Synthesis, antitubercular activity and mechanism of resistance of highly effective thiacetazone analogues

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 bhydroxyacyl-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

Thiacetazone, an Antitubercular Drug that Inhibits Cyclopropanation of Cell Wall Mycolic Acids in Mycobacteria

Background. Mycolic acids are a complex mixture of branched, long-chain fatty acids, representing key components of the highly hydrophobic mycobacterial cell wall. Pathogenic mycobacteria carry mycolic acid sub-types that contain cyclopropane rings. Double bonds at specific sites on mycolic acid precursors are modified by the action of cyclopropane mycolic acid synthases (CMASs). The latter belong to a family of S-adenosyl-methionine-dependent methyl transferases, of which several have been well studied in Mycobacterium tuberculosis, namely, MmaA1 through A4, PcaA and CmaA2. Cyclopropanated mycolic acids are key factors participating in cell envelope permeability, host immunomodulation and persistence of M. tuberculosis. While several antitubercular agents inhibit mycolic acid synthesis, to date, the CMASs have not been shown to be drug targets. Methodology/Principle Findings. We have employed various complementary approaches to show that the antitubercular drug, thiacetazone (TAC), and its chemical analogues, inhibit mycolic acid cyclopropanation. Dramatic changes in the content and ratio of mycolic acids in the vaccine strainMycobacterium bovis BCG, as well as in the related pathogenic speciesMycobacterium marinum were observed after treatment with the drugs. Combination of thin layer chromatography, mass spectrometry and Nuclear Magnetic Resonance (NMR) analyses of mycolic acids purified fromdrug-treated mycobacteria showed a significant loss of cyclopropanation in both the a- and oxygenated mycolate sub-types. Additionally, High-Resolution Magic Angle Spinning (HR-MAS) NMR analyses on whole cells was used to detect cell wall-associated mycolates and to quantify the cyclopropanation status of the cell envelope. Further, overexpression of cmaA2, mmaA2 or pcaA in mycobacteria partially reversed the effects of TAC and its analogue on mycolic acid cyclopropanation, suggesting that the drugs act directly on CMASs. Conclusions/Significance. This is a first report on them echanism of action of TAC, demonstrating the CMASs as its cellular targets in mycobacteria. The implications of this study may be important for the design of alternative strategies for tuberculosis treatment

PCT/GB2009/000064 Weight Reducing Compounds (Tech1660)

The present invention relates to compounds which find use as weight reducing agents, and find use in treating obesity and/or excess adiposity

Strategies and challenges involved in the discovery of new chemical entities during early-stage tuberculosis drug discovery

There is an increasing flow of new antituberculosis chemical entities entering the tuberculosis drug development pipeline. Although this is encouraging, the current number of compounds is too low to meet the demanding criteria required for registration, shorten treatment duration, treat drug-resistant infection, and address pediatric tuberculosis cases. More new chemical entities are needed urgently to supplement the pipeline and ensure that more drugs and regimens enter clinical practice. Most drug discovery projects under way exploit enzyme systems deemed essential in a specific Mycobacterium tuberculosis biosynthetic pathway or develop chemical scaffolds identified by phenotypic screening of compound libraries, specific pharmacophores or chemical clusters, and natural products. Because the development of a compound for treating tuberculosis is even longer than for treating other infection indications, the identification of selective, potent, and safe chemical entities early in the drug development process is essential to ensure that the pipeline is filled with new candidates that have the best chance to reach the clinic

Dose-response effects of TAC and related analogues on mycolic acid biosynthesis in <i>M. tuberculosis.</i>

<p>The inhibitory effect on the incorporation of [2-¹⁴C]acetate was assayed by labeling in the presence of increasing concentrations of TAC, <b>15 </b>or <b>16</b>. The corresponding fatty acid methyl esters (FAME) and mycolic acid methyl esters (MAME) were extracted and equal counts were loaded onto a TLC plate. (<b>A</b>) <b>1D TLC profile of MAMEs.</b> Radiolabeled lipids (40,000 cpm) were resolved with hexane/ethyl acetate (19/1, v/v, 2 runs) and exposed overnight to a film. (<b>B</b>) <b>2D TLC profile of MAMEs.</b> Radiolabeled lipids (30, 000 cpm) were resolved with hexane/ethyl acetate (19/1, v/v, 2 runs) in the first dimension and petroleum ether/diethyl ether (17/3, v/v, 3 runs) in the second direction on 10% silver nitrate-impregnated plates and exposed for two days to a film. Α, α-mycolates; keto, ketomycolates, methoxy, methoxymycolates. Arrowheads indicate positions of the unsaturated mycolic acid species. OAME, oleic acid methyl ester.</p

Susceptibility and genetic mutations associated to TAC resistance in <i>M. tuberculosis</i> strains selected on high concentrations of TAC or SRI-224.

<p>Susceptibility and genetic mutations associated to TAC resistance in <i>M. tuberculosis</i> strains selected on high concentrations of TAC or SRI-224.</p

Mycolic acid profile of the parental <i>M. tuberculosis</i> strain and independent TAC-resistant derived mutants.

<p>FAMEs and MAMEs were extracted from exponentially growing cultures and resolved by single dimension TLC in hexane/ethyl acetate (19/1, v/v) prior to visualization using molybdophosphoric acid and charring. α, α-mycolic acids; methoxy, methoxy-mycolic acids; keto, keto-mycolic acids.</p

Oligonucleotides used in this study.

<p>F and R stand for forward and reverse, respectively.</p