stimulated calcium mobilisation by FLIPRAntagonistic activity against human CXCR2 in neutrophils assessed as blockade of GROalpha stimulated calcium mobilisation by FLIPR
Antagonistic activity against human CXCR2 in neutrophils assessed as blockade of GROalpha stimulated calcium mobilisation by FLIPRAntagonistic activity against human CXCR2 in neutrophils assessed as blockade of GROalpha stimulated calcium mobilisation by FLIPR
Antagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assayAntagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assay
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Antagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assayAntagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assay
Antagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assayAntagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assay
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assayAntagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assay
Antagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assayAntagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assay
Antagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assayAntagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assay
Antagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assayAntagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assay
Antagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assayAntagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assay
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assayAntagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assay
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assayAntagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assay
Antagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assayAntagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assay
Antagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assayAntagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assay
Antagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assayAntagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assay
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Calcium Fluorescence Assay: The assay is based on the detection of intracellular calcium changes detected by the selective, calcium-chelating dye, Fluo-4 (Molecular Probes). A large fluorescence intensity increase is observed upon calcium association with Fluo-4. The dye is delivered to the cell interior using an acetoxymethylester form of Fluo-4, where the intracellular esterase activity results in the charged species being released and trapped within the cytoplasm of the cell. hence, influx of calcium to this cytoplasmic pocket, via release from intracellular pools and the phospholipase C cascade can be detected. By co-expressing the CXCR2 receptor and the promiscuous Galpha 16 protein, activation of this chemokine receptor is directed into this phospholipase C cascade resulting in intracellular calcium mobilization.The CHOK1 cells stably transfected with human CXCR2 and the promiscuous Galpha 16 protein were maintained in a log phase of growth at 37 C. and 5% CO2 in the following media: Iscove's.Calcium Fluorescence Assay: The assay is based on the detection of intracellular calcium changes detected by the selective, calcium-chelating dye, Fluo-4 (Molecular Probes). A large fluorescence intensity increase is observed upon calcium association with Fluo-4. The dye is delivered to the cell interior using an acetoxymethylester form of Fluo-4, where the intracellular esterase activity results in the charged species being released and trapped within the cytoplasm of the cell. hence, influx of calcium to this cytoplasmic pocket, via release from intracellular pools and the phospholipase C cascade can be detected. By co-expressing the CXCR2 receptor and the promiscuous Galpha 16 protein, activation of this chemokine receptor is directed into this phospholipase C cascade resulting in intracellular calcium mobilization.The CHOK1 cells stably transfected with human CXCR2 and the promiscuous Galpha 16 protein were maintained in a log phase of growth at 37 C. and 5% CO2 in the following media: Iscove's.
Calcium Fluorescence Assay: The assay is based on the detection of intracellular calcium changes detected by the selective, calcium-chelating dye, Fluo-4 (Molecular Probes). A large fluorescence intensity increase is observed upon calcium association with Fluo-4. The dye is delivered to the cell interior using an acetoxymethylester form of Fluo-4, where the intracellular esterase activity results in the charged species being released and trapped within the cytoplasm of the cell. hence, influx of calcium to this cytoplasmic pocket, via release from intracellular pools and the phospholipase C cascade can be detected. By co-expressing the CXCR2 receptor and the promiscuous Galpha 16 protein, activation of this chemokine receptor is directed into this phospholipase C cascade resulting in intracellular calcium mobilization.The CHOK1 cells stably transfected with human CXCR2 and the promiscuous Galpha 16 protein were maintained in a log phase of growth at 37 C. and 5% CO2 in the following media: Iscove's.Calcium Fluorescence Assay: The assay is based on the detection of intracellular calcium changes detected by the selective, calcium-chelating dye, Fluo-4 (Molecular Probes). A large fluorescence intensity increase is observed upon calcium association with Fluo-4. The dye is delivered to the cell interior using an acetoxymethylester form of Fluo-4, where the intracellular esterase activity results in the charged species being released and trapped within the cytoplasm of the cell. hence, influx of calcium to this cytoplasmic pocket, via release from intracellular pools and the phospholipase C cascade can be detected. By co-expressing the CXCR2 receptor and the promiscuous Galpha 16 protein, activation of this chemokine receptor is directed into this phospholipase C cascade resulting in intracellular calcium mobilization.The CHOK1 cells stably transfected with human CXCR2 and the promiscuous Galpha 16 protein were maintained in a log phase of growth at 37 C. and 5% CO2 in the following media: Iscove's.
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assayAntagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assay
Antagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assayAntagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assay
Antagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assayAntagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assay
Antagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assayAntagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assay
Antagonist activity at CXCR2 (unknown origin) by [35S]-GTPgammaS binding assayAntagonist activity at CXCR2 (unknown origin) by [35S]-GTPgammaS binding assay
Antagonist activity at CXCR2 (unknown origin) by [35S]-GTPgammaS binding assayAntagonist activity at CXCR2 (unknown origin) by [35S]-GTPgammaS binding assay
Inhibition of CXCR2 (unknown origin) expressed in HEK293 coexpressing snap-CXCR2-LGBit and beta-arrestin2-SMBit using furimazine as substrate preincubated for 1 hr followed by substrate addition and measured after 5 mins by bioluminescence based NanoBiT assayInhibition of CXCR2 (unknown origin) expressed in HEK293 coexpressing snap-CXCR2-LGBit and beta-arrestin2-SMBit using furimazine as substrate preincubated for 1 hr followed by substrate addition and measured after 5 mins by bioluminescence based NanoBiT assay
Inhibition of CXCR2 (unknown origin) expressed in HEK293 coexpressing snap-CXCR2-LGBit and beta-arrestin2-SMBit using furimazine as substrate preincubated for 1 hr followed by substrate addition and measured after 5 mins by bioluminescence based NanoBiT assayInhibition of CXCR2 (unknown origin) expressed in HEK293 coexpressing snap-CXCR2-LGBit and beta-arrestin2-SMBit using furimazine as substrate preincubated for 1 hr followed by substrate addition and measured after 5 mins by bioluminescence based NanoBiT assay
Inhibition of CXCR2 (unknown origin) expressed in HEK293 coexpressing snap-CXCR2-LGBit and beta-arrestin2-SMBit using furimazine as substrate preincubated for 1 hr followed by substrate addition and measured after 5 mins by bioluminescence based NanoBiT assayInhibition of CXCR2 (unknown origin) expressed in HEK293 coexpressing snap-CXCR2-LGBit and beta-arrestin2-SMBit using furimazine as substrate preincubated for 1 hr followed by substrate addition and measured after 5 mins by bioluminescence based NanoBiT assay
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assayAntagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assay
Calcium Fluorescence Assay: The assay is based on the detection of intracellular calcium changes detected by the selective, calcium-chelating dye, Fluo-4 (Molecular Probes). A large fluorescence intensity increase is observed upon calcium association with Fluo-4. The dye is delivered to the cell interior using an acetoxymethylester form of Fluo-4, where the intracellular esterase activity results in the charged species being released and trapped within the cytoplasm of the cell. hence, influx of calcium to this cytoplasmic pocket, via release from intracellular pools and the phospholipase C cascade can be detected. By co-expressing the CXCR2 receptor and the promiscuous Galpha 16 protein, activation of this chemokine receptor is directed into this phospholipase C cascade resulting in intracellular calcium mobilization.The CHOK1 cells stably transfected with human CXCR2 and the promiscuous Galpha 16 protein were maintained in a log phase of growth at 37 C. and 5% CO2 in the following media: Iscove's.Calcium Fluorescence Assay: The assay is based on the detection of intracellular calcium changes detected by the selective, calcium-chelating dye, Fluo-4 (Molecular Probes). A large fluorescence intensity increase is observed upon calcium association with Fluo-4. The dye is delivered to the cell interior using an acetoxymethylester form of Fluo-4, where the intracellular esterase activity results in the charged species being released and trapped within the cytoplasm of the cell. hence, influx of calcium to this cytoplasmic pocket, via release from intracellular pools and the phospholipase C cascade can be detected. By co-expressing the CXCR2 receptor and the promiscuous Galpha 16 protein, activation of this chemokine receptor is directed into this phospholipase C cascade resulting in intracellular calcium mobilization.The CHOK1 cells stably transfected with human CXCR2 and the promiscuous Galpha 16 protein were maintained in a log phase of growth at 37 C. and 5% CO2 in the following media: Iscove's.
Calcium Fluorescence Assay: The assay is based on the detection of intracellular calcium changes detected by the selective, calcium-chelating dye, Fluo-4 (Molecular Probes). A large fluorescence intensity increase is observed upon calcium association with Fluo-4. The dye is delivered to the cell interior using an acetoxymethylester form of Fluo-4, where the intracellular esterase activity results in the charged species being released and trapped within the cytoplasm of the cell. hence, influx of calcium to this cytoplasmic pocket, via release from intracellular pools and the phospholipase C cascade can be detected. By co-expressing the CXCR2 receptor and the promiscuous Galpha 16 protein, activation of this chemokine receptor is directed into this phospholipase C cascade resulting in intracellular calcium mobilization.The CHOK1 cells stably transfected with human CXCR2 and the promiscuous Galpha 16 protein were maintained in a log phase of growth at 37 C. and 5% CO2 in the following media: Iscove's.Calcium Fluorescence Assay: The assay is based on the detection of intracellular calcium changes detected by the selective, calcium-chelating dye, Fluo-4 (Molecular Probes). A large fluorescence intensity increase is observed upon calcium association with Fluo-4. The dye is delivered to the cell interior using an acetoxymethylester form of Fluo-4, where the intracellular esterase activity results in the charged species being released and trapped within the cytoplasm of the cell. hence, influx of calcium to this cytoplasmic pocket, via release from intracellular pools and the phospholipase C cascade can be detected. By co-expressing the CXCR2 receptor and the promiscuous Galpha 16 protein, activation of this chemokine receptor is directed into this phospholipase C cascade resulting in intracellular calcium mobilization.The CHOK1 cells stably transfected with human CXCR2 and the promiscuous Galpha 16 protein were maintained in a log phase of growth at 37 C. and 5% CO2 in the following media: Iscove's.
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysisAntagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysis
Antagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysisAntagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysis
Antagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysisAntagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysis
Antagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysisAntagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysis
Antagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assayAntagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assay
Antagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assayAntagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assay
Antagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced intracellular Ca2+ release by fluorescence based calcium flux assayAntagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced intracellular Ca2+ release by fluorescence based calcium flux assay
Antagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced intracellular Ca2+ release by fluorescence based calcium flux assayAntagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced intracellular Ca2+ release by fluorescence based calcium flux assay
Antagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced intracellular Ca2+ release by fluorescence based calcium flux assayAntagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced intracellular Ca2+ release by fluorescence based calcium flux assay
Calcium Fluorescence Assay: The assay is based on the detection of intracellular calcium changes detected by the selective, calcium-chelating dye, Fluo-4 (Molecular Probes). A large fluorescence intensity increase is observed upon calcium association with Fluo-4. The dye is delivered to the cell interior using an acetoxymethylester form of Fluo-4, where the intracellular esterase activity results in the charged species being released and trapped within the cytoplasm of the cell. hence, influx of calcium to this cytoplasmic pocket, via release from intracellular pools and the phospholipase C cascade can be detected. By co-expressing the CXCR2 receptor and the promiscuous Galpha 16 protein, activation of this chemokine receptor is directed into this phospholipase C cascade resulting in intracellular calcium mobilization.The CHOK1 cells stably transfected with human CXCR2 and the promiscuous Galpha 16 protein were maintained in a log phase of growth at 37 C. and 5% CO2 in the following media: Iscove's.Calcium Fluorescence Assay: The assay is based on the detection of intracellular calcium changes detected by the selective, calcium-chelating dye, Fluo-4 (Molecular Probes). A large fluorescence intensity increase is observed upon calcium association with Fluo-4. The dye is delivered to the cell interior using an acetoxymethylester form of Fluo-4, where the intracellular esterase activity results in the charged species being released and trapped within the cytoplasm of the cell. hence, influx of calcium to this cytoplasmic pocket, via release from intracellular pools and the phospholipase C cascade can be detected. By co-expressing the CXCR2 receptor and the promiscuous Galpha 16 protein, activation of this chemokine receptor is directed into this phospholipase C cascade resulting in intracellular calcium mobilization.The CHOK1 cells stably transfected with human CXCR2 and the promiscuous Galpha 16 protein were maintained in a log phase of growth at 37 C. and 5% CO2 in the following media: Iscove's.
Calcium Fluorescence Assay: The assay is based on the detection of intracellular calcium changes detected by the selective, calcium-chelating dye, Fluo-4 (Molecular Probes). A large fluorescence intensity increase is observed upon calcium association with Fluo-4. The dye is delivered to the cell interior using an acetoxymethylester form of Fluo-4, where the intracellular esterase activity results in the charged species being released and trapped within the cytoplasm of the cell. hence, influx of calcium to this cytoplasmic pocket, via release from intracellular pools and the phospholipase C cascade can be detected. By co-expressing the CXCR2 receptor and the promiscuous Galpha 16 protein, activation of this chemokine receptor is directed into this phospholipase C cascade resulting in intracellular calcium mobilization.The CHOK1 cells stably transfected with human CXCR2 and the promiscuous Galpha 16 protein were maintained in a log phase of growth at 37 C. and 5% CO2 in the following media: Iscove's.Calcium Fluorescence Assay: The assay is based on the detection of intracellular calcium changes detected by the selective, calcium-chelating dye, Fluo-4 (Molecular Probes). A large fluorescence intensity increase is observed upon calcium association with Fluo-4. The dye is delivered to the cell interior using an acetoxymethylester form of Fluo-4, where the intracellular esterase activity results in the charged species being released and trapped within the cytoplasm of the cell. hence, influx of calcium to this cytoplasmic pocket, via release from intracellular pools and the phospholipase C cascade can be detected. By co-expressing the CXCR2 receptor and the promiscuous Galpha 16 protein, activation of this chemokine receptor is directed into this phospholipase C cascade resulting in intracellular calcium mobilization.The CHOK1 cells stably transfected with human CXCR2 and the promiscuous Galpha 16 protein were maintained in a log phase of growth at 37 C. and 5% CO2 in the following media: Iscove's.
Antagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assayAntagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assay
Antagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assayAntagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assay
Inhibition of CXCR2 (unknown origin) expressed in HEK293 coexpressing snap-CXCR2-LGBit and beta-arrestin2-SMBit using furimazine as substrate preincubated for 1 hr followed by substrate addition and measured after 5 mins by bioluminescence based NanoBiT assayInhibition of CXCR2 (unknown origin) expressed in HEK293 coexpressing snap-CXCR2-LGBit and beta-arrestin2-SMBit using furimazine as substrate preincubated for 1 hr followed by substrate addition and measured after 5 mins by bioluminescence based NanoBiT assay
Inhibition of CXCR2 (unknown origin) expressed in HEK293 coexpressing snap-CXCR2-LGBit and beta-arrestin2-SMBit using furimazine as substrate preincubated for 1 hr followed by substrate addition and measured after 5 mins by bioluminescence based NanoBiT assayInhibition of CXCR2 (unknown origin) expressed in HEK293 coexpressing snap-CXCR2-LGBit and beta-arrestin2-SMBit using furimazine as substrate preincubated for 1 hr followed by substrate addition and measured after 5 mins by bioluminescence based NanoBiT assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysisAntagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysis
Antagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysisAntagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysis
Calicum Fluorescence Assay: The assay is based on the detection of intracellular calcium changes detected by the selective, calcium-chelating dye, Fluo-4 (Molecular Probes).Calicum Fluorescence Assay: The assay is based on the detection of intracellular calcium changes detected by the selective, calcium-chelating dye, Fluo-4 (Molecular Probes).
Calicum Fluorescence Assay: The assay is based on the detection of intracellular calcium changes detected by the selective, calcium-chelating dye, Fluo-4 (Molecular Probes).Calicum Fluorescence Assay: The assay is based on the detection of intracellular calcium changes detected by the selective, calcium-chelating dye, Fluo-4 (Molecular Probes).
Antagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysisAntagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysis
Antagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysisAntagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysis
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assayAntagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assay
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysisAntagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysis
Antagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysisAntagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysis
Antagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assayAntagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Antagonist activity at CXCR2 assessed as inhibition of neutrophil CD11b up-regulationAntagonist activity at CXCR2 assessed as inhibition of neutrophil CD11b up-regulation
Antagonist activity at CXCR2 assessed as inhibition of neutrophil CD11b up-regulationAntagonist activity at CXCR2 assessed as inhibition of neutrophil CD11b up-regulation
Antagonist activity at CXCR2 assessed as inhibition of neutrophil CD11b up-regulationAntagonist activity at CXCR2 assessed as inhibition of neutrophil CD11b up-regulation
Antagonist activity at CXCR2 assessed as inhibition of neutrophil CD11b up-regulationAntagonist activity at CXCR2 assessed as inhibition of neutrophil CD11b up-regulation
Antagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysisAntagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysis
Antagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysisAntagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysis
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assayAntagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assay
Antagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assayAntagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assay
Antagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assayAntagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assay
Antagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assayAntagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assay
Antagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assayAntagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assay
Antagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assayAntagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assay
Inhibition of CXCR2 (unknown origin) expressed in HEK293 coexpressing snap-CXCR2-LGBit and beta-arrestin2-SMBit using furimazine as substrate preincubated for 1 hr followed by substrate addition and measured after 5 mins by bioluminescence based NanoBiT assayInhibition of CXCR2 (unknown origin) expressed in HEK293 coexpressing snap-CXCR2-LGBit and beta-arrestin2-SMBit using furimazine as substrate preincubated for 1 hr followed by substrate addition and measured after 5 mins by bioluminescence based NanoBiT assay
Inhibition of CXCR2 (unknown origin) expressed in HEK293 coexpressing snap-CXCR2-LGBit and beta-arrestin2-SMBit using furimazine as substrate preincubated for 1 hr followed by substrate addition and measured after 5 mins by bioluminescence based NanoBiT assayInhibition of CXCR2 (unknown origin) expressed in HEK293 coexpressing snap-CXCR2-LGBit and beta-arrestin2-SMBit using furimazine as substrate preincubated for 1 hr followed by substrate addition and measured after 5 mins by bioluminescence based NanoBiT assay
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assayAntagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assay
Antagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assayAntagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysisAntagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysis
Antagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysisAntagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysis
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assayAntagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assay
Antagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assayAntagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assay
Antagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assayAntagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity at CXCR2 assessed as inhibition of neutrophil shape changeAntagonist activity at CXCR2 assessed as inhibition of neutrophil shape change
Antagonist activity at CXCR2 assessed as inhibition of neutrophil shape changeAntagonist activity at CXCR2 assessed as inhibition of neutrophil shape change
Antagonist activity at CXCR2 assessed as inhibition of neutrophil shape changeAntagonist activity at CXCR2 assessed as inhibition of neutrophil shape change
Antagonist activity at CXCR2 assessed as inhibition of neutrophil shape changeAntagonist activity at CXCR2 assessed as inhibition of neutrophil shape change
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assayAntagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assay
Antagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assayAntagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assay
Antagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assayAntagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assay
Antagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assayAntagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assay
Antagonist activity at CXCR2 receptor expressed in CHOK1 cells assessed as human IL8-induced calcium mobilization by FLIPRAntagonist activity at CXCR2 receptor expressed in CHOK1 cells assessed as human IL8-induced calcium mobilization by FLIPR
Antagonist activity at CXCR2 receptor expressed in CHOK1 cells assessed as human IL8-induced calcium mobilization by FLIPRAntagonist activity at CXCR2 receptor expressed in CHOK1 cells assessed as human IL8-induced calcium mobilization by FLIPR
Antagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assayAntagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assay
Antagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assayAntagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assay
Antagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assayAntagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assay
Antagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assayAntagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assay
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assayAntagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assay
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Antagonist activity at CXCL8-activated CXCR2 (unknown origin) overexpressed in human U2OS cells preincubated for 30 mins followed by CXCL8 addition measured after 5 hrs by beta-lactamase reporter gene assayAntagonist activity at CXCL8-activated CXCR2 (unknown origin) overexpressed in human U2OS cells preincubated for 30 mins followed by CXCL8 addition measured after 5 hrs by beta-lactamase reporter gene assay
Antagonist activity at CXCL8-activated CXCR2 (unknown origin) overexpressed in human U2OS cells preincubated for 30 mins followed by CXCL8 addition measured after 5 hrs by beta-lactamase reporter gene assayAntagonist activity at CXCL8-activated CXCR2 (unknown origin) overexpressed in human U2OS cells preincubated for 30 mins followed by CXCL8 addition measured after 5 hrs by beta-lactamase reporter gene assay
Antagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assayAntagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assay
Antagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assayAntagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assay
Antagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assayAntagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assay
Antagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assayAntagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assay
Antagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assayAntagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assay
Antagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assayAntagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assay
Antagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assayAntagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assay
Antagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assayAntagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assay
Antagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assayAntagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assay
Antagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assayAntagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assay
Antagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assayAntagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assay
Antagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assayAntagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assay
Antagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysisAntagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysis
Antagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysisAntagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysis
Antagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced intracellular Ca2+ release by fluorescence based calcium flux assayAntagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced intracellular Ca2+ release by fluorescence based calcium flux assay
Antagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced intracellular Ca2+ release by fluorescence based calcium flux assayAntagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced intracellular Ca2+ release by fluorescence based calcium flux assay
Antagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced intracellular Ca2+ release by fluorescence based calcium flux assayAntagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced intracellular Ca2+ release by fluorescence based calcium flux assay
Antagonist activity at CXCL8-activated CXCR2 (unknown origin) overexpressed in human U2OS cells preincubated for 30 mins followed by CXCL8 addition measured after 5 hrs by beta-lactamase reporter gene assayAntagonist activity at CXCL8-activated CXCR2 (unknown origin) overexpressed in human U2OS cells preincubated for 30 mins followed by CXCL8 addition measured after 5 hrs by beta-lactamase reporter gene assay
Antagonist activity at CXCL8-activated CXCR2 (unknown origin) overexpressed in human U2OS cells preincubated for 30 mins followed by CXCL8 addition measured after 5 hrs by beta-lactamase reporter gene assayAntagonist activity at CXCL8-activated CXCR2 (unknown origin) overexpressed in human U2OS cells preincubated for 30 mins followed by CXCL8 addition measured after 5 hrs by beta-lactamase reporter gene assay
Antagonist activity at CXCR2 receptor expressed in CHOK1 cells assessed as human IL8-induced calcium mobilization by FLIPRAntagonist activity at CXCR2 receptor expressed in CHOK1 cells assessed as human IL8-induced calcium mobilization by FLIPR
Antagonist activity at CXCR2 receptor expressed in CHOK1 cells assessed as human IL8-induced calcium mobilization by FLIPRAntagonist activity at CXCR2 receptor expressed in CHOK1 cells assessed as human IL8-induced calcium mobilization by FLIPR
Antagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assayAntagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysisAntagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysis
Antagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysisAntagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysis
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assayAntagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assay
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assayAntagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assayAntagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assay
Antagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assayAntagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assay
Antagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysisAntagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysis
Antagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysisAntagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysis
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assayAntagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assay
Antagonist activity at CXCR2 expressed in CHO cells assessed as inhibition of IL-8-induced chemotaxisAntagonist activity at CXCR2 expressed in CHO cells assessed as inhibition of IL-8-induced chemotaxis
Antagonist activity at CXCR2 expressed in CHO cells assessed as inhibition of IL-8-induced chemotaxisAntagonist activity at CXCR2 expressed in CHO cells assessed as inhibition of IL-8-induced chemotaxis
Antagonist activity at CXCR2 receptor expressed in CHOK1 cells assessed as human IL8-induced calcium mobilization by FLIPRAntagonist activity at CXCR2 receptor expressed in CHOK1 cells assessed as human IL8-induced calcium mobilization by FLIPR
Antagonist activity at CXCR2 receptor expressed in CHOK1 cells assessed as human IL8-induced calcium mobilization by FLIPRAntagonist activity at CXCR2 receptor expressed in CHOK1 cells assessed as human IL8-induced calcium mobilization by FLIPR
Antagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assayAntagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assay
Antagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assayAntagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assay
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assayAntagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assay
Antagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assayAntagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assay
Antagonist activity at CXCR2 (unknown origin) stably expressed in HEK293 cells co-expressing Galpha16 assessed as reduction in IL-8-induced intracellular calcium change incubated for 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity at CXCR2 (unknown origin) stably expressed in HEK293 cells co-expressing Galpha16 assessed as reduction in IL-8-induced intracellular calcium change incubated for 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity at CXCR2 (unknown origin) stably expressed in HEK293 cells co-expressing Galpha16 assessed as reduction in IL-8-induced intracellular calcium change incubated for 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity at CXCR2 (unknown origin) stably expressed in HEK293 cells co-expressing Galpha16 assessed as reduction in IL-8-induced intracellular calcium change incubated for 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity at CXCR2 in human neutrophils assessed as reduction in GROalpha stimulated intracellular calcium mobilisation by FLIPR assayAntagonist activity at CXCR2 in human neutrophils assessed as reduction in GROalpha stimulated intracellular calcium mobilisation by FLIPR assay
Antagonist activity at CXCR2 in human neutrophils assessed as reduction in GROalpha stimulated intracellular calcium mobilisation by FLIPR assayAntagonist activity at CXCR2 in human neutrophils assessed as reduction in GROalpha stimulated intracellular calcium mobilisation by FLIPR assay
Antagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assayAntagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assay
Antagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assayAntagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assay
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysisAntagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysis
Antagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysisAntagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysis
Antagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assayAntagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assay
Antagonist activity at CXCR2 receptor expressed in CHOK1 cells assessed as human IL8-induced calcium mobilization by FLIPRAntagonist activity at CXCR2 receptor expressed in CHOK1 cells assessed as human IL8-induced calcium mobilization by FLIPR
Antagonist activity at CXCR2 receptor expressed in CHOK1 cells assessed as human IL8-induced calcium mobilization by FLIPRAntagonist activity at CXCR2 receptor expressed in CHOK1 cells assessed as human IL8-induced calcium mobilization by FLIPR
Antagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assayAntagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assay
Antagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assayAntagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assayAntagonist activity at CXCR2 (unknown origin) expressed in CHO-K1 cells co-expressing Galpha-16 assessed as inhibition of IL8-induced calcium mobilization measured after 10 mins by Fluo-4AM dye based FLIPR assay
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assayAntagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assay
Antagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assayAntagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assay
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Antagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assayAntagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assay
Antagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assayAntagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assay
Antagonist activity at CXCR2 receptor expressed in CHOK1 cells assessed as human IL8-induced calcium mobilization by FLIPRAntagonist activity at CXCR2 receptor expressed in CHOK1 cells assessed as human IL8-induced calcium mobilization by FLIPR
Antagonist activity at CXCR2 receptor expressed in CHOK1 cells assessed as human IL8-induced calcium mobilization by FLIPRAntagonist activity at CXCR2 receptor expressed in CHOK1 cells assessed as human IL8-induced calcium mobilization by FLIPR
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at CXCR2 expressed in CHO cells assessed as inhibition of IL-8-induced chemotaxisAntagonist activity at CXCR2 expressed in CHO cells assessed as inhibition of IL-8-induced chemotaxis
Antagonist activity at CXCR2 expressed in CHO cells assessed as inhibition of IL-8-induced chemotaxisAntagonist activity at CXCR2 expressed in CHO cells assessed as inhibition of IL-8-induced chemotaxis
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assayAntagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assay
Antagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assayAntagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assay
Antagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assayAntagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assay
Antagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assayAntagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assay
Antagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assayAntagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CVCL8-induced [35S]GTPgammaS binding after 60 minsAntagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CVCL8-induced [35S]GTPgammaS binding after 60 mins
Antagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CVCL8-induced [35S]GTPgammaS binding after 60 minsAntagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CVCL8-induced [35S]GTPgammaS binding after 60 mins
Antagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CVCL8-induced [35S]GTPgammaS binding after 60 minsAntagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CVCL8-induced [35S]GTPgammaS binding after 60 mins
Antagonist activity at CXCR2 receptor expressed in CHOK1 cells assessed as human IL8-induced calcium mobilization by FLIPRAntagonist activity at CXCR2 receptor expressed in CHOK1 cells assessed as human IL8-induced calcium mobilization by FLIPR
Antagonist activity at CXCR2 receptor expressed in CHOK1 cells assessed as human IL8-induced calcium mobilization by FLIPRAntagonist activity at CXCR2 receptor expressed in CHOK1 cells assessed as human IL8-induced calcium mobilization by FLIPR
Calcium Fluorescence Assay: The assay is based on the detection of intracellular calcium changes detected by the selective, calcium-chelating dye, Fluo-4 (Molecular Probes). A large fluorescence intensity increase is observed upon calcium association with Fluo-4. The dye is delivered to the cell interior using an acetoxymethylester form of Fluo-4, where the intracellular esterase activity results in the charged species being released and trapped within the cytoplasm of the cell. hence, influx of calcium to this cytoplasmic pocket, via release from intracellular pools and the phospholipase C cascade can be detected. By co-expressing the CXCR2 receptor and the promiscuous Galpha 16 protein, activation of this chemokine receptor is directed into this phospholipase C cascade resulting in intracellular calcium mobilization.The CHOK1 cells stably transfected with human CXCR2 and the promiscuous Galpha 16 protein were maintained in a log phase of growth at 37 C. and 5% CO2 in the following media: Iscove's.Calcium Fluorescence Assay: The assay is based on the detection of intracellular calcium changes detected by the selective, calcium-chelating dye, Fluo-4 (Molecular Probes). A large fluorescence intensity increase is observed upon calcium association with Fluo-4. The dye is delivered to the cell interior using an acetoxymethylester form of Fluo-4, where the intracellular esterase activity results in the charged species being released and trapped within the cytoplasm of the cell. hence, influx of calcium to this cytoplasmic pocket, via release from intracellular pools and the phospholipase C cascade can be detected. By co-expressing the CXCR2 receptor and the promiscuous Galpha 16 protein, activation of this chemokine receptor is directed into this phospholipase C cascade resulting in intracellular calcium mobilization.The CHOK1 cells stably transfected with human CXCR2 and the promiscuous Galpha 16 protein were maintained in a log phase of growth at 37 C. and 5% CO2 in the following media: Iscove's.
