High throughput fluorescence polarization: A homogeneous alternative to radioligand binding for cell surface receptors

High throughput fluorescence polarization (FP) assays are described that of fer a nonradioactive, homogeneous, and low-cost alternative to radioligand binding assays for cell surface receptors (G protein-coupled receptors and ligand-gated ion channels). FP assays were shown to work across a range of both peptide (vasopressin V-1a and delta-opioid) and nonpeptide (beta(1)-ad renoceptor, 5-hydroxytryptamine(3)) receptors, Structure-activity relations hips were investigated at beta(1)-receptors and were found to be consistent with radioligand binding assays. FP was shown to tolerate up to 5% DMSO wi th no loss in sensitivity or signal window. From a random set of 1,280 comp ounds, 1.9% were found to significantly interfere with FP measurement. If f luorescent or quenching compounds were eliminated (3% of ail compounds), le ss than 0.4% of compounds were found to interfere with FP measurement. Assa ys could be run in 384-well plates with little loss of signal window or sen sitivity compared to 96-well plate assays, New advances in FP measurement h ave therefore enabled FP to offer a high throughput alternative to radiolig and binding for cell surface receptors.