Development of a small molecule probe targeting non-replicating M. tuberculosis

描述(申请人提供)：结核病(TB)仍然是一项全球公共卫生挑战，每年感染全球近900万人，并导致100多万人死亡。结核病控制工作的两个主要障碍包括(1)治愈结核病所需的长期药物方案和(2)人类免疫力无法有效清除结核分枝杆菌(Mtb)感染，导致潜伏感染。据估计，世界上三分之一的人口患有结核病，这提供了一个巨大的蓄水池，可以从那里重新激活为急性疾病。迫切需要能够简化或缩短急性和潜伏性结核病感染的现有疗程的新药，以帮助提高患者的依从性，从而限制耐药结核病的出现。目前，成功治疗活动性、有症状的结核病感染需要持续至少6个月的复杂药物方案，而潜伏感染需要3个月的多种药物治疗或长达9个月的单一药物治疗。据认为，我们目前的抗生素对结核病的缓慢清除是由于宿主组织中的细菌亚群采用了耐药、非复制的生理状态。更好地消除这些持久的、不可复制的细菌的药物方案可以缩短所需的 活动性感染和潜伏性感染的治疗时间。因此，识别杀死非复制型结核分枝杆菌的小分子将是非常有价值的，首先对于我们理解非复制型生理状态的生物学，然后对于开发更有效、可能更短的结核病治疗方法。为了找到这样的分子，我们将营养剥夺模型应用于高通量的化学筛选，以识别能够杀死碳匮乏的、非复制的结核分枝杆菌的化合物。从最初的恶二唑击中化合物，我们已经开发出ML338作为一种小分子探针，选择性地针对非复制的结核分枝杆菌。由于饥饿反应已被证明对动物模型中的结核病持久性是必要的，ML338不仅是一个有价值的工具，用于确定结核分枝杆菌在非复制状态下的基本功能和脆弱性，而且还用于测试体内感染期间非复制状态的相关性。我们将首先努力识别分子靶标和 ML338的作用机理。目标识别将需要一系列平行的方法，包括分离耐药突变的遗传方法以及确定结合伙伴的基于亲和力的分析。了解ML338的作用机制是至关重要的，因为它将揭示非复制型结核分枝杆菌的一个或多个脆弱途径。此外，我们将利用ML338作为工具，使用小鼠感染模型来探索非复制状态与结核病化疗的相关性。

DESCRIPTION (provided by applicant): Tuberculosis (TB) remains a global public health challenge, infecting almost nine million people worldwide each year and killing over a million. Two major obstacles to TB control efforts include (1) the protracted drug regimens required to cure TB and (2) the inability of human immunity to effectively clear M. tuberculosis (Mtb) infection, resulting in latent infection. It is estimated that 1/3 of the world's population has laent TB, providing an enormous reservoir from which reactivation to acute disease can occur. New drugs that can simplify or shorten the existing treatment courses for both acute and latent TB infection are badly needed to help improve patient compliance and thus limit the emergence of drug resistant TB. Currently, successful treatment of active, symptomatic TB infection requires a complex drug regimen lasting at least six months, while latent infection requires multi-drug therapy for 3 months or mono-therapy for as long as 9 months. It is believed that this slow clearance of TB by our current antibiotics is due to subpopulations of bacteria within the host tissues that have adopted a drug-tolerant, non-replicating physiological state. Drug regimens that are better at eliminating these persistent, non-replicating bacilli could shorten the required duration of therapy for both active and latent infection. Thus, the identification of small molecules that kill non-replicating Mtb would be invaluable, first to our understanding of the biology of the non-replicating physiological state, and then to the development of more effective, potentially shorter course TB therapy. To find such molecules, we adapted a nutrient deprivation model to high-throughput chemical screening in order to identify compounds that kill carbon-starved, non-replicating Mtb. From an initial oxadiazole hit compound, we have developed ML338 as a small molecule probe that selectively targets non-replicating Mtb bacilli. Because the starvation response has been shown to be necessary for TB persistence in animal models, ML338 represents a valuable tool for not only identifying essential functions and vulnerabilities of the Mtb bacilli in the non-replicating state, but also for testing the relevance of the non-replicating state in vivo during infection. We will first work to identify the molecular target and mechanism of action of ML338. Target identification will entail a series of parallel approaches, including both genetic approaches to isolate resistant mutants as well as affinity-based assays to determine binding partners. Understanding the mechanism of action of ML338 is critical, as it will reveal a vulnerable pathway or pathways in non-replicating Mtb. In addition, we will leverage ML338 as a tool to explore the relevance of the non-replicating state to TB chemotherapy using a mouse infection model.