Research article

A covalent peptide inhibitor of RGS4 identified in a focused one-bead, one compound library screen

Rebecca A Roof^1,3 , David L Roman^1,4 , Samuel T Clements¹ , Katarzyna Sobczyk-Kojiro² , Levi L Blazer¹ , Shodai Ota¹ , Henry I Mosberg² and Richard R Neubig¹

¹Department of Pharmacology, University of Michigan,1301 MSRB III SPC 5632, 1150 W. Medical Center Dr, Ann Arbor, MI 48109, USA

²Department of Medicinal Chemistry, University of Michigan, 428 Church St, Ann Arbor, MI 48109, USA

³Current address: Molecular Neuropharmacology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 5625 Fishers Ln, Bethesda, MD 20852, USA

⁴Current address: College of Pharmacy, University of Iowa, 115 S. Grand Ave., Iowa City, IA 52242, USA

author email corresponding author email

BMC Pharmacology 2009, 9:9doi:10.1186/1471-2210-9-9

Published:	22 May 2009

Abstract

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.

Results

Peptide 5nd (Tyr-Trp-c [Cys-Lys-Gly-Leu-Cys]-Lys-NH₂, S-S) blocks the RGS4-Gα_o interaction with an IC₅₀ of 28 μM. It forms a covalent, dithiothreitol (DTT) sensitive adduct with a mass consistent with the incorporation of one peptide per RGS. Peptide 5nd activity is abolished by either changing its disulfide bridge to a methylene dithioether bridge, which cannot form disulfide bridges to the RGS, or by removing all cysteines from the RGS protein. However, no single cysteine in RGS4 is completely necessary or sufficient for 5nd activity.

Conclusion

Though it has some RGS selectivity, 5nd appears to be a partially random cysteine modifier. These data suggest that it inhibits RGS4 by forming disulfide bridges with the protein.