Oxygen Sensors and P450 Monooxygenases in Mycobacertium tuberculosis

描述(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该病的潜伏期；以及(C)缩短疗程，这与第二个目标有关。在这里，我们建议表征结核分枝杆菌的DEVS/DosR和冗余的DOST/DosR双组分调控系统。这些调控系统控制着一个由大约50个基因组成的面板，这些基因是由低氧浓度或没有暴露的环境诱导的。结核分枝杆菌的厌氧状态被认为与潜伏感染中发现的相似(但不完全相同)，并作为它的模型。我们已经发现DEVS和DOST感受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的结合与激酶中组氨酸残基的自动磷酸化联系在一起的机制。将进行高通量筛选以确定DEVS/DOST激酶的抑制剂，因为抑制剂提供了治疗这种潜伏性疾病的可能途径。将继续进行结构-活性分布的确定和抑制剂的优化。在一个相关但不同的努力中，我们建议克隆、表达和鉴定结核分枝杆菌的P450酶补体。临床上使用的针对真菌中P450酶的唑类药物即使在潜伏状态下也能抑制分枝杆菌的生长，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，我们已经有了另外三个结核分枝杆菌家族的成员。每种酶都将通过光谱和结晶学方法进行表征，并将确定其与唑类药物的亲和力。将使用各种技术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”)结核分枝杆菌中的每一种P450酶来进行研究。除了确定细菌生长和毒力的相关变化外，还将通过DNA微阵列研究来分析每个P450酶中断的后果。因此，该项目包括两个依赖血红素和氧气的结核分枝杆菌系统，这两个系统为开发有效对抗该疾病潜伏期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.