The RGS protein inhibitor CCG-4986 is a covalent modifier of the RGS4 Galpha-interaction face

Biochim Biophys Acta. 2007 Sep;1774(9):1213-20. doi: 10.1016/j.bbapap.2007.06.002. Epub 2007 Jun 29.


Regulator of G-protein signaling (RGS) proteins accelerate GTP hydrolysis by Galpha subunits and are thus crucial to the timing of G protein-coupled receptor (GPCR) signaling. Small molecule inhibition of RGS proteins is an attractive therapeutic approach to diseases involving dysregulated GPCR signaling. Methyl-N-[(4-chlorophenyl)sulfonyl]-4-nitrobenzenesulfinimidoate (CCG-4986) was reported as a selective RGS4 inhibitor, but with an unknown mechanism of action [D.L. Roman, J.N. Talbot, R.A. Roof, R.K. Sunahara, J.R. Traynor, R.R. Neubig, Identification of small-molecule inhibitors of RGS4 using a high-throughput flow cytometry protein interaction assay, Mol. Pharmacol. 71 (2007) 169-75]. Here, we describe its mechanism of action as covalent modification of RGS4. Mutant RGS4 proteins devoid of surface-exposed cysteine residues were characterized using surface plasmon resonance and FRET assays of Galpha binding, as well as single-turnover GTP hydrolysis assays of RGS4 GAP activity, demonstrating that cysteine-132 within RGS4 is required for sensitivity to CCG-4986 inhibition. Sensitivity to CCG-4986 can be engendered within RGS8 by replacing the wildtype residue found in this position to cysteine. Mass spectrometry analysis identified a 153-Dalton fragment of CCG-4986 as being covalently attached to the surface-exposed cysteines of the RGS4 RGS domain. We conclude that the mechanism of action of the RGS protein inhibitor CCG-4986 is via covalent modification of Cys-132 of RGS4, likely causing steric hindrance with the all-helical domain of the Galpha substrate.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Sequence
  • Cysteine / antagonists & inhibitors
  • Cysteine / chemistry
  • Fluorescence Resonance Energy Transfer
  • Humans
  • Models, Molecular
  • Molecular Sequence Data
  • RGS Proteins / antagonists & inhibitors*
  • RGS Proteins / chemistry*
  • RGS Proteins / genetics
  • Sequence Alignment
  • Sulfonamides / chemistry
  • Sulfonamides / pharmacology*


  • RGS Proteins
  • Sulfonamides
  • methyl N-((4-chlorophenyl)sulfonyl)-4-nitrobenzenesulfinimidoate
  • RGS4 protein
  • Cysteine