title: Targeting the Dbl and dock-family RhoGEFs: a yeast-based assay to identify cell-active inhibitors of Rho-controlled pathways.
abstract: The Ras-like superfamily of low molecular weight GTPases is made of five major families (Arf/Sar, Rab, Ran, Ras, and Rho), highly conserved across evolution. This is in keeping with their roles in basic cellular functions (endo/exocytosis, vesicular trafficking, nucleocytoplasmic trafficking, cell signaling, proliferation and apoptosis, gene regulation, F-actin dynamics), whose alterations are associated with various types of diseases, in particular cancer, neurodegenerative, cardiovascular, and infectious diseases. For these reasons, Ras-like pathways are of great potential in therapeutics and identifying inhibitors that decrease signaling activity is under intense research. Along this line, guanine exchange factors (GEFs) represent attractive targets. GEFs are proteins that promote the active GTP-bound state of GTPases and represent the major entry points whereby extracellular cues are converted into Ras-like signaling. We previously developed the yeast exchange assay (YEA), an experimental setup in the yeast in which activity of a mammalian GEF can be monitored by auxotrophy and color reporter genes. This assay was further engineered for medium-throughput screening of GEF inhibitors, which can readily select for cell-active and specific compounds. We report here on the successful identification of inhibitors against Dbl and CZH/DOCK-family members, GEFs for Rho GTPases, and on the experimental setup to screen for inhibitors of GEFs of the Arf family. We also discuss on inhibitors developed using virtual screening (VS), which target the GEF/GTPase interface with high efficacy and specificity. We propose that using VS and YEA in combination may represent a method of choice for identifying specific and cell-active GEF inhibitors.
authors.affiliation: Montpellier University, Montpellier, France; Centre de Recherche de Biochimie Macromoléculaire, CNRS, UMR5237, Montpellier, France. Electronic address: anne.blangy@crbm.cnrs.fr.
authors.forename: Anne
authors.initials: A
authors.lastname: Blangy
authors.affiliation: Montpellier University, Montpellier, France; Centre de Recherche de Biochimie Macromoléculaire, CNRS, UMR5237, Montpellier, France.
authors.forename: Philippe
authors.initials: P
authors.lastname: Fort
mesh_terms: D000818:Animals; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D020662:Guanine Nucleotide Exchange Factors / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006801:Humans; D051379:Mice; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D064067:Rho Guanine Nucleotide Exchange Factors / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D020798:Two-Hybrid System Techniques
chemical_list: D004791:Enzyme Inhibitors; D020662:Guanine Nucleotide Exchange Factors; C057811:MCF2 protein, human; C512843:Mcf2 protein, mouse; D011518:Proto-Oncogene Proteins; D064067:Rho Guanine Nucleotide Exchange Factors
keywords: Cell-active inhibitors; Dbl and CZH/DOCK domains; GEF/GTPase complex interface; GTPases of the Rho and Arf families; Guanine nucleotide exchange factors; Ras-like signaling pathways; Trio and DOCK5 exchange factors; Virtual screening; Yeast exchange assay; Yeast two-hybrid system