title: Kinetic characterization and molecular docking of novel allosteric inhibitors of aminoglycoside phosphotransferases.
abstract: Bacterial antibiotic resistance often leads to treatment failure which may have serious consequences, especially in critically sick patients. Resistance to aminoglycosides is mainly due to the expression of antibiotic-modifying enzymes. One important mechanism of aminoglycoside modification is the ATP/GTP-dependent O-phosphorylation catalyzed by aminoglycoside phosphotransferases, APHs. The aim of this study is to identify specific inhibitors of APHs that could restore bacterial susceptibility to aminoglycosides.
  
  
  
  We focused on the search for allosteric inhibitors that bind to small cavities of the protein and block the enzyme function by perturbing its dynamics.
  
  
  
  From normal mode analysis, a cavity of variable volume belonging to a large groove which splits the protein into two parts was chosen as target. By molecular docking, we screened a large library of commercially available compounds. Seventeen of the highest ranked compounds were tested by in vitro kinetic experiments in order to evaluate their ability to inhibit APHs. Site-directed mutagenesis was carried out with the aim of confirming the inhibition mechanism determined kinetically and the interactions with the protein predicted by in silico studies. These interactions were also confirmed by the use of structurally-related molecules.
  
  
  
  Two compounds showed interesting inhibition properties, and one was able to block two different classes of APH.
  
  
  
  This study gives new insights into the inhibition of APHs by such allosteric inhibitors, and provides the basis for the future development of combined therapies, antibiotic plus APH inhibitor, which may reverse the resistance to aminoglycosides in a clinical context.
authors.affiliation: CNRS FRE 3689, Université de Montpellier, Centre d'études d'agents Pathogènes et Biotechnologies pour la Santé (CPBS), F-34293 Montpellier, France.
authors.forename: Nadia
authors.initials: N
authors.lastname: Leban
authors.affiliation: CNRS FRE 3689, Université de Montpellier, Centre d'études d'agents Pathogènes et Biotechnologies pour la Santé (CPBS), F-34293 Montpellier, France.
authors.forename: Elise
authors.initials: E
authors.lastname: Kaplan
authors.affiliation: CNRS FRE 3689, Université de Montpellier, Centre d'études d'agents Pathogènes et Biotechnologies pour la Santé (CPBS), F-34293 Montpellier, France.
authors.forename: Laurent
authors.initials: L
authors.lastname: Chaloin
authors.affiliation: INSERM U1058, Université de Montpellier, Centre Hospitalier Régional Universitaire de Montpellier, Département de Bactériologie-Virologie, Montpellier, France.
authors.forename: Sylvain
authors.initials: S
authors.lastname: Godreuil
authors.affiliation: CNRS FRE 3689, Université de Montpellier, Centre d'études d'agents Pathogènes et Biotechnologies pour la Santé (CPBS), F-34293 Montpellier, France. Electronic address: lionne@cbs.cnrs.fr.
authors.forename: Corinne
authors.initials: C
authors.lastname: Lionne
mesh_terms: D000494:Allosteric Regulation / Q000187:drug effects; D000617:Aminoglycosides / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D007700:Kinetics; D062105:Molecular Docking Simulation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D000617:Aminoglycosides; D004791:Enzyme Inhibitors; D017853:Phosphotransferases (Alcohol Group Acceptor)
keywords: Allosteric inhibitors; Antibiotic resistance; Docking; Inhibition modes; Protein dynamics; Steady state kinetics