Inhibition of CX3CL1-stimulated CX3CR1 in human HTLA cells pre-incubated for 20 mins measured on day 4 by beta arrestin-recruitment mediated luciferase reporter gene assayInhibition of CX3CL1-stimulated CX3CR1 in human HTLA cells pre-incubated for 20 mins measured on day 4 by beta arrestin-recruitment mediated luciferase reporter gene assay
Inhibition of CX3CL1-stimulated CX3CR1 in human HTLA cells pre-incubated for 20 mins with centrifuged compound solution measured on day 4 by beta arrestin-recruitment mediated luciferase reporter gene assayInhibition of CX3CL1-stimulated CX3CR1 in human HTLA cells pre-incubated for 20 mins with centrifuged compound solution measured on day 4 by beta arrestin-recruitment mediated luciferase reporter gene assay
Antagonist activity in CXCR1 (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 CXCR1 (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 CXCR1 (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 CXCR1 (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 CXCR1 (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 CXCR1 (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
Inhibitory concentration against interleukin-8 receptor of human neutrophils by using [125I]IL-8 (0.125 nM) as radioligandInhibitory concentration against interleukin-8 receptor of human neutrophils by using [125I]IL-8 (0.125 nM) as radioligand
Inhibitory concentration against interleukin-8 receptor of human neutrophils by using [125I]IL-8 (0.125 nM) as radioligandInhibitory concentration against interleukin-8 receptor of human neutrophils by using [125I]IL-8 (0.125 nM) as radioligand
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 CX3CL1-stimulated CX3CR1 in human HTLA cells pre-incubated for 20 mins with centrifuged compound solution measured on day 4 by beta arrestin-recruitment mediated luciferase reporter gene assayInhibition of CX3CL1-stimulated CX3CR1 in human HTLA cells pre-incubated for 20 mins with centrifuged compound solution measured on day 4 by beta arrestin-recruitment mediated luciferase reporter gene 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).
Displacement of [125I]hCXCL8 from human CXCR1 receptor expressed in BaF3 cellsDisplacement of [125I]hCXCL8 from human CXCR1 receptor expressed in BaF3 cells
Inhibition of binding of IL-8 to membranes of cloned CXC chemokine receptor 1 expressed in CHO Cells using [125I]IL-8 radioligandInhibition of binding of IL-8 to membranes of cloned CXC chemokine receptor 1 expressed in CHO Cells using [125I]IL-8 radioligand
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).
Inhibitory concentration against interleukin-8 receptor of human neutrophils by using [125I]IL-8 (0.125 nM) as radioligandInhibitory concentration against interleukin-8 receptor of human neutrophils by using [125I]IL-8 (0.125 nM) as radioligand
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 wild type CXCR1 transfected in mouse L1.2 cells assessed as inhibition of CXCL8-induced cell migration pretreated for 15 mins measured after 4 hrsInhibition of wild type CXCR1 transfected in mouse L1.2 cells assessed as inhibition of CXCL8-induced cell migration pretreated for 15 mins measured after 4 hrs
Inhibition of wild type CXCR1 transfected in mouse L1.2 cells assessed as inhibition of CXCL8-induced cell migration pretreated for 15 mins measured after 4 hrsInhibition of wild type CXCR1 transfected in mouse L1.2 cells assessed as inhibition of CXCL8-induced cell migration pretreated for 15 mins measured after 4 hrs
Displacement of human recombinant [125I]IL8 from CXCR1 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR1 receptor expressed in CHO cells
Inhibitory concentration against interleukin-8 receptor of human neutrophils by using [125I]IL-8 (0.125 nM) as radioligandInhibitory concentration against interleukin-8 receptor of human neutrophils by using [125I]IL-8 (0.125 nM) as radioligand
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 CXCR1 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR1 receptor expressed in CHO cells
Displacement of human recombinant [125I]IL8 from CXCR1 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR1 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).
Concentration required to inhibit [125I]-IL-8 binding towards C-X-C chemokine receptor type 1 of human expressed in CHO cellsConcentration required to inhibit [125I]-IL-8 binding towards C-X-C chemokine receptor type 1 of human expressed in CHO cells
Antagonist activity at CXCR1 (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 CXCR1 (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
Concentration required to inhibit [125I]-IL-8 binding towards C-X-C chemokine receptor type 1 of human expressed in CHO cellsConcentration required to inhibit [125I]-IL-8 binding towards C-X-C chemokine receptor type 1 of human expressed in CHO cells
Antagonist activity at CXCR1 (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 CXCR1 (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
Displacement of [125I]CXCL8 from CXCR1 receptor in human PMN assessed as myeloperoxidase releaseDisplacement of [125I]CXCL8 from CXCR1 receptor in human PMN assessed as myeloperoxidase release
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]hCXCL8 from human CXCR1 receptor expressed in BaF3 cellsDisplacement of [125I]hCXCL8 from human CXCR1 receptor expressed in BaF3 cells
Antagonist activity in CXCR1 (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 CXCR1 (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
Inhibition of binding of IL-8 to membranes of cloned CXC chemokine receptor 1 expressed in CHO Cells using [125I]IL-8 radioligandInhibition of binding of IL-8 to membranes of cloned CXC chemokine receptor 1 expressed in CHO Cells using [125I]IL-8 radioligand
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: 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 CX3CL1-stimulated CX3CR1 in human HTLA cells pre-incubated for 20 mins measured on day 4 by beta arrestin-recruitment mediated luciferase reporter gene assayInhibition of CX3CL1-stimulated CX3CR1 in human HTLA cells pre-incubated for 20 mins measured on day 4 by beta arrestin-recruitment mediated luciferase reporter gene 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).
Antagonist activity in CXCR1 (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 CXCR1 (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
Inhibitory concentration against interleukin-8 receptor of human neutrophils by using [125I]IL-8 (0.125 nM) as radioligandInhibitory concentration against interleukin-8 receptor of human neutrophils by using [125I]IL-8 (0.125 nM) as radioligand
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 CXCR1 (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 CXCR1 (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 CXCR1 (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 CXCR1 (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 CXCR1 (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 CXCR1 (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
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: 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]IL-8 from CXCR1 (unknown origin) stably expressed in CHO cellsDisplacement of [125I]IL-8 from CXCR1 (unknown origin) stably expressed in CHO cells
Inhibition of binding of IL-8 to membranes of cloned CXC chemokine receptor 1 expressed in CHO Cells using [125I]IL-8 radioligandInhibition of binding of IL-8 to membranes of cloned CXC chemokine receptor 1 expressed in CHO Cells using [125I]IL-8 radioligand
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]hCXCL8 from human CXCR1 receptor expressed in BaF3 cellsDisplacement of [125I]hCXCL8 from human CXCR1 receptor expressed in BaF3 cells
Antagonist activity at CXCR1 (unknown origin) expressed in HEK293 cells assessed as suppression of IL-8-induced inhibition of forskolin-induced cAMP formation preincubated for 15 mins followed by forskolin and IL-8 stimulation and measured after 15 mins by cAMP-d2 and Eu-Anti-cAMP based fluorescence assayAntagonist activity at CXCR1 (unknown origin) expressed in HEK293 cells assessed as suppression of IL-8-induced inhibition of forskolin-induced cAMP formation preincubated for 15 mins followed by forskolin and IL-8 stimulation and measured after 15 mins by cAMP-d2 and Eu-Anti-cAMP based fluorescence assay
