Isoxyl (ISO) and thiacetazone (TAC) are two antitubercular prodrugs that abolish mycolic acid biosynthesis and kill Mycobacterium tuberculosis (Mtb) through the inhibition of the essential type II fatty acid synthase (FAS-II) dehydratase HadAB. While mutations preventing ISO and TAC either from being converted to their active form or from covalently modifying their target are the most frequent spontaneous mutations associated with high-level resistance to both drugs, the molecular mechanisms underlying the high-level ISO and TAC resistance of Mtb strains harboring missense mutations in the second, nonessential, FAS-II dehydratase HadBC have remained unexplained. Using a combination of genetic, biochemical, and biophysical approaches and molecular dynamics simulation, we here show that all four reported resistance mutations in the HadC subunit of HadBC alter the stability and/or specific activity of the enzyme, allowing it in two cases (HadBCV85I and HadBCK157R) to compensate for a deficiency in HadAB in whole Mtb bacilli. The analysis of the mycolic acid profiles of Mtb strains expressing the mutated forms of HadC further points to alterations in the activity of the mycolic acid biosynthetic complex and suggests an additional contributing resistance mechanism whereby HadC mutations may reduce the accessibility of HadAB to ISO and TAC. Collectively, our results highlight the importance of developing optimized inhibitors of the dehydration step of FAS-II capable of inhibiting both dehydratases simultaneously, a goal that may be achievable given the structural resemblance of the two enzymes and their reliance on the same catalytic subunit HadB.

中文翻译：

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与结核分枝杆菌中II型脂肪酸合酶脱水步骤的抑制有关的抗性机制。

异氧基（ISO）和噻乙acet（TAC）是两种抗结核前药，它们通过抑制必需的II型脂肪酸合酶（FAS-II）脱水酶HadAB来消除霉菌酸的生物合成并杀死结核分枝杆菌（Mtb）。虽然阻止ISO和TAC转化为活性形式或共价修饰靶标的突变是与两种药物均具有高水平耐药性相关的最常见的自发突变，但Mtb的高水​​平ISO和TAC耐药性的分子机制在第二个非必需的FAS-II脱水酶HadBC中带有错义突变的菌株仍无法解释。结合了遗传，生物化学和生物物理方法以及分子动力学模拟，我们在这里显示，HadBC的HadC亚基中所有四个已报道的抗性突变均会改变该酶的稳定性和/或比活性，从而使其在两种情况下（HadBCV85I和HadBCK157R）能够补偿整个Mtb杆菌中HadAB的缺乏。表达突变形式的HadC的Mtb菌株的霉菌酸谱分析进一步指出了霉菌酸生物合成复合物活性的变化，并提示了另外的抗药性机制，其中HadC突变可降低HadAB对ISO和TAC的可及性。总的来说，我们的结果突出了开发优化的FAS-II脱水步骤抑制剂的重要性，该抑制剂能够同时抑制两种脱水酶，