Peptide Inhibitors of Regulator of G Protein Signaling 4 (RGS4): Rational and Combinatorial Approaches.

Abstract

Regulators of G protein signaling (RGSs) accelerate GTP hydrolysis by Gα subunits and profoundly inhibit signaling by G protein-coupled receptors (GPCRs). The distinct expression patterns and pathophysiologic regulation of RGS proteins suggest that inhibitors may have therapeutic potential. The Mosberg and Neubig labs previously reported the design of a constrained peptide inhibitor of RGS4 (YJ34: Ac-Val-Lys-c[Cys-Thr-Gly-Ile-Cys]-Glu-NH2, S-S) based on the structure of the Gαi switch 1 region, however, its mechanism of action was not established. In Chapter 2, the mechanism and structure-activity relationship (SAR) of YJ34 were investigated. Some features were found to be necessary for YJ34 activity, including a N-terminal acetyl group, a C-terminal amide, a Gly at position 5, Cys at positions 3 and 7 and a disulfide bridge. In Chapter 3, a focused one-bead, one-compound (OBOC) peptide library was screened for RGS4 inhibitors. Each peptide in the library contained the above mentioned features that are thought to be required for the function of YJ34. The other 5 amino acids were randomized among the 19 natural amino acids (except Cys) to afford 2.5 million possible peptide sequences. From this screen, two novel inhibitors of RGS4 in a single turnover GTPase assay were found: 2ad (Ac-Gly-Thr-c[Cys-Phe-Gly-Thr-Cys]-Trp-NH2, S-S) and 2nd (Gly-Thr-c[Cys-Phe-Gly-Thr-Cys]-Trp-NH2, S-S). Chapter 4 describes a third hit, 9nd (Tyr-Trp-[Cys-Lys-Gly-Leu-Cys]-Lys-NH2, S-S), which blocks the RGS4-Gαo interaction in the flow cytometry protein interaction assay (FCPIA). Interestingly, this peptide binds RGS4 covalently in a dithiothreitol (DTT)-sensitive manner, suggesting that a disulfide bridge is formed between RGS4 and peptide 9nd. Although it has RGS4 selectivity (RGS4>RGS8>RGS16>RGS19), it appears that the mechanism of action is through non-selective cysteine modification. There are some interesting observations from the project. First, our focused library yielded binding peptides with both the anticipated and unanticipated mechanisms. Second, RGS4 appears to be more sensitive to cysteine modification than other RGS proteins. Third, in the library hits, there is a correlation between activity in the FCPIA assay and hydrophobicity.Ph.D.PharmacologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/60887/1/myersra_1.pd