Synthesis, Antitubercular Activity and Mechanism of Resistance of Highly Effective Thiacetazone Analogues

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.

Resistance to Thiacetazone Derivatives Active against Mycobacterium abscessus Involves Mutations in the MmpL5 Transcriptional Repressor MAB_4384

“…The experiments described below involve the D6, D15, and D17 compounds, but larger numbers of analogues will be required to develop a robust structure-activity relationship for the thiosemicarbazone class of anti-M. abscessus agents. Nevertheless, structural activity requirements seem to be apparent for activity against M. abscessus that are similar to those for activity against M. tuberculosis (20). Modifications at the para position of the benzene ring (R3 position [ Table 1]) suggest that a good balance between size, lipophilicity, and flexibility is required for activity with active compounds D6, D15, and D17 ( Fig.…”

“…In an independent round of selection, another set of six D15-resistant strains were derived from the parental R strain, designated D15_R1 to D15_R6, all exhibiting high resistance to D15 (MIC of Ͼ200 g/ml) ( Table 4). Since resistance to TAC in M. tuberculosis has been associated with mutations either in the EthA activator (14) or in the HadABC drug target (20,31), PCR amplification and sequencing of the ethA MAB and hadABC MAB loci were performed in all 11 mutants. These sequences failed to reveal mutations, thus ruling out the involvement of these genes in the drug resistance phenotype.…”

“…This emphasizes that TAC analogues can be synthesized with improved potency not only against M. tuberculosis but also against NTM. Since a simple modification of the parental TAC scaffold to create SRI-224 led to greater potency, the effect of further simple structural modifications was investigated, leading to a second generation of TAC analogues (20). Among these compounds, two exhibited MICs 10-fold lower than the parental molecule and inhibited mycolic acid biosynthesis in M. tuberculosis (20).…”

“…Since a simple modification of the parental TAC scaffold to create SRI-224 led to greater potency, the effect of further simple structural modifications was investigated, leading to a second generation of TAC analogues (20). Among these compounds, two exhibited MICs 10-fold lower than the parental molecule and inhibited mycolic acid biosynthesis in M. tuberculosis (20). This prompted us to investigate the potential antimicrobial efficacy of these TAC derivatives against M. abscessus.…”

Síntese e caracterização de complexos de metais da primeira série do bloco d com tiossemicarbazonas para investigar seu potencial contra Mycobacterium tuberculosis

“…Diferentemente das demais, a pirazinamida não possui uma atividade direta ao alvo. No entanto, a alta concentração de ácido pirazinóico no meio intracelular abaixa drasticamente o pH a ponto de inativar enzimas fundamentais para o metabolismo celular 7 .…”