TB DRUGS VIA INHIBITORS OF CELL WALL SYNTHETIC ENZYMES

PO-1的主旨是开发针对结核分枝杆菌细胞壁的新型结核病药物。项目1和2主要研究抑制霉菌酸的合成，项目3主要研究脂类聚异戊二烯载体的合成。本项目4通过针对阿拉伯半乳糖多糖（AG）来完善P01，该多糖将蜡质霉菌酸层固定在内部肽聚糖上。AG是经证实的药物靶点，因为它是乙胺丁醇的靶点。此外，我们有一个耻垢分枝杆菌的TS突变体，该突变体由结核分枝杆菌基因编码AG生物合成靶酶鼠李糖转移酶（rhamnosyl transferase, WbbL）。在不允许的温度下处理后，非互补的TS突变体不再存活。早期的研究通过确定AG的结构和生物合成途径，克隆和表达五种必需的生物合成酶，以及开发适合微滴板筛选的检测方法，已将AG作为药物靶点。在此，我们建议通过筛选AG生物合成酶试验中的潜在抑制剂来鉴定AG生物合成抑制剂。检测包括：形成dTDP-rha所需的三种酶（rmb - d），形成UDP-Galf所需的酶（Gif）；鼠李糖转移酶（WbbL）；形成十烯丙基磷酸- d -阿拉伯糖的酶；在一个需要一些发展的系统中，阿拉伯糖基和半乳糖呋喃基转移酶。许多潜在抑制剂的互补来源将被使用。这些包括：1)已知抑制结核分枝杆菌生长的化合物，但作用方式尚不清楚；2)由合作有机化学家根据某些酶的晶体结构设计的化合物；3)已知抑制结核分枝杆菌生长的天然产物混合物，以及整个细菌将进一步研究。复利权通常归原所有者所有。我们的任务是利用屏幕、基础科学以及与x射线晶体学家和有机化学家的合作，使化合物尽可能地向前发展。最后，我们将为PO1的其他组分提供筛选专业知识和实际化合物，包括开发脱氧醛糖-5磷酸合成酶和甾醇去甲基化酶（项目3），霉菌基转移酶（项目1）和霉菌基冷凝酶（项目2）的抑制剂。

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).