A 1,536-Well [35S]GTPγS Scintillation Proximity Binding Assay for Ultra-High-Throughput Screening of an Orphan Gαi-Coupled GPCR
| Autor: | Jason Cassaday, Berta Strulovici, Xiaoqing Shi, Eric N. Johnson, Marc Ferrer, Priya Kunapuli |
|---|---|
| Rok vydání: | 2008 |
| Předmět: |
Guanosine 5'-O-(3-Thiotriphosphate)
High-throughput screening Ligand binding assay Chinese hamster ovary cell GTPgammaS Guanosine CHO Cells GTP-Binding Protein alpha Subunits Gi-Go Biology Sulfur Radioisotopes Molecular biology Receptors G-Protein-Coupled chemistry.chemical_compound Cricetulus Scintillation proximity assay chemistry Cricetinae Drug Discovery Animals Scintillation Counting Molecular Medicine G protein-coupled receptor |
| Zdroj: | ASSAY and Drug Development Technologies. 6:327-337 |
| ISSN: | 1557-8127 1540-658X |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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