title: Characterization of DprE1-Mediated Benzothiazinone Resistance in Mycobacterium tuberculosis.
abstract: Benzothiazinones (BTZs) are a class of compounds found to be extremely potent against both drug-susceptible and drug-resistant Mycobacterium tuberculosis strains. The potency of BTZs is explained by their specificity for their target decaprenylphosphoryl-d-ribose oxidase (DprE1), in particular by covalent binding of the activated form of the compound to the critical cysteine 387 residue of the enzyme. To probe the role of C387, we used promiscuous site-directed mutagenesis to introduce other codons at this position into dprE1 of M. tuberculosis The resultant viable BTZ-resistant mutants were characterized in vitro, ex vivo, and biochemically to gain insight into the effects of these mutations on DprE1 function and on M. tuberculosis Five different mutations (C387G, C387A, C387S, C387N, and C387T) conferred various levels of resistance to BTZ and exhibited different phenotypes. The C387G and C387N mutations resulted in a lower growth rate of the mycobacterium on solid medium, which could be attributed to the significant decrease in the catalytic efficiency of the DprE1 enzyme. All five mutations rendered the mycobacterium less cytotoxic to macrophages. Finally, differences in the potencies of covalent and noncovalent DprE1 inhibitors in the presence of C387 mutations were revealed by enzymatic assays. As expected from the mechanism of action, the covalent inhibitor PBTZ169 only partially inhibited the mutant DprE1 enzymes compared to the near-complete inhibition with a noncovalent DprE1 inhibitor, Ty38c. This study emphasizes the importance of the C387 residue for DprE1 activity and for the killing action of covalent inhibitors such as BTZs and other recently identified nitroaromatic inhibitors.
authors.affiliation: Global Health Institute, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.
authors.forename: Caroline Shi-Yan
authors.initials: CS
authors.lastname: Foo
authors.affiliation: Global Health Institute, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.|Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland.
authors.forename: Benoit
authors.initials: B
authors.lastname: Lechartier
authors.affiliation: Global Health Institute, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.
authors.forename: Gaëlle S
authors.initials: GS
authors.lastname: Kolly
authors.affiliation: Global Health Institute, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Boy-Röttger
authors.affiliation: Global Health Institute, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.|UCB Biopharma, Braine L'Alleud, Belgium.
authors.forename: João
authors.initials: J
authors.lastname: Neres
authors.affiliation: Global Health Institute, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.|1st Department of Internal Medicine, University of Cologne, Cologne, Germany.
authors.forename: Jan
authors.initials: J
authors.lastname: Rybniker
authors.affiliation: Global Health Institute, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.
authors.forename: Andréanne
authors.initials: A
authors.lastname: Lupien
authors.affiliation: Global Health Institute, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.
authors.forename: Claudia
authors.initials: C
authors.lastname: Sala
authors.affiliation: Global Health Institute, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.
authors.forename: Jérémie
authors.initials: J
authors.lastname: Piton
authors.affiliation: Global Health Institute, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland stewart.cole@epfl.ch.
authors.forename: Stewart T
authors.initials: ST
authors.lastname: Cole
mesh_terms: D000429:Alcohol Oxidoreductases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D000995:Antitubercular Agents / Q000737:chemistry / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D015870:Gene Expression; D006801:Humans; D008264:Macrophages / Q000382:microbiology; D062105:Molecular Docking Simulation; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D020102:Mycobacterium smegmatis / Q000187:drug effects / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D010641:Phenotype; D010879:Piperazines / Q000737:chemistry / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013843:Thiazines / Q000737:chemistry / Q000494:pharmacology; D018088:Tuberculosis, Multidrug-Resistant / Q000382:microbiology
chemical_list: D000995:Antitubercular Agents; D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D010879:Piperazines; D013843:Thiazines; C000592783:macozinone; D000429:Alcohol Oxidoreductases; C583625:DprE1 protein, Mycobacterium tuberculosis; D003545:Cysteine