Abstract
Defining the pharmacological target(s) of currently used drugs and developing new analogues with greater potency are both important aspects of the search for agents that are effective against drug-sensitive and drug-resistant Mycobacterium tuberculosis. Thiacetazone (TAC) is an anti-tubercular drug that was formerly used in conjunction with isoniazid, but removed from the antitubercular chemotherapeutic arsenal due to toxic side effects. However, several recent studies have linked the mechanisms of action of TAC to mycolic acid metabolism and TAC-derived analogues have shown increased potency against M. tuberculosis. To obtain new insights into the molecular mechanisms of TAC resistance, we isolated and analyzed 10 mutants of M. tuberculosis that were highly resistant to TAC. One strain was found to be mutated in the methyltransferase MmaA4 at Gly101, consistent with its lack of oxygenated mycolic acids. All remaining strains harbored missense mutations in either HadA (at Cys61) or HadC (at Val85, Lys157 or Thr123), which are components of the β-hydroxyacyl-ACP dehydratase complex that participates in the mycolic acid elongation step. Separately, a library of 31 new TAC analogues was synthesized and evaluated against M. tuberculosis. Two of these compounds, 15 and 16, exhibited minimal inhibitory concentrations 10-fold lower than the parental molecule, and inhibited mycolic acid biosynthesis in a dose-dependent manner. Moreover, overexpression of HadAB HadBC or HadABC in M. tuberculosis led to high level resistance to these compounds, demonstrating that their mode of action is similar to that of TAC. In summary, this study uncovered new mutations associated with TAC resistance and also demonstrated that simple structural optimization of the TAC scaffold was possible and may lead to a new generation of TAC-derived drug candidates for the potential treatment of tuberculosis as mycolic acid inhibitors.
Highlights
Drug-resistant Mycobacterium tuberculosis is an increasing threat to global health [1,2]
To continue our studies on the antitubercular activity of TAC, we selected TAC-resistant M. tuberculosis mutants by plating actively-growing cultures on Middlebrook 7H10 media supplemented with oleic aciddextrose-catalase (OADC) and either TAC or SRI-224 at concentrations that were 10 to 40 times higher than the minimum inhibitory concentration (MIC99)
The ten mutants that were obtained, labeled MTTR2 through MTTR28, are listed in Table 2, which indicates the concentration of the drug on which these were selected and shows that most mutants exhibited comparably high levels of resistance to both TAC and SRI-224, with minimal inhibitory concentration (MIC) values $20 mg/ml compared to the MIC of 0.25 mg/ml for the parental strain, as previously reported [23]
Summary
Drug-resistant Mycobacterium tuberculosis is an increasing threat to global health [1,2].
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