title: Novel GPCR screening approach: indirect identification of S1P receptor agonists in antagonist screening using a calcium assay.
abstract: To elucidate the physiological function of sphingosine 1-phosphate receptors 1-3 (S1P1-3) we aimed to identify selective ligands for these GPCRs. S1P2 and S1P3 are coupled to Gq, and are, therefore, linked to the phospholipase C/IP3/calcium pathway. S1P1 is solely coupled to Gi and was artificially linked to calcium signaling by coexpression of Galpha 16. The three receptors desensitized on challenge of cells with an agonist (i.e., agonists appeared as antagonists in a second calcium measurement). We screened a compound library for inhibitors of S1P-stimulated calcium signals, and we could identify agonists and antagonists with a single measurement. Agonism and antagonism were confirmed by recording compound-and S1P-induced calcium signals from the same assay well. For the three receptors, we found a reciprocal correlation of agonism and "apparent" antagonism of agonists. In addition, agonists indirectly discovered by this approach do not promote calcium mobilization through endogenous GPCRs.
authors.affiliation: Discovery Technologies, Novartis Institutes for BioMedical Research, Basel, Switzerland. sandra.siehler@novartis.com
authors.forename: Sandra
authors.initials: S
authors.lastname: Siehler
authors.forename: Danilo
authors.initials: D
authors.lastname: Guerini
mesh_terms: D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D008024:Ligands; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D049349:Receptors, Lysosphingolipid / Q000819:agonists* / Q000037:antagonists & inhibitors; D014162:Transfection
chemical_list: D008024:Ligands; D043562:Receptors, G-Protein-Coupled; D049349:Receptors, Lysosphingolipid; D002118:Calcium