title: High-throughput screening for small-molecule inhibitors of LARG-stimulated RhoA nucleotide binding via a novel fluorescence polarization assay.
abstract: Guanine nucleotide exchange factors (GEFs) stimulate guanine nucleotide exchange and the subsequent activation of Rho-family proteins in response to extracellular stimuli acting upon cytokine, tyrosine kinase, adhesion, integrin, and G-protein-coupled receptors (GPCRs). Upon Rho activation, several downstream events occur, such as morphological and cytoskeletal changes, motility, growth, survival, and gene transcription. The leukemia-associated RhoGEF (LARG) is a member of the regulators of G-protein signaling homology domain (RH) family of GEFs originally identified as a result of chromosomal translocation in acute myeloid leukemia. Using a novel fluorescence polarization guanine nucleotide-binding assay using BODIPY-Texas Red-GTPgammaS (BODIPY-TR-GTPgammaS), the authors performed a 10,000-compound high-throughput screen for inhibitors of LARG-stimulated RhoA nucleotide binding. Five compounds identified from the high-throughput screen were confirmed in a nonfluorescent radioactive guanine nucleotide-binding assay measuring LARG-stimulated [( 35)S] GTPgammaS binding to RhoA, thus ruling out nonspecific fluorescent effects. All 5 compounds selectively inhibited LARG-stimulated RhoA [( 35)S] GTPgammaS binding but had little to no effect on RhoA or Galpha( o) [(35)S] GTPgammaS binding. Therefore, these 5 compounds should serve as promising starting points for the development of small-molecule inhibitors of LARG-mediated nucleotide exchange as both pharmacological tools and therapeutics. In addition, the fluorescence polarization guanine nucleotide-binding assay described here should serve as a useful approach for both high-throughput screening and general biological applications.
authors.affiliation: Department of Pharmacology, University of Michigan Medical Center, Ann Arbor, Michigan 48109-0632, USA.
authors.forename: Chris R
authors.initials: CR
authors.lastname: Evelyn
authors.forename: Timothy
authors.initials: T
authors.lastname: Ferng
authors.forename: Rafael J
authors.initials: RJ
authors.lastname: Rojas
authors.forename: Martha J
authors.initials: MJ
authors.lastname: Larsen
authors.forename: John
authors.initials: J
authors.lastname: Sondek
authors.forename: Richard R
authors.initials: RR
authors.lastname: Neubig
mesh_terms: D000465:Algorithms; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005454:Fluorescence Polarization / Q000379:methods; D006131:Growth Inhibitors / Q000302:isolation & purification* / Q000494:pharmacology; D020662:Guanine Nucleotide Exchange Factors / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism / Q000493:pharmacokinetics; D006801:Humans; D008954:Models, Biological; D011485:Protein Binding / Q000187:drug effects; D064067:Rho Guanine Nucleotide Exchange Factors; D054852:Small Molecule Libraries / Q000032:analysis*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D020742:rhoA GTP-Binding Protein / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: C577222:ARHGEF12 protein, human; D006131:Growth Inhibitors; D020662:Guanine Nucleotide Exchange Factors; D064067:Rho Guanine Nucleotide Exchange Factors; D054852:Small Molecule Libraries; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D020742:rhoA GTP-Binding Protein