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

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• 作者：Adam J. Kimple ; Francis S. Willard ; Patrick M. Giguè ; re ; Christopher A. Johnston ; Viorel Mocanu ; David P. Siderovski
• 关键词：CCG-4986 ; RGS4 ; RGS protein inhibitor ; Regulator of G-protein signaling ; Thiol adduct
• 刊名：Biochimica et Biophysica Acta - Proteins and Proteomics
• 出版年：2007
• 期刊代码：161_15709639
• 类别：bio
• 出版时间：September 2007
• 卷：1774
• 期：9
• 页码：1213-1220
• 文件大小：796 K

摘要

Regulator of G-protein signaling (RGS) proteins accelerate GTP hydrolysis by Gα 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 Gα 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 Gα substrate.