Structural Characterization of the M. tuberculosis Thioredoxin System

结核分枝杆菌硫氧还蛋白系统的结构表征

• 批准号: 8366857
• 负责人: DANIEL S SEM
• 金额: $ 38.27万
• 依托单位: CONCORDIA UNIVERSITY WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-06-01 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8366857
• 关键词: 3-Dimensional; Address; Affinity; Alveolar Macrophages; Antitubercular Agents; Bacteria; Binding; Biological Assay; Cell physiology; Cells; Chemicals; Complex; Data; Disease; Disulfides; Docking; Drug Delivery Systems; Environment; Enzyme Inhibition; Enzymes; Escherichia coli; Foundations; Genus Mycobacterium; Glutathione; Homeostasis; Homologous Gene; Human; Immune; Infection; Minimum Inhibitory Concentration measurement; Mycobacterium Infections; Mycobacterium smegmatis; Mycobacterium tuberculosis; NADP; Outcome; Oxidation-Reduction; Play; Property; Proteins; Publications; Relative (related person); Role; Solutions; Structural Models; Structure; Sulfhydryl Compounds; System; Testing; Therapeutic Agents; Thioredoxin; Titrations; Tuberculosis; base; chemical genetics; cofactor; design; drug development; drug discovery; inhibitor/antagonist; macrophage; mycobacterial; novel therapeutics; small molecule; thioredoxin reductase; tuberculosis drugs

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.

描述（由申请人提供）：硫氧还蛋白系统无处不在，并且在维持细胞中硫醇/二硫化物氧化还原稳态方面起着至关重要的作用。最近已经提出，结核分枝杆菌（M. TB）硫氧还蛋白系统是抗结核药物（TB）药物发育的一个靶标，它破坏了其保护大麻菌的TB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是良好的，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）和一个硫氧还蛋白还原酶（TRXR）组成。针对该系统的药物发现工作受到TRXA和TRXB的可用结构及其与TRXR的复合物的缺乏的阻碍。此外，三个M. TB硫氧还蛋白的差异作用尚不清楚，其中一些出版物表明TRXA甚至可能是非功能性的。该项目的目的是定义三个M. TB硫氧还蛋白的不同结构/功能特性，并确定TRXA是否真正隐秘。我们将使用这些结构为基于结构的整个M. TB硫氧还蛋白系统或特定复合物的抑制剂的基础。预期的结果和影响：该项目将产生M. TB硫氧还蛋白系统的结构表征，而抑制剂作为功能和抗TB药物铅的化学遗传探针。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：两个氧化还原状态（硫醇/二硫化物）中的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的结构模型（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（两个氧化还原状态）。 （b）确定TRXR结合诱导的TRXN的结构变化。 （c）确定由TRXN结合引起的NADPH辅因子（与TRXR结合）的动力学变化。 （d）基于NMR化学位移扰动的两个TRXN/TRXR复合物的结构模型，用于模仿催化循环中的各种死端复合物。 3。确定结核分枝杆菌TRXR/TRX系统的抑制剂。 （a）使用三维结构模型进行结核分枝杆菌TRXR/TRXN系统（以及可比的Smegmatis结构），计算码头化合物以鉴定候选抑制剂，（b）测试候选抑制剂，两种NMR结合（滴答）和酶抑制量<50次的测试，以及（C），以确定相互抑制的测试量，以及（C）。 （最小抑制浓度）最初用Smegmatis（然后是结核分枝杆菌）进行测定。 公共卫生相关性：这项研究将从结核分枝杆菌（结核病的病因）中产生硫氧还蛋白酶系统的全面结构表征。由于结核分枝杆菌硫氧还蛋白系统可保护其免受人类免疫细胞的氧化攻击，因此目前正在将其作为有希望的新药靶标。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，并有可能提供用于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。