A homogeneous high throughput nonradioactive method for measurement of functional activity of Gs-coupled receptors in membranes.
A method is described for measuring the activity of G(s)-coupled receptors in a nonradioactive homogeneous membrane-based assay. This method has several major advantages over currently used methods for measuring functional activity of G(s)-coupled receptors. The assay is high throughput (>150,000 data points/day using a single reader). Dimethyl sulfoxide tolerance is high ( approximately 10%). Compared to complex cell-based assays, there is limited potential for nonspecific compound action. This resulted in low compound hit rates in robustness screening, where hit rates from a simulated screen were 1.0% (antagonist screen) and 0.1% (agonist screen). No continuous cell culture is required for the assay, reducing cell culture overheads and allowing the screen to run every day. Automation is simple and requires no temperature- or humidity-controlled incubation. No radioactivity is required. The method relies on measurement of cyclic AMP (cAMP) generation by fluorescence polarization assay using commercially available reagents. Membranes (1-2 microg protein per well, containing anti-cAMP antibody) are transferred to 384-well plates containing 1 microl test compound. For antagonist screens, agonist is added 15 min later. After 30 min incubation at room temperature, one further assay reagent (fluorescein-cAMP in a buffer containing detergent) is added. The signal may be read after 1 h and is stable for greater than 12 h. Typical Z' for the assay is approximately 0.5.
Allen
Michael
M
Systems Research, GlaxoSmithKline, Stevenage, Herts, UK. ma16490@gsk.com
Hall
David
D
Collins
Barbara
B
Moore
Keith
K
D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000818:Animals; D001331:Automation; D001709:Biotechnology / Q000379:methods*; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D019204:GTP-Binding Proteins / Q000378:metabolism*; D020128:Inhibitory Concentration 50; D007545:Isoproterenol / Q000494:pharmacology; D007700:Kinetics; D011433:Propranolol / Q000494:pharmacology; D013696:Temperature; D013997:Time Factors; D014662:Vasoconstrictor Agents / Q000494:pharmacology; D014667:Vasopressins / Q000494:pharmacology
D000318:Adrenergic beta-Agonists; D014662:Vasoconstrictor Agents; D014667:Vasopressins; D005576:Colforsin; D011433:Propranolol; D000242:Cyclic AMP; D019204:GTP-Binding Proteins; D007545:Isoproterenol