Calcium Fluorescence Assay: The assay is based on the detection of intracellular calcium changes detected by the selective, calcium-chelating dye, Fluo-4 (Molecular Probes). A large fluorescence intensity increase is observed upon calcium association with Fluo-4. The dye is delivered to the cell interior using an acetoxymethylester form of Fluo-4, where the intracellular esterase activity results in the charged species being released and trapped within the cytoplasm of the cell. hence, influx of calcium to this cytoplasmic pocket, via release from intracellular pools and the phospholipase C cascade can be detected. By co-expressing the CXCR2 receptor and the promiscuous Galpha 16 protein, activation of this chemokine receptor is directed into this phospholipase C cascade resulting in intracellular calcium mobilization.The CHOK1 cells stably transfected with human CXCR2 and the promiscuous Galpha 16 protein were maintained in a log phase of growth at 37 C. and 5% CO2 in the following media: Iscove's.Calcium Fluorescence Assay: The assay is based on the detection of intracellular calcium changes detected by the selective, calcium-chelating dye, Fluo-4 (Molecular Probes). A large fluorescence intensity increase is observed upon calcium association with Fluo-4. The dye is delivered to the cell interior using an acetoxymethylester form of Fluo-4, where the intracellular esterase activity results in the charged species being released and trapped within the cytoplasm of the cell. hence, influx of calcium to this cytoplasmic pocket, via release from intracellular pools and the phospholipase C cascade can be detected. By co-expressing the CXCR2 receptor and the promiscuous Galpha 16 protein, activation of this chemokine receptor is directed into this phospholipase C cascade resulting in intracellular calcium mobilization.The CHOK1 cells stably transfected with human CXCR2 and the promiscuous Galpha 16 protein were maintained in a log phase of growth at 37 C. and 5% CO2 in the following media: Iscove's.
Calicum Fluorescence Assay: The assay is based on the detection of intracellular calcium changes detected by the selective, calcium-chelating dye, Fluo-4 (Molecular Probes).Calicum Fluorescence Assay: The assay is based on the detection of intracellular calcium changes detected by the selective, calcium-chelating dye, Fluo-4 (Molecular Probes).
Calicum Fluorescence Assay: The assay is based on the detection of intracellular calcium changes detected by the selective, calcium-chelating dye, Fluo-4 (Molecular Probes).Calicum Fluorescence Assay: The assay is based on the detection of intracellular calcium changes detected by the selective, calcium-chelating dye, Fluo-4 (Molecular Probes).
Antagonist activity at CXCR2 expressed in CHO cells assessed as inhibition of IL-8-induced chemotaxisAntagonist activity at CXCR2 expressed in CHO cells assessed as inhibition of IL-8-induced chemotaxis
Antagonist activity at CXCR2 expressed in CHO cells assessed as inhibition of IL-8-induced chemotaxisAntagonist activity at CXCR2 expressed in CHO cells assessed as inhibition of IL-8-induced chemotaxis
Antagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assayAntagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assay
Antagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assayAntagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assay
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Antagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysisAntagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysis
Antagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysisAntagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysis
Antagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assayAntagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assay
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assayAntagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assay
Antagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assayAntagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assay
Antagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assayAntagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assay
Antagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assayAntagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assay
Antagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assayAntagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assay
Antagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysisAntagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysis
Antagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysisAntagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysis
Antagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assayAntagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assay
Antagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assayAntagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assay
Antagonist activity at CXCR2 expressed in CHO cells assessed as inhibition of IL-8-induced chemotaxisAntagonist activity at CXCR2 expressed in CHO cells assessed as inhibition of IL-8-induced chemotaxis
Antagonist activity at CXCR2 expressed in CHO cells assessed as inhibition of IL-8-induced chemotaxisAntagonist activity at CXCR2 expressed in CHO cells assessed as inhibition of IL-8-induced chemotaxis
Antagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assayAntagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assay
Antagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assayAntagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assay
Antagonist activity at CXCL8-activated CXCR2 (unknown origin) overexpressed in human U2OS cells preincubated for 30 mins followed by CXCL8 addition measured after 5 hrs by beta-lactamase reporter gene assayAntagonist activity at CXCL8-activated CXCR2 (unknown origin) overexpressed in human U2OS cells preincubated for 30 mins followed by CXCL8 addition measured after 5 hrs by beta-lactamase reporter gene assay
Antagonist activity at CXCL8-activated CXCR2 (unknown origin) overexpressed in human U2OS cells preincubated for 30 mins followed by CXCL8 addition measured after 5 hrs by beta-lactamase reporter gene assayAntagonist activity at CXCL8-activated CXCR2 (unknown origin) overexpressed in human U2OS cells preincubated for 30 mins followed by CXCL8 addition measured after 5 hrs by beta-lactamase reporter gene assay
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Inhibition of CXCR2 (unknown origin) expressed in HEK293T cells cotransfected with GFP-p22F assessed as reduction in IL-8-induced cAMP incubated for 10 mins followed by IL-8 stimulation and measured after 10 mins by luminescence based assayInhibition of CXCR2 (unknown origin) expressed in HEK293T cells cotransfected with GFP-p22F assessed as reduction in IL-8-induced cAMP incubated for 10 mins followed by IL-8 stimulation and measured after 10 mins by luminescence based assay
Inhibition of CXCR2 (unknown origin) expressed in HEK293T cells cotransfected with GFP-p22F assessed as reduction in IL-8-induced cAMP incubated for 10 mins followed by IL-8 stimulation and measured after 10 mins by luminescence based assayInhibition of CXCR2 (unknown origin) expressed in HEK293T cells cotransfected with GFP-p22F assessed as reduction in IL-8-induced cAMP incubated for 10 mins followed by IL-8 stimulation and measured after 10 mins by luminescence based assay
Antagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assayAntagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assay
Antagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assayAntagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assay
Antagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assayAntagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assay
Antagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assayAntagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assay
Antagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assayAntagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assay
Antagonist activity at GFP-tagged CXCR2 (unknown origin) expressed in 293T cells assessed as suppression of IL-8-induced inhibition of forskolin-stimulated cAMP accumulation preincubated for 10 mins followed by IL-8 addition and measured after 10 mins by cAMP assayAntagonist activity at GFP-tagged CXCR2 (unknown origin) expressed in 293T cells assessed as suppression of IL-8-induced inhibition of forskolin-stimulated cAMP accumulation preincubated for 10 mins followed by IL-8 addition and measured after 10 mins by cAMP assay
Antagonist activity at GFP-tagged CXCR2 (unknown origin) expressed in 293T cells assessed as suppression of IL-8-induced inhibition of forskolin-stimulated cAMP accumulation preincubated for 10 mins followed by IL-8 addition and measured after 10 mins by cAMP assayAntagonist activity at GFP-tagged CXCR2 (unknown origin) expressed in 293T cells assessed as suppression of IL-8-induced inhibition of forskolin-stimulated cAMP accumulation preincubated for 10 mins followed by IL-8 addition and measured after 10 mins by cAMP assay
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Calcium Fluorescence Assay: The assay is based on the detection of intracellular calcium changes detected by the selective, calcium-chelating dye, Fluo-4 (Molecular Probes). A large fluorescence intensity increase is observed upon calcium association with Fluo-4. The dye is delivered to the cell interior using an acetoxymethylester form of Fluo-4, where the intracellular esterase activity results in the charged species being released and trapped within the cytoplasm of the cell. hence, influx of calcium to this cytoplasmic pocket, via release from intracellular pools and the phospholipase C cascade can be detected. By co-expressing the CXCR2 receptor and the promiscuous Galpha 16 protein, activation of this chemokine receptor is directed into this phospholipase C cascade resulting in intracellular calcium mobilization.The CHOK1 cells stably transfected with human CXCR2 and the promiscuous Galpha 16 protein were maintained in a log phase of growth at 37 C. and 5% CO2 in the following media: Iscove's.Calcium Fluorescence Assay: The assay is based on the detection of intracellular calcium changes detected by the selective, calcium-chelating dye, Fluo-4 (Molecular Probes). A large fluorescence intensity increase is observed upon calcium association with Fluo-4. The dye is delivered to the cell interior using an acetoxymethylester form of Fluo-4, where the intracellular esterase activity results in the charged species being released and trapped within the cytoplasm of the cell. hence, influx of calcium to this cytoplasmic pocket, via release from intracellular pools and the phospholipase C cascade can be detected. By co-expressing the CXCR2 receptor and the promiscuous Galpha 16 protein, activation of this chemokine receptor is directed into this phospholipase C cascade resulting in intracellular calcium mobilization.The CHOK1 cells stably transfected with human CXCR2 and the promiscuous Galpha 16 protein were maintained in a log phase of growth at 37 C. and 5% CO2 in the following media: Iscove's.
Calcium Fluorescence Assay: The assay is based on the detection of intracellular calcium changes detected by the selective, calcium-chelating dye, Fluo-4 (Molecular Probes). A large fluorescence intensity increase is observed upon calcium association with Fluo-4. The dye is delivered to the cell interior using an acetoxymethylester form of Fluo-4, where the intracellular esterase activity results in the charged species being released and trapped within the cytoplasm of the cell. hence, influx of calcium to this cytoplasmic pocket, via release from intracellular pools and the phospholipase C cascade can be detected. By co-expressing the CXCR2 receptor and the promiscuous Galpha 16 protein, activation of this chemokine receptor is directed into this phospholipase C cascade resulting in intracellular calcium mobilization.The CHOK1 cells stably transfected with human CXCR2 and the promiscuous Galpha 16 protein were maintained in a log phase of growth at 37 C. and 5% CO2 in the following media: Iscove's.Calcium Fluorescence Assay: The assay is based on the detection of intracellular calcium changes detected by the selective, calcium-chelating dye, Fluo-4 (Molecular Probes). A large fluorescence intensity increase is observed upon calcium association with Fluo-4. The dye is delivered to the cell interior using an acetoxymethylester form of Fluo-4, where the intracellular esterase activity results in the charged species being released and trapped within the cytoplasm of the cell. hence, influx of calcium to this cytoplasmic pocket, via release from intracellular pools and the phospholipase C cascade can be detected. By co-expressing the CXCR2 receptor and the promiscuous Galpha 16 protein, activation of this chemokine receptor is directed into this phospholipase C cascade resulting in intracellular calcium mobilization.The CHOK1 cells stably transfected with human CXCR2 and the promiscuous Galpha 16 protein were maintained in a log phase of growth at 37 C. and 5% CO2 in the following media: Iscove's.
Antagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysisAntagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysis
Antagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysisAntagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysis
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Antagonist activity at CXCR2 expressed in CHO cells assessed as inhibition of IL-8-induced chemotaxisAntagonist activity at CXCR2 expressed in CHO cells assessed as inhibition of IL-8-induced chemotaxis
Antagonist activity at CXCR2 expressed in CHO cells assessed as inhibition of IL-8-induced chemotaxisAntagonist activity at CXCR2 expressed in CHO cells assessed as inhibition of IL-8-induced chemotaxis
Antagonist activity at CXCR2 expressed in CHO cells assessed as inhibition of IL-8-induced chemotaxisAntagonist activity at CXCR2 expressed in CHO cells assessed as inhibition of IL-8-induced chemotaxis
Antagonist activity at CXCR2 expressed in CHO cells assessed as inhibition of IL-8-induced chemotaxisAntagonist activity at CXCR2 expressed in CHO cells assessed as inhibition of IL-8-induced chemotaxis
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assayAntagonist activity at CXCR2 in human CD4 cells by fluorescence based calcium mobilization assay
Antagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assayAntagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assay
Antagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assayAntagonist activity at CXCR2 in human PMNs assessed as inhibition of CXCL1-induced intracellular Ca2+ release by fluorescence based calcium flux assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assayAntagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assay
Antagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assayAntagonist activity against human recombinant CXCR2 receptor expressed in CHO cell membranes by SPA based [35S]GTPgammaS binding assay
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Antagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA methodAntagonist activity at human recombinant CXCR2 receptor expressed in CHO cells assessed as inhibition of IL8-induced [35S]GTPgammaS binding by SPA method
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysisAntagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysis
Antagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysisAntagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysis
Antagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysisAntagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysis
Antagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysisAntagonist activity at CXCR2 (unknown origin) transfected in RBL cells assessed as inhibition of IL-8-mediated intracellular calcium release preincubated for 30 mins followed by IL-8 addition by FLUO-4AM-based fluorescent microplate reader analysis
Antagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assayAntagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assayAntagonist activity in CXCR2 (unknown origin) expressed in human HEK293 cells co-expressing Galpha16 assessed as reduction in calcium immobilization pretreated with Fluo-4AM for 45 mins followed by compound addition and measured after 10 mins by Fluo-4AM dye based fluorescence assay
Antagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assayAntagonist activity at human CXCR2 expressed in HEK293 cells assessed as inhibition of CXCL8-induced beta-arrestin recruitment preincubated for 30 mins followed by addition of agonist incubated for 90 mins by beta arrestin assay
Antagonistic activity against human CXCR2 in neutrophils assessed as blockade of GROalpha stimulated calcium mobilisation by FLIPRAntagonistic activity against human CXCR2 in neutrophils assessed as blockade of GROalpha stimulated calcium mobilisation by FLIPR
Antagonistic activity against human CXCR2 in neutrophils assessed as blockade of GROalpha stimulated calcium mobilisation by FLIPRAntagonistic activity against human CXCR2 in neutrophils assessed as blockade of GROalpha stimulated calcium mobilisation by FLIPR
Antagonist activity at CXCR2 receptor in HEK293 cells assessed as mobilization of intracellular calcium by FLIPR analysisAntagonist activity at CXCR2 receptor in HEK293 cells assessed as mobilization of intracellular calcium by FLIPR analysis
Antagonist activity at CXCR2 receptor in HEK293 cells assessed as mobilization of intracellular calcium by FLIPR analysisAntagonist activity at CXCR2 receptor in HEK293 cells assessed as mobilization of intracellular calcium by FLIPR analysis
Antagonist activity at CXCR2 receptor in HEK293 cells assessed as mobilization of intracellular calcium by FLIPR analysisAntagonist activity at CXCR2 receptor in HEK293 cells assessed as mobilization of intracellular calcium by FLIPR analysis
Antagonist activity at CXCR2 receptor in HEK293 cells assessed as mobilization of intracellular calcium by FLIPR analysisAntagonist activity at CXCR2 receptor in HEK293 cells assessed as mobilization of intracellular calcium by FLIPR analysis
Antagonist activity at CXCR2 receptor in HEK293 cells assessed as mobilization of intracellular calcium by FLIPR analysisAntagonist activity at CXCR2 receptor in HEK293 cells assessed as mobilization of intracellular calcium by FLIPR analysis
Antagonist activity at CXCR2 receptor in HEK293 cells assessed as mobilization of intracellular calcium by FLIPR analysisAntagonist activity at CXCR2 receptor in HEK293 cells assessed as mobilization of intracellular calcium by FLIPR analysis
Antagonist activity at CXCR2 receptor in HEK293 cells assessed as mobilization of intracellular calcium by FLIPR analysisAntagonist activity at CXCR2 receptor in HEK293 cells assessed as mobilization of intracellular calcium by FLIPR analysis
Antagonist activity at CXCR2 receptor in HEK293 cells assessed as mobilization of intracellular calcium by FLIPR analysisAntagonist activity at CXCR2 receptor in HEK293 cells assessed as mobilization of intracellular calcium by FLIPR analysis
Antagonist activity at CXCR2 receptor in HEK293 cells assessed as mobilization of intracellular calcium by FLIPR analysisAntagonist activity at CXCR2 receptor in HEK293 cells assessed as mobilization of intracellular calcium by FLIPR analysis
Antagonist activity at CXCR2 receptor in HEK293 cells assessed as mobilization of intracellular calcium by FLIPR analysisAntagonist activity at CXCR2 receptor in HEK293 cells assessed as mobilization of intracellular calcium by FLIPR analysis
Antagonist activity at CXCR2 receptor in HEK293 cells assessed as mobilization of intracellular calcium by FLIPR analysisAntagonist activity at CXCR2 receptor in HEK293 cells assessed as mobilization of intracellular calcium by FLIPR analysis
Antagonist activity at CXCR2 receptor in HEK293 cells assessed as mobilization of intracellular calcium by FLIPR analysisAntagonist activity at CXCR2 receptor in HEK293 cells assessed as mobilization of intracellular calcium by FLIPR analysis
Antagonist activity at CXCR2 receptor in HEK293 cells assessed as mobilization of intracellular calcium by FLIPR analysisAntagonist activity at CXCR2 receptor in HEK293 cells assessed as mobilization of intracellular calcium by FLIPR analysis
Antagonist activity at CXCR2 receptor in HEK293 cells assessed as mobilization of intracellular calcium by FLIPR analysisAntagonist activity at CXCR2 receptor in HEK293 cells assessed as mobilization of intracellular calcium by FLIPR analysis
Antagonist activity at CXCR2 receptor in HEK293 cells assessed as mobilization of intracellular calcium by FLIPR analysisAntagonist activity at CXCR2 receptor in HEK293 cells assessed as mobilization of intracellular calcium by FLIPR analysis
Antagonist activity at CXCR2 receptor in HEK293 cells assessed as mobilization of intracellular calcium by FLIPR analysisAntagonist activity at CXCR2 receptor in HEK293 cells assessed as mobilization of intracellular calcium by FLIPR analysis
Antagonist activity at CXCR2 receptor in HEK293 cells assessed as mobilization of intracellular calcium by FLIPR analysisAntagonist activity at CXCR2 receptor in HEK293 cells assessed as mobilization of intracellular calcium by FLIPR analysis
Antagonist activity at CXCR2 receptor in HEK293 cells assessed as mobilization of intracellular calcium by FLIPR analysisAntagonist activity at CXCR2 receptor in HEK293 cells assessed as mobilization of intracellular calcium by FLIPR analysis
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor after 60 mins by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor after 60 mins by scintillation counting analysis
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor after 60 mins by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor after 60 mins by scintillation counting analysis
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assayDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assay
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assayDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assay
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assayDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assay
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assayDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assay
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assayDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assay
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assayDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader methodAntagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader method
Antagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader methodAntagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader method
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader methodAntagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader method
Antagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader methodAntagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader method
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Concentration required to inhibit [125I]IL-8 binding towards C-X-C chemokine receptor type 2 of human expressed in CHO cellsConcentration required to inhibit [125I]IL-8 binding towards C-X-C chemokine receptor type 2 of human expressed in CHO cells
Concentration required to inhibit [125I]IL-8 binding towards C-X-C chemokine receptor type 2 of human expressed in CHO cellsConcentration required to inhibit [125I]IL-8 binding towards C-X-C chemokine receptor type 2 of human expressed in CHO cells
Displacement of [125I]IL-8 from CHO cell membranes expressing human CX3C chemokine receptor 2Displacement of [125I]IL-8 from CHO cell membranes expressing human CX3C chemokine receptor 2
Displacement of [125I]IL-8 from CHO cell membranes expressing human CX3C chemokine receptor 2Displacement of [125I]IL-8 from CHO cell membranes expressing human CX3C chemokine receptor 2
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
Antagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysisAntagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysis
Antagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysisAntagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysis
Antagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysisAntagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysis
Antagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysisAntagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysis
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader methodAntagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader method
Antagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader methodAntagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader method
Binding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-induced calcium response incubated for 10 mins followed by addition of CXCL8 AF647 measured by FLIPR Tetra system methodBinding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-induced calcium response incubated for 10 mins followed by addition of CXCL8 AF647 measured by FLIPR Tetra system method
Binding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-induced calcium response incubated for 10 mins followed by addition of CXCL8 AF647 measured by FLIPR Tetra system methodBinding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-induced calcium response incubated for 10 mins followed by addition of CXCL8 AF647 measured by FLIPR Tetra system method
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
Antagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysisAntagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysis
Antagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysisAntagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysis
Antagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader methodAntagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader method
Antagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader methodAntagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader method
Evaluated using an SPA assay with recombinant human [125I]IL-8 and membranes prepared from Sf9 cells expressing human CXCR2Evaluated using an SPA assay with recombinant human [125I]IL-8 and membranes prepared from Sf9 cells expressing human CXCR2
Evaluated using an SPA assay with recombinant human [125I]IL-8 and membranes prepared from Sf9 cells expressing human CXCR2Evaluated using an SPA assay with recombinant human [125I]IL-8 and membranes prepared from Sf9 cells expressing human CXCR2
Inhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranesInhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranes
Inhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranesInhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranes
Antagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader methodAntagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader method
Antagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader methodAntagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader method
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 membranes by SPADisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 membranes by SPA
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 membranes by SPADisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 membranes by SPA
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 membranes by SPADisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 membranes by SPA
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 membranes by SPADisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 membranes by SPA
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 membranes by SPADisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 membranes by SPA
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 membranes by SPADisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 membranes by SPA
Inhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranesInhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranes
Inhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranesInhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranes
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
Binding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-binding incubated for 15 mins followed by addition of CXCL8 AF647 measured after 30 mins by flow cytometry analysisBinding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-binding incubated for 15 mins followed by addition of CXCL8 AF647 measured after 30 mins by flow cytometry analysis
Binding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-binding incubated for 15 mins followed by addition of CXCL8 AF647 measured after 30 mins by flow cytometry analysisBinding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-binding incubated for 15 mins followed by addition of CXCL8 AF647 measured after 30 mins by flow cytometry analysis
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
Inhibition of binding of IL-8 to membranes of cloned CXC chemokine receptor 2 expressed in CHO Cells using [125I]IL-8 radioligandInhibition of binding of IL-8 to membranes of cloned CXC chemokine receptor 2 expressed in CHO Cells using [125I]IL-8 radioligand
Inhibition of binding of IL-8 to membranes of cloned CXC chemokine receptor 2 expressed in CHO Cells using [125I]IL-8 radioligandInhibition of binding of IL-8 to membranes of cloned CXC chemokine receptor 2 expressed in CHO Cells using [125I]IL-8 radioligand
Binding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-induced calcium response incubated for 10 mins followed by addition of CXCL8 AF647 measured by FLIPR Tetra system methodBinding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-induced calcium response incubated for 10 mins followed by addition of CXCL8 AF647 measured by FLIPR Tetra system method
Binding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-induced calcium response incubated for 10 mins followed by addition of CXCL8 AF647 measured by FLIPR Tetra system methodBinding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-induced calcium response incubated for 10 mins followed by addition of CXCL8 AF647 measured by FLIPR Tetra system method
Compound binding was evaluated using an SPA assay with recombinant human [125I]IL-8 and membranes prepared from Sf9 cells expressing human C-X-C chemokine receptor type 2Compound binding was evaluated using an SPA assay with recombinant human [125I]IL-8 and membranes prepared from Sf9 cells expressing human C-X-C chemokine receptor type 2
Compound binding was evaluated using an SPA assay with recombinant human [125I]IL-8 and membranes prepared from Sf9 cells expressing human C-X-C chemokine receptor type 2Compound binding was evaluated using an SPA assay with recombinant human [125I]IL-8 and membranes prepared from Sf9 cells expressing human C-X-C chemokine receptor type 2
Displacement of [125I]IL-8 from CHO cell membranes expressing human CX3C chemokine receptor 2Displacement of [125I]IL-8 from CHO cell membranes expressing human CX3C chemokine receptor 2
Displacement of [125I]IL-8 from CHO cell membranes expressing human CX3C chemokine receptor 2Displacement of [125I]IL-8 from CHO cell membranes expressing human CX3C chemokine receptor 2
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
Displacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assayDisplacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assay
Displacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assayDisplacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assay
Displacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assayDisplacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assay
Displacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assayDisplacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assay
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the beta -arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 beta -arrestin line results in the recruitment of beta -arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation.>>J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with beta -arrestin 2, a beta -arrestin 2 recruitment test for CXCR2 or CXCR1 based on beta -galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the beta -arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 beta -arrestin line results in the recruitment of beta -arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation.>>J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with beta -arrestin 2, a beta -arrestin 2 recruitment test for CXCR2 or CXCR1 based on beta -galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the beta -arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 beta -arrestin line results in the recruitment of beta -arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation.>>J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with beta -arrestin 2, a beta -arrestin 2 recruitment test for CXCR2 or CXCR1 based on beta -galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the beta -arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 beta -arrestin line results in the recruitment of beta -arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation.>>J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with beta -arrestin 2, a beta -arrestin 2 recruitment test for CXCR2 or CXCR1 based on beta -galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
Concentration required to inhibit [125I]IL-8 binding towards C-X-C chemokine receptor type 2 of human expressed in CHO cellsConcentration required to inhibit [125I]IL-8 binding towards C-X-C chemokine receptor type 2 of human expressed in CHO cells
Concentration required to inhibit [125I]IL-8 binding towards C-X-C chemokine receptor type 2 of human expressed in CHO cellsConcentration required to inhibit [125I]IL-8 binding towards C-X-C chemokine receptor type 2 of human expressed in CHO cells
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
Concentration required to inhibit [125I]IL-8 binding towards C-X-C chemokine receptor type 2 of human expressed in CHO cellsConcentration required to inhibit [125I]IL-8 binding towards C-X-C chemokine receptor type 2 of human expressed in CHO cells
Concentration required to inhibit [125I]IL-8 binding towards C-X-C chemokine receptor type 2 of human expressed in CHO cellsConcentration required to inhibit [125I]IL-8 binding towards C-X-C chemokine receptor type 2 of human expressed in CHO cells
Inhibition of CXCR2-mediated chemotaxis in Ba/F3 cells expressing human CXCR2Inhibition of CXCR2-mediated chemotaxis in Ba/F3 cells expressing human CXCR2
Inhibition of CXCR2-mediated chemotaxis in Ba/F3 cells expressing human CXCR2Inhibition of CXCR2-mediated chemotaxis in Ba/F3 cells expressing human CXCR2
Displacement of [125I]-CXCL8 from human CXCR2 transfected in CHO-K1 cell membrane incubated for 45 mins by scintillation proximity binding assayDisplacement of [125I]-CXCL8 from human CXCR2 transfected in CHO-K1 cell membrane incubated for 45 mins by scintillation proximity binding assay
Displacement of [125I]-CXCL8 from human CXCR2 transfected in CHO-K1 cell membrane incubated for 45 mins by scintillation proximity binding assayDisplacement of [125I]-CXCL8 from human CXCR2 transfected in CHO-K1 cell membrane incubated for 45 mins by scintillation proximity binding assay
Displacement of [125I]-CXCL8 from human CXCR2 transfected in CHO-K1 cell membrane incubated for 45 mins by scintillation proximity binding assayDisplacement of [125I]-CXCL8 from human CXCR2 transfected in CHO-K1 cell membrane incubated for 45 mins by scintillation proximity binding assay
Displacement of [125I]-CXCL8 from human CXCR2 transfected in CHO-K1 cell membrane incubated for 45 mins by scintillation proximity binding assayDisplacement of [125I]-CXCL8 from human CXCR2 transfected in CHO-K1 cell membrane incubated for 45 mins by scintillation proximity binding assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader methodAntagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader method
Antagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader methodAntagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader method
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assayDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assay
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assayDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assay
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 membranes by SPADisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 membranes by SPA
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 membranes by SPADisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 membranes by SPA
Displacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assayDisplacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assay
Displacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assayDisplacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assay
Displacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assayDisplacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assay
Displacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assayDisplacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assay
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
Displacement of [125I]IL-8 from CHO cell membranes expressing human CX3C chemokine receptor 2 (CXCR2 filter mat binding assay)Displacement of [125I]IL-8 from CHO cell membranes expressing human CX3C chemokine receptor 2 (CXCR2 filter mat binding assay)
Displacement of [125I]IL-8 from CHO cell membranes expressing human CX3C chemokine receptor 2 (CXCR2 filter mat binding assay)Displacement of [125I]IL-8 from CHO cell membranes expressing human CX3C chemokine receptor 2 (CXCR2 filter mat binding assay)
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
Antagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysisAntagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysis
Antagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysisAntagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysis
Antagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysisAntagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysis
Antagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysisAntagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysis
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Binding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-binding incubated for 15 mins followed by addition of CXCL8 AF647 measured after 30 mins by flow cytometry analysisBinding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-binding incubated for 15 mins followed by addition of CXCL8 AF647 measured after 30 mins by flow cytometry analysis
Binding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-binding incubated for 15 mins followed by addition of CXCL8 AF647 measured after 30 mins by flow cytometry analysisBinding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-binding incubated for 15 mins followed by addition of CXCL8 AF647 measured after 30 mins by flow cytometry analysis
Binding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-binding incubated for 15 mins followed by addition of CXCL8 AF647 measured after 30 mins by flow cytometry analysisBinding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-binding incubated for 15 mins followed by addition of CXCL8 AF647 measured after 30 mins by flow cytometry analysis
