Structural Characterization of the M. tuberculosis Thioredoxin System

描述（由申请人提供）：硫氧还蛋白系统无处不在，在维持细胞内硫醇/二硫化物氧化还原稳态中发挥着重要作用。结核分枝杆菌 (M. tb) 硫氧还蛋白系统最近被建议作为抗结核 (TB) 药物开发的目标，通过破坏其保护结核分枝杆菌免受巨噬细胞氧化攻击的能力。但是，虽然来自某些细菌（例如大肠杆菌）和人类的硫氧还蛋白系统已被充分表征，但分枝杆菌系统却没有。 M.tb 系统由三种硫氧还蛋白（TrxA、TrxB 和 TrxC）和一种硫氧还蛋白还原酶 (TrxR) 组成。由于缺乏 TrxA 和 TrxB 及其与 TrxR 的复合物的可用结构，针对该系统的药物发现工作受到阻碍。此外，三种 M. tb 硫氧还蛋白的不同作用尚不清楚，一些出版物表明 TrxA 甚至可能没有功能。该项目的目标是定义三种 M.tb 硫氧还蛋白的不同结构/功能特性，并确定 TrxA 是否真正神秘。我们将使用这些结构作为整个结核分枝杆菌硫氧还蛋白系统或特定复合物的抑制剂的基于结构的鉴定的基础。预期成果和影响：该项目将产生结核分枝杆菌硫氧还蛋白系统的结构特征，以及作为功能化学遗传探针的抑制剂和抗结核药物先导物。我们的目标是： 1. 确定所有结核分枝杆菌硫氧还蛋白的溶液结构。确定 4 个 NMR 结构：两种氧化还原态（硫醇/二硫化物）的 TrxA 和 TrxB，并与我们的 TrxC 结构进行比较。 2. 确定结核分枝杆菌 TrxR/TrxN（其中 N= A、B）复合物的结构模型，并与 TrxR/TrxC 进行比较。 (a) 确定 TrxR 是否以及如何结合两个 TrxN（均为氧化还原态）。 (b) 确定 TrxN 中由 TrxR 结合引起的结构变化。 (c) 确定 TrxN 结合诱导的 NADPH 辅因子（与 TrxR 结合）的动态变化。 (d) 基于 NMR 化学位移扰动构建两个 TrxN/TrxR 配合物的结构模型，用于模拟催化循环中的中间体的各种死端配合物。 3. 鉴定结核分枝杆菌 TrxR/Trx 系统的抑制剂。 (a) 使用结核分枝杆菌 TrxR/TrxN 系统的 3 维结构模型（以及类似的耻垢分枝杆菌结构），通过计算对接化合物来识别候选抑制剂，(b) 在 NMR 结合（滴定）和酶抑制测定中测试候选抑制剂，以确定亲和力，以及 (c) 最初使用耻垢分枝杆菌进行 MIC（最低抑制浓度）测定中测试 Kd < 50 mM 的化合物（然后是结核分枝杆菌）。 公共健康相关性：这项研究将对结核病病原体结核分枝杆菌的硫氧还蛋白酶系统进行全面的结构表征。由于结核分枝杆菌硫氧还蛋白系统可以保护其免受人类免疫细胞的氧化攻击，因此目前它被视为一个有前途的新药物靶点。因此，该项目的结构数据将用于指导基于结构的抑制剂鉴定，有可能提供治疗分枝杆菌感染的新治疗剂，以及化学遗传探针以增加对该酶系统的了解。

DESCRIPTION (provided by applicant): The thioredoxin system is ubiquitous, and plays an essential role in maintaining thiol/disulfide redox homeostasis in cells. The Mycobacterium tuberculosis (M. tb) thioredoxin system has recently been suggested as a target for anti-tuberculosis (TB) drug development, by disrupting its ability to protect M. tb from the oxidative attacks of macrophages. But, while thioredoxin systems from some bacteria (ex. E. coli) and human is well-characterized, the mycobacterial system is not. The M. tb system is comprised of three thioredoxins (TrxA, TrxB and TrxC) and one thioredoxin reductase (TrxR). Drug discovery efforts targeting this system are hindered by the lack of available structures for TrxA and TrxB and of their complexes with TrxR. Furthermore, the differential roles of the three M. tb thioredoxins are not known, with some publications suggesting that TrxA may even be non-functional. The objective of this project is to define different structural/functional properties o the three M. tb thioredoxins, and to determine if TrxA is truly cryptic. We will use these structures a a foundation for structure-based identification of inhibitors of the entire M. tb thioredoxin syste, or specific complexes. Expected outcomes and impact: This project will produce a structural characterization of the M. tb thioredoxin system, and inhibitors as chemical genetic probes of function and anti-TB drug leads. Our Aims Are to: 1. Determine solution structures for all M. tuberculosis thioredoxins. Determine 4 NMR structures: TrxA and TrxB in both redox states (thiol/disulfide), and compare to our TrxC structures. 2. Determine structural models of M. tuberculosis TrxR/TrxN (where N= A, B) complexes, and compare to TrxR/TrxC. (a) Establish if and how TrxR binds the two TrxN's (both redox states). (b) Determine structural changes in TrxN, induced by TrxR binding. (c) Determine dynamics changes in NADPH cofactor (bound to TrxR), induced by TrxN binding. (d) Construct structural models for the two TrxN/TrxR complexes based on NMR chemical shift perturbations, for various dead end complexes to mimic intermediates in the catalytic cycle. 3. Identify inhibitors of the M. tuberculosis TrxR/Trx system. (a) Using the 3-dimensional structural models for the M. tuberculosis TrxR/TrxN system (and comparable M. smegmatis structures), computationally dock compounds to identify candidate inhibitors, (b) test candidate inhibitors in both NMR binding (titration) and enzymatic inhibition assays, to determine affinity, and (c) test compounds that have Kd < 50 mM in MIC (minimum inhibitory concentration) assays initially with M. smegmatis (then M. tuberculosis). PUBLIC HEALTH RELEVANCE: This study will yield a comprehensive structural characterization of the thioredoxin enzyme system from Mycobacterium tuberculosis, the causative agent of tuberculosis. Since the Mycobacterium tuberculosis thioredoxin system protects it from the oxidative attacks of human immune cells, it is currently being pursued as a promising new drug target. The structural data from this project will therefore be used to guide structure-based identification of inhibitors, potentially providing new therapeutic agents for treating mycobacterial infections, as well as chemical genetic probes to increase understanding of this enzyme system.