title: Identification of small-molecule inhibitors of RGS4 using a high-throughput flow cytometry protein interaction assay.
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 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.
authors.affiliation: Department of Pharmacology, University of Michigan Medical School, 1150 W. Medical Center Drive, 1303 MSRB III, Ann Arbor, MI 41809, USA.
authors.forename: David L
authors.initials: DL
authors.lastname: Roman
authors.forename: Jeffery N
authors.initials: JN
authors.lastname: Talbot
authors.forename: Rebecca A
authors.initials: RA
authors.lastname: Roof
authors.forename: Roger K
authors.initials: RK
authors.lastname: Sunahara
authors.forename: John R
authors.initials: JR
authors.lastname: Traynor
authors.forename: Richard R
authors.initials: RR
authors.lastname: Neubig
mesh_terms: D019921:Biotinylation; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005434:Flow Cytometry; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D021122:Protein Subunits / Q000378:metabolism; D020710:RGS Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D021122:Protein Subunits; D020710:RGS Proteins; C098799:RGS4 protein; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate