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Antimicrobial Agents and Chemotherapy, November 2007, p. 3824-3829, Vol. 51, No. 11
0066-4804/07/$08.00+0     doi:10.1128/AAC.00433-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Isoxyl Activation Is Required for Bacteriostatic Activity against Mycobacterium tuberculosis{triangledown}

Jana Korduláková,1,{ddagger} Yves L. Janin,2 Avraham Liav,3 Nathalie Barilone,1 Tiago Dos Vultos,1 Jean Rauzier,1 Patrick J. Brennan,3 Brigitte Gicquel,1 and Mary Jackson1*

Unité de Génétique Mycobactérienne,1 Unité de Chimie Organique, URA 2128 CNRS-Institut Pasteur, Institut Pasteur, Paris, France,2 Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, Colorado 805233

Received 29 March 2007/ Returned for modification 28 April 2007/ Accepted 25 August 2007

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.

* Corresponding author. Present address: Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523-1682. Phone: (970) 491 3582. Fax: (970) 491 1815. E-mail: mary.jackson{at}colostate.edu

{triangledown} Published ahead of print on 4 September 2007.

{ddagger} Present address: Department of Biochemistry, Faculty of Natural Sciences, Comenius University, Mlynska dolina CH-1, 84215 Bratislava, Slovak Republic.

Antimicrobial Agents and Chemotherapy, November 2007, p. 3824-3829, Vol. 51, No. 11
0066-4804/07/$08.00+0     doi:10.1128/AAC.00433-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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