Background: 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. We recently described a focused one-bead, one-compound (OBOC) library screen to identify peptide inhibitors of RGS4. Here we extend our observations to include another peptide with a different mechanism of action.

RGS proteins accelerate the hydrolysis of GTP by the Gα subunit of the heterotrimeric G‐protein and therefore quicken the inactivation of G‐protein signaling. The distinct expression patterns and pathophysiologic regulation of RGS proteins suggest that inhibitors of their activity may have therapeutic potential.The objective of this study is to identify peptide inhibitors of RGS4. We synthesized a focused One‐Bead, One‐Compound peptide library that retains structural features known to be necessary for the activity of a previously identified peptide. The library of 2.5 million peptides was screened and peptide‐beads with increased binding to a fluorescently labelled RGS4 were isolated. One of these hit peptides (9, Tyr‐Trp‐[Cys‐Lys‐Leu‐Cys]‐Lys‐NH2, S‐S) blocked the interaction between biotin‐RGS4 on avidin beads and a fluorescent Gαo with an IC50 of 28 μM in a Flow Cytometry Protein‐Protein Interaction Assay. It had RGS selectivity with the following order of inhibition RGS4>RGS8>RGS16≫RGS19>RGS7. Surprisingly, inhibition of RGS4 was irreversible. A methylene dithioether bridged peptide did not inhibit RGS4/Gαo interactions, suggesting that peptide 9 was forming a disulfide bond to RGS4. Future directions include determining which cysteine in the RGS is modified using mutagenesis and mass spectrometry. Supported by 5R01DA003910‐21 to HIM.