Antagonist activity at CXCR1 (unknown origin) stably expressed in HEK293 cells assessed as reduction in IL-8-induced intracellular calcium change incubated for 15 mins followed by IL-8-stimulation and measured after 15 mins by cAMP-d2/Eu-Anti-cAM based fluorescence assayAntagonist activity at CXCR1 (unknown origin) stably expressed in HEK293 cells assessed as reduction in IL-8-induced intracellular calcium change incubated for 15 mins followed by IL-8-stimulation and measured after 15 mins by cAMP-d2/Eu-Anti-cAM based fluorescence assay
Inhibition of CX3CL1-stimulated CX3CR1 in human HTLA cells pre-incubated for 20 mins measured on day 4 by beta arrestin-recruitment mediated luciferase reporter gene assayInhibition of CX3CL1-stimulated CX3CR1 in human HTLA cells pre-incubated for 20 mins measured on day 4 by beta arrestin-recruitment mediated luciferase reporter gene assay
Inhibition of CX3CL1-stimulated CX3CR1 in human HTLA cells pre-incubated for 20 mins measured on day 4 by beta arrestin-recruitment mediated luciferase reporter gene assayInhibition of CX3CL1-stimulated CX3CR1 in human HTLA cells pre-incubated for 20 mins measured on day 4 by beta arrestin-recruitment mediated luciferase reporter gene assay
Inhibition of CX3CL1-stimulated CX3CR1 in human HTLA cells pre-incubated for 20 mins with Tween-80-treated compound solution measured on day 4 by beta arrestin-recruitment mediated luciferase reporter gene assayInhibition of CX3CL1-stimulated CX3CR1 in human HTLA cells pre-incubated for 20 mins with Tween-80-treated compound solution measured on day 4 by beta arrestin-recruitment mediated luciferase reporter gene 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).
Inhibition of CX3CL1-stimulated CX3CR1 in human HTLA cells pre-incubated for 20 mins measured on day 4 by beta arrestin-recruitment mediated luciferase reporter gene assayInhibition of CX3CL1-stimulated CX3CR1 in human HTLA cells pre-incubated for 20 mins measured on day 4 by beta arrestin-recruitment mediated luciferase reporter gene assay
Inhibitory concentration against interleukin-8 receptor of human neutrophils by using [125I]IL-8 (0.125 nM) as radioligandInhibitory concentration against interleukin-8 receptor of human neutrophils by using [125I]IL-8 (0.125 nM) as radioligand
Displacement of human recombinant [125I]IL8 from CXCR1 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR1 receptor expressed in CHO cells
Antagonist activity in CXCR1 (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 CXCR1 (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 CXCR1 (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 CXCR1 (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
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 CX3CL1-stimulated CX3CR1 in human HTLA cells pre-incubated for 20 mins with Tween-80-treated compound solution measured on day 4 by beta arrestin-recruitment mediated luciferase reporter gene assayInhibition of CX3CL1-stimulated CX3CR1 in human HTLA cells pre-incubated for 20 mins with Tween-80-treated compound solution measured on day 4 by beta arrestin-recruitment mediated luciferase reporter gene assay
Inhibitory concentration against interleukin-8 receptor of human neutrophils by using [125I]IL-8 (0.125 nM) as radioligandInhibitory concentration against interleukin-8 receptor of human neutrophils by using [125I]IL-8 (0.125 nM) as radioligand
Inhibition of binding of IL-8 to membranes of cloned CXC chemokine receptor 1 expressed in CHO Cells using [125I]IL-8 radioligandInhibition of binding of IL-8 to membranes of cloned CXC chemokine receptor 1 expressed in CHO Cells using [125I]IL-8 radioligand
Antagonist activity in CXCR1 (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 CXCR1 (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 CXCR1 assessed as inhibition of CXCL8 binding by cell based assayAntagonist activity at CXCR1 assessed as inhibition of CXCL8 binding by cell based assay
Inhibitory concentration against interleukin-8 receptor of human neutrophils by using [125I]IL-8 (0.125 nM) as radioligandInhibitory concentration against interleukin-8 receptor of human neutrophils by using [125I]IL-8 (0.125 nM) as radioligand
Inhibition of CX3CL1-stimulated CX3CR1 in human HTLA cells pre-incubated for 20 mins measured on day 4 by beta arrestin-recruitment mediated luciferase reporter gene assayInhibition of CX3CL1-stimulated CX3CR1 in human HTLA cells pre-incubated for 20 mins measured on day 4 by beta arrestin-recruitment mediated luciferase reporter gene 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).
