M. tuberculosis Adenosine Kinase and Design of Antimicrobial Nucleoside Analogs

描述（由申请方提供）：结核分枝杆菌是一种导致结核病的机会致病菌，在全球范围内导致显著的发病率和死亡率。多药耐药（MDR）菌株的出现和艾滋病相关的结核病感染导致迫切需要开发用于结核病治疗的新的和有效的治疗剂。腺苷激酶（AK）是M.结核杆菌，并且最近被鉴定为负责一组对M.结核我们的长期目标是开发新的抗分枝杆菌药物，利用特异性抑制嘌呤补救途径在M。结核我们假设选择性抑制M.结核病AK或酶对核苷类似物的特异性活化可用于MDRM的治疗干预。肺结核感染。这一假设基于以下观察。1)纯化的M.结核杆菌AK磷酸化了一组腺苷类似物，包括2-甲基腺苷，并且磷酸化是它们活化以变得对M具有选择性活性所必需的。结核2)M.结核病AK与人类同源物的序列同一性低于20%;因此，其结构可能与人类AK显着不同。此外，M.结核病嘌呤补救途径不是目前任何结核病治疗的靶点;因此，抑制该途径可能被证明是一种新的MDR-TB治疗干预。基于这些观察，本建议的实验重点是解开M的三维结构。结核分枝杆菌AK的结构与功能关系，并利用这种关系开发新的抗分枝杆菌药物。我们最近测定了M的晶体结构。结核杆菌AK与腺苷复合。这种独特的细菌AK的结构揭示了功能性二聚体结构，活性位点构象和病原体和人AK之间的活性位点的显着结构差异。本研究的主要目的是：（1）。确定了M.结核分枝杆菌AK的不同形式，以了解酶反应机制参与腺苷磷酸化和腺苷类似物的生物激活在M。结核2)识别M.结核特异性底物和AK的抑制剂，使用基于结构和活性的方法的组合。3)评价先导化合物的生物活性，确定M.结核病AK与铅腺苷类似物。这些研究将为抗分枝杆菌核苷类似物的开发提供分子基础。

DESCRIPTION (provided by applicant): Mycobacterium tuberculosis is an opportunistic pathogen that causes TB, which is responsible for significant morbidity and mortality worldwide. The emergence of multi-drug resistance (MDR) strains and the AIDS-associated TB infection has led to a critical need for the development of new and effective therapeutic agents for the TB treatment. Adenosine kinase (AK) is a key enzyme in the purine salvage pathway of M. tuberculosis and has recently been identified to be responsible for the bioactivation of a group of nucleoside analogs that are active against M. tuberculosis. Our long term goal is to develop new antimycobacterial agents by exploiting specific inhibition of the purine salvage pathway in M. tuberculosis. We hypothesize that selective inhibition of M. tuberculosis AK or specific activation of nucleoside analogs by the enzyme can be exploited for therapeutic intervention of MDR M. tuberculosis infection. This hypothesis is based on the following observations. 1) Purified M. tuberculosis AK phosphorylated a group of adenosine analogs, including 2-methyladenosine, and the phosphorylation is required for their activation to become selectively active against M. tuberculosis. 2) M. tuberculosis AK share less than 20% sequence identity with the human homolog; therefore, its structure is likely to be significantly different from that of human AK. In addition, the M. tuberculosis purine salvage pathway is not the target of any current TB therapy; therefore, inhibition of this pathway may prove to be a novel therapeutic intervention to MDR-TB. Based on these observations, the experimental focus of this proposal is to unravel the three-dimensional structure of M. tuberculosis AK, explore the structure function relationship of the enzyme, and exploit this relationship for the development of new antimycobacterial agents. We have recently determined the crystal structure of M. tuberculosis AK in complex with adenosine. The structure of this unique bacterial AK revealed a functional dimer structure, the active site conformation and significant structural differences in the active sites between the pathogen and human AK. Our specific aims of this proposed research are: 1). to determine the crystal structure of M. tuberculosis AK in different forms for understanding the enzyme reaction mechanism involved in adenosine phosphorylation and bioactivation of adenosine analogs in M. tuberculosis. 2) To identify M. tuberculosis -specific substrates and inhibitors of AK using a combination of structure- and activity-based approaches. 3) To evaluate the biological activities of the lead compounds and determine the complex structure of M. tuberculosis AK with a lead adenosine analog. These studies will provide molecular basis for development of anti-mycobacterial nucleoside analogs.