Numerous existing drugs target G-protein coupled receptors, and genomics research has revealed numerous new family members that may be important in disease therapies. The ability to assay novel compounds for effects on the immediate downstream signaling from these receptors therefore provides a promising general approach to discovery of new drugs. We propose to develop binding assays that provide the sensitivity and throughput of radioactive assays, without radiation hazards, at significantly lower cost and higher convenience. Quantitation by fluorescence polarization, validated in prior work, will enable a homogeneous assay. Non-homogeneous assays require filtration and washing steps ill-suited to the highly automated environment of modern drug discovery that is based on the screening of hundreds of thousands to millions of chemical compounds. In Phase I, we will prepare fluorescent analogs of GTP and demonstrate specific binding to G-proteins by fluorescence polarization. We will use purified receptors expressed as recombinant proteins: human formyl peptide receptor with associated heterotrimeric G-protein complex, and a fusion protein of the beta2- adienoreceptor with G-alpha. In Phase II, we will optimize these fluorescence polarization assays for commercial use in receptor membrane preparations that are used in high throughput screening, and demonstrate performance superior to radioactive methods. PROPOSED COMMERCIAL APPLICATIONS: Homogeneous assays utilizing FP and novel probes will greatly facilitate the new drug discovery process, lowering costs and increasing throughput. The applicant company is a leader in instrumentation for high throughput screening, with instruments placed in most major drug screening facilities. The proposed assays will be marketed for use with these instruments.
