Molecular Mechanisms for Inhibition of Regulators of G-protein Signaling by Small Molecules.

Abstract

Regulator of G-protein signaling (RGS) proteins potently suppress G-protein coupled receptor (GPCR) signal transduction by accelerating GTP hydrolysis on activated heterotrimeric Gα proteins. RGS proteins have become attractive targets for the purpose of manipulating GPCR-mediated cellular responses. The RGS family comprises thirty-seven proteins with differential expression patterns throughout the body. RGS4 is enriched in the CNS and has been proposed as a therapeutic target for treatment of neurological disorders including epilepsy and Parkinson's disease. Therefore, our lab has focused on the identification of small molecule inhibitors of RGS4. To date, all small molecule inhibitors of RGS proteins function through covalent modification of cysteine residues, yet substantial specificity has been observed for RGS4 over other closely related homologs. The work in this thesis details the molecular mechanism of inhibition by a potent RGS4 inhibitor (CCG-50014; IC50 = 30 nM), and reveals the importance of RGS4 dynamics in the exposure of key cysteine residues that upon binding the inhibitor prevent the protein from reaching native conformations. Elucidating this mechanism has allowed us to propose a novel cryptic site (C-site) that is formed around the buried cysteine residues, and may be more druggable than previously proposed sites on RGS4. In addition, new chemical scaffolds have been identified using a cell-based high-throughput screen that also inhibit RGS4 through a cysteine-dependent mechanism, but are significantly reversible in contrast to the first cell-active RGS4 inhibitors. Furthermore, I employed IP6 as a derivative of an endogenous negative regulator, PIP3, to map the binding site on RGS4 and the corresponding effects on protein stability and function. This study shows the direct interactions of a non-covalent small molecule with an allosteric site on the RGS4 structure, and provides insight into the mechanism of endogenous regulation of RGS4. In conclusion, the studies described within this thesis provide new pharmacological tools for the study of RGS function in a cellular context, and describe in detail the molecular interactions of both endogenous and pharmacological modulators of RGS4. These results provide the theoretical framework to pursue drug discovery strategies that are expected to significantly advance the field of RGS drug discovery.PHDPharmacologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/107061/1/storaska_1.pd