Structural Characterization of the M. tuberculosis Thioredoxin System

描述(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/二硫代氧化还原平衡方面起着重要作用。结核分枝杆菌硫氧还蛋白系统最近被认为是抗结核药物开发的靶点，因为它破坏了保护结核分枝杆菌免受巨噬细胞氧化攻击的能力。但是，虽然一些细菌(例如，)的硫氧还蛋白系统大肠杆菌)和人类是很好的特征，分枝杆菌系统不是。结核分枝杆菌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。由于TrxA和TrxB及其与TrxR的络合物缺乏可用的结构，针对该系统的药物开发工作受到阻碍。此外，三种结核分枝杆菌硫氧还蛋白的不同作用尚不清楚，一些文献表明TrxA甚至可能是非功能性的。该项目的目标是确定三种结核分枝杆菌硫氧还蛋白的不同结构/功能性质，并确定TrxA是否真的神秘。我们将使用这些结构作为基于结构的识别整个结核分枝杆菌硫氧还蛋白系统或特定复合体的抑制剂的基础。预期结果和影响：该项目将产生结核分枝杆菌硫氧还蛋白系统的结构特征，以及作为功能和抗结核药物先导的化学遗传探针的抑制剂。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。确定了4种核磁共振结构：TrxA和TrxB在两种氧化还原状态(硫醇/二硫键)，并与我们的TrxC结构进行了比较。2.确定结核分枝杆菌TrxR/TrxN(其中N=A、B)复合体的结构模型，并与TrxR/TrxC进行比较。(A)确定TrxR是否以及如何结合两个TrxN(都是氧化还原状态)。(B)确定TrxR结合引起的TrxN的结构变化。(C)测定TrxN结合诱导的NADPH辅因子(与TrxR结合)的动态变化。(D)基于核磁共振化学位移扰动，构建两个TrxN/TrxR配合物的结构模型，以模拟催化循环中的中间体。3.确定结核分枝杆菌TrxR/Trx系统的抑制剂。(A)利用结核分枝杆菌TrxR/TrxN系统(以及可比的耻垢分枝杆菌结构)的三维结构模型，通过计算对接化合物来确定候选抑制剂，(B)在核磁共振结合(滴定)和酶抑制试验中测试候选抑制剂，以确定亲和力，以及(C)测试最初与耻垢分枝杆菌(然后是结核分枝杆菌)进行MIC(最低抑菌浓度)检测中Kd&lt；50 mM的化合物。 公共卫生相关性：这项研究将产生结核分枝杆菌硫氧还蛋白酶系统的全面结构特征，结核杆菌是结核病的病原体。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，它目前正被视为一种有前途的新药靶点。因此，该项目的结构数据将用于指导基于结构的抑制剂的鉴定，有可能为治疗分枝杆菌感染提供新的治疗剂，以及化学遗传探针，以增加对这一酶系统的了解。

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.