title: Isoxyl activation is required for bacteriostatic activity against Mycobacterium tuberculosis.
abstract: Isoxyl (ISO), a thiourea derivative that was successfully used for the clinical treatment of tuberculosis during the 1960s, is an inhibitor of the synthesis of oleic and mycolic acids in Mycobacterium tuberculosis. Its effect on oleic acid synthesis has been shown to be attributable to its inhibitory activity on the stearoyl-coenzyme A desaturase DesA3, but its enzymatic target(s) in the mycolic acid pathway remains to be identified. With the goal of elucidating the mode of action of ISO, we have isolated a number of spontaneous ISO-resistant mutants of M. tuberculosis and undertaken their genotypic characterization. We report here the characterization of a subset of these strains carrying mutations in the monooxygenase gene ethA. Through complementation studies, we demonstrate for the first time that the EthA-mediated oxidation of ISO is absolutely required for this prodrug to inhibit its lethal enzymatic target(s) in M. tuberculosis. An analysis of the metabolites resulting from the in vitro transformation of ISO by purified EthA revealed the occurrence of a formimidamide allowing the formulation of an activation pathway in which the oxidation of ISO catalyzed by EthA is followed by chemical transformations involving extrusion or elimination and, finally, hydrolysis.
authors.affiliation: Unité de Génétique Mycobactérienne, URA 2128 CNRS-Institute Pasteur, Paris, France.
authors.forename: Jana
authors.initials: J
authors.lastname: Korduláková
authors.forename: Yves L
authors.initials: YL
authors.lastname: Janin
authors.forename: Avraham
authors.initials: A
authors.lastname: Liav
authors.forename: Nathalie
authors.initials: N
authors.lastname: Barilone
authors.forename: Tiago
authors.initials: T
authors.lastname: Dos Vultos
authors.forename: Jean
authors.initials: J
authors.lastname: Rauzier
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Brennan
authors.forename: Brigitte
authors.initials: B
authors.lastname: Gicquel
authors.forename: Mary
authors.initials: M
authors.lastname: Jackson
mesh_terms: D000995:Antitubercular Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism; D015394:Molecular Structure; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D009171:Mycolic Acids / Q000378:metabolism; D013890:Thiourea / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*