Oxygen Sensors and P450 Monooxygenases in Mycobacertium tuberculosis

描述(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要是：(A)开发针对结核分枝杆菌耐药菌株的药物，(B)开发有效的方法来治疗疾病的潜伏期，以及(C)缩短疗程，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该环境中的生存至关重要。在支持期满期间，我们已经鉴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，胆汁-4-烯-3-酮，当这些P450酶被敲除时会积聚。该中间体在几种碳源上抑制结核分枝杆菌的生长。我们建议确定胆甾醇-4-烯-3-酮的作用部位和作用机制，因为它是一个潜在的药物靶点。此外，我们将致力于开发参与胆固醇侧链降解的两种主要酶的机理抑制剂，因为它们的失活不仅会阻止胆固醇的利用，还会导致胆碱-4-烯-3-酮的积累。在这个项目的第二个方面，我们将表征和定义两种与毒力相关的P450酶的生物学作用，这两种酶被提议氧化甲基支链碳氢化合物。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。在二十种P450酶中，我们现在知道四种酶的底物和功能。我们已经克隆了剩下的16个，部分鉴定了4个，并拥有一个的结构。集体证据表明，结核分枝杆菌中的P450酶具有特定的生物学作用，而不是参与异体代谢。在一个相关但不同的努力中，我们将进一步描述结核分枝杆菌冗余的DOSS/DOST/DOSR二元调控系统的特征。这些传感器控制着一个由大约50个基因组成的调节子，这些基因是由低氧、一氧化氮或一氧化碳诱导的。相关的代谢转变被认为类似于启动结核分枝杆菌休眠状态的代谢转变。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响应，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的身份作为OFF-(O2)或ON-(NO，CO)信号转移到传感器的激活域的机制来完成对该系统的定义。我们还将进行高通量搜索，寻找DOSS/DOST激酶的抑制剂，这些抑制剂可能为开发有效对抗结核分枝杆菌潜伏期的药物提供先导化合物。拟议的工作基于尖端技术，包括对结核分枝杆菌突变体和基因敲除的蛋白质组、脂组和代谢组学分析，用13C和19F标记的特定位点结合的氨基酸对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病生物学中的作用的化学生物学研究。 结核分支杆菌。

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.