We found a match
Your institution may have rights to this item. Sign in to continue.
- Title
A 1,536-Well [35S]GTPγS Scintillation Proximity Binding Assay for Ultra-High-Throughput Screening of an Orphan Gαi-Coupled GPCR.
- Authors
Eric N. Johnson; Xiaoqing Shi; Jason Cassaday; Marc Ferrer; Berta Strulovici; Priya Kunapuli
- Abstract
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 μMusing 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.
- Subjects
MEMBRANE proteins; BIOLOGICAL membranes; G proteins; DRUG development; CYCLIC adenylic acid
- Publication
Assay & Drug Development Technologies, 2008, Vol 6, Issue 3, p327
- ISSN
1540-658X
- Publication type
Article
- DOI
10.1089/adt.2007.113