Abstract
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 α-subunits (Gα) and thus shorten the time course and reduce the magnitude of G-protein α- and βγ-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 Gα-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/Gαo binding with 3 to 5 μM potency. It binds to RGS4, inhibits RGS4 stimulation of Gαo GTPase activity in vitro, and prevents RGS4 regulation of μ-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/Gα protein-protein interactions with small molecules as a novel means to modulate GPCR-mediated signaling processes.
Footnotes
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↵1 The RGS4-short was found to be indistinguishable from a full-length 6X-His-tagged RGS4 with regard to inhibition of activity or Gαo binding by CCG-4986.
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This work was supported by National Institutes of Health grants GM039561 (to R.R.N.), F32-GM076821 (to D.L.R.), DA004087 (to J.R.T.), and T32-DA007268 (to J.R.T. and J.N.T.).
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ABBREVIATIONS: GPCR, G-protein-coupled receptor; FCPIA, flow cytometry protein interaction assay; RGS, regulator of G-protein signaling; GAP, GTPase accelerating protein; CCG-4986, methyl-N-[(4-chlorophenyl)sulfonyl]-4-nitrobenzenesulfinimidoate; CCG-2046, 3-methyl-3-propylcyclopropane-1,1,2,2-tetracarbonitrile; RT, room temperature; DMSO, dimethyl sulfoxide; cv, column volume; DAMGO, [d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin; BCB, bead-coupling buffer; AC, adenylyl cyclase; buffer A, HEPES and dithiothreitol; [35S]GTPγS, guanosine 5′-O-(3-[35S]thio)triphosphate.
- Received July 9, 2006.
- Accepted September 27, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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