Abstract: Members of the superfamily of seven transmembrane receptors, known as G protein-coupled receptors (GPCRs), are important targets for many therapeutic areas in drug discovery. A homogeneous guanosine 5′-O-(3-[35S]thio)triphosphate ([35S]GTPγS) scintillation proximity assay (SPA) binding assay targeting a Gαi-coupled GPCR recombinantly expressed in membranes of Chinese hamster ovary (CHO) cells was developed and miniaturized into 1,536-well plate format. The primary ultra-high-throughput screen of the entire compound collection was accomplished on the Kalypsys (San Diego, CA) robotic platform at a concentration of 8 μM using the 1,536-well [35S]GTPγS SPA binding functional assay. The signal-to-noise ratio of the primary screen was approximately 2.1-fold, and the plate coefficient of variation for the compound field was approximately 11%. The hit rate from the primary screen for receptor agonists at >35% activity was approximately 0.3%. Primary hits were cherry-picked, confirmed in triplicate, counterscreened against untransfected CHO cell membranes, and further analyzed in a cyclic AMP functional assay, resulting in 34 leads for optimization.
