Structural Characterization of the M. tuberculosis Thioredoxin System

描述（由申请人提供）：硫氧还蛋白系统是普遍存在的，在维持细胞中硫醇/二硫化物氧化还原稳态中发挥重要作用。结核分枝杆菌（Mycobacterium tuberculosis，M.结核分枝杆菌硫氧还蛋白系统通过破坏其保护结核分枝杆菌的能力而成为抗结核药物开发的靶点。结核病从巨噬细胞的氧化攻击。但是，虽然硫氧还蛋白系统从一些细菌（例如。E.大肠杆菌）和人的系统是良好表征的，而分枝杆菌系统不是。分枝tb系统由三个硫氧还蛋白（TrxA、TrxB和TrxC）和一个硫氧还蛋白还原酶（TrxR）组成。靶向该系统的药物发现努力受到缺乏TrxA和TrxB及其与TrxR的复合物的可用结构的阻碍。此外，三个M的不同作用。硫氧还蛋白是未知的，一些出版物表明TrxA甚至可能是无功能的。本项目的目标是确定三个M的不同结构/功能特性。tb硫氧还蛋白，并确定是否TrxA是真正的隐蔽。我们将利用这些结构为基于结构鉴定整个M的抑制剂奠定基础。TB硫氧还蛋白系统或特定复合物。预期成果和影响：本项目将对M.结核病硫氧还蛋白系统，以及作为功能化学基因探针和抗结核药物先导的抑制剂。我们的目标是：1.确定所有M的溶液结构。结核硫氧还蛋白确定4个NMR结构：两种氧化还原状态（硫醇/二硫化物）下的TrxA和TrxB，并与我们的TrxC结构进行比较。 2.确定了M.结核病TrxR/TrxN（其中N= A，B）复合物，并与TrxR/TrxC进行比较。(a)确定TrxR是否以及如何结合两个TrxN（两种氧化还原状态）。(b)确定由TrxR结合诱导的TrxN结构变化。(c)测定由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，构建两种TrxN/TrxR复合物的结构模型，用于模拟催化循环中的中间体的各种死端复合物。 3.鉴定M.结核病TrxR/Trx系统。(a)利用M.结核分枝杆菌TrxR/TrxN系统（和可比较的M. smeglycin结构），计算对接化合物以鉴定候选抑制剂，（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，和（c）在MIC（最小抑制浓度）测定中具有Kd < 50 mM的测试化合物，最初用M. megalovirus（然后M.肺结核）。 公共卫生相关性：这项研究将产生一个全面的结构表征的硫氧还蛋白酶系统从结核分枝杆菌，结核病的病原体。由于结核分枝杆菌硫氧还蛋白系统保护其免受人类免疫细胞的氧化攻击，因此目前正被视为一个有前途的新药物靶标。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，可能为治疗分枝杆菌感染提供新的治疗药物，以及化学遗传探针，以增加对这种酶系统的了解。

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.