Development of Small Molecule RGS Inhibitors as a Mechanism to Modulate G-Protein Signaling.

Abstract

Regulator of G-protein Signaling (RGS) proteins are important regulatory molecules in the transduction of G-Protein Coupled Receptor (GPCR) signaling. They function by directly binding to G alpha subunits and accelerating GTP hydrolysis, thus potently inhibiting GPCR signaling. We and others have proposed that small molecule inhibitors of RGS proteins may provide a novel mechanism for therapeutic intervention in diseases stemming from deficiencies in GPCR signaling. This thesis details the identification and characterization of two novel classes of small molecule RGS inhibitors with unique properties. These compounds were identified from a series of high throughput screens performed by myself and others in our laboratory. The CCG-63802 class of molecules includes the first examples of reversible inhibitors of RGS4. These compounds can inhibit the in vitro binding and activity of several RGS proteins with IC50 values in the 3-100 micromolar range. They function by binding to RGS4 near a site thought to be important for allosteric regulation by endogenous acidic phospholipids. The second class of molecules, typified by CCG-50014, includes the most potent RGS4 inhibitors identified to date. This compound irreversibly inhibits RGS4 with nanomolar potency (IC50 30±6 nM) by covalently interacting with at least one cysteine on the RGS. In spite of the thiol dependence of these compounds, several members of this class can inhibit RGS binding and activity on G protein alpha subunits in living cells. Future work with these compounds is focused upon testing their activity in a variety of isolated organ and whole-animal studies. It is hoped that these compounds will provide a foundation for the development of new, more active RGS inhibitors with potential clinical and/or research utility.