Oxygen Sensors and P450 Monooxygenases in Mycobacertium tuberculosis

结核分枝杆菌中的氧传感器和 P450 单加氧酶

• 批准号: 7294665
• 负责人: Paul R Ortiz De Montellano
• 金额: $ 40.69万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-01 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7294665
• 关键词: Acquired Immunodeficiency Syndrome; Aerobic; Affinity; Antitubercular Agents; Area; Attenuated; Azoles; Bacillus (bacterium); Binding; Biochemical; Biological; Cessation of life; Clinic; Commercial Sectors; Complement; Condition; Cytochrome P450; DNA Microarray Chip; DNA Microarray format; Development; Disease; Disruption; Drug Delivery Systems; Drug Design; Drug resistance; Enzymes; Evaluation; Family; Gases; Genes; Genome; Genus Mycobacterium; Goals; Growth; Hand; Health; Heel; Heme; Heme Group; Heme Iron; Hemeproteins; Histidine; In Vitro; Incidence; Individual; International; Knock-out; Label; Laboratories; Lead; Length; Libraries; Ligands; Link; Literature; Methods; Mixed Function Oxygenases; Modeling; Mus; Mycobacterium tuberculosis; Organism; Outcome; Oxygen; Persons; Pharmaceutical Preparations; Phosphotransferases; Physiological; Population; Prevalence; Public Health; Publishing; Regulon; Relative (related person); Research; Research Personnel; Resistance; Resistance development; Role; Route; Scourge; Screening procedure; Sensory; Signal Transduction; Site; Sterilization for infection control; Structure; System; Techniques; Testing; Therapeutic; Therapeutic Agents; Thinking; Translating; Tuberculosis; United States; United States National Institutes of Health; Update; Virulence; Work; base; cost; drug development; experience; fungus; high throughput screening; in vivo; inhibitor/antagonist; interest; latent infection; member; men; mortality; mycobacterial; programs; protein-histidine kinase; receptor; sensor; synergism; tuberculosis drugs; tuberculosis treatment

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.

描述（由申请人提供）：目前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，这与第二个目标相关。在这里，我们建议描述结核分枝杆菌的 DevS/DosR 和冗余 DosT/DosR 双组分调节系统。这些调节系统控制由低氧浓度或 NO 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中发现的厌氧状态相似（但不相同），并可作为其模型。我们发现 DevS 和 DosT 传感器激酶在其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磷酸化联系起来的机制。将进行高通量筛选来鉴定 DevS / DosT 激酶的抑制剂，因为抑制剂为潜在疾病的治疗提供了可能的途径。将继续确定抑制剂的结构-活性特征和优化。在一项相关但独特的工作中，我们建议克隆、表达和表征结核分枝杆菌的 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会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。文献中已经描述了其中两种 P450 酶，并且我们已经掌握了该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。将使用多种技术来寻找每种酶的相关底物。还将通过单独破坏（“敲除”）结核分枝杆菌中的每种 P450 酶来研究 P450 酶的作用。除了确定细菌生长和毒力的相关变化之外，还将通过 DNA 微阵列研究来分析每次 P450 酶破坏的后果。因此，该项目涵盖结核分枝杆菌的两个血红素和氧依赖性系统，为开发有效对抗潜在疾病的药物提供了新途径。