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

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 Gαactivity. Efforts to identify small molecules capable of inhibiting the protein-protein interactions between activated Gαsubunits 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 Gα-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 Gα-RGS complex formation and Gαenzymatic 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.