Developing TB Cell Wall Enzyme Drug Targets

在本P01的项目3中，我们专注于开发和执行基于微量滴定板的酶测定法，用于M.肺结核的生存能力。在整个P01中，已经开发了一个精炼循环，其中化合物由化合物开发模块合成，并由化合物分析模块分析。将化合物的生物活性报告给化合物开发模块，并重复该循环，直到产生对MDR-TB有效且对小鼠中的TB也有效的先导物。我们在项目3中的任务之一是确定化合物开发模块制备的"循环就绪酶"抑制剂的IC50和Ki（如适用）。目前，这些"循环就绪酶"包括参与dTDP-鼠李糖合成的两种酶 (RmlC和RmlD）和酶UDP-吡喃半乳糖苷酶（Glf）。通过"循环就绪酶 目标"我们的意思是他们已经有了针对他们开发的分析方法，确定了晶体结构，并通过筛选化学文库确定了可用作起点的抑制剂。项目3的另一个主要任务是开发四种额外的酶，以便它们也可以被我们的精炼周期所针对（即成为“周期就绪”）。这些酶是GImU，其通过两个单独的反应法呢基二磷酸合成酶、十异戊烯基二磷酸合成酶和各种半乳糖呋喃糖基转移酶合成UDP-GlcNAc。对于这些酶，将开发基于微量滴定板的测定，并筛选化学文库以鉴定初始命中。然后，化合物开发模块将使用该命中与酶形成共晶体，并在精制循环的背景下设计和合成越来越多的活性抑制剂。

In project 3 of this P01, we focus on the development and execution of micro titer plate based enzyme assays for cell wall synthetic enzymes that are required by M. tuberculosis for viability. In the over all P01 a refinement cycle has been developed in which compounds are synthesized by a compound development module and analyzed by a compound analysis module. The biological activities of the compounds are reported to the compound development module and the cycle repeated until leads that are effective against MDR-TB and also effective against TB in mice are produced. One of our tasks in project 3 is to determine IC50's and, as appropriate, Ki's of inhibitors of "cycle ready enzymes" made by the compound development module. At present, these "cycle ready enzymes" include two enzymes involved in the synthesis of dTDP-rhamnose (RmlC and RmlD) and the enzyme UDP-galactopyranose mutase (GIf). By "cycle ready enzyme targets" we mean that they have already had the assays developed for them, crystal structures determined, and inhibitors that can be used as starting points identified by screening chemical libraries. Another major task of project 3 is to develop four additional enzymes so that they can also be targeted by our refinement cycle (i.e. become "cycle ready"). These enzymes are GImU which synthesizes UDP-GIcNAc via two separate reactions, farnesyl diphosphate synthetase, decaprenyl diphosphate synthetase, and various galactofuranosyl transferases. For these enzymes microtiter plate based assays will be developed and chemical libraries screened to identify initial hits. The compound development module will then use this hits to form co-crystals with the enzymes and design and synthesize increasingly more active inhibitors in the context of the refinement cycle.