A 1,536-well [(35)S]GTPgammaS scintillation proximity binding assay for ultra-high-throughput screening of an orphan galphai-coupled GPCR.
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-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) scintillation proximity assay (SPA) binding assay targeting a Galphai-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 muM using the 1,536-well [(35)S]GTPgammaS 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.
Department of Automated Biotechnology, Merck Research Laboratories, North Wales, PA 19454, USA. eric_johnson4@merck.com
Eric N
EN
Johnson
Xiaoqing
X
Shi
Jason
J
Cassaday
Marc
M
Ferrer
Berta
B
Strulovici
Priya
P
Kunapuli
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000032:analysis*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000032:analysis*; D012588:Scintillation Counting / Q000379:methods*; D013462:Sulfur Radioisotopes
D043562:Receptors, G-Protein-Coupled; D013462:Sulfur Radioisotopes; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019206:GTP-Binding Protein alpha Subunits, Gi-Go