Binding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-binding incubated for 15 mins followed by addition of CXCL8 AF647 measured after 30 mins by flow cytometry analysisBinding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-binding incubated for 15 mins followed by addition of CXCL8 AF647 measured after 30 mins by flow cytometry analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assayDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assay
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assayDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assay
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assayDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assay
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assayDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assay
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assayDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assay
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assayDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assay
Binding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-binding incubated for 15 mins followed by addition of CXCL8 AF647 measured after 30 mins by flow cytometry analysisBinding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-binding incubated for 15 mins followed by addition of CXCL8 AF647 measured after 30 mins by flow cytometry analysis
Binding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-binding incubated for 15 mins followed by addition of CXCL8 AF647 measured after 30 mins by flow cytometry analysisBinding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-binding incubated for 15 mins followed by addition of CXCL8 AF647 measured after 30 mins by flow cytometry analysis
Evaluated using an SPA assay with recombinant human [125I]IL-8 and membranes prepared from Sf9 cells expressing human CXCR2Evaluated using an SPA assay with recombinant human [125I]IL-8 and membranes prepared from Sf9 cells expressing human CXCR2
Evaluated using an SPA assay with recombinant human [125I]IL-8 and membranes prepared from Sf9 cells expressing human CXCR2Evaluated using an SPA assay with recombinant human [125I]IL-8 and membranes prepared from Sf9 cells expressing human CXCR2
Evaluated using an SPA assay with recombinant human [125I]IL-8 and membranes prepared from Sf9 cells expressing human CXCR2Evaluated using an SPA assay with recombinant human [125I]IL-8 and membranes prepared from Sf9 cells expressing human CXCR2
Evaluated using an SPA assay with recombinant human [125I]IL-8 and membranes prepared from Sf9 cells expressing human CXCR2Evaluated using an SPA assay with recombinant human [125I]IL-8 and membranes prepared from Sf9 cells expressing human CXCR2
Inhibition of CXCR2 (unknown origin) expressed in PathHunter celline by beta-arrestin assayInhibition of CXCR2 (unknown origin) expressed in PathHunter celline by beta-arrestin assay
Inhibition of CXCR2 (unknown origin) expressed in PathHunter celline by beta-arrestin assayInhibition of CXCR2 (unknown origin) expressed in PathHunter celline by beta-arrestin assay
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 membranes by SPADisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 membranes by SPA
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 membranes by SPADisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 membranes by SPA
Displacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assayDisplacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assay
Displacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assayDisplacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assay
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 membranes by SPADisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 membranes by SPA
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 membranes by SPADisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 membranes by SPA
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assayDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assay
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assayDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assay
Displacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assayDisplacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assay
Displacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assayDisplacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assay
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 membranes by SPADisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 membranes by SPA
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 membranes by SPADisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 membranes by SPA
Displacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assayDisplacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assay
Displacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assayDisplacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assay
Inhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranesInhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranes
Inhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranesInhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranes
Inhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranesInhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranes
Inhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranesInhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranes
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
Displacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cellsDisplacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cells
Displacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cellsDisplacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cells
Displacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cellsDisplacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cells
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
Displacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assayDisplacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assay
Displacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assayDisplacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assay
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the beta -arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 beta -arrestin line results in the recruitment of beta -arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation.>>J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with beta -arrestin 2, a beta -arrestin 2 recruitment test for CXCR2 or CXCR1 based on beta -galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the beta -arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 beta -arrestin line results in the recruitment of beta -arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation.>>J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with beta -arrestin 2, a beta -arrestin 2 recruitment test for CXCR2 or CXCR1 based on beta -galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the beta -arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 beta -arrestin line results in the recruitment of beta -arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation.>>J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with beta -arrestin 2, a beta -arrestin 2 recruitment test for CXCR2 or CXCR1 based on beta -galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the beta -arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 beta -arrestin line results in the recruitment of beta -arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation.>>J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with beta -arrestin 2, a beta -arrestin 2 recruitment test for CXCR2 or CXCR1 based on beta -galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
Binding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-induced calcium response incubated for 10 mins followed by addition of CXCL8 AF647 measured by FLIPR Tetra system methodBinding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-induced calcium response incubated for 10 mins followed by addition of CXCL8 AF647 measured by FLIPR Tetra system method
Binding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-induced calcium response incubated for 10 mins followed by addition of CXCL8 AF647 measured by FLIPR Tetra system methodBinding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-induced calcium response incubated for 10 mins followed by addition of CXCL8 AF647 measured by FLIPR Tetra system method
Antagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysisAntagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysis
Antagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysisAntagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysis
Antagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysisAntagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysis
Antagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysisAntagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysis
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader methodAntagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader method
Antagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader methodAntagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader method
Antagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader methodAntagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader method
Antagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader methodAntagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader method
Antagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader methodAntagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader method
Antagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader methodAntagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader method
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader methodAntagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader method
Antagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader methodAntagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader method
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assayDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assay
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assayDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assay
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
Displacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assayDisplacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assay
Displacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assayDisplacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assay
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
Displacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cellsDisplacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cells
Displacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cellsDisplacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cells
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Displacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assayDisplacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assay
Displacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assayDisplacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assay
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
Inhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranesInhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranes
Inhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranesInhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranes
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
Binding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-binding incubated for 15 mins followed by addition of CXCL8 AF647 measured after 30 mins by flow cytometry analysisBinding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-binding incubated for 15 mins followed by addition of CXCL8 AF647 measured after 30 mins by flow cytometry analysis
Binding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-binding incubated for 15 mins followed by addition of CXCL8 AF647 measured after 30 mins by flow cytometry analysisBinding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-binding incubated for 15 mins followed by addition of CXCL8 AF647 measured after 30 mins by flow cytometry analysis
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Displacement of [125I]IL-8 from human CXCR2 expressed in mouse BaF3 cells by liquid scintillation countingDisplacement of [125I]IL-8 from human CXCR2 expressed in mouse BaF3 cells by liquid scintillation counting
Displacement of [125I]IL-8 from human CXCR2 expressed in mouse BaF3 cells by liquid scintillation countingDisplacement of [125I]IL-8 from human CXCR2 expressed in mouse BaF3 cells by liquid scintillation counting
Displacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assayDisplacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assay
Displacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assayDisplacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assay
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
Antagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysisAntagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysis
Antagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysisAntagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysis
Antagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysisAntagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysis
Antagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysisAntagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysis
Antagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysisAntagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysis
Antagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysisAntagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysis
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
Antagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader methodAntagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader method
Antagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader methodAntagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader method
Antagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader methodAntagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader method
Antagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader methodAntagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader method
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assayDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assay
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assayDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assay
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assayDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assay
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assayDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assay
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Inhibition of CXCR2-mediated chemotaxis in Ba/F3 cells expressing human CXCR2Inhibition of CXCR2-mediated chemotaxis in Ba/F3 cells expressing human CXCR2
Inhibition of CXCR2-mediated chemotaxis in Ba/F3 cells expressing human CXCR2Inhibition of CXCR2-mediated chemotaxis in Ba/F3 cells expressing human CXCR2
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
Displacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cellsDisplacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cells
Displacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cellsDisplacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cells
Displacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cellsDisplacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cells
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
Inhibition of binding of IL-8 to membranes of cloned CXC chemokine receptor 2 expressed in CHO Cells using [125I]IL-8 radioligandInhibition of binding of IL-8 to membranes of cloned CXC chemokine receptor 2 expressed in CHO Cells using [125I]IL-8 radioligand
Inhibition of binding of IL-8 to membranes of cloned CXC chemokine receptor 2 expressed in CHO Cells using [125I]IL-8 radioligandInhibition of binding of IL-8 to membranes of cloned CXC chemokine receptor 2 expressed in CHO Cells using [125I]IL-8 radioligand
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
Displacement of [125I]GRO-alpha from human recombinant CXCR2 receptor expressed in CHO cellsDisplacement of [125I]GRO-alpha from human recombinant CXCR2 receptor expressed in CHO cells
Displacement of [125I]GRO-alpha from human recombinant CXCR2 receptor expressed in CHO cellsDisplacement of [125I]GRO-alpha from human recombinant CXCR2 receptor expressed in CHO cells
Inhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranesInhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranes
Inhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranesInhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranes
Inhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranesInhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranes
Inhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranesInhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranes
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
Displacement of [125I]CXCL1 from CXCR2 receptor in human PMN assessed as myeloperoxidase releaseDisplacement of [125I]CXCL1 from CXCR2 receptor in human PMN assessed as myeloperoxidase release
Displacement of [125I]CXCL1 from CXCR2 receptor in human PMN assessed as myeloperoxidase releaseDisplacement of [125I]CXCL1 from CXCR2 receptor in human PMN assessed as myeloperoxidase release
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the beta -arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 beta -arrestin line results in the recruitment of beta -arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation.>>J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with beta -arrestin 2, a beta -arrestin 2 recruitment test for CXCR2 or CXCR1 based on beta -galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the beta -arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 beta -arrestin line results in the recruitment of beta -arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation.>>J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with beta -arrestin 2, a beta -arrestin 2 recruitment test for CXCR2 or CXCR1 based on beta -galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the beta -arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 beta -arrestin line results in the recruitment of beta -arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation.>>J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with beta -arrestin 2, a beta -arrestin 2 recruitment test for CXCR2 or CXCR1 based on beta -galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the beta -arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 beta -arrestin line results in the recruitment of beta -arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation.>>J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with beta -arrestin 2, a beta -arrestin 2 recruitment test for CXCR2 or CXCR1 based on beta -galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
Displacement of 125I-IL-8 from CXCR2 (unknown origin) expressed in CHO cell membrane incubated for 1 hr by liquid scintillation counter analysisDisplacement of 125I-IL-8 from CXCR2 (unknown origin) expressed in CHO cell membrane incubated for 1 hr by liquid scintillation counter analysis
Displacement of 125I-IL-8 from CXCR2 (unknown origin) expressed in CHO cell membrane incubated for 1 hr by liquid scintillation counter analysisDisplacement of 125I-IL-8 from CXCR2 (unknown origin) expressed in CHO cell membrane incubated for 1 hr by liquid scintillation counter analysis
Displacement of 125I-IL-8 from CXCR2 (unknown origin) expressed in CHO cell membrane incubated for 1 hr by liquid scintillation counter analysisDisplacement of 125I-IL-8 from CXCR2 (unknown origin) expressed in CHO cell membrane incubated for 1 hr by liquid scintillation counter analysis
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Inhibition of binding of IL-8 to membranes of cloned CXC chemokine receptor 2 expressed in CHO Cells using [125I]IL-8 radioligandInhibition of binding of IL-8 to membranes of cloned CXC chemokine receptor 2 expressed in CHO Cells using [125I]IL-8 radioligand
Inhibition of binding of IL-8 to membranes of cloned CXC chemokine receptor 2 expressed in CHO Cells using [125I]IL-8 radioligandInhibition of binding of IL-8 to membranes of cloned CXC chemokine receptor 2 expressed in CHO Cells using [125I]IL-8 radioligand
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assayDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assay
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assayDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assay
Displacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assayDisplacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assay
Displacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assayDisplacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assay
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
Inhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranesInhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranes
Inhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranesInhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranes
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Displacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cellsDisplacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cells
Displacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cellsDisplacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cells
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
Displacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cellsDisplacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cells
Displacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cellsDisplacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cells
Displacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assayDisplacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assay
Displacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assayDisplacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assay
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
Inhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranesInhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranes
Inhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranesInhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranes
Displacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cellsDisplacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cells
Displacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cellsDisplacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cells
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Binding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-induced calcium response incubated for 10 mins followed by addition of CXCL8 AF647 measured by FLIPR Tetra system methodBinding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-induced calcium response incubated for 10 mins followed by addition of CXCL8 AF647 measured by FLIPR Tetra system method
Binding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-induced calcium response incubated for 10 mins followed by addition of CXCL8 AF647 measured by FLIPR Tetra system methodBinding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-induced calcium response incubated for 10 mins followed by addition of CXCL8 AF647 measured by FLIPR Tetra system method
Binding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-induced calcium response incubated for 10 mins followed by addition of CXCL8 AF647 measured by FLIPR Tetra system methodBinding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-induced calcium response incubated for 10 mins followed by addition of CXCL8 AF647 measured by FLIPR Tetra system method
Binding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-induced calcium response incubated for 10 mins followed by addition of CXCL8 AF647 measured by FLIPR Tetra system methodBinding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-induced calcium response incubated for 10 mins followed by addition of CXCL8 AF647 measured by FLIPR Tetra system method
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
Inhibition of binding of IL-8 to membranes of cloned CXC chemokine receptor 2 expressed in CHO Cells using [125I]IL-8 radioligandInhibition of binding of IL-8 to membranes of cloned CXC chemokine receptor 2 expressed in CHO Cells using [125I]IL-8 radioligand
Inhibition of binding of IL-8 to membranes of cloned CXC chemokine receptor 2 expressed in CHO Cells using [125I]IL-8 radioligandInhibition of binding of IL-8 to membranes of cloned CXC chemokine receptor 2 expressed in CHO Cells using [125I]IL-8 radioligand
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader methodAntagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader method
Antagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader methodAntagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader method
Antagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader methodAntagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader method
Antagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader methodAntagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader method
Antagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader methodAntagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader method
Antagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader methodAntagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader method
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
Binding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-binding incubated for 15 mins followed by addition of CXCL8 AF647 measured after 30 mins by flow cytometry analysisBinding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-binding incubated for 15 mins followed by addition of CXCL8 AF647 measured after 30 mins by flow cytometry analysis
