Targeting MtrAB of M. tuberculosis

描述（由申请人提供）：结核病（TB）对全球健康产生巨大影响。世界卫生组织估计全世界三分之一的人患有潜伏性结核病感染，每年有一千六百万例活动性疾病和近两百万例由结核分枝杆菌（MTB）引起的死亡.结核病感染是异质性的，目前的药物对所有MTB细菌的效果并不相同。补充结核病治疗武器库的努力远远落后于日益增长的需求，特别是对多重耐药（MDR）或广泛耐药（XDR）结核病患者。 在细菌中，许多对环境线索的关键反应是由蛋白质对组成的传感器激酶，其在组氨酸上自动磷酸化，然后将这种磷酸转移到其同源反应调节蛋白上的天冬氨酸。MTB组氨酸-天冬氨酸磷酸化系统是潜在的有吸引力的药物靶标，原因有两个：首先，在MTB中的双组分系统中，至少一种MtrAB对于即使在丰富培养基中的活力也是必不可少的，而其他系统在体内毒力或持久性中起关键作用。其次，反应调节剂上的磷酸受体位点之间的高度保守性，以及哺乳动物中不存在此类蛋白质，表明开发泛特异性和无毒的抑制剂是可能的。我们的策略是集中在MTB的一个重要的2CR，MtrAB，并将信号传导靶向反应调节剂，而不是在激酶内，克服了在其他细菌的2CR方法中遇到的问题。 除了目标验证，我们的工作预计将产生至少一种适合扩展到临床前先导化合物的化学支架。未来的计划将把目标扩展到更大的分枝杆菌2CR反应调节剂家族。具体来说，在这个概念验证工作中，我们计划：具体目标1.开发必需MTB 2CR MtrAB抑制剂的测定方法，并将其用于HTS。我们计划采用一种创新的体外磷酸转移试验，合成一组候选报告底物，并对其进行评估。将选择一种报告底物进行特异性目标2。具体目标2。执行MtrAB磷酸转移高通量筛选（HTS）。使用所选择的合成报告底物，我们将对100，000个化合物文库进行HTS，所述化合物文库由从Sequella收集和从McMaster University的HTS设施获得的化合物组成。预计命中率约为0.1 - 0.2%，从而为特定目标3提供100-200个新结构。3.优先级和扩大从特定目标2击中脚手架。我们将选择最有希望的结构并进行体外二次筛选。有限的药物化学扩展将产生2-3个不同的支架，适合在随后的研究阶段进行研究。 公共卫生相关性：结核病（TB）对全球健康产生巨大影响，补充TB治疗武器库的努力远远落后于日益增长的需求，特别是对于多重（MDR）或广泛（XDR）耐药TB患者。我们已经选择了一个有前途的新的药物靶点，并建议开发一种筛选方法，以确定具有显着潜力的分子作为新的候选药物用于治疗结核病，耐多药结核病和广泛耐药结核病。

DESCRIPTION (provided by applicant): Tuberculosis (TB) has a massive impact on global health. The World Health Organization estimates that one- third of all people worldwide harbor latent TB infections, with sixteen million cases of active disease and nearly two million deaths caused by Mycobacterium tuberculosis (MTB) each year. TB infections are heterogeneous, and current medicines do not work equally well on all MTB bacteria. Efforts to replenish the TB treatment arsenal lag far behind the growing need, particularly for patients with multi- (MDR) or extensively- (XDR) drug-resistant-TB. In bacteria, many critical responses to environmental cues are performed by protein pairs comprised of a sensor kinase that auto-phosphorylates on a histidine and then transfers this phosphate to an aspartate on its cognate response regulator protein. The MTB histidine-aspartate phosphorelay systems are potentially attractive drug targets for two major reasons: First, of the two-component systems in MTB, at least one, MtrAB, is essential for viability even in rich media while others play key roles in virulence or persistence in vivo. Second, the high conservation among phosphoacceptor sites on the response regulators, and the absence of such proteins in mammals, suggest that developing pan-specific and non-toxic inhibitors may be possible. Our strategy is to focus here on the one essential 2CR of MTB, MtrAB, and target the signaling to the response regulator rather than within the kinase, overcoming problems encountered in approaches with 2CRs of other bacteria. In addition to target validation, our work is anticipated to produce at least one chemical scaffold suitable for expansion into a preclinical lead compound. Future plans will expand the targets to the larger family of mycobacterial 2CR response regulators. Specifically in this proof-of-concept work we plan to: Specific Aim 1. Develop an assay for inhibitors of the essential MTB 2CR MtrAB, and format it for HTS. We plan to adapt an innovative in vitro phospho-transfer assay, synthesize a panel of candidate reporter substrates, and evaluate them. One reporter substrate will be selected to conduct Specific Aim 2. Specific Aim 2. Execute an MtrAB phospho-transfer high-throughput screen (HTS). Using the selected synthetic reporter substrate we will conduct an HTS on a 100,000 compound library comprised of compounds obtained from both the Sequella collection and from McMaster University's HTS facility. An anticipated hit rate of ca 0.1 -0.2 % is anticipated, thus providing 100-200 novel structures for Specific Aim 3. 3. Prioritize and expand hit scaffolds from Specific Aim 2. We will select the most promising structures and run in vitro secondary screens. A limited medicinal chemistry expansion will then produce 2-3 distinct scaffolds suitable for pursuit in a subsequent research phase. PUBLIC HEALTH RELEVANCE: Tuberculosis (TB) has a massive impact on global health, and efforts to replenish the TB treatment arsenal lag far behind the growing need, particularly for patients with multi- (MDR) or extensively- (XDR) drug-resistant-TB. We have chosen a promising new drug target, and propose to develop a screening method to identify molecules with significant potential as new drug candidates for the treatment of TB, MDR-TB, and XDR-TB.