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J. Biol. Chem., Vol. 278, Issue 52, 53123-53130, December 26, 2003

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Unique Mechanism of Action of the Thiourea Drug Isoxyl on Mycobacterium tuberculosis^*

Benjawan Phetsuksiri, Mary Jackson¶, Hataichanok Scherman, Michael McNeil, Gurdyal S. Besra||, Alain R. Baulard**, Richard A. Slayden, Andrea E. DeBarber, Clifton E. Barry, III, Mark S. Baird, Dean C. Crick, and Patrick J. Brennan¶¶

From the Department of Microbiology, Pathology, and Immunology, Colorado State University, Fort Collins, Colorado 80523-1682, NIAID, National Institutes of Health, Rockville, Maryland 20852, and Department of Chemistry, University of Wales, Bangor, LL57 2UW Wales

The thiourea isoxyl (thiocarlide; 4,4'-diisoamyloxydiphenylthiourea) is known to be an effective anti-tuberculosis drug, active against a range of multidrug-resistant strains of Mycobacterium tuberculosis and has been used clinically. Little was known of its mode of action. We now demonstrate that isoxyl results in a dose-dependent decrease in the synthesis of oleic and, consequently, tuberculostearic acid in M. tuberculosis with complete inhibition at 3 µg/ml. Synthesis of mycolic acid was also affected. The anti-bacterial effect of isoxyl was partially reversed by supplementing growth medium with oleic acid. The specificity of this inhibition pointed to a 9-stearoyl desaturase as the drug target. Development of a cell-free assay for 9-desaturase activity allowed direct demonstration of the inhibition of oleic acid synthesis by isoxyl. Interestingly, sterculic acid, a known inhibitor of 9-desaturases, emulated the effect of isoxyl on oleic acid synthesis but did not affect mycolic acid synthesis, demonstrating the lack of a relationship between the two effects of the drug. The three putative fatty acid desaturases in the M. tuberculosis genome, desA1, desA2, and desA3, were cloned and expressed in Mycobacterium bovis BCG. Cell-free assays and whole cell labeling demonstrated increased 9-desaturase activity and oleic acid synthesis only in the desA3-overexpressing strain and an increase in the minimal inhibitory concentration for isoxyl, indicating that DesA3 is the target of the drug. These results validate membrane-bound 9-desaturase, DesA3, as a new therapeutic target, and the thioureas as anti-tuberculosis drugs worthy of further development.

Received for publication, October 13, 2003

^* This work was funded by NIAID, National Institutes of Health Grants AI-18357 and AI-38087 (a Research Project Agreement from the National Cooperative Drug Discovery Groups for the Treatment of Opportunistic Infections in AIDS). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of a Thai government scholarship and Thailand Research Fund Grant PDF/60/2544. Present address: The Sasakawa Research Center, Leprosy Division, Dept. of Communicable Disease Control, Ministry of Public Health, Nonthaburi Province, Thailand 11000.

^¶ Recipient of a post-doctoral fellowship from the Heiser Program for Research in Leprosy and Tuberculosis. Present address: Institut Pasteur, Unité de Génétique Mycobactérienne, 75724 Paris Cedex 15, France.

^|| Present address: School of Biosciences, University of Birmingham, Birmingham, UK.

^** Present address: INSERM U447, Institut Pasteur de Lille, 59019 Lille, France.

^¶¶ To whom correspondence should be addressed. Tel.: 970-491-6700; Fax: 970-491-1815; E-mail: Patrick.Brennan{at}ColoState.edu.

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