Inhibition of Fatty Acid Biosynthesis in M. Tuberculosis

结核分枝杆菌中脂肪酸生物合成的抑制

• 批准号: 6680800
• 负责人: PETER J TONGE
• 金额: $ 25.2万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2005-01-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6680800
• 关键词: Mycobacterium tuberculosis; Raman spectrometry; X ray crystallography; affinity labeling; antitubercular agents; bacterial proteins; drug design /synthesis /production; drug screening /evaluation; enzyme inhibitors; enzyme mechanism; ethers; fatty acid biosynthesis; fatty acid synthase; isoniazid; microarray technology; protein protein interaction; site directed mutagenesis

The long term goal of this proposal is to generate novel inhibitors of fatty acid biosynthesis in Mycobacterium tuberculosis. It is hypothesized that such compounds will have antimycobacterial activity and will provide a appropriate starting point for generating drugs to treat multi-drug resistant tuberculosis. The proposal has two Aims. Specific Aim 1 focuses on the design and synthesis of inhibitors that target InhA, the enoyl reductase FASII enzyme. This enzyme is one of the putative targets for isoniazid, a frontline antituberculosis drug. Novel compounds will be synthesized based on the diphenyl ether skeleton of triclosan, an inhibitor of enoyl reductases in M. tuberculosis and other bacteria. Inhibitor design will utilize X-ray crystallography, Raman spectroscopy and computational approaches. Compounds will be tested using enzyme kinetics, the antimycobacterial activity will be assessed using MICs and the intracellular mode of action of the compounds will be evaluated using DNA microarrays and photoaffinity labeling. Specific Aim 2 will investigate the mechanism of action of isoniazid and will test the hypothesis that proteinprotein interactions within the mycobacterium modulate the sensitivity of InhA and other FASII enzymes to isoniazid. The FASII enzyme complex from M. tuberculosis will be purified and the activity and sensitivity of each enzyme component toward FAS inhibitors will be evaluated. Characterization of the FASII complex will reveal the identity of the dehydrase enzyme and the FASII complex will be reconstituted in vitro using recombinantly expressed proteins. In addition, pull-down experiments will be used to identify other InhA protein binding partners and characterization of the FASI enzyme complex from M. tuberculosis will be initiated.

该建议的长期目标是产生结核分枝杆菌中脂肪酸生物合成的新型抑制剂。假设这种化合物将具有抗菌活性，并将为生成药物治疗多药耐多药的结核病提供合适的起点。 该提案有两个目标。 具体目标1的重点是靶向INHA（Enoyl还原酶FASII酶）的抑制剂的设计和合成。该酶是前线抗结核药物Isoniazid的推定靶标之一。新颖的化合物将根据三氯生的二苯基醚骨架合成，三氯糖剂是结核分枝杆菌和其他细菌中的enoyl还原酶的抑制剂。抑制剂设计将利用X射线晶体学，拉曼光谱和计算方法。将使用酶动力学对化合物进行测试，将使用MICS评估抗菌活性，并将使用DNA微阵列和光性标记评估化合物的细胞内作用方式。 具体的目标2将研究异念珠菌的作用机理，并将检验以下假设：分枝杆菌中蛋白蛋白质相互作用调节INHA和其他FASII酶对异辛氮化的敏感性。将纯化来自结核分枝杆菌的FASII酶复合物，并评估每个酶成分对FAS抑制剂的活性和敏感性。 FASII复合物的表征 将揭示脱水酶的身份，而FASII复合物将在体外使用重组表达的蛋白在体外重构。此外，将使用下拉实验来识别其他INHA蛋白结合伴侣，并将启动FASI酶复合物的表征。