Premium
Characterization and mechanistic investigation of CCG‐4986, a small molecule RGS4 inhibitor
Author(s) -
Roman David L.,
Talbot Jeffery N.,
Roof Rebecca A.,
Sunahara Roger K.,
Traynor John R.,
Neubig Richard R.
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a431
Subject(s) - g protein , biology , docking (animal) , receptor , chemistry , pharmacology , microbiology and biotechnology , biochemistry , medicine , nursing
Regulators of G protein signaling (RGS) proteins accelerate the intrinsic GTPase activity of GTP‐bound Gα subunits, promoting the reformation of inactive Gαβγ heterotrimer. Our aim is to demonstrate the feasibility of RGS proteins as novel pharmacological targets for modulating G protein coupled receptor signaling. We recently reported (Mol Pharm, in press ) the discovery of CCG‐4986 ( methyl N‐[(4‐chlorophenyl)sulfonyl]‐4‐nitrobenzenesulfinimidoate ), a small molecule inhibitor of RGS4 identified in a high throughput screen for Gα o /RGS4 binding. CCG‐4986 binds directly to RGS4 with a Kd of ~3 uM as demonstrated by quenching of RGS4 intrinsic fluorescence. It inhibits the RGS4/Gα interaction, functionally inhibits RGS4 GTPase accelerating activity, and inhibits RGS4 action on μ‐opioid receptor mediated adenylate cyclase regulation in permeabilized C6 cells. CCG‐4986 exhibits specificity as it does not inhibit Gα binding or GTPase acceleration by RGS7 or RGS8, the most similar RGS4 family member. Surprisingly, the actions of CCG‐4986 were found to be irreversible, as washing steps do not reduce the inhibition of RGS4 by CCG‐4986. Furthermore, high concentrations of sulfhydryl reagents can reverse the inhibition suggesting covalent modification of cysteines on RGS4. This should facilitate studies to determine the site of action of CCG‐4986 on RGS4 and identification of additional inhibitor compounds by computer‐based docking studies. Supported by NIH GM39561 (RRN), DA0004087 (JRT) and GM076821 (DLR).