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Mol Pharmacol. 2007 Jan;71(1):169-75. Epub 2006 Sep 29.

Identification of small-molecule inhibitors of RGS4 using a high-throughput flow cytometry protein interaction assay.

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  • 1Department of Pharmacology, University of Michigan Medical School, 1150 W. Medical Center Drive, 1303 MSRB III, Ann Arbor, MI 41809, USA.


Regulators of G-protein signaling (RGS) proteins are important components of signal transduction pathways initiated through G-protein-coupled receptors (GPCRs). RGS proteins accelerate the intrinsic GTPase activity of G-protein alpha-subunits (Galpha) and thus shorten the time course and reduce the magnitude of G-protein alpha- and betagamma-subunit signaling. Inhibiting RGS action has been proposed as a means to enhance the activity and specificity of GPCR agonist drugs, but pharmacological targeting of protein-protein interactions has typically been difficult. The aim of this project was to identify inhibitors of RGS4. Using a Luminex 96-well plate bead analyzer and a novel flow-cytometric protein interaction assay to assess Galpha-RGS interactions in a high-throughput screen, we identified the first small-molecule inhibitor of an RGS protein. Of 3028 compounds screened, 1, methyl N-[(4-chlorophenyl)sulfonyl]-4-nitrobenzenesulfinimidoate (CCG-4986), inhibited RGS4/Galpha(o) binding with 3 to 5 muM potency. It binds to RGS4, inhibits RGS4 stimulation of Galpha(o) GTPase activity in vitro, and prevents RGS4 regulation of mu-opioid-inhibited adenylyl cyclase activity in permeabilized cells. Furthermore, CCG-4986 is selective for RGS4 and does not inhibit RGS8. Thus, we demonstrate the feasibility of targeting RGS/Galpha protein-protein interactions with small molecules as a novel means to modulate GPCR-mediated signaling processes.

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