M. tuberculosis Adenosine Kinase and Design of Antimicrobial Nucleoside Analogs

描述（由申请人提供）：结核分枝杆菌是一种导致结核病的机会性病原体，在世界范围内造成严重的发病率和死亡率。耐多药（MDR）菌株的出现和艾滋病相关结核感染导致迫切需要开发新的有效的结核病治疗药物。腺苷激酶（AK）是结核分枝杆菌嘌呤回收途径中的一个关键酶，最近被确定为负责一组对结核分枝杆菌有活性的核苷类似物的生物激活。我们的长期目标是通过利用结核分枝杆菌嘌呤回收途径的特异性抑制来开发新的抗细菌药物。我们假设该酶选择性抑制结核分枝杆菌AK或特异性激活核苷类似物可用于耐多药结核分枝杆菌感染的治疗干预。这一假设是基于以下观察。1)纯化的结核分枝杆菌AK磷酸化了一组腺苷类似物，包括2-甲基腺苷，磷酸化是激活它们对结核分枝杆菌具有选择性活性所必需的。2)结核分枝杆菌AK与人类同源序列的同源性小于20%；因此，它的结构可能与人类的AK有很大的不同。此外，结核分枝杆菌嘌呤挽救途径不是目前任何结核病治疗的目标；因此，抑制这一途径可能被证明是耐多药结核病的一种新的治疗干预措施。在此基础上，本课题的实验重点是揭示结核分枝杆菌AK酶的三维结构，探索其结构功能关系，并利用这种关系开发新的抗细菌药物。我们最近测定了结核分枝杆菌AK与腺苷复合物的晶体结构。该细菌AK具有功能性二聚体结构、活性位点构象以及与人类AK在结构上的显著差异。我们提出的这项研究的具体目的是：1)确定结核分枝杆菌不同形式AK的晶体结构，以了解结核分枝杆菌中参与腺苷磷酸化和腺苷类似物生物活化的酶反应机制。2)结合基于结构和活性的方法鉴定结核分枝杆菌AK特异性底物和抑制剂。3)评价先导化合物的生物活性，确定结核分枝杆菌AK与一先导腺苷类似物的复合物结构。这些研究将为开发抗分枝杆菌核苷类似物提供分子基础。

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.