Inhibitory concentration against interleukin-8 receptor of human neutrophils by using [125I]IL-8 (0.125 nM) as radioligandInhibitory concentration against interleukin-8 receptor of human neutrophils by using [125I]IL-8 (0.125 nM) as radioligand
Displacement of [125I]IL-8 from CXCR1 in human neutrophils incubated for 3 hrs by gamma counting methodDisplacement of [125I]IL-8 from CXCR1 in human neutrophils incubated for 3 hrs by gamma counting method
Antagonist activity in CXCR1 (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 CXCR1 (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
Displacement of [125I]hCXCL8 from human CXCR1 receptor expressed in BaF3 cellsDisplacement of [125I]hCXCL8 from human CXCR1 receptor expressed in BaF3 cells
Displacement of human recombinant [125I]IL8 from CXCR1 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR1 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 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 CXCR1 (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 CXCR1 (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
Inhibitory concentration against interleukin-8 receptor of human neutrophils by using [125I]IL-8 (0.125 nM) as radioligandInhibitory concentration against interleukin-8 receptor of human neutrophils by using [125I]IL-8 (0.125 nM) as radioligand
Displacement of [125I]hCXCL8 from human CXCR1 receptor expressed in BaF3 cellsDisplacement of [125I]hCXCL8 from human CXCR1 receptor expressed in BaF3 cells
Displacement of [125I]hCXCL8 from human CXCR1 receptor expressed in BaF3 cellsDisplacement of [125I]hCXCL8 from human CXCR1 receptor expressed in BaF3 cells
Displacement of [125I]IL-8 from CXCR1 (unknown origin) stably expressed in CHO cellsDisplacement of [125I]IL-8 from CXCR1 (unknown origin) stably expressed in CHO cells
Inhibition of binding of IL-8 to membranes of cloned CXC chemokine receptor 1 expressed in CHO Cells using [125I]IL-8 radioligandInhibition of binding of IL-8 to membranes of cloned CXC chemokine receptor 1 expressed in CHO Cells using [125I]IL-8 radioligand
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).
Inhibitory concentration against interleukin-8 receptor of human neutrophils by using [125I]IL-8 (0.125 nM) as radioligandInhibitory concentration against interleukin-8 receptor of human neutrophils by using [125I]IL-8 (0.125 nM) as radioligand
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 CXCR1 (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 CXCR1 (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 CXCR1 (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 CXCR1 (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
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).
Inhibitory concentration against interleukin-8 receptor of human neutrophils by using [125I]IL-8 (0.125 nM) as radioligandInhibitory concentration against interleukin-8 receptor of human neutrophils by using [125I]IL-8 (0.125 nM) as radioligand
Concentration required to inhibit [125I]-IL-8 binding towards C-X-C chemokine receptor type 1 of human expressed in CHO cellsConcentration required to inhibit [125I]-IL-8 binding towards C-X-C chemokine receptor type 1 of human expressed in CHO cells
Concentration required to inhibit [125I]-IL-8 binding towards C-X-C chemokine receptor type 1 of human expressed in CHO cellsConcentration required to inhibit [125I]-IL-8 binding towards C-X-C chemokine receptor type 1 of human 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).
Displacement of [125I]hCXCL8 from human CXCR1 receptor expressed in BaF3 cellsDisplacement of [125I]hCXCL8 from human CXCR1 receptor expressed in BaF3 cells
Antagonist activity at CXCR1 (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 CXCR1 (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
Inhibitory concentration against interleukin-8 receptor of human neutrophils by using [125I]IL-8 (0.125 nM) as radioligandInhibitory concentration against interleukin-8 receptor of human neutrophils by using [125I]IL-8 (0.125 nM) as radioligand
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 CXCR1 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR1 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).