Binding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-binding incubated for 15 mins followed by addition of CXCL8 AF647 measured after 30 mins by flow cytometry analysisBinding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-binding incubated for 15 mins followed by addition of CXCL8 AF647 measured after 30 mins by flow cytometry analysis
Displacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assayDisplacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assay
Displacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assayDisplacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assay
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
Antagonist activity at CXCR2 (unknown origin) expressed in human U2OS cells cp-expressing betaarrestin-2/TEV protease and beta lactamase assessed as effect on beta-arrestin2 recruitment preincubated for 30 mins followed by IL-8 addition and further incubated for 5 hrs subsequently adding CCF4-AM staining and measured after 2 hrs by Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in human U2OS cells cp-expressing betaarrestin-2/TEV protease and beta lactamase assessed as effect on beta-arrestin2 recruitment preincubated for 30 mins followed by IL-8 addition and further incubated for 5 hrs subsequently adding CCF4-AM staining and measured after 2 hrs by Tango assay
Antagonist activity at CXCR2 (unknown origin) expressed in human U2OS cells cp-expressing betaarrestin-2/TEV protease and beta lactamase assessed as effect on beta-arrestin2 recruitment preincubated for 30 mins followed by IL-8 addition and further incubated for 5 hrs subsequently adding CCF4-AM staining and measured after 2 hrs by Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in human U2OS cells cp-expressing betaarrestin-2/TEV protease and beta lactamase assessed as effect on beta-arrestin2 recruitment preincubated for 30 mins followed by IL-8 addition and further incubated for 5 hrs subsequently adding CCF4-AM staining and measured after 2 hrs by Tango assay
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assayDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assay
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assayDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assay
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
Antagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysisAntagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysis
Antagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysisAntagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysis
Antagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysisAntagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysis
Antagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysisAntagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysis
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader methodAntagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader method
Antagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader methodAntagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader method
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
Displacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assayDisplacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assay
Displacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assayDisplacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assay
Displacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assayDisplacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assay
Displacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assayDisplacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assay
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
Binding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-induced calcium response incubated for 10 mins followed by addition of CXCL8 AF647 measured by FLIPR Tetra system methodBinding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-induced calcium response incubated for 10 mins followed by addition of CXCL8 AF647 measured by FLIPR Tetra system method
Binding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-induced calcium response incubated for 10 mins followed by addition of CXCL8 AF647 measured by FLIPR Tetra system methodBinding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-induced calcium response incubated for 10 mins followed by addition of CXCL8 AF647 measured by FLIPR Tetra system method
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 membranes by SPADisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 membranes by SPA
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 membranes by SPADisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 membranes by SPA
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
Binding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-binding incubated for 15 mins followed by addition of CXCL8 AF647 measured after 30 mins by flow cytometry analysisBinding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-binding incubated for 15 mins followed by addition of CXCL8 AF647 measured after 30 mins by flow cytometry analysis
Binding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-binding incubated for 15 mins followed by addition of CXCL8 AF647 measured after 30 mins by flow cytometry analysisBinding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-binding incubated for 15 mins followed by addition of CXCL8 AF647 measured after 30 mins by flow cytometry analysis
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assayDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assay
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assayDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assay
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assayDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assay
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assayDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assay
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
Evaluated using an SPA assay with recombinant human [125I]IL-8 and membranes prepared from Sf9 cells expressing human CXCR2Evaluated using an SPA assay with recombinant human [125I]IL-8 and membranes prepared from Sf9 cells expressing human CXCR2
Evaluated using an SPA assay with recombinant human [125I]IL-8 and membranes prepared from Sf9 cells expressing human CXCR2Evaluated using an SPA assay with recombinant human [125I]IL-8 and membranes prepared from Sf9 cells expressing human CXCR2
Inhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranesInhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranes
Inhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranesInhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranes
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assayDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assay
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assayDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Displacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cellsDisplacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cells
Displacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cellsDisplacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cells
Displacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cellsDisplacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cells
Displacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cellsDisplacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cells
Displacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cellsDisplacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cells
Displacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cellsDisplacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cells
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Antagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubationAntagonist activity at human recombinant Gal4-VP16 fused-CXCR2 assessed as inhibition of CXCL1-mediated lactamase reporter gene expression after overnight incubation
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
Displacement of [125I]IL-8 from CXCR2 in human neutrophils incubated for 3 hrs by gamma counting methodDisplacement of [125I]IL-8 from CXCR2 in human neutrophils incubated for 3 hrs by gamma counting method
Displacement of [125I]IL-8 from CXCR2 in human neutrophils incubated for 3 hrs by gamma counting methodDisplacement of [125I]IL-8 from CXCR2 in human neutrophils incubated for 3 hrs by gamma counting method
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
Inhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranesInhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranes
Inhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranesInhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranes
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
Antagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysisAntagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysis
Antagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysisAntagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysis
Antagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysisAntagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysis
Antagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysisAntagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysis
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader methodAntagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader method
Antagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader methodAntagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader method
Antagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader methodAntagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader method
Antagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader methodAntagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader method
Antagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader methodAntagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader method
Antagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader methodAntagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader method
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
Displacement of [125I]IL-8 from CHO cell membranes expressing human CX3C chemokine receptor 2Displacement of [125I]IL-8 from CHO cell membranes expressing human CX3C chemokine receptor 2
Displacement of [125I]IL-8 from CHO cell membranes expressing human CX3C chemokine receptor 2Displacement of [125I]IL-8 from CHO cell membranes expressing human CX3C chemokine receptor 2
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
Evaluated using an SPA assay with recombinant human [125I]IL-8 and membranes prepared from Sf9 cells expressing human CXCR2Evaluated using an SPA assay with recombinant human [125I]IL-8 and membranes prepared from Sf9 cells expressing human CXCR2
Evaluated using an SPA assay with recombinant human [125I]IL-8 and membranes prepared from Sf9 cells expressing human CXCR2Evaluated using an SPA assay with recombinant human [125I]IL-8 and membranes prepared from Sf9 cells expressing human CXCR2
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
Displacement of [125I]IL-8 from CXCR2 (unknown origin) stably expressed in CHO cellsDisplacement of [125I]IL-8 from CXCR2 (unknown origin) stably expressed in CHO cells
Displacement of [125I]IL-8 from CXCR2 (unknown origin) stably expressed in CHO cellsDisplacement of [125I]IL-8 from CXCR2 (unknown origin) stably expressed in CHO cells
Displacement of [125I]IL-8 from CXCR2 (unknown origin) stably expressed in CHO cellsDisplacement of [125I]IL-8 from CXCR2 (unknown origin) stably expressed in CHO cells
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
Inhibition of binding of IL-8 to membranes of cloned CXC chemokine receptor 2 expressed in CHO Cells using [125I]IL-8 radioligandInhibition of binding of IL-8 to membranes of cloned CXC chemokine receptor 2 expressed in CHO Cells using [125I]IL-8 radioligand
Inhibition of binding of IL-8 to membranes of cloned CXC chemokine receptor 2 expressed in CHO Cells using [125I]IL-8 radioligandInhibition of binding of IL-8 to membranes of cloned CXC chemokine receptor 2 expressed in CHO Cells using [125I]IL-8 radioligand
Inhibition of binding of IL-8 to membranes of cloned CXC chemokine receptor 2 expressed in CHO Cells using [125I]IL-8 radioligandInhibition of binding of IL-8 to membranes of cloned CXC chemokine receptor 2 expressed in CHO Cells using [125I]IL-8 radioligand
Displacement of [125I]IL-8 from human CXCR2 expressed in mouse BaF3 cells by liquid scintillation countingDisplacement of [125I]IL-8 from human CXCR2 expressed in mouse BaF3 cells by liquid scintillation counting
Displacement of [125I]IL-8 from human CXCR2 expressed in mouse BaF3 cells by liquid scintillation countingDisplacement of [125I]IL-8 from human CXCR2 expressed in mouse BaF3 cells by liquid scintillation counting
Displacement of [125I]GRO-alpha from human recombinant CXCR2 receptor expressed in CHO cellsDisplacement of [125I]GRO-alpha from human recombinant CXCR2 receptor expressed in CHO cells
Displacement of [125I]GRO-alpha from human recombinant CXCR2 receptor expressed in CHO cellsDisplacement of [125I]GRO-alpha from human recombinant CXCR2 receptor expressed in CHO cells
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assayDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assay
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assayDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assay
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assayDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assay
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assayDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assay
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 membranes by SPADisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 membranes by SPA
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 membranes by SPADisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 membranes by SPA
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
Inhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranesInhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranes
Inhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranesInhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranes
Inhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranesInhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranes
Inhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranesInhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranes
Inhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranesInhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranes
Inhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranesInhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranes
Inhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranesInhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranes
Inhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranesInhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranes
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
Displacement of [125I]CXCL1 from CXCR2 receptor in human PMN assessed as myeloperoxidase releaseDisplacement of [125I]CXCL1 from CXCR2 receptor in human PMN assessed as myeloperoxidase release
Displacement of [125I]CXCL1 from CXCR2 receptor in human PMN assessed as myeloperoxidase releaseDisplacement of [125I]CXCL1 from CXCR2 receptor in human PMN assessed as myeloperoxidase release
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
Antagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysisAntagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysis
Antagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysisAntagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysis
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader methodAntagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader method
Antagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader methodAntagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader method
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 membranes by SPADisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 membranes by SPA
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 membranes by SPADisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 membranes by SPA
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
Antagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysisAntagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysis
Antagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysisAntagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysis
Antagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysisAntagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysis
Antagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysisAntagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysis
Antagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysisAntagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysis
Antagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysisAntagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysis
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assayDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assay
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assayDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assay
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Inhibition of CXCR2-mediated chemotaxis in Ba/F3 cells expressing human CXCR2Inhibition of CXCR2-mediated chemotaxis in Ba/F3 cells expressing human CXCR2
Inhibition of CXCR2-mediated chemotaxis in Ba/F3 cells expressing human CXCR2Inhibition of CXCR2-mediated chemotaxis in Ba/F3 cells expressing human CXCR2
Displacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cellsDisplacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cells
Displacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cellsDisplacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cells
Antagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysisAntagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysis
Antagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysisAntagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysis
Displacement of [125I]-GRO-alpha from human recombinant CXCR2 receptor expressed in CHO cell membranesDisplacement of [125I]-GRO-alpha from human recombinant CXCR2 receptor expressed in CHO cell membranes
Displacement of [125I]-GRO-alpha from human recombinant CXCR2 receptor expressed in CHO cell membranesDisplacement of [125I]-GRO-alpha from human recombinant CXCR2 receptor expressed in CHO cell membranes
Displacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assayDisplacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assay
Displacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assayDisplacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assay
Evaluated using an SPA assay with recombinant human [125I]IL-8 and membranes prepared from Sf9 cells expressing human CXCR2Evaluated using an SPA assay with recombinant human [125I]IL-8 and membranes prepared from Sf9 cells expressing human CXCR2
Evaluated using an SPA assay with recombinant human [125I]IL-8 and membranes prepared from Sf9 cells expressing human CXCR2Evaluated using an SPA assay with recombinant human [125I]IL-8 and membranes prepared from Sf9 cells expressing human CXCR2
Inhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranesInhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranes
Inhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranesInhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranes
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
Antagonist activity at CXCR2 in human neutrophils assessed as reduction in GRO-alpha mediated chemotaxisAntagonist activity at CXCR2 in human neutrophils assessed as reduction in GRO-alpha mediated chemotaxis
Antagonist activity at CXCR2 in human neutrophils assessed as reduction in GRO-alpha mediated chemotaxisAntagonist activity at CXCR2 in human neutrophils assessed as reduction in GRO-alpha mediated chemotaxis
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Inhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranesInhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranes
Inhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranesInhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranes
Inhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranesInhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranes
Inhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranesInhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranes
Binding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-binding incubated for 15 mins followed by addition of CXCL8 AF647 measured after 30 mins by flow cytometry analysisBinding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-binding incubated for 15 mins followed by addition of CXCL8 AF647 measured after 30 mins by flow cytometry analysis
Binding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-binding incubated for 15 mins followed by addition of CXCL8 AF647 measured after 30 mins by flow cytometry analysisBinding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-binding incubated for 15 mins followed by addition of CXCL8 AF647 measured after 30 mins by flow cytometry analysis
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Binding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-binding incubated for 15 mins followed by addition of CXCL8 AF647 measured after 30 mins by flow cytometry analysisBinding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-binding incubated for 15 mins followed by addition of CXCL8 AF647 measured after 30 mins by flow cytometry analysis
Binding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-binding incubated for 15 mins followed by addition of CXCL8 AF647 measured after 30 mins by flow cytometry analysisBinding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-binding incubated for 15 mins followed by addition of CXCL8 AF647 measured after 30 mins by flow cytometry analysis
Displacement of [125I]-GRO-alpha from human recombinant CXCR2 receptor expressed in CHO cell membranesDisplacement of [125I]-GRO-alpha from human recombinant CXCR2 receptor expressed in CHO cell membranes
Displacement of [125I]-GRO-alpha from human recombinant CXCR2 receptor expressed in CHO cell membranesDisplacement of [125I]-GRO-alpha from human recombinant CXCR2 receptor expressed in CHO cell membranes
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
Binding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-binding incubated for 15 mins followed by addition of CXCL8 AF647 measured after 30 mins by flow cytometry analysisBinding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-binding incubated for 15 mins followed by addition of CXCL8 AF647 measured after 30 mins by flow cytometry analysis
Binding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-binding incubated for 15 mins followed by addition of CXCL8 AF647 measured after 30 mins by flow cytometry analysisBinding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-binding incubated for 15 mins followed by addition of CXCL8 AF647 measured after 30 mins by flow cytometry analysis
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
Inhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranesInhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranes
Inhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranesInhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranes
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assayDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assay
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assayDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assay
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Inhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranesInhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranes
Inhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranesInhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranes
Inhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranesInhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranes
Inhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranesInhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranes
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
Evaluated using an SPA assay with recombinant human [125I]IL-8 and membranes prepared from Sf9 cells expressing human CXCR2Evaluated using an SPA assay with recombinant human [125I]IL-8 and membranes prepared from Sf9 cells expressing human CXCR2
Evaluated using an SPA assay with recombinant human [125I]IL-8 and membranes prepared from Sf9 cells expressing human CXCR2Evaluated using an SPA assay with recombinant human [125I]IL-8 and membranes prepared from Sf9 cells expressing human CXCR2
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 membranes by SPADisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 membranes by SPA
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 membranes by SPADisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 membranes by SPA
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assayDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assay
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assayDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assay
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assayDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assay
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assayDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assay
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assayDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assay
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assayDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assay
Displacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cellsDisplacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cells
Displacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cellsDisplacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cells
