Mycobacterium tubercolosis Adenosine Kinase Design Antimicrobial Nucleoside Anal

描述(申请人提供)：结核分枝杆菌是一种引起结核病的机会性病原体，它在全球范围内导致严重的发病率和死亡率。多药耐药(MDR)菌株的出现和艾滋病相关的结核病感染导致了对开发新的有效的结核病治疗药物的迫切需求。腺苷激酶(AK)是结核分枝杆菌嘌呤挽救途径中的一个关键酶，最近被发现负责一组核苷类似物的生物激活，这些类似物具有抗结核活性。我们的长期目标是通过利用对结核分枝杆菌嘌呤挽救途径的特异性抑制来开发新的抗分枝杆菌药物。我们推测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多药耐药结核分枝杆菌感染的治疗干预。这一假设基于以下观察结果。1)纯化的结核分枝杆菌AK使包括2-甲基腺苷在内的一组腺苷类似物磷酸化，而磷酸化是激活它们成为选择性抗结核分枝杆菌活性所必需的。2)结核分枝杆菌AK与人类同源物的序列同源性不到20%；因此，其结构可能与人类AK显著不同。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靶点；因此，抑制这一途径可能被证明是一种新的耐多药结核病治疗干预措施。基于这些观察，本方案的实验重点是解开结核分枝杆菌AK的三维结构，探索该酶的结构和功能关系，并利用这种关系开发新的抗分枝杆菌药物。我们最近测定了结核分枝杆菌AK与腺苷的络合物的晶体结构。这种独特的细菌AK的结构揭示了功能二聚体结构、活性部位构象以及病原菌与人AK活性部位的显著结构差异。我们提出的研究的具体目的是：1)。测定不同形式的结核分枝杆菌AK的晶体结构，以了解参与结核分枝杆菌腺苷磷酸化和腺苷类似物生物活性的酶反应机制。2)结合基于结构和活性的方法，鉴定结核分枝杆菌AK的特异性底物和抑制剂。3)评价先导化合物的生物活性，并用腺苷类似物确定结核分枝杆菌AK的络合物结构。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.