Mode of Action of Thiorcarlide in Mycobacterium Tuberculosis

描述（申请人提供）：硫卡内酯（THC），一种硫脲，抑制结核分枝杆菌（M. tuberculosis）中分枝菌酸和脂肪酸的合成。tb）。我们的证据表明THC对脂肪酸合成的独特作用模式，即THC对膜相关硬脂酰-CoA（9）去饱和酶DesA 3活性的抑制作用可归因于对油酸合成的抑制。尽管THC在分枝菌酸生物合成途径中具有至少一种其他酶靶，但这种酶仍有待鉴定。由于我们的初步数据表明，四氢大麻酚对棒状杆菌和诺卡氏菌属的分枝菌酸合成没有抑制作用，对金分枝杆菌的FAS-Ⅰ和FAS-Ⅱ系统的活性也没有抑制作用，因此该药物的第二个靶点可能与其他抗结核药物（如异烟肼和乙硫异烟胺）不同，可能是一种将早期官能团引入分枝菌酸的部分分枝菌酸链的酶。分枝杆菌基因组tb可能编码7种与环氧化物水解酶相关的蛋白质，其中一些似乎参与分枝菌酸中官能团的引入，并且对THC和其他更有效的脲/硫脲抑制剂敏感。这些酶将被纯化，并在体外直接测试它们被THC抑制。为了更广泛的遗传方法，我们已经分离出一系列的THC和尿素/THC（交叉）抗性突变体的M。结核病，并着手表征突变负责赋予高水平的耐药性，这些药物。将确定新确定的THC目标的重要性和功能。将确定药物最相关靶点的三维结构，并开发无细胞测定法，以筛选抑制剂，包括我们现有的尿素和硫脲衍生物库，并在后期促进以化学合成和产品扩展测试形式的药物化学方法。THC曾经被接受用于治疗结核病，但由于不良的吸收动力学和低生物利用度而失去了青睐。这种方法旨在克服这些方面。从更基本的角度来看，阐明THC的作用模式也代表了一个独特的机会，以确定负责在分枝菌酸的部分分枝菌酸链中引入双键和含氧功能的未知酶。公共卫生相关性：在耐多药结核和广泛耐药结核威胁的背景下，迫切需要抗结核新药。我们建议阐明硫卡利特（thiocarlide）的作用方式，硫卡利特是20世纪60年代结核病临床治疗的一部分，并利用这些知识开发具有相同作用方式但药理学性质大大改善的新型治疗药物。

DESCRIPTION (provided by applicant): Thiocarlide (THC), a thiourea, inhibits the synthesis of mycolic acids and fatty acids in Mycobacterium tuberculosis (M. tb). Our evidence indicates a unique mode of action of THC on fatty acid synthesis, namely, the inhibition of the synthesis of oleic acid attributable to the inhibitory effect of THC on the activity of the membrane-associated stearoyl-CoA ( 9) desaturase DesA3. Although THC has at least one other enzymatic target in the mycolic acid biosynthetic pathway, this(ese) enzyme(s) remain(s) to be identified. Since our preliminary data indicate that THC has no inhibitory effect on mycolic acid synthesis in Corynebacterium and Nocardia species and on the activity of the FAS-I and FAS-II systems from Mycobacterium aurum, the second target of the drug is likely to differ from that of other anti-TB drugs such as isoniazid and ethionamide and could be an enzyme introducing early functional groups into the meromycolate chain of mycolic acids. The genome of M. tb potentially encodes seven proteins related to epoxide hydrolases, some of which appear to be involved in the introduction of functional groups in mycolic acids and to be sensitive to THC and other more potent urea/thiourea inhibitors. These enzymes will be purified and their inhibition by THC directly tested in vitro. Towards a broader genetic approach, we have isolated a series of THC- and urea/THC (cross-)resistant mutants of M. tb and undertaken to characterize the mutations responsible for conferring high level of resistance to these drugs. The essentiality and function of the newly identified target(s) of THC will be established. The three-dimensional structure of the most relevant targets of the drug will be determined and cell-free assays developed to allow screening of inhibitors, including our existing libraries of urea and thioureas derivatives, and facilitate at a later stage a medicinal chemistry approach in the form of chemical synthesis and extended testing of products. THC was once acceptable for treatment of tuberculosis but lost favor due to untoward absorption kinetics and low bioavailability. This approach is designed to overcome these aspects. From a more fundamental point of view, the elucidation of the mode of action of THC also represents a unique opportunity to identify the as yet unknown enzymes responsible for the introduction of double bonds and oxygenated functions in the meromycolate chain of mycolic acids. PUBLIC HEALTH RELEVANCE: In the context of the threat of MDR-TB and XDR-TB, new drugs against tuberculosis are urgently needed. We propose to elucidate the mode of action of thiocarlide, a drug that was part of the clinical treatment of tuberculosis in the 1960s, and to exploit this knowledge to develop novel therapeutic agents with the same mode of action but much improved pharmacological properties.