Mechanisms of Susceptibility and Resistance of Mycobacterium tuberculosis to Isoxyl and Thiacetazone

项目摘要 由结核分枝杆菌(Mtb)引起的结核病(TB)仍然是一个主要的公共卫生问题 在全球范围内，艾滋病毒携带者中最常见的表现疾病占四分之一 与艾滋病毒/艾滋病相关的死亡。结核分枝杆菌多重耐药菌株的不断上升使其发展 开发新的有效的抗结核药物是一个高度优先的问题。在此背景下，重新审视分子作用机制 针对与一线抗结核药物相同的经过充分验证的生物合成途径的已建立的抗结核药物 结核病原体，但在一个明显的催化步骤，从而绕过广泛的耐药机制，可能有 对耐药结核病的治疗产生重大影响。两种以前用于临床治疗的此类药物 Tb，异氧基(ISO)和硫代乙酮(TAC)是我们研究的对象。 ISO和TAC是含有硫代氨基甲酰胺的前体药物，需要通过 MTB黄素依赖的单加氧酶乙醇胺用于杀菌活性。激活后，ISO和TAC抑制 真菌酸的生物合成，从而取消结核分枝杆菌外膜的形成，并导致 细胞死亡。我们最近发现了II型脂肪酸合成酶(Fas-II)伸长的脱水步骤 循环是指ISO和TAC都抑制霉菌酸生物合成的点。具体来说，我们发现 ISO和TAC通过共价修饰必需的Fas-II脱水酶来阻断Fas-II延长周期， 哈达布。尽管取得了这一重要突破，但ISO和TAC是否也抑制了(非 Fas-II脱水酶HADBC及其错义突变的机制(S) 结核分枝杆菌对这两种药物的耐药性增加16到32倍仍未解释(S)。 这一应用是为了完善我们对结核分枝杆菌的易感和抗性机制的理解。 ISO和TAC的长期目标是开发新的Fas-II脱水步骤抑制剂 减少了耐药频率，并大大提高了效力、特异性和药理学特征 ISO和TAC。具体地说，我们在目标1下建议确定ISO和TAC是否在体外抑制HadBC 并在整个MTB细胞中，在目标2中，定义HadC错义的分子机制 突变会对这两种药物产生抗药性。

Project Summary Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a major public health problem worldwide and the most common presenting illness among people living with HIV accounting for one in four HIV/AIDS-associated deaths. The continuing rise of multidrug-resistant strains of Mtb makes the development of new effective anti-TB drugs a high priority. In this context, re-examining the molecular mechanism of action of established antitubercular drugs that target the same well-validated biosynthetic pathway as front-line anti- TB agents but at a distinct catalytic step, thereby bypassing widespread resistance mechanisms, could have a major impact on the treatment of drug-resistant TB. Two such drugs previously used in the clinical treatment of TB, Isoxyl (ISO) and Thiacetazone (TAC), are the object of our investigations. ISO and TAC are thiocarbamide-containing prodrugs that require activation of their thiocarbonyl moiety by the Mtb flavin-dependent monooxygenase EthA for bactericidal activity. Following activation, ISO and TAC inhibit the biosynthesis of mycolic acids thereby abolishing the formation of the outer membrane of Mtb and leading to cell death. We recently identified the dehydration step of the type II fatty acid synthase (FAS-II) elongation cycle as the point at which both ISO and TAC inhibit the biosynthesis of mycolic acids. Specifically, we found that ISO and TAC block the FAS-II elongation cycle by covalently modifying the essential FAS-II dehydratase, HadAB. Despite this important breakthrough, it is still unclear whether ISO and TAC also inhibit the (non- essential) FAS-II dehydratase HadBC, and the mechanism(s) through which missense mutations in HadC increase 16 to 32-fold the resistance of Mtb to both drugs remain(s) unexplained. This application is to complete our understanding of the mechanisms of susceptibility and resistance of Mtb to ISO and TAC with the long-term goal of developing novel inhibitors of the dehydration step of FAS-II with reduced resistance frequencies and much improved potency, specificity and pharmacological features than ISO and TAC. Specifically, we propose under Aim 1 to determine whether ISO and TAC inhibit HadBC in vitro and in whole Mtb cells and, in Aim 2, to define the molecular mechanisms through which HadC missense mutations confer resistance to both drugs.