Oxygen Sensors and P450 Monooxygenases in Mycobacertium tuberculosis

描述（由申请人提供）：在结核病治疗方面取得进展的三个最重要的研究需求是(a)开发针对耐药结核分枝杆菌菌株的药物，(b)开发治疗疾病潜伏状态的有效方法，以及(c)缩短治疗过程，这与第二个目标有关。胆固醇对巨噬细胞的感染和分枝杆菌在这种环境下的存活至关重要。在支持到期期间，我们已经确定了三种能够启动胆固醇分解代谢的结核分枝杆菌细胞色素P450酶。我们的研究已经确定了一种中间产物，当这些P450酶被敲除时，胆-4-en-3- 1会积累起来。这种中间体在几种碳源上抑制结核分枝杆菌的生长。由于胆-4-烯-3- 1是一种潜在的药物靶点，我们建议确定其作用部位和机制。此外，我们将开发两种参与胆固醇侧链降解的初级酶的机制抑制剂，因为它们的失活不仅会阻止胆固醇的利用，还会导致胆固醇-4-烯-3- 1的积累。在该项目的第二个方面，我们将描述和定义两种与毒性相关的P450酶的生物学作用，这两种酶被提议氧化甲基支化烃链。在我们P450研究的第三个方面，我们将推进我们的工作，以确定其他结核分枝杆菌P450酶的结构，底物和作用。在20种P450酶中，我们现在知道了4种的底物和功能。我们克隆了剩下的16个，其中4个具有部分特征，结构为1。这些证据表明，P450酶在结核分枝杆菌中具有特定的生物学作用，而不是参与外源代谢。在一项相关但不同的工作中，我们将进一步描述结核分枝杆菌的冗余DosS/DosT/DosR双组分监管系统。这些传感器控制由缺氧、一氧化氮或一氧化碳诱导的约50个基因的调控。相关的代谢变化被认为与结核分枝杆菌休眠状态的启动相似。我们已经证明了传感器是血红素蛋白，定义了对各种气体的不同反应，并确定了DosT的晶体结构。我们现在建议通过分析与血红素铁原子结合的气体身份作为关闭（O2）或打开（NO， CO）信号转移到传感器的激酶结构域的机制来完成系统的定义。我们还将进行高通量搜索DosS / DosT激酶抑制剂，这些抑制剂可能为开发有效对抗结核分枝杆菌潜伏状态的药物提供先导化合物。建议的工作依赖于尖端技术，包括对结核分枝杆菌突变体和敲除的蛋白质组学、脂质组学和代谢组学分析，对13C-和19f标记位点特异性结合氨基酸的蛋白质构象变化的核磁共振研究，以及类固醇和其他因素在结核分枝杆菌生物学中的作用的化学生物学研究

DESCRIPTION (provided by applicant): The three most important research needs for progress in the treatment of tuberculosis are to (a) develop drugs against drug resistant strains of Mycobacterium tuberculosis, (b) develop effective approaches to treat the latent states of the disease, and (c) shorten the course of therapy, which is related to the second goal. Cholesterol is crucial for infection of macrophages and survival of the mycobacteria in that environment. In the expiring period of support, we have identified three M. tuberculosis cytochrome P450 enzymes able to initiate cholesterol catabolism. Our studies have identified an intermediate, cholest-4-en-3-one that accumulates when these P450 enzymes are knocked out. This intermediate inhibits growth of MTB on several carbon sources. We propose to identify the site and mechanism of action of cholest-4-en-3-one, as it is a potential drug target. Furthermore, we will undertake the development of mechanism-based inhibitors of the two primary enzymes involved in degradation of the cholesterol side-chain, as their inactivation will not only block cholesterol utilization but will lead to accumulation of cholest-4-en-3-one. In a second facet of this project, we will characterize and define the biological roles of two P450 enzymes related to virulence that are proposed to oxidize methyl-branched hydrocarbon chains. In a third facet of our P450 studies, we will advance our work to define the structures, substrates, and roles of the other M. tuberculosis P450 enzymes. Of the twenty P450 enzymes, we now know the substrates and functions of four. We have cloned the remaining 16, partially characterized four, and have the structure of one. The collective evidence indicates that the P450 enzymes in MTB have specific biological roles rather than being involved in xenobiotic metabolism. In a related but distinct effort, we will further characterize the redundant DosS/DosT/DosR two-component regulatory systems of M. tuberculosis. These sensors control a regulon of approximately 50 genes that is induced by hypoxia, NO, or CO. The associated metabolic shift is thought to be similar to that which initiates the dormant state of M. tuberculosis. We have demonstrated that the sensors are heme proteins, defined the differential response to the various gases, and determined the crystal structure of DosT. We now propose to complete definition of the system by analyzing the mechanism by which the identity of the gas binding to the heme iron atom is transferred to the kinase domain of the sensor as either an off- (O2) or on- (NO, CO) signal. We will also perform a high-throughput search for inhibitors of the DosS / DosT kinases that may provide lead compounds for the development of agents effective against the latent states of M. tuberculosis. The proposed work rests on cutting-edge techniques, including proteomic, lipidomic, and metabolomic analysis of M. tuberculosis mutants and knockouts, NMR studies of protein conformational changes with 13C- and 19F-labeled site-specifically incorporated amino acids, and chemical biological studies of the roles of steroids and other factors in the biology of M. tuberculosis. PUBLIC HEALTH RELEVANCE: Tuberculosis is world-wide scourge, as shown by the fact that (a) one-third of the globe's population is infected with Mycobacterium tuberculosis and approximately two million people die of TB each year, (b) each second a new person is infected with TB, and (c) 5-10% of the individuals infected with latent (dormant) TB will become actively sick during their lifetime. The emergence of drug resistant strains, including strains resistant to all available drugs, has refocused attention on this disease. New targets for drug design efforts are urgently needed and the cytochrome P450 enzymes and gas sensors of Mycobacterium tuberculosis have emerged as novel targets for the development of tuberculosis therapeutic agents.