The RGS protein inhibitor CCG-4986 is a covalent modifier of the RGS4 Galpha-interaction face.
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.
Kimple
Adam J
AJ
Department of Pharmacology, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7365, USA.
Willard
Francis S
FS
Giguère
Patrick M
PM
Johnston
Christopher A
CA
Mocanu
Viorel
V
Siderovski
David P
DP
D000595:Amino Acid Sequence; D003545:Cysteine / Q000037:antagonists & inhibitors / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020710:RGS Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics; D016415:Sequence Alignment; D013449:Sulfonamides / Q000737:chemistry / Q000494:pharmacology*
D020710:RGS Proteins; D013449:Sulfonamides; C523871:methyl N-((4-chlorophenyl)sulfonyl)-4-nitrobenzenesulfinimidoate; C098799:RGS4 protein; D003545:Cysteine