Oxygen Sensors and P450 Monooxygenases in Mycobacertium tuberculosis

描述（由申请人提供）：结核病治疗进展的三个最重要的研究需求是（a）开发针对结核分枝杆菌耐药菌株的药物，（B）开发治疗疾病潜伏状态的有效方法，以及（c）缩短疗程，这与第二个目标有关。 胆固醇对于巨噬细胞的感染和分枝杆菌在该环境中的存活至关重要。在支持到期期间，我们确定了三个M。结核病细胞色素P450酶能够启动胆固醇催化剂。我们的研究已经确定了一个中间体，胆甾-4-烯-3-酮，当这些P450酶被敲除时积累。该中间体抑制MTB在几种碳源上的生长。我们建议确定胆甾-4-烯-3-酮的作用位点和机制，因为它是一个潜在的药物靶点。此外，我们将开发参与胆固醇侧链降解的两种主要酶的基于机制的抑制剂，因为它们的失活不仅会阻止胆固醇的利用，而且会导致胆甾-4-烯-3-酮的积累。在这个项目的第二个方面，我们将描述和定义两个P450酶的生物学作用，这些酶与毒性有关，被提议用于氧化甲基支链烃链。在我们的P450研究的第三个方面，我们将推进我们的工作，以确定其他M的结构，底物和作用。结核病P450酶。在20种P450酶中，我们现在知道了4种酶的底物和功能。我们克隆了剩下的16个，部分表征了4个，并具有1个的结构。集体的证据表明，MTB中的P450酶具有特定的生物学作用，而不是参与异生物质代谢。 在相关但不同的努力中，我们将进一步表征M的冗余DoS/DoS/DosR双组分调节系统。结核这些传感器控制着由缺氧、NO或CO诱导的大约50个基因的调节子。相关的代谢转变被认为与启动M的休眠状态相似。结核我们已经证明，传感器是血红素蛋白，定义了各种气体的差分响应，并确定了晶体结构的DoS。我们现在建议通过分析机制来完成系统的定义，通过该机制将与血红素铁原子结合的气体的身份转移到传感器的激酶结构域，作为关闭（O2）或打开（NO，CO）信号。我们还将进行高通量的DoS/DoS T激酶抑制剂的研究，这些抑制剂可能为开发有效对抗M潜伏状态的药物提供先导化合物。结核 这项工作的提出依赖于尖端技术，包括蛋白质组学，脂质组学和代谢组学分析的M。结核病突变体和基因敲除，用13 C和19 F标记的位点特异性掺入的氨基酸进行的蛋白质构象变化的NMR研究，以及类固醇和其他因子在结核病生物学中的作用的化学生物学研究。 M.结核 公共卫生相关性：结核病是世界性的祸害，如以下事实所示：（a）地球仪人口的三分之一感染结核分枝杆菌，并且每年约有200万人死于结核病，（B）每秒钟就有一个新的人感染结核病，以及（c）感染潜伏（休眠）结核病的个体中的5-10%将在其一生中变为活动性疾病。耐药菌株的出现，包括耐 所有可用的药物，使人们重新关注这种疾病。药物设计工作的新目标是迫切需要的，结核分枝杆菌的细胞色素P450酶和气体传感器已成为结核病治疗剂开发的新目标。

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.