Evaluation of the Selectivity and Cysteine Dependence of Inhibitors across the Regulator of G Protein–Signaling Family

Abstract

Since their discovery more than 20 years ago, regulators of G protein-signaling (RGS) proteins have received considerable attention as potential drug targets because of their ability to modulate G activity. Efforts to identify small molecules capable of inhibiting the protein-protein interactions between activated G subunits and RGS proteins have yielded a substantial number of inhibitors, especially toward the well studied RGS4. These efforts also determined that many of these small molecules inhibit the protein-protein interactions through covalent modification of cysteine residues within the RGS domain that are located distal to the G-binding interface. As some of these cysteine residues are highly conserved within the RGS family, many of these inhibitors display activity toward multiple RGS family members. In this work, we sought to determine the selectivity of these small-molecule inhibitors against 12 RGS proteins, as well as against the cysteine-null mutants for 10 of these proteins. Using both biochemical and cell-based methods to assess G-RGS complex formation and G enzymatic activity, we found that several previously identified RGS4 inhibitors were active against other RGS members, such as RGS14, with comparable or greater potency. Additionally, for every compound tested, activity was dependent on the presence of cysteine residues. This work defines the selectivity of commercially available RGS inhibitors and provides insight into the RGS family members for which drug discovery efforts may be most likely to succeed.

Regulator of G-protein signaling (RGS) proteins as drug targets: Progress and future potentials

“…This examination revealed the significant activity of some compounds for RGS proteins that were not used during the compound's initial discovery. For example, CCG-50014 was identified as the most potent RGS4 inhibitor (106), inhibiting RGS4 with an IC 50 of 30 nM; but, surprisingly, it was found to inhibit RGS14 with an IC 50 of 8 nM (112). Furthermore, we found that of the 13 inhibitors, at least one new RGS was found to be inhibited by the small molecules tested (112).…”

“…The report by Hayes et al (112) used both biochemical and cellular protein-protein interaction assays to perform selectivity analysis with a number of previously identified RGS smallmolecule inhibitors against a wide range of RGS proteins. This examination revealed the significant activity of some compounds for RGS proteins that were not used during the compound's initial discovery.…”

“…For example, CCG-50014 was identified as the most potent RGS4 inhibitor (106), inhibiting RGS4 with an IC 50 of 30 nM; but, surprisingly, it was found to inhibit RGS14 with an IC 50 of 8 nM (112). Furthermore, we found that of the 13 inhibitors, at least one new RGS was found to be inhibited by the small molecules tested (112). This led to identification of additional functions for previously published small-molecule inhibitors, providing new information on novel RGS-inhibitor pairs.…”

Regulator of G-Protein Signaling (RGS) Protein Modulation of Opioid Receptor Signaling as a Potential Target for Pain Management

“…Similarly, CCG-50014 inhibits both RGS4 and RGS19 and is selective for these RGS proteins over RGS8 and RGS16 (Blazer et al, 2011), None of the small molecule inhibitors identified to date act at RGS6 or RGS7 (Hayes et al, 2018) which lack a cysteine in the RH domain, although it is worth noting that RGS9 does have a cysteine in the same position at the sensitive cysteine in RGS19. Of interest is that studies have now shown CCG-50014 also inhibits RGS1,5,14, and 17 and in fact is most potent at RGS 14 (Hayes et al, 2018). This could be significant given the, albeit limited, knowledge on the role of RGS14 in morphine antinociception (Garzón et al, 2001;Rodríguez-Muñoz et al, 2007a).…”

“…For example, CCG-203769 which inhibits RGS4, is 10-fold less potent at inhibiting RGS19, but has a very high selectivity over other members of the RGS family (Blazer et al, 2015). Similarly, CCG-50014 inhibits both RGS4 and RGS19 and is selective for these RGS proteins over RGS8 and RGS16 (Blazer et al, 2011), None of the small molecule inhibitors identified to date act at RGS6 or RGS7 (Hayes et al, 2018) which lack a cysteine in the RH domain, although it is worth noting that RGS9 does have a cysteine in the same position at the sensitive cysteine in RGS19. Of interest is that studies have now shown CCG-50014 also inhibits RGS1,5,14, and 17 and in fact is most potent at RGS 14 (Hayes et al, 2018).…”