Kinetic characterization and molecular docking of novel allosteric inhibitors of aminoglycoside phosphotransferases

• Published in: Biochimica et biophysica acta. General subjects Vol. 1861; no. 1; pp. 3464 - 3473
• Main Authors: Leban, Nadia, Kaplan, Elise, Chaloin, Laurent, Godreuil, Sylvain, Lionne, Corinne
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.01.2017; Elsevier
• Subjects: Allosteric inhibitors; Allosteric Regulation - drug effects; Aminoglycosides - metabolism; Antibiotic resistance; Biochemistry; Biochemistry, Molecular Biology; Biophysics; Crystallography, X-Ray; Docking; Enzyme Inhibitors - chemistry; Enzyme Inhibitors - pharmacology; Inhibition modes; Kinetics; Life Sciences; Molecular biology; Molecular Docking Simulation; Phosphotransferases (Alcohol Group Acceptor) - antagonists & inhibitors; Phosphotransferases (Alcohol Group Acceptor) - metabolism; Protein dynamics; Steady state kinetics; GOLD; Steady state kinetics; AAC; ANT; Antibiotic resistance; AME; APH; Allosteric inhibitors; Docking; Protein dynamics; Inhibition modes; aminoglycoside Ophosphotransferase; aminoglycoside-modifying enzyme; Genetic Optimization for Ligand Docking allosteric inhibitors; aminoglycoside Nacetyltransferase; aminoglycoside O-nucleotidyltransferase
• ISSN: 0304-4165; 1872-8006
• DOI: 10.1016/j.bbagen.2016.09.012

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. •We identified allosteric APH inhibitors that bind to small protein cavities•The strategy is to block the enzyme function by perturbing its dynamics•Basis for combined therapies development that reverse aminoglycoside resistance