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

结核病（TB）仍然是全球公共卫生挑战，每年感染全球近900万人，造成100多万人死亡。结核病控制工作的两个主要障碍包括（1）治疗结核病所需的长期药物治疗方案和（2）人体免疫力不能有效清除M。结核病（Mtb）感染，导致潜伏感染。据估计，世界人口的1/3患有潜伏性结核病，这提供了一个巨大的库，可以发生急性疾病的再激活。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治疗疗程的新药，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、症状性TB感染需要持续至少6个月的复杂药物方案，而潜伏性感染需要3个月的多药治疗或长达9个月的单药治疗。据信，我们目前的抗生素对TB的这种缓慢清除是由于宿主组织内的细菌亚群已经采取了耐药性、非复制性生理状态。更好地消除这些持久性，非复制性杆菌的药物方案可以缩短所需的时间。 活动性和潜伏性感染的治疗持续时间。因此，鉴定杀死非复制型结核分枝杆菌的小分子将是非常宝贵的，首先是我们对非复制型生理状态的生物学的理解，然后是开发更有效、可能更短疗程的结核病治疗。为了找到这样的分子，我们采用了营养剥夺模型进行高通量化学筛选，以确定杀死碳饥饿，非复制结核分枝杆菌的化合物。从最初的恶二唑命中化合物，我们已经开发了ML 338作为选择性靶向非复制型结核分枝杆菌的小分子探针。由于饥饿反应已被证明是TB在动物模型中持续存在所必需的，ML 338代表了一种有价值的工具，不仅用于鉴定非复制状态下Mtb杆菌的基本功能和脆弱性，而且用于测试感染期间体内非复制状态的相关性。我们将首先确定分子目标， ML 338的作用机制。靶标鉴定将需要一系列平行的方法，包括分离抗性突变体的遗传方法以及确定结合伴侣的基于亲和力的测定。了解ML 338的作用机制至关重要，因为它将揭示非复制型Mtb中的一个或多个脆弱途径。此外，我们将利用ML 338作为工具，使用小鼠感染模型探索非复制状态与TB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.