title: In vitro assays to characterize inhibitors of the activation of small G proteins by their guanine nucleotide exchange factors.
abstract: Guanine nucleotide exchange factors (GEFs) are essential regulators of the spatiotemporal conditions of small GTP-binding protein (SMG) activation. Their cellular activities combine the biochemical stimulation of GDP/GTP exchange, which leads to the active conformation of the SMG, to the detection of upstream signals and, in some cases, interaction with downstream effectors. Inhibition of GEF activities by small molecules has become recently a very active field, both for understanding biology with the tools of chemistry and because GEFs are emerging as therapeutic targets. The natural compound brefeldin A (BFA) was the first inhibitor of a GEF to be characterized, and several inhibitors of SMG activation have since been discovered using a variety of screening methods. An essential step toward their use in basic research or as leads in therapeutics is the characterization of their mechanism of inhibition. GEFs function according to a multistep mechanism, involving transient ternary (nucleotide-bound) and binary (nucleotide-free) intermediates. This mechanism thereby offers many opportunities for blockage, but a thorough analysis is necessary to define the inhibition mechanism and the steps of the reaction that are affected by the inhibitor. Here, based on the case study of how BFA inhibits the activation of Arf activation by Sec7 domains, we describe a flowchart of assays to decipher the mechanism of inhibitors of the activation of SMGs by their GEFs.
authors.affiliation: Laboratoire d'Enzymologie et Biochimie Structurales, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France.
authors.forename: Jean-Christophe
authors.initials: JC
authors.lastname: Zeeh
authors.forename: Bruno
authors.initials: B
authors.lastname: Antonny
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Cherfils
authors.forename: Mahel
authors.initials: M
authors.lastname: Zeghouf
mesh_terms: D020727:ADP-Ribosylation Factors / Q000378:metabolism; D020126:Brefeldin A / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D007700:Kinetics; D020559:Monomeric GTP-Binding Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
chemical_list: D020662:Guanine Nucleotide Exchange Factors; D020126:Brefeldin A; D020727:ADP-Ribosylation Factors; D020559:Monomeric GTP-Binding Proteins