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A Homogeneous High Throughput Nonradioactive Method for Measurement of Functional Activity of Gs-Coupled Receptors in Membranes

Systems Research, GlaxoSmithKline, Stevenage, Herts, UK

David Hall

In Vitro Pharmacology, GlaxoSmithKline, Stevenage, Herts, UK

Barbara Collins

Systems Research, GlaxoSmithKline, Stevenage, Herts, UK

Keith Moore

Systems Research, GlaxoSmithKline, Stevenage, Herts, UK

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 (~10%). Compared to complex cell-based assays, there is limited potential for non-specific 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 µg protein per well, containing anti-cAMP antibody) are transferred to 384-well plates containing 1 µl 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 ~0.5.

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