TB DRUGS VIA INHIBITORS OF CELL WALL SYNTHETIC ENZYMES

PO-1的主要目的是开发新的结核病药物来对抗结核分枝杆菌的细胞壁。项目1和2的重点是抑制霉酚酸的合成，项目3的重点是合成脂类聚异戊二烯载体。本项目4通过靶向阿拉伯半乳聚糖(AG)来完成P01，这种多糖将蜡质霉菌酸层固定在内肽多聚糖上。AG是一个被证实的药物靶标，因为它是乙胺丁醇的靶标。此外，我们还有一个耻垢分枝杆菌的TS突变体，它由编码AG生物合成靶酶鼠李糖基转移酶(WBBL)的结核分枝杆菌基因补充。在不允许的温度下处理后，未互补的TS突变体不再存活。早期的研究已经通过确定AG的结构和生物合成途径，通过克隆和表达五种必需的生物合成酶，以及通过开发适合于微滴度平板筛选的方法来开发AG作为药物靶点。在此，我们建议通过在AG生物合成酶分析中筛选潜在的抑制剂来识别AG生物合成的抑制剂。这些分析包括：形成dTDP-RHA所需的三种酶(RmIB-D)，形成UDP-GALF所需的酶(GIF)；鼠李糖转移酶(WBBL)；形成十烯基磷酰D-阿拉伯糖的酶；以及在需要一些开发的系统中，阿拉伯糖基和半乳呋喃糖基转移酶。将使用许多潜在抑制剂的互补来源。这些包括：1)已知可抑制结核分枝杆菌生长的化合物(作用模式未知)，2)由合作有机化学家根据某些酶的晶体结构知识设计的化合物，3)已知可抑制结核分枝杆菌生长的天然产品混合物，以及将进一步研究的整个细菌。复合权利通常将保留在原始所有者手中。这项任务是为了确保屏幕、基础科学以及与X射线结晶学家和有机化学家的合作被用来使化合物尽可能地向前发展。最后，我们将为PO1的其他成分提供筛选专业知识和实际化合物，包括开发脱氧硫糖-5磷酸合成酶和类固醇去甲基酶的抑制剂(项目3)、真菌转移酶(项目1)和真菌缩合酶(项目2)。

The thrust of the PO-1 is the development of new TB drugs against the cell wall of M. tuberculosis. Projects 1 & 2 focus on inhibiting mycolic acid synthesis and project 3 on the synthesis of the lipid polyisoprene carriers. This Project 4 rounds out the P01 by targeting the polysaccharide arabinogalactan (AG), which anchors the waxy mycolic acid layer to the inner peptidoglycan. AG is a proven drug target as it is the target of ethambutol. Also, we have a TS mutant of M. smegmatis that is complemented by the M. tuberculosis gene encoding the AG biosynthetic target enzyme, rhamnosyl transferase (WbbL). The non-complemented TS mutant is no longer viable after treatment at the non-permissive temperature. Earlier research has developed AG as a drug target by defining its structure and biosynthetic pathway, by cloning and expressing five essential biosynthetic enzymes, and by developing assays amenable to microtiter plate screening. Herein we propose to identify inhibitors of AG biosynthesis by screening potential inhibitors in AG biosynthetic enzyme assays. The assays include: the three enzymes (RmIB-D) required to form dTDP-rha, the enzyme (Gif) required to form UDP-Galf; the rhamnosyl transferase (WbbL); the enzymes which form decaprenylphosphoryl-D-arabinose; and, in a system requiring some development, the arabinosyl and galactofuranosyl transferases. Many complementary sources of potential inhibitors will be used. These include: 1) compounds known to inhibit the growth of M. tuberculosis where the mode of action is unknown, 2) compounds designed by a collaborating organic chemist based on knowledge of the crystal structures of some of the enzymes, 3) natural product mixtures which are known to inhibit growth of M. tuberculosis, and whole bacteria will be further pursued. Compound rights will usually remain with the original owner. The mission is to see that the screens, basic science, and collaborations with X-ray crystallographers and organic chemists are used to bring compounds as far forward as possible. Finally we will provide both the screening expertise and the actual compounds to other components of the PO1 including the development of inhibitors of deoxyxylulose-5 phosphate synthetase and the sterol demethylase (Project 3), the mycolyl transferase (Project 1) and mycolyl condensation enzyme (Project 2).