Antagonist activity at CXCR1 (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 CXCR1 (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
Inhibition of binding of IL-8 to membranes of cloned CXC chemokine receptor 1 expressed in CHO Cells using [125I]IL-8 radioligandInhibition of binding of IL-8 to membranes of cloned CXC chemokine receptor 1 expressed in CHO Cells using [125I]IL-8 radioligand
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).
Displacement of [125I]hCXCL8 from human CXCR1 receptor expressed in BaF3 cellsDisplacement of [125I]hCXCL8 from human CXCR1 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).
Displacement of [125I]hCXCL8 from human CXCR1 receptor expressed in BaF3 cellsDisplacement of [125I]hCXCL8 from human CXCR1 receptor expressed in BaF3 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).
Antagonist activity in CXCR1 (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 CXCR1 (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
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).
Inhibitory concentration against interleukin-8 receptor of human neutrophils by using [125I]IL-8 (0.125 nM) as radioligandInhibitory concentration against interleukin-8 receptor of human neutrophils by using [125I]IL-8 (0.125 nM) as radioligand
Displacement of [125I]CXCL8 from CXCR1 receptor in human PMN assessed as myeloperoxidase releaseDisplacement of [125I]CXCL8 from CXCR1 receptor in human PMN assessed as myeloperoxidase release
Antagonist activity at CXCR1 (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 CXCR1 (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 CXCR1 (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 CXCR1 (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
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 1 expressed in CHO Cells using [125I]IL-8 radioligandInhibition of binding of IL-8 to membranes of cloned CXC chemokine receptor 1 expressed in CHO Cells using [125I]IL-8 radioligand
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 CXCR1 (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 CXCR1 (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
Concentration required to inhibit [125I]-IL-8 binding towards C-X-C chemokine receptor type 1 of human expressed in CHO cellsConcentration required to inhibit [125I]-IL-8 binding towards C-X-C chemokine receptor type 1 of human 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).
Inhibitory concentration against interleukin-8 receptor of human neutrophils by using [125I]IL-8 (0.125 nM) as radioligandInhibitory concentration against interleukin-8 receptor of human neutrophils by using [125I]IL-8 (0.125 nM) as radioligand
Antagonist activity at CXCR1 (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 CXCR1 (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
Inhibition of CX3CL1-stimulated CX3CR1 in human HTLA cells pre-incubated for 20 mins measured on day 4 by beta arrestin-recruitment mediated luciferase reporter gene assayInhibition of CX3CL1-stimulated CX3CR1 in human HTLA cells pre-incubated for 20 mins measured on day 4 by beta arrestin-recruitment mediated luciferase reporter gene assay
Inhibition of CX3CL1-stimulated CX3CR1 in human HTLA cells pre-incubated for 20 mins measured on day 4 by beta arrestin-recruitment mediated luciferase reporter gene assayInhibition of CX3CL1-stimulated CX3CR1 in human HTLA cells pre-incubated for 20 mins measured on day 4 by beta arrestin-recruitment mediated luciferase reporter gene 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).
Inhibition of binding of IL-8 to membranes of cloned CXC chemokine receptor 1 expressed in CHO Cells using [125I]IL-8 radioligandInhibition of binding of IL-8 to membranes of cloned CXC chemokine receptor 1 expressed in CHO Cells using [125I]IL-8 radioligand
Antagonist activity in CXCR1 (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 CXCR1 (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 CXCR1 (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 CXCR1 (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
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).
