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Research ArticleArticle

Identification of Small-Molecule Inhibitors of RGS4 Using a High-Throughput Flow Cytometry Protein Interaction Assay

David L. Roman, Jeffery N. Talbot, Rebecca A. Roof, Roger K. Sunahara, John R. Traynor and Richard R. Neubig
Molecular Pharmacology January 2007, 71 (1) 169-175; DOI: https://doi.org/10.1124/mol.106.028670
David L. Roman
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Jeffery N. Talbot
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Rebecca A. Roof
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Roger K. Sunahara
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John R. Traynor
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Richard R. Neubig
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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

  • ↵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.

  • 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.).

  • 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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Molecular Pharmacology: 71 (1)
Molecular Pharmacology
Vol. 71, Issue 1
1 Jan 2007
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Research ArticleArticle

Identification of Small-Molecule Inhibitors of RGS4 Using a High-Throughput Flow Cytometry Protein Interaction Assay

David L. Roman, Jeffery N. Talbot, Rebecca A. Roof, Roger K. Sunahara, John R. Traynor and Richard R. Neubig
Molecular Pharmacology January 1, 2007, 71 (1) 169-175; DOI: https://doi.org/10.1124/mol.106.028670

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Research ArticleArticle

Identification of Small-Molecule Inhibitors of RGS4 Using a High-Throughput Flow Cytometry Protein Interaction Assay

David L. Roman, Jeffery N. Talbot, Rebecca A. Roof, Roger K. Sunahara, John R. Traynor and Richard R. Neubig
Molecular Pharmacology January 1, 2007, 71 (1) 169-175; DOI: https://doi.org/10.1124/mol.106.028670
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