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，全世界有三分之一的人患有潜伏性结核感染，每年有1600万例活动性疾病和近200万人死于结核分枝杆菌（MTB）。结核病感染是异质性的，目前的药物对所有结核分枝杆菌的效果并不相同。补充结核病治疗药库的努力远远落后于日益增长的需求，特别是对耐多药（MDR）或广泛耐药（XDR）结核病患者的需求。在细菌中，对环境线索的许多关键反应是由由一种传感器激酶组成的蛋白质对执行的，该激酶在组氨酸上自动磷酸化，然后将该磷酸盐转移到其同源反应调节蛋白上的天冬氨酸上。MTB组氨酸-天冬氨酸磷接力系统是潜在的有吸引力的药物靶点，主要有两个原因：首先，在MTB的双组分系统中，至少有一种MtrAB即使在富媒体中也对生存至关重要，而其他系统在体内的毒力或持久性中起关键作用。其次，反应调节因子上磷受体位点的高度保守性，以及哺乳动物中缺乏此类蛋白，表明开发泛特异性和无毒抑制剂是可能的。我们的策略是专注于MTB的一个基本2CR， MtrAB，并将信号传导到反应调节因子而不是激酶内，克服了在其他细菌的2CR方法中遇到的问题。除了目标验证之外，我们的工作预计将产生至少一种适合扩展为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：开发一种检测基本MTB 2CR MtrAB抑制剂的方法，并将其格式化为HTS。我们计划采用一种创新的体外磷转移试验，合成一组候选报告底物，并对其进行评估。将选择一个报告基板进行Specific Aim 2。具体目标2。执行MtrAB磷光传输高通量屏幕（HTS）。使用选定的合成报告底物，我们将对从Sequella收集和麦克马斯特大学HTS设施获得的化合物组成的100,000个化合物库进行HTS。预计命中率约为0.1 - 0.2%，从而为Specific Aim 3提供100-200个新结构。3. 优先考虑并扩大特定目标2中的命中支架。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种不同的支架，适合在随后的研究阶段进行。

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.