Inhibition of CX3CL1-stimulated CX3CR1 in human HTLA cells pre-incubated for 20 mins with centrifuged compound solution measured on day 4 by beta arrestin-recruitment mediated luciferase reporter gene assayInhibition of CX3CL1-stimulated CX3CR1 in human HTLA cells pre-incubated for 20 mins with centrifuged compound solution measured on day 4 by beta arrestin-recruitment mediated luciferase reporter gene assay
Inhibition of CX3CL1-stimulated CX3CR1 in human HTLA cells pre-incubated for 20 mins measured on day 4 by beta arrestin-recruitment mediated luciferase reporter gene assayInhibition of CX3CL1-stimulated CX3CR1 in human HTLA cells pre-incubated for 20 mins measured on day 4 by beta arrestin-recruitment mediated luciferase reporter gene assay
Inhibition of CX3CL1-stimulated CX3CR1 in human HTLA cells pre-incubated for 20 mins measured on day 4 by beta arrestin-recruitment mediated luciferase reporter gene assayInhibition of CX3CL1-stimulated CX3CR1 in human HTLA cells pre-incubated for 20 mins measured on day 4 by beta arrestin-recruitment mediated luciferase reporter gene assay
Inhibition of CX3CL1-stimulated CX3CR1 in human HTLA cells pre-incubated for 20 mins with centrifuged compound solution measured on day 4 by beta arrestin-recruitment mediated luciferase reporter gene assayInhibition of CX3CL1-stimulated CX3CR1 in human HTLA cells pre-incubated for 20 mins with centrifuged compound solution measured on day 4 by beta arrestin-recruitment mediated luciferase reporter gene 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 Assay: The 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 in CXCR1 (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 CXCR1 (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
Inhibitory concentration against interleukin-8 receptor of human neutrophils by using [125I]IL-8 (0.125 nM) as radioligandInhibitory concentration against interleukin-8 receptor of human neutrophils by using [125I]IL-8 (0.125 nM) as radioligand
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).
Antagonist activity in CXCR1 (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 CXCR1 (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
Displacement of [125I]hCXCL8 from human CXCR1 receptor expressed in BaF3 cellsDisplacement of [125I]hCXCL8 from human CXCR1 receptor expressed in BaF3 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 Assay: The 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: 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).
Inhibitory concentration against interleukin-8 receptor of human neutrophils by using [125I]IL-8 (0.125 nM) as radioligandInhibitory concentration against interleukin-8 receptor of human neutrophils by using [125I]IL-8 (0.125 nM) as radioligand
Concentration required to inhibit [125I]-IL-8 binding towards C-X-C chemokine receptor type 1 of human expressed in CHO cellsConcentration required to inhibit [125I]-IL-8 binding towards C-X-C chemokine receptor type 1 of human 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).
Antagonist activity at CXCR1 (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 CXCR1 (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
Inhibitory concentration against interleukin-8 receptor of human neutrophils by using [125I]IL-8 (0.125 nM) as radioligandInhibitory concentration against interleukin-8 receptor of human neutrophils by using [125I]IL-8 (0.125 nM) as radioligand
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: 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).
Inhibitory concentration against interleukin-8 receptor of human neutrophils by using [125I]IL-8 (0.125 nM) as radioligandInhibitory concentration against interleukin-8 receptor of human neutrophils by using [125I]IL-8 (0.125 nM) as radioligand
Antagonist activity at CXCR1 (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 CXCR1 (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
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 CXCR1 (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 CXCR1 (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
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).
