CELL WALL BIOGENESIS: TARGET FOR NEW ANTI-TB DRUGS

细胞壁生物发生：新抗结核药物的目标

• 批准号: 3148759
• 负责人: Michael R McNeil
• 金额: $ 36.74万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-30 至 1997-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3148759
• 关键词: Mycobacterium tuberculosis; antitubercular agents; arabinose; carbohydrate biosynthesis; cell wall; disease /disorder model; drug design /synthesis /production; drug resistance; drug screening /evaluation; electroporation; enzyme inhibitors; enzyme substrate analog; ethambutol; genetic library; isoniazid; laboratory mouse; microorganism disease chemotherapy; microorganism genetics; microorganism growth; molecular cloning; molecular site; mutant; nonhuman therapy evaluation; tuberculosis

The lack of fundamental knowledge of the biochemistry and genetics of Mycobacterium tuberculosis and the lack of a variety of drugs against tuberculosis have become serious problems in the face of increased prevalence and multiple drug resistance. Basic studies on the cell wall of mycobacteria emerging from this laboratory have revealed the presence of a unique tetramycolated penta-D-arabinoside (McNeil et al. , J. Biol, Chem, 266, 13217-13223, 1991). This unit is essential for the structural integrity and permeability characteristics of the cell wall of M. tuberculosis and provides a germane target for a new generation of anti-tuberculosis drugs. Neither mycolic acids nor D-arabinose are present in the host, and two effective antimycobacterials, isoniazid (INH) and ethambutol, are known to inhibit the biogenesis of mycolylarabinoside. In this proposal, we present a synergistic balance of fundamental biochemical and genetic research coupled with the design, chemical synthesis, and antimicrobial testing of new enzyme antagonists. Specifically, we will elucidate the pathway for arabinan biosynthesis, develop assays for relevant enzymes, and clone the genes for particularly promising drug targets. To accomplish this, we have developed an active enzyme system capable of converting ribulose-5-phosphate to arabinose-5-phosphate and beyond, to cell wall arabinan. In parallel studies, we will chemically synthesize transition state analogs and substrate analogs of key enzymes. The effect of these inhibitors on enzymatic conversions will be determined separately from their effect on whole bacteria, allowing compounds which inhibit enzymes but are unable to penetrate the bacteria to be recognized and then chemically modified to allow for permeation. As warranted, the effects of the new compounds on bacterial growth in mice will be determined. As well as the new antagonists, INH and ethambutol will be studied. Preliminary results have identified the general site of action of ethambutol on arabinan synthesis; the specific enzymes involved will be identified. In this regard, genes encoding for resistance to ethambutol have been cloned; recent evidence suggests that one of the ethambutol-resistance determinants is involved in arabinan biosynthesis. The cloning of genes encoding for INH resistance is proposed. In addition, enzymes susceptible to INH will be identified using an enzyme system capable of synthesizing mycolic acids from 14C labeled medium chain fatty acids. Finally, the ability of novel, tailored inhibitors such as cyclopropene-containing analogs to inhibit mycolic acid synthesis will be explored.

缺乏生物化学和遗传学的基本知识， 结核分枝杆菌缺乏多种药物防治 结核病已经成为严重的问题， 流行和多重耐药性。 细胞壁的基础研究 实验室发现的分枝杆菌 一种独特的四分枝杆菌化的五-D-阿拉伯糖苷（McNeil等人，J. Biol， Chem，266，13217-13223，1991）。 该单元对于结构至关重要 的细胞壁的完整性和渗透性特征。 结核病，并提供了一个密切的目标，新一代的 抗结核药物。 分枝菌酸和D-阿拉伯糖都不是 和两种有效的抗分枝杆菌药物异烟肼 (INH)和乙胺丁醇，已知可抑制 分枝石松苷。 在这个建议中，我们提出了一个协同平衡， 基础生物化学和遗传学研究与设计相结合， 新酶拮抗剂的化学合成和抗微生物测试。 具体来说，我们将阐明阿拉伯聚糖生物合成的途径， 开发相关酶的检测方法，并克隆基因， 有希望的药物靶点 为了实现这一目标，我们开发了一种积极的 能够将核酮糖-5-磷酸转化为 阿拉伯糖-5-磷酸和更高，与细胞壁阿拉伯聚糖结合。 并行 研究，我们将化学合成过渡态类似物， 关键酶的底物类似物。 这些抑制剂对 酶促转化将与它们对 整个细菌，允许化合物抑制酶，但不能 穿透细菌进行识别，然后进行化学修饰 以允许渗透。 正如所保证的，新化合物的效果 对小鼠体内细菌生长的影响。 以及新的 拮抗剂，INH和乙胺丁醇将被研究。 初步结果 已确定乙胺丁醇对阿拉伯聚糖的一般作用部位 合成;将鉴定所涉及的特定酶。 在这 在这方面，已经克隆了编码乙胺丁醇抗性的基因; 最近的证据表明，乙胺丁醇耐药性之一 决定簇参与阿拉伯聚糖的生物合成。 基因克隆 编码的INH耐药性的建议。 此外，酶 将使用能够 由14 C标记的中链脂肪酸合成分枝菌酸。 最后，新的、定制的抑制剂，如 抑制分枝菌酸合成的含环丙沙星的类似物将被 探讨了