The GPCR family mediates the physiological actions of neurotransmitters and hormones in the body and is a target for the therapeutic actions of 50% of FDA approved drugs. GPCRs can exist in multiple forms. Biochemical evidence suggests that GPCRs can form oligomers, either homomeric or heteromeric. Recent studies have indicated that some GPCR hetero-oligomers have different ligand specificity and functional responses than the monomers or homo-oligomers. If true, this would open up an entirely new field of drug research and discovery because it would offer the possibility of identifying drugs targeting specific GPCR complexes that do not recognize the monomers. Such oligomer-directed drugs might produce desired pharmacological actions lacking in drugs acting at monomers with tissue or cellular selectivity not possible with drug discovery approaches presently in use in the pharmaceutical industry. We propose to develop a novel technology that can be employed for high throughput screening (HTS) of drugs against GPCR oligomers. This approach will be a fusion of technologies developed by Patobios and DiscoveRx. Patobios has shown that insertion of a nuclear localization signal (NLS) into GPCRs directs the receptor away from the cell membrane to the cytosol (O'Dowd et al, 2005). High-affinity binding of ligands to the GPCR causes retention of the GPCR in the cell membrane providing a readout of ligand binding independent of functional activation. In preliminary studies we showed internalization of hetero-oligomers, in which one GPCR is tagged with an NLS and a different GPCR is not. However, if ligand binds to the receptor not tagged with the NLS, the complex is retained in the cell membrane providing a readout of ligand binding to GPCR hetero-oligomers. DiscoveRx has developed a sensitive enzyme fragment complementation (EFC) technology to detect surface expression of GPCR in a HTS format using a luminescent readout (Eglen and Singh, 2003; Dunlop and Eglen, 2004). Combining these technologies will provide an assay to screen for drugs uniquely binding to hetero-oligomers and lead to the discovery and development of a new generation of GPCR-based drugs.
