Oxygen Sensors and P450 Monooxygenases in Mycobacertium tuberculosis

描述（由申请人提供）：目前对结核病治疗进展的三个最大需求是（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。在这里，我们建议描述M的DevS/DosR和冗余DosT/DosR双组分调节系统的特征。结核这些调控系统控制着一组由低氧浓度或NO暴露诱导的约50个基因。M.结核病被认为类似于我们已经发现DevS和DoS T传感器激酶在其感觉域中具有血红素基团，并且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的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鉴定DevS / DosT激酶的抑制剂，因为抑制剂提供了治疗潜伏性疾病的可能途径。将继续进行抑制剂的构效关系测定和优化。在一个相关的，但不同的努力，我们建议克隆，表达和表征的P450酶补体的M。结核临床上使用的靶向真菌中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M.结核病P450酶未知。文献中描述了两种P450酶，我们已经有了这种M.肺结核家族在手上每种酶将通过光谱学和晶体学方法表征，并确定其对唑类药物的亲和力。将使用各种技术来寻找每种酶的相关底物。P450酶的作用也将通过单独破坏（“敲除”）M中的每种P450酶来研究。结核除了确定细菌生长和毒力的相关变化外，还将通过DNA微阵列研究分析每种P450酶破坏的后果。因此，该项目包括两个血红素和氧依赖系统的M。结核病，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.