A homogeneous high throughput nonradioactive method for measurement of functional activity of GS-coupled receptors in membranes

Abstract

A method is described for measuring the activity of GS-coupled receptors in a nonradioactive homogeneous membrane-based assay. This method has several major advantages over currently used methods for measuring functional activity of GS-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 rain 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.