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Displacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cellsDisplacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cells
Displacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cellsDisplacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cells
Displacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cellsDisplacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cells
Displacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cellsDisplacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cells
Antagonist activity at CXCR2 in C57 mouse whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 1 hr followed by GROalpha stimulation measured after 10 mins by FITC-staining based FACS analysisAntagonist activity at CXCR2 in C57 mouse whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 1 hr followed by GROalpha stimulation measured after 10 mins by FITC-staining based FACS analysis
Antagonist activity at CXCR2 in C57 mouse whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 1 hr followed by GROalpha stimulation measured after 10 mins by FITC-staining based FACS analysisAntagonist activity at CXCR2 in C57 mouse whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 1 hr followed by GROalpha stimulation measured after 10 mins by FITC-staining based FACS analysis
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 membranes by SPADisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 membranes by SPA
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 membranes by SPADisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 membranes by SPA
Displacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assayDisplacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assay
Displacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assayDisplacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
Antagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysisAntagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysis
Antagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysisAntagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysis
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
Displacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assayDisplacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assay
Displacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assayDisplacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assay
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
Displacement of [125I]IL-8 from human CXCR2 expressed in mouse BaF3 cells by liquid scintillation countingDisplacement of [125I]IL-8 from human CXCR2 expressed in mouse BaF3 cells by liquid scintillation counting
Displacement of [125I]IL-8 from human CXCR2 expressed in mouse BaF3 cells by liquid scintillation countingDisplacement of [125I]IL-8 from human CXCR2 expressed in mouse BaF3 cells by liquid scintillation counting
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
Displacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assayDisplacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assay
Displacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assayDisplacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assay
Inhibition of binding of IL-8 to membranes of cloned CXC chemokine receptor 2 expressed in CHO Cells using [125I]IL-8 radioligandInhibition of binding of IL-8 to membranes of cloned CXC chemokine receptor 2 expressed in CHO Cells using [125I]IL-8 radioligand
Inhibition of binding of IL-8 to membranes of cloned CXC chemokine receptor 2 expressed in CHO Cells using [125I]IL-8 radioligandInhibition of binding of IL-8 to membranes of cloned CXC chemokine receptor 2 expressed in CHO Cells using [125I]IL-8 radioligand
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
Displacement of [125I]IL-8 from CXCR2 (unknown origin) stably expressed in CHO cellsDisplacement of [125I]IL-8 from CXCR2 (unknown origin) stably expressed in CHO cells
Displacement of [125I]IL-8 from CXCR2 (unknown origin) stably expressed in CHO cellsDisplacement of [125I]IL-8 from CXCR2 (unknown origin) stably expressed in CHO cells
Displacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cellsDisplacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cells
Displacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cellsDisplacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cells
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Inhibition of binding of IL-8 to membranes of cloned CXC chemokine receptor 2 expressed in CHO Cells using [125I]IL-8 radioligandInhibition of binding of IL-8 to membranes of cloned CXC chemokine receptor 2 expressed in CHO Cells using [125I]IL-8 radioligand
Inhibition of binding of IL-8 to membranes of cloned CXC chemokine receptor 2 expressed in CHO Cells using [125I]IL-8 radioligandInhibition of binding of IL-8 to membranes of cloned CXC chemokine receptor 2 expressed in CHO Cells using [125I]IL-8 radioligand
Displacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cellsDisplacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cells
Displacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cellsDisplacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cells
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader methodAntagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader method
Antagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader methodAntagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader method
Antagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader methodAntagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader method
Antagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader methodAntagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader method
Displacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cellsDisplacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cells
Displacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cellsDisplacement of [125I]hCXCL8 from human CXCR2 receptor expressed in BaF3 cells
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assayDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assay
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assayDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assay
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
Concentration required to inhibit [125I]IL-8 binding towards C-X-C chemokine receptor type 2 of human expressed in CHO cellsConcentration required to inhibit [125I]IL-8 binding towards C-X-C chemokine receptor type 2 of human expressed in CHO cells
Concentration required to inhibit [125I]IL-8 binding towards C-X-C chemokine receptor type 2 of human expressed in CHO cellsConcentration required to inhibit [125I]IL-8 binding towards C-X-C chemokine receptor type 2 of human expressed in CHO cells
Antagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysisAntagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysis
Antagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysisAntagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysis
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assayDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assay
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assayDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assay
Antagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysisAntagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysis
Antagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysisAntagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysis
Antagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysisAntagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysis
Antagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysisAntagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysis
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader methodAntagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader method
Antagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader methodAntagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader method
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
Antagonist activity at CXCR2 in human U2OS cells assessed as effect on beta-arrestin2 recruitment by CCF4-AM staining based Tango assayAntagonist activity at CXCR2 in human U2OS cells assessed as effect on beta-arrestin2 recruitment by CCF4-AM staining based Tango assay
Antagonist activity at CXCR2 in human U2OS cells assessed as effect on beta-arrestin2 recruitment by CCF4-AM staining based Tango assayAntagonist activity at CXCR2 in human U2OS cells assessed as effect on beta-arrestin2 recruitment by CCF4-AM staining based Tango assay
Antagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader methodAntagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader method
Antagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader methodAntagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader method
Displacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assayDisplacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assay
Displacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assayDisplacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assay
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
Displacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assayDisplacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assay
Displacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assayDisplacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assay
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
Displacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assayDisplacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assay
Displacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assayDisplacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assay
Binding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-induced calcium response incubated for 10 mins followed by addition of CXCL8 AF647 measured by FLIPR Tetra system methodBinding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-induced calcium response incubated for 10 mins followed by addition of CXCL8 AF647 measured by FLIPR Tetra system method
Binding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-induced calcium response incubated for 10 mins followed by addition of CXCL8 AF647 measured by FLIPR Tetra system methodBinding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-induced calcium response incubated for 10 mins followed by addition of CXCL8 AF647 measured by FLIPR Tetra system method
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Inhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranesInhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranes
Inhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranesInhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranes
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
Antagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysisAntagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysis
Antagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysisAntagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysis
Antagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysisAntagonist activity at CXCR2 in human whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 15 mins followed by GROalpha stimulation measured after 15 mins by FITC-staining based FACS analysis
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
Concentration required to inhibit [125I]IL-8 binding towards C-X-C chemokine receptor type 2 of human expressed in CHO cellsConcentration required to inhibit [125I]IL-8 binding towards C-X-C chemokine receptor type 2 of human expressed in CHO cells
Concentration required to inhibit [125I]IL-8 binding towards C-X-C chemokine receptor type 2 of human expressed in CHO cellsConcentration required to inhibit [125I]IL-8 binding towards C-X-C chemokine receptor type 2 of human expressed in CHO cells
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assayDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assay
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assayDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assay
Antagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader methodAntagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader method
Antagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader methodAntagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader method
Antagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader methodAntagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader method
Antagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader methodAntagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader method
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 membranes by SPADisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 membranes by SPA
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 membranes by SPADisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 membranes by SPA
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
Displacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assayDisplacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assay
Displacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assayDisplacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assay
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
Inhibition of CXCR2 (unknown origin) expressed in PathHunter celline by beta-arrestin assayInhibition of CXCR2 (unknown origin) expressed in PathHunter celline by beta-arrestin assay
Inhibition of CXCR2 (unknown origin) expressed in PathHunter celline by beta-arrestin assayInhibition of CXCR2 (unknown origin) expressed in PathHunter celline by beta-arrestin assay
Inhibition of CXCR2 (unknown origin) expressed in PathHunter celline by beta-arrestin assayInhibition of CXCR2 (unknown origin) expressed in PathHunter celline by beta-arrestin assay
Inhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranesInhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranes
Inhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranesInhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranes
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
Antagonist activity at CXCR2 (unknown origin) expressed in human U2OS cells cp-expressing betaarrestin-2/TEV protease and beta lactamase assessed as effect on beta-arrestin2 recruitment preincubated for 30 mins followed by IL-8 addition and further incubated for 5 hrs subsequently adding CCF4-AM staining and measured after 2 hrs by Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in human U2OS cells cp-expressing betaarrestin-2/TEV protease and beta lactamase assessed as effect on beta-arrestin2 recruitment preincubated for 30 mins followed by IL-8 addition and further incubated for 5 hrs subsequently adding CCF4-AM staining and measured after 2 hrs by Tango assay
Antagonist activity at CXCR2 (unknown origin) expressed in human U2OS cells cp-expressing betaarrestin-2/TEV protease and beta lactamase assessed as effect on beta-arrestin2 recruitment preincubated for 30 mins followed by IL-8 addition and further incubated for 5 hrs subsequently adding CCF4-AM staining and measured after 2 hrs by Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in human U2OS cells cp-expressing betaarrestin-2/TEV protease and beta lactamase assessed as effect on beta-arrestin2 recruitment preincubated for 30 mins followed by IL-8 addition and further incubated for 5 hrs subsequently adding CCF4-AM staining and measured after 2 hrs by Tango assay
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
Inhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranesInhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranes
Inhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranesInhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranes
Binding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-induced calcium response incubated for 10 mins followed by addition of CXCL8 AF647 measured by FLIPR Tetra system methodBinding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-induced calcium response incubated for 10 mins followed by addition of CXCL8 AF647 measured by FLIPR Tetra system method
Binding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-induced calcium response incubated for 10 mins followed by addition of CXCL8 AF647 measured by FLIPR Tetra system methodBinding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-induced calcium response incubated for 10 mins followed by addition of CXCL8 AF647 measured by FLIPR Tetra system method
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
Binding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-induced calcium response incubated for 10 mins followed by addition of CXCL8 AF647 measured by FLIPR Tetra system methodBinding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-induced calcium response incubated for 10 mins followed by addition of CXCL8 AF647 measured by FLIPR Tetra system method
Binding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-induced calcium response incubated for 10 mins followed by addition of CXCL8 AF647 measured by FLIPR Tetra system methodBinding affinity to human CXCR2 expressed in human U87 cells assessed as inhibition of CXCL8 AF647-induced calcium response incubated for 10 mins followed by addition of CXCL8 AF647 measured by FLIPR Tetra system method
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assayAntagonist activity at TEV protease cleavage site linked GAL4-VP16-fused recombinant human CXCR2 expressed in cells assessed as inhibition of TEV protease tagged human CXCL1-stimulated beta-arrestin recruitment by CCF4-AM-fluorescence based FRET assay
Antagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader methodAntagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader method
Antagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader methodAntagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader method
Antagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader methodAntagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader method
Antagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader methodAntagonist activity at human recombinant CXCR2 assessed as inhibition of CXCL1-induced neutrophil chemotaxis after 45 mins by calcein-AM dye based plate reader method
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
Displacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysisDisplacement of [125I]IL-8 from human recombinant CXCR2 receptor expressed in HEK293 cells by scintillation counting analysis
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
Antagonist activity at CXCR2 in C57 mouse whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 1 hr followed by GROalpha stimulation measured after 10 mins by FITC-staining based FACS analysisAntagonist activity at CXCR2 in C57 mouse whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 1 hr followed by GROalpha stimulation measured after 10 mins by FITC-staining based FACS analysis
Antagonist activity at CXCR2 in C57 mouse whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 1 hr followed by GROalpha stimulation measured after 10 mins by FITC-staining based FACS analysisAntagonist activity at CXCR2 in C57 mouse whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 1 hr followed by GROalpha stimulation measured after 10 mins by FITC-staining based FACS analysis
Antagonist activity at CXCR2 in C57 mouse whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 1 hr followed by GROalpha stimulation measured after 10 mins by FITC-staining based FACS analysisAntagonist activity at CXCR2 in C57 mouse whole blood assessed as inhibition of GROalpha-stimulated CD11b upregulation preincubated for 1 hr followed by GROalpha stimulation measured after 10 mins by FITC-staining based FACS analysis
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
Inhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranesInhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranes
Inhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranesInhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranes
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
Inhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranesInhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranes
Inhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranesInhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranes
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assayDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assay
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assayDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 cells by SPA assay
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
Inhibition of binding of IL-8 to membranes of cloned CXC chemokine receptor 2 expressed in CHO Cells using [125I]IL-8 radioligandInhibition of binding of IL-8 to membranes of cloned CXC chemokine receptor 2 expressed in CHO Cells using [125I]IL-8 radioligand
Inhibition of binding of IL-8 to membranes of cloned CXC chemokine receptor 2 expressed in CHO Cells using [125I]IL-8 radioligandInhibition of binding of IL-8 to membranes of cloned CXC chemokine receptor 2 expressed in CHO Cells using [125I]IL-8 radioligand
Displacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assayDisplacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assay
Displacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assayDisplacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assay
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
Antagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assayAntagonist activity at CXCR2 (unknown origin) expressed in U2OS cells cotransfected with beta-arrestin/TEV protease fusion protein and beta-lactamase assessed as inhibition of CXCL8-induced beta arrestin2 recruitment preincubated for 30 mins followed by CXCL8 stimulation and measured after 5 hrs by FRET based fluorescence/Tango assay
Displacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assayDisplacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assay
Displacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assayDisplacement of europium labeled-human IL8 from human cloned CXCR2 expressed in Sf9 membrane with Galphai3-beta-1-gamma-2 by DELFIA binding assay
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
Inhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranesInhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranes
Inhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranesInhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranes
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 membranes by SPADisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 membranes by SPA
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 membranes by SPADisplacement of [125I]IL8 from human recombinant CXCR2 expressed in HEK293 membranes by SPA
Evaluated using an SPA assay with recombinant human [125I]IL-8 and membranes prepared from Sf9 cells expressing human CXCR2Evaluated using an SPA assay with recombinant human [125I]IL-8 and membranes prepared from Sf9 cells expressing human CXCR2
Evaluated using an SPA assay with recombinant human [125I]IL-8 and membranes prepared from Sf9 cells expressing human CXCR2Evaluated using an SPA assay with recombinant human [125I]IL-8 and membranes prepared from Sf9 cells expressing human CXCR2
Concentration required to inhibit [125I]IL-8 binding towards C-X-C chemokine receptor type 2 of human expressed in CHO cellsConcentration required to inhibit [125I]IL-8 binding towards C-X-C chemokine receptor type 2 of human expressed in CHO cells
Concentration required to inhibit [125I]IL-8 binding towards C-X-C chemokine receptor type 2 of human expressed in CHO cellsConcentration required to inhibit [125I]IL-8 binding towards C-X-C chemokine receptor type 2 of human expressed in CHO cells
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
Inhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranesInhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranes
Inhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranesInhibition of binding of [125I]IL8 to human recombinant CXC chemokine receptor 2 (CXCR2) expressed in HEK 293 membranes
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
Displacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR2 receptor expressed in CHO cells
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
Displacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cellsDisplacement of [125I]IL8 from human recombinant CXCR2 expressed in CHO cells
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).In Vitro Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the .beta.-arrestin recruitment type after receptor activation.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 .beta.-arrestin line results in the recruitment of .beta.-arrestin (Richardson et al. 2003 Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911.)In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with .beta.-arrestin 2, a .beta.-arrestin 2 recruitment test for CXCR2 or CXCR1 based on of .beta.-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).In Vitro Inhibition Assay: An in vitro assay showed inhibition of CXCR2-mediated intracellular calcium release. Briefly, human neutrophils were suspended in HBSS- (without Ca2+ and Mg2+) containing 10 mM HEPES and FLIPR Calcium 3 dye (3.1x107 cells in total volume 1.7 mL). Cells were aliquoted (200 uL of the cell suspension per tube, 8 tubes total) and 2 uL of the designated compound (with appropriate dilutions) were added to each of 6 tubes. As controls, 2 uL of DMSO (1% final concentration) were added to 2 other tubes. Cells were incubated for 30 min at 37 C. After dye loading, tubes were centrifuged at 6,000 rpm for 1 min, supernatant was removed and the cell pellet was re-suspended in 200 uL of HBSS+ (with Ca2+ and Mg2+) containing 10 mM HEPES. The test compound or DMSO (control) was added again at the same concentrations that were used during cell loading. The cell suspension was aliquoted into a 96-well Reading Plate (Corning) in a volume of 90 uL (105 cells/well).
