FLUORESCENT G PROTEINS FOR GPCR DRUG DISCOVERY

DESCRIPTION (provided by applicant): G protein coupled receptors (GPCRs) constitute the largest family of membrane proteins in the human genome and the largest group of targets for drug discovery. Yet the success rate for developing new effective drugs for specific GPCRs has been disappointing in spite of remarkable scientific advances in the field over the past 20 years. Drug development for GPCRs is frequently limited by insufficient functional information obtained at the level of high throughput screening and lead optimization. High throughput assays for GPCRs are generally cell based and designed for sensitivity, often at the cost of physiologically relevant functional information such as efficacy and G protein coupling specificity. In contrast, assays that directly measure the coupling of receptors to G proteins (such as GTP?S binding assays) provide valuable functional information, but are less amenable to high throughput screening. Moreover, these assays are generally limited to Gi coupled receptors because of relatively low signal to noise for Gs and Gq coupled receptors. The goal of this Phase I proposal is to develop more robust and versatile fluorescence-based assays that directly measure coupling of GPCRs to G proteins. The technical foundation of this proposal is the use of membrane tethered G protein alpha subunits (Tet-Ga) that couple more efficiently to GPCRs than native G proteins. Moreover, they can be efficiently and economically purified and reconstituted with membranes containing GPCRs. We will take advantage of this patented technology to develop sensitive, fluorescence-based assays for use in high-throughput screening and lead development. These assays will have higher commercial value for pharmaceutical companies. Phase I Specific Aims: 1. Generate a modified, functional Tet-Gas with no reactive cysteines. 2. Generate modified G proteins having single reactive cysteines to act as conformationally sensitive reporters. 3. Demonstrate changes in fluorescence properties of labeled, purified Tet-Gas upon activation by a GPCR. If Phase I goals are successful, we will seek Phase II funding to develop high-throughput assays for commercial use.