Development of nonseparation binding and functional assays for G protein-coupled receptors for high throughput screening:: Pharmacological characterization of the immobilized CCR5 receptor on FlashPlate®

G protein-coupled receptors (GPCRs) represent a very important class of drug targets. The development of microformatted nonseparation assays constitute a key step in the process of assay development for high throughput drug screening (HTS), We have developed a microformatted nonseparation assay for membrane preparations containing the CCR5 GPCR using FlashPlate(R) microplates (Packard Instrument Company, Meriden, CT). The pharmacodynamic (radioligand-binding) and functional (agonist-stimulated [S-35]GTP gamma S binding) properties of this receptor observed in FlashPlate-based assays were compared with standard filtration assays. Saturation binding experiments performed using either assay platform revealed identical K-d for [I-125]-MIP-1 beta (0.7 nM). Comparable signal-to-noise ratios (SNR), similar affinities (K-i), and identical order of potency (RANTES congruent to MIP-1 beta > MIP-1 alpha) were observed following competition binding assays in both platforms. In functional assays, the order of potency for different agonists were similar in both platforms with RANTES congruent to MIP-1 beta greater than or equal to MIP-1 alpha, which correspond to the relative affinities determined for the three ligands in competition binding experiments. Because similar pharmacologic properties were obtained in both FlashPlate microplates and standard filtration platforms, we conclude that FlashPlate microplates could provide a valuable nonseparation platform for primary and secondary HTS for this and possibly other GPCRs.