The RGS protein inhibitor CCG-4986 is a covalent modifier of the RGS4 Gα-interaction face
| Autor: | Francis S. Willard, Adam J. Kimple, David P. Siderovski, Viorel Mocanu, Christopher A. Johnston, Patrick M. Giguère |
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| Rok vydání: | 2007 |
| Předmět: |
Models
Molecular GTPase-activating protein Molecular Sequence Data Biophysics CCG-4986 GTPase Biochemistry Article Analytical Chemistry RGS4 chemistry.chemical_compound Fluorescence Resonance Energy Transfer medicine Humans Amino Acid Sequence Cysteine Molecular Biology G protein-coupled receptor Sulfonamides biology Small molecule chemistry Mechanism of action biology.protein medicine.symptom Sequence Alignment RGS Proteins |
| Zdroj: | Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics. 1774:1213-1220 |
| ISSN: | 1570-9639 |
| DOI: | 10.1016/j.bbapap.2007.06.002 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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