Oxygen Sensors and P450 Monooxygenases in Mycobacertium tuberculosis

描述（由申请人提供）：目前在结核病治疗方面取得进展的三个最大需求是：(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。在这里，我们提出表征结核分枝杆菌的DevS/DosR和冗余的DosT/DosR双组分调控系统。这些调节系统控制着由低氧浓度或一氧化氮暴露诱导的大约50个基因。结核分枝杆菌的厌氧状态被认为与潜伏感染相似（但不完全相同），并作为其模型。我们已经发现DevS和DosT传感器激酶在它们的感觉域中有一个血红素基团，并将研究这些传感器的结构，它们对O2， CO和NO的敏感性，以及将气体与血红素基团结合与激酶中组氨酸残基的自磷酸化联系起来的机制。将进行高通量筛选以确定DevS / DosT激酶的抑制剂，因为抑制剂为治疗潜伏性疾病提供了可能的途径。确定抑制剂的结构-活性分布和优化将继续进行。在一个相关但不同的努力中，我们提出克隆，表达和表征结核分枝杆菌的P450酶补体。临床使用的针对真菌P450酶的唑类药物即使在潜伏状态下也能减弱分枝杆菌的生长，但20种结核分枝杆菌P450酶的底物和生物学作用尚不清楚。文献中已经描述了两种P450酶，并且我们已经掌握了这个结核分枝杆菌家族的另外三个成员。每种酶将通过光谱和晶体学方法进行表征，并确定其对唑类药物的亲和力。为每一种酶寻找相关底物将采用多种技术。P450酶的作用也将通过单独破坏（“敲除”）结核分枝杆菌中的每个P450酶来研究。除了确定细菌生长和毒力的相关变化外，每个P450酶破坏的后果将通过DNA微阵列研究进行分析。因此，该项目包括两个依赖血红素和氧的结核分枝杆菌系统，为开发有效对抗潜伏形式的疾病的药物提供了新的途径。

DESCRIPTION (provided by applicant): The three greatest current 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. Here we propose to characterize the DevS/DosR and the redundant DosT/DosR two-component regulatory systems of M. tuberculosis. These regulatory systems control a panel of approximately 50 genes that is induced by low oxygen concentrations or NO exposure. The anaerobic state of M. tuberculosis is thought to resemble (but not be identical to) that found in latent infections and serves as model for it. We have found that the DevS and DosT sensor kinases have a heme group in their sensory domain and will investigate the structure of these sensors, their sensitivity to O2, CO, and NO, and the mechanisms that link binding of a gas to the heme group with autophosphorylation of a histidine residue in the kinase. A high-throughput screen will be performed to identify inhibitors of the DevS / DosT kinases, as inhibitors offer a possible route to treatment of the latent disease. Determination of structure-activity profiles and optimization of the inhibitors will be pursued. In a related but distinct effort, we propose to clone, express, and characterize the P450 enzyme complement of M. tuberculosis. Clinically used azole drugs that target P450 enzymes in fungi attenuate mycobacterial growth even in the latent state, but the substrates and biological roles of the twenty M. tuberculosis P450 enzymes are not known. Two of the P450 enzymes have been described in the literature, and we already have three further members of this M. tuberculosis family in hand. Each enzyme will be characterized by spectroscopic and crystallographic methods, and its affinity for azole drugs will be determined. A search for the relevant substrates for each enzyme will be pursued using a variety of techniques. The role of the P450 enzymes will also be investigated by individually disrupting ("knocking out") each P450 enzyme in M. tuberculosis. The consequences of each P450 enzyme disruption will be analyzed by DNA microarray studies in addition to determination of the associated changes in bacterial growth and virulence. The project thus encompasses two heme- and oxygen-dependent system of M. tuberculosis that offer new avenues to the development of drugs effective against the latent form of the disease.