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EthA, a Common Activator of Thiocarbamide-Containing Drugs Acting on Different Mycobacterial Targets

Lynn G. Dover,^1, Anuradha Alahari,^2, Paul Gratraud,² Jessica M. Gomes,¹ Veemal Bhowruth,¹ Robert C. Reynolds,³ Gurdyal S. Besra,^1* and Laurent Kremer^2*

School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom,¹ Laboratoire de Dynamique Moléculaire des Interactions Membranaires Normales et Pathologiques, UMR 5235 CNRS, Université de Montpellier II, case 107, Place Eugène Bataillon, 34095 Montpellier Cedex 05, France,² Drug Discovery Division, Southern Research Institute, P.O. Box 55305, Birmingham, Alabama 35225-5305³

Received 28 August 2006/ Returned for modification 30 November 2006/ Accepted 21 December 2006

Many of the current antimycobacterial agents require some form of cellular activation unmasking reactive groups, which in turn will bind to their specific targets. Therefore, understanding the mechanisms of activation of current antimycobacterials not only helps to decipher mechanisms of drug resistance but may also facilitate the development of alternative activation strategies or of analogues that do not require such processes. Herein, through the use of genetically defined strains of Mycobacterium bovis BCG we provide evidence that EthA, previously shown to activate ethionamide, also converts isoxyl (ISO) and thiacetazone (TAC) into reactive species. These results were further supported by the development of an in vitro assay using purified recombinant EthA, which allowed direct assessment of the metabolism of ISO. Interestingly, biochemical analysis of [¹⁴C]acetate-labeled cultures suggested that all of these EthA-activated drugs inhibit mycolic acid biosynthesis via different mechanisms through binding to specific targets. This report is also the first description of the molecular mechanism of action of TAC, a thiosemicarbazone antimicrobial agent that is still used in the treatment of tuberculosis as a second-line drug in many developing countries. Altogether, the results suggest that EthA is a common activator of thiocarbamide-containing drugs. The broad specificity of EthA can now be used to improve the activation process of these drugs, which may help overcome the toxicity problems associated with clinical thiocarbamide use.

Published ahead of print on 12 January 2007.

Both authors contributed equally.

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