CELL WALL BIOGENESIS--TARGET FOR NEW ANTI-TB DRUGS

缺乏生物化学和遗传学的基础知识 结核分枝杆菌和缺乏多种抗结核药物 面对日益严重的结核病，结核病已经成为严重的问题 流行率和多重耐药性。细胞壁的基础研究 从这个实验室出现的分枝杆菌已经揭示了 一种独特的四聚D-阿拉伯糖苷(McNeil等人)，J.Biol， 化学，13217-13223,1991年)。这个单元是结构上必不可少的 M.细胞壁的完整性和通透性特征。 结核病，为新一代结核病提供了一个密切相关的靶子 抗结核药物。真菌酸和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，已知抑制生物发生。 霉菌糖苷。在这项建议中，我们提出了一种协同平衡 基础生化和基因研究与设计相结合， 新的酶拮抗剂的化学合成和抗菌试验。 具体地说，我们将阐明阿拉伯胶生物合成的途径， 建立相关酶的检测方法，并克隆特定的基因 前景看好的药物靶点。为了实现这一目标，我们开发了一种主动 能够将核酮糖-5-磷酸转化为 阿拉伯糖-5-磷酸及以上，至细胞壁阿拉伯糖。并行的 研究，我们将化学合成过渡态类似物和 关键酶的底物类似物。这些抑制剂对血管紧张素转换酶的影响 酶转化率的测定将与其对 整个细菌，允许抑制酶的化合物，但不能 穿透被识别的细菌，然后进行化学修饰 以允许渗透。正如所保证的，新化合物的影响 对小鼠体内细菌生长的影响将被确定。以及新的 将研究拮抗剂异烟肼和乙胺丁醇。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；将确定涉及的特定酶。在这 在这方面，编码乙胺丁醇抗性的基因已经被克隆； 最近的证据表明，乙胺丁醇的耐药性之一 决定因素与阿拉伯胶的生物合成有关。基因的克隆 提出了异烟肼抗性的编码方法。此外，酶 对异烟肼敏感的将使用一种酶系统进行鉴定 由~(14)C标记的中链脂肪酸合成霉菌酸。 最后，新的、量身定做的抑制剂的能力 含有环丙烯的类似物将抑制霉菌酸的合成 探索过了。

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.