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Development of a Homogeneous MAP Kinase Reporter Gene Screen for the Identification of Agonists and Antagonists at the CXCR1 Chemokine Receptor

Molecular Discovery, Glaxo Wellcome Medicines Research Centre, Hert-fordshire, UK

Duncan P. Martin

Receptor Pharmacology, Glaxo Wellcome Medicines Research Centre, Hert-fordshire, UK

Sarah V. Scott

Molecular Discovery, Glaxo Wellcome Medicines Research Centre, Hert-fordshire, UK

Sue H. Brown

Molecular Pharmacology Research Units, Glaxo Wellcome Medicines Research Centre, Hert-fordshire, UK

Neil Fraser

Molecular Pharmacology Research Units, Glaxo Wellcome Medicines Research Centre, Hert-fordshire, UK

Celestine O'Shaughnessy

Molecular Pharmacology Research Units, Glaxo Wellcome Medicines Research Centre, Hert-fordshire, UK

Isabel J.M. Beresford

Receptor Pharmacology, Glaxo Wellcome Medicines Research Centre, Hert-fordshire, UK

Agonist activity at G protein-coupled receptors (GPCRs) that regulate heterotrimeric G proteins of the G_i/o or Gaq families has been shown to result in activation of the mitogen-activated protein (MAP) kinase cascade. To facilitate compound screening for these classes of GPCR, we have developed a reporter gene that detects the activation of the ternary complex transcription factor Sapla following MAP kinase activation. In contrast to other reporter gene assays for G_i/o-coupled GPCRs, the MAP kinase reporter generates an increase in signal in the presence of agonist. The reporter gene has been transfected into Chinese hamster ovary cells to generate a "host" reporter gene-containing cell line. The G_i-coupled human CXCR1 chemokine receptor was subsequently transfected into this cell line in order to develop a 384-well format screen for both agonists and antagonists of this receptor. Agonists activated the reporter gene with the expected rank order of potency and with similar concentration dependence as seen with the regulation of other signal transduction cascades in mammalian cells: interleukin-8 (IL-8) (pEC₅₀ = 7.0 ± 0.1) > GCP-2 (pEC₅₀ = 6.3 ± 0.1) > NAP-2 (pEC₅₀ < 6). CXCR1-mediated activation of MAP kinase was inhibited by pertussis toxin and the MEK inhibitor PD98059, demonstrating that receptor activation of MAP kinase is due to pertussis toxin-sensitive G/_i/o-family G proteins to cause the activation of MEK kinase. Using the 384-well format, assay performance was unaffected by solvent concentrations of 0.5% ethanol, 0.15% glycerol, or 1% DMSO. Signal crosstalk between adjacent wells was less than 1%. The assay exhibited a Z factor of 0.53 and a coefficient of variation of response to repeated application of IL-8 (100 nM) of 15.9%.

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