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).In Vitro Affinity Assay: The in vitro affinity of the compounds of the present invention for the CXCR1 and CXCR2 receptors was determined on a functional test of the β-arrestin recruitment after receptor activation type.It was demonstrated that the activation by CXCL8 of the CXCR2 receptor in cells of the PathHunter HEK293-CXCR2 line or of the CXCR1 receptor in cells of the U2OS h CXCR1 β-arrestin line results in the recruitment of β-arrestin (Richardson, R. M., R. J. Marjoram, L. S. Barak, R. Snyderman. 2003. Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. J. Immunol. 170: 2904-2911).In order to evaluate the direct interaction of the CXCR2 or CXCR1 receptor with β-arrestin 2, a β-arrestin 2 recruitment test for CXCR2 or CXCR1 based on β-galactosidase enzyme complementation (Olson K R, Eglen R M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol. 2007 February; 5(1); 137-44), as established by DiscoveRx Corporation was used. The stimulation of these two cell lines with CXCL8 (10 nM) induces β-arrestin 2 recruitment, as indicated by a significant increase in the induction factor. All the CXCR2 antagonists were tested in a dose-dependent manner and the concentration corresponding to 50% inhibition of the response was determined (IC50=half inhibition concentration).
Binding affinity to human CXCR2 assessed as dissociation constant incubated for 6 to 24 hrs by radioligand binding assayBinding affinity to human CXCR2 assessed as dissociation constant incubated for 6 to 24 hrs by radioligand binding assay
Binding affinity to human CXCR2 assessed as dissociation constant incubated for 6 to 24 hrs by radioligand binding assayBinding affinity to human CXCR2 assessed as dissociation constant incubated for 6 to 24 hrs by radioligand binding assay
Displacement of [3H]Sch527123 from human CXCR2 assessed as dissociation constant measured every 30 secs for 48 hrs by radioligand binding assayDisplacement of [3H]Sch527123 from human CXCR2 assessed as dissociation constant measured every 30 secs for 48 hrs by radioligand binding assay
Displacement of [3H]Sch527123 from human CXCR2 assessed as dissociation constant measured every 30 secs for 48 hrs by radioligand binding assayDisplacement of [3H]Sch527123 from human CXCR2 assessed as dissociation constant measured every 30 secs for 48 hrs by radioligand binding assay
Displacement of [3H]Sch527123 from human CXCR2 assessed as dissociation constant measured every 30 secs for 48 hrs by radioligand binding assayDisplacement of [3H]Sch527123 from human CXCR2 assessed as dissociation constant measured every 30 secs for 48 hrs by radioligand binding assay
Binding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as dissociation constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assayBinding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as dissociation constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assay
Binding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as dissociation constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assayBinding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as dissociation constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assay
Binding affinity to CXCR2 (unknown origin) (28 to 99 residues) expressed in HEK293 cell membrane assessed as dissociation constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assayBinding affinity to CXCR2 (unknown origin) (28 to 99 residues) expressed in HEK293 cell membrane assessed as dissociation constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assay
Binding affinity to CXCR2 (unknown origin) (28 to 99 residues) expressed in HEK293 cell membrane assessed as dissociation constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assayBinding affinity to CXCR2 (unknown origin) (28 to 99 residues) expressed in HEK293 cell membrane assessed as dissociation constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assay
Binding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as dissociation constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assayBinding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as dissociation constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assay
Binding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as dissociation constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assayBinding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as dissociation constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assay
Binding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as dissociation constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assayBinding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as dissociation constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assay
Binding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as dissociation constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assayBinding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as dissociation constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assay
Binding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as dissociation constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assayBinding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as dissociation constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assay
Binding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as dissociation constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assayBinding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as dissociation constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assay
Binding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assayBinding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assay
Binding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assayBinding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assay
Binding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cells assessed as dissociation constant using furimazine as substrate preincubated for 1 hr followed by substrate addition for mins and measured every hour for 3 hrs by luminescence based NanoBRET fluorescent binding assayBinding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cells assessed as dissociation constant using furimazine as substrate preincubated for 1 hr followed by substrate addition for mins and measured every hour for 3 hrs by luminescence based NanoBRET fluorescent binding assay
Binding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cells assessed as dissociation constant using furimazine as substrate preincubated for 1 hr followed by substrate addition for mins and measured every hour for 3 hrs by luminescence based NanoBRET fluorescent binding assayBinding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cells assessed as dissociation constant using furimazine as substrate preincubated for 1 hr followed by substrate addition for mins and measured every hour for 3 hrs by luminescence based NanoBRET fluorescent binding assay
Binding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cells assessed as dissociation constant using furimazine as substrate preincubated for 1 hr followed by substrate addition for mins and measured every hour for 3 hrs by luminescence based NanoBRET fluorescent binding assayBinding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cells assessed as dissociation constant using furimazine as substrate preincubated for 1 hr followed by substrate addition for mins and measured every hour for 3 hrs by luminescence based NanoBRET fluorescent binding assay
Binding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cells assessed as dissociation constant using furimazine as substrate preincubated for 1 hr followed by substrate addition for mins and measured every hour for 3 hrs by luminescence based NanoBRET fluorescent binding assayBinding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cells assessed as dissociation constant using furimazine as substrate preincubated for 1 hr followed by substrate addition for mins and measured every hour for 3 hrs by luminescence based NanoBRET fluorescent binding assay
Binding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cells assessed as dissociation constant using furimazine as substrate preincubated for 1 hr followed by substrate addition for mins and measured every hour for 3 hrs by luminescence based NanoBRET fluorescent binding assayBinding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cells assessed as dissociation constant using furimazine as substrate preincubated for 1 hr followed by substrate addition for mins and measured every hour for 3 hrs by luminescence based NanoBRET fluorescent binding assay
Binding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cells assessed as dissociation constant using furimazine as substrate preincubated for 1 hr followed by substrate addition for mins and measured every hour for 3 hrs by luminescence based NanoBRET fluorescent binding assayBinding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cells assessed as dissociation constant using furimazine as substrate preincubated for 1 hr followed by substrate addition for mins and measured every hour for 3 hrs by luminescence based NanoBRET fluorescent binding assay
Binding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cells assessed as dissociation constant using furimazine as substrate preincubated for 1 hr followed by substrate addition for mins and measured every hour for 3 hrs by luminescence based NanoBRET fluorescent binding assayBinding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cells assessed as dissociation constant using furimazine as substrate preincubated for 1 hr followed by substrate addition for mins and measured every hour for 3 hrs by luminescence based NanoBRET fluorescent binding assay
Binding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cells assessed as dissociation constant using furimazine as substrate preincubated for 1 hr followed by substrate addition for mins and measured every hour for 3 hrs by luminescence based NanoBRET fluorescent binding assayBinding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cells assessed as dissociation constant using furimazine as substrate preincubated for 1 hr followed by substrate addition for mins and measured every hour for 3 hrs by luminescence based NanoBRET fluorescent binding assay
Binding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as dissociation constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assayBinding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as dissociation constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assay
Binding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as dissociation constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assayBinding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as dissociation constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assay
Binding affinity to CXCR2 (unknown origin) assessed as inhibition constant by NanoBERT competition assayBinding affinity to CXCR2 (unknown origin) assessed as inhibition constant by NanoBERT competition assay
Binding affinity to CXCR2 (unknown origin) assessed as inhibition constant by NanoBERT competition assayBinding affinity to CXCR2 (unknown origin) assessed as inhibition constant by NanoBERT competition assay
Binding affinity to CXCR2 (unknown origin) assessed as inhibition constant by NanoBERT competition assayBinding affinity to CXCR2 (unknown origin) assessed as inhibition constant by NanoBERT competition assay
Binding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assayBinding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assay
Binding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assayBinding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assay
Binding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assayBinding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assay
Binding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assayBinding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assay
Binding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assayBinding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assay
Binding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assayBinding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assay
Binding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assayBinding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assay
Binding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assayBinding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assay
Binding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assayBinding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assay
Binding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assayBinding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assay
Binding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assayBinding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assay
Binding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assayBinding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assay
Binding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assayBinding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assay
Binding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assayBinding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assay
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]IL-8 from human CXCR2 expressed in mouse BaF3 cells by liquid scintillation countingDisplacement of [125I]IL-8 from human CXCR2 expressed in mouse BaF3 cells by liquid scintillation counting
Displacement of [125I]IL-8 from human CXCR2 expressed in mouse BaF3 cells by liquid scintillation countingDisplacement of [125I]IL-8 from human CXCR2 expressed in mouse BaF3 cells by liquid scintillation counting
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]IL-8 from human CXCR2 expressed in mouse BaF3 cells by liquid scintillation countingDisplacement of [125I]IL-8 from human CXCR2 expressed in mouse BaF3 cells by liquid scintillation counting
Displacement of [125I]IL-8 from human CXCR2 expressed in mouse BaF3 cells by liquid scintillation countingDisplacement of [125I]IL-8 from human CXCR2 expressed in mouse BaF3 cells by liquid scintillation counting
Displacement of [I-125]-interleukin-8 against human recombinant CXCR2 expressed in CHO-K1 cells incubated for 60 mins by radio ligand binding assayDisplacement of [I-125]-interleukin-8 against human recombinant CXCR2 expressed in CHO-K1 cells incubated for 60 mins by radio ligand binding assay
Displacement of [125I]IL-8 from human CXCR2 expressed in mouse BaF3 cells by liquid scintillation countingDisplacement of [125I]IL-8 from human CXCR2 expressed in mouse BaF3 cells by liquid scintillation counting
Displacement of [125I]IL-8 from human CXCR2 expressed in mouse BaF3 cells by liquid scintillation countingDisplacement of [125I]IL-8 from human CXCR2 expressed in mouse BaF3 cells by liquid scintillation counting
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]IL-8 from human CXCR2 expressed in mouse BaF3 cells by liquid scintillation countingDisplacement of [125I]IL-8 from human CXCR2 expressed in mouse BaF3 cells by liquid scintillation counting
Displacement of [125I]IL-8 from human CXCR2 expressed in mouse BaF3 cells by liquid scintillation countingDisplacement of [125I]IL-8 from human CXCR2 expressed in mouse BaF3 cells by liquid scintillation counting
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Binding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assayBinding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assay
Binding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assayBinding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assay
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Binding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assayBinding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assay
Binding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assayBinding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assay
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]IL-8 from human CXCR2 expressed in mouse BaF3 cells by liquid scintillation countingDisplacement of [125I]IL-8 from human CXCR2 expressed in mouse BaF3 cells by liquid scintillation counting
Displacement of [125I]IL-8 from human CXCR2 expressed in mouse BaF3 cells by liquid scintillation countingDisplacement of [125I]IL-8 from human CXCR2 expressed in mouse BaF3 cells by liquid scintillation counting
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Binding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assayBinding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assay
Binding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assayBinding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assay
Binding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assayBinding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assay
Displacement of [125I]IL-8 from human CXCR2 expressed in mouse BaF3 cells by liquid scintillation countingDisplacement of [125I]IL-8 from human CXCR2 expressed in mouse BaF3 cells by liquid scintillation counting
Displacement of [125I]IL-8 from human CXCR2 expressed in mouse BaF3 cells by liquid scintillation countingDisplacement of [125I]IL-8 from human CXCR2 expressed in mouse BaF3 cells by liquid scintillation counting
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Binding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assayBinding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assay
Binding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assayBinding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assay
Binding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assayBinding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assay
Binding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assayBinding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assay
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [I-125]-interleukin-8 against human recombinant CXCR2 expressed in CHO-K1 cells incubated for 60 mins by radio ligand binding assayDisplacement of [I-125]-interleukin-8 against human recombinant CXCR2 expressed in CHO-K1 cells incubated for 60 mins by radio ligand binding assay
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]IL-8 from human CXCR2 expressed in mouse BaF3 cells by liquid scintillation countingDisplacement of [125I]IL-8 from human CXCR2 expressed in mouse BaF3 cells by liquid scintillation counting
Displacement of [125I]IL-8 from human CXCR2 expressed in mouse BaF3 cells by liquid scintillation countingDisplacement of [125I]IL-8 from human CXCR2 expressed in mouse BaF3 cells by liquid scintillation counting
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Binding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assayBinding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assay
Binding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assayBinding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assay
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]IL-8 from human CXCR2 expressed in mouse BaF3 cells by liquid scintillation countingDisplacement of [125I]IL-8 from human CXCR2 expressed in mouse BaF3 cells by liquid scintillation counting
Displacement of [125I]IL-8 from human CXCR2 expressed in mouse BaF3 cells by liquid scintillation countingDisplacement of [125I]IL-8 from human CXCR2 expressed in mouse BaF3 cells by liquid scintillation counting
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Binding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assayBinding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assay
Binding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assayBinding affinity to NanoLuc fused Snap CXCR2 (unknown origin) expressed in HEK293 cell membrane assessed as inhibition constant using furimazine as substrate incubated with substrate for 5 mins and measured every 15 secs for 60 mins by luminescence based NanoBRET fluorescent binding assay
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Displacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysisDisplacement of [125I]-IL8 from human CXCR2 transfected in HEK293 cells after 4 hrs by microbeta counting analysis
Measuring displacement of CXCL8 binding to recombinant CXCR2 in HEK cell membrane preparations.Measuring displacement of CXCL8 binding to recombinant CXCR2 in HEK cell membrane preparations.
Measuring displacement of CXCL8 binding to recombinant CXCR2 in HEK cell membrane preparations.Measuring displacement of CXCL8 binding to recombinant CXCR2 in HEK cell membrane preparations.
Measuring displacement of CXCL8 binding to recombinant CXCR2 in HEK cell membrane preparations.Measuring displacement of CXCL8 binding to recombinant CXCR2 in HEK cell membrane preparations.