Displacement of human recombinant [125I]IL8 from CXCR1 receptor expressed in CHO cellsDisplacement of human recombinant [125I]IL8 from CXCR1 receptor expressed in CHO cells
Inhibitory concentration against interleukin-8 receptor of human neutrophils by using [125I]IL-8 (0.125 nM) as radioligandInhibitory concentration against interleukin-8 receptor of human neutrophils by using [125I]IL-8 (0.125 nM) as radioligand
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: 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]hCXCL8 from human CXCR1 receptor expressed in BaF3 cellsDisplacement of [125I]hCXCL8 from human CXCR1 receptor expressed in BaF3 cells
Inhibition of CX3CL1-stimulated CX3CR1 in human HTLA cells pre-incubated for 20 mins with Tween-80-treated compound solution measured on day 4 by beta arrestin-recruitment mediated luciferase reporter gene assayInhibition of CX3CL1-stimulated CX3CR1 in human HTLA cells pre-incubated for 20 mins with Tween-80-treated compound solution measured on day 4 by beta arrestin-recruitment mediated luciferase reporter gene assay
Inhibition of CX3CL1-stimulated CX3CR1 in human HTLA cells pre-incubated for 20 mins with Tween-80-treated compound solution measured on day 4 by beta arrestin-recruitment mediated luciferase reporter gene assayInhibition of CX3CL1-stimulated CX3CR1 in human HTLA cells pre-incubated for 20 mins with Tween-80-treated compound solution measured on day 4 by beta arrestin-recruitment mediated luciferase reporter gene assay
Displacement of [125I]hCXCL8 from human CXCR1 receptor expressed in BaF3 cellsDisplacement of [125I]hCXCL8 from human CXCR1 receptor expressed in BaF3 cells
Inhibitory concentration against interleukin-8 receptor of human neutrophils by using [125I]IL-8 (0.125 nM) as radioligandInhibitory concentration against interleukin-8 receptor of human neutrophils by using [125I]IL-8 (0.125 nM) as radioligand
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 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 human CXCR1 expressed in HEK293 cells assessed as inhibition of CXCL8-induced intracellular Ca2+ release by fluorescence based calcium flux assayAntagonist activity at human CXCR1 expressed in HEK293 cells assessed as inhibition of CXCL8-induced intracellular Ca2+ release by fluorescence based calcium flux 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).
Inhibitory concentration against interleukin-8 receptor of human neutrophils by using [125I]IL-8 (0.125 nM) as radioligandInhibitory concentration against interleukin-8 receptor of human neutrophils by using [125I]IL-8 (0.125 nM) as radioligand
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).
Displacement of [125I]hCXCL8 from human CXCR1 receptor expressed in BaF3 cellsDisplacement of [125I]hCXCL8 from human CXCR1 receptor expressed in BaF3 cells
Antagonist activity in CXCR1 (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 CXCR1 (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
Inhibition of CXCR1-mediated chemotaxis in Ba/F3 cells expressing human CXCR1Inhibition of CXCR1-mediated chemotaxis in Ba/F3 cells expressing human CXCR1
Inhibitory concentration against interleukin-8 receptor of human neutrophils by using [125I]IL-8 (0.125 nM) as radioligandInhibitory concentration against interleukin-8 receptor of human neutrophils by using [125I]IL-8 (0.125 nM) as radioligand
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 Assay: The 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 CXCR1 expressed in mouse BaF3 cells by liquid scintillation countingDisplacement of [125I]IL-8 from human CXCR1 expressed in mouse BaF3 cells by liquid scintillation counting
Displacement of [125I]IL-8 from human CXCR1 expressed in mouse BaF3 cells by liquid scintillation countingDisplacement of [125I]IL-8 from human CXCR1 expressed in mouse BaF3 cells by liquid scintillation counting
Displacement of [125I]IL-8 from human CXCR1 expressed in mouse BaF3 cells by liquid scintillation countingDisplacement of [125I]IL-8 from human CXCR1 expressed in mouse BaF3 cells by liquid scintillation counting
Displacement of [125I]IL-8 from human CXCR1 expressed in mouse BaF3 cells by liquid scintillation countingDisplacement of [125I]IL-8 from human CXCR1 expressed in mouse BaF3 cells by liquid scintillation counting
Measuring displacement of CXCL8 binding to recombinant CXCR1 in HEK cell membrane preparations.Measuring displacement of CXCL8 binding to recombinant CXCR1 in HEK cell membrane preparations.