A novel "shock and kill" strategy for eliminating Mtb persisters in the CD4 T-cell-deficient host

 描述(由申请人提供) 目前可用于治疗活动性结核病(TB)感染的漫长而复杂的多药疗法导致了医学上的不坚持和新出现的多药耐药(MDR)和广泛耐药(XDR)结核病问题，在艾滋病毒合并感染的情况下，这些问题尤其致命。此外，艾滋病毒携带者即使在成功完成抗结核治疗后，复发的风险也会增加。根除结核病感染所需的长期治疗被认为反映了结核分枝杆菌(Mtb) 在宿主内持续处于非复制状态，其特征是对杀菌药物具有抗生素耐受性，杀菌药物主要针对积极分裂的结核杆菌。过度磷酸化鸟苷((P)ppGpp)是由严格反应酶relA合成的，在细菌生长抑制和抗生素耐受中发挥着重要作用。在不同的体外和体内应激条件下，RELA对于结核分枝杆菌的长期存活是必不可少的。 然而，在缺乏细胞免疫的宿主中，是否需要RelA才能使结核分枝杆菌持续存在，这是一个重要的知识缺口。最近，我们发现RelA缺乏的Mtb在应激条件下表现出持续的复制和新陈代谢，最终导致细菌死亡，以及在营养饥饿期间和慢性感染小鼠的肺部对异烟肼的易感性增加。与葛兰素史克的Tres Cantos发展中国家疾病开放实验室(GSK-DDW)合作，我们筛选了一个包含200多万种化合物的再抑制和全细胞杀伤试验文库，并进行了初步的细胞毒性研究。总共鉴定了178个RELA抑制剂候选化合物，代表18个独特的支架，到目前为止测试的39个化合物中有12个化合物显示出RELA特异性活性和与异烟肼的协同作用，以对抗营养缺乏的结核分枝杆菌。这一建议的中心假设是，通过抑制严格反应酶relA来释放对结核分枝杆菌生长和复制的分子“刹车”是一种安全有效的策略，可以防止结核分枝杆菌的持久性，降低药物耐受性，并增强CD4T细胞缺陷宿主对杀菌药物的敏感性。具体地说，我们将使用全细胞营养饥饿分析来筛选热门节目。符合物理化学和毒性标准的靶向特定候选者将优先用于免疫能力良好的C57BL/6J小鼠的安全性/PK研究。在这个小鼠模型中，最多3个有利的RELA抑制剂候选者将在剂量范围研究中单独和结合一线方案进行测试，这是CD4tm1Mak小鼠的背景菌株，将用于验证RERA作为CD4T细胞缺陷宿主中Mtb持久者的靶标。我们新的“休克和杀死”策略有望缩短感染艾滋病毒和未感染艾滋病毒的宿主对药物敏感和耐药结核病的治疗时间。除了改善服药依从性和减少产生耐药性的可能性外，治疗活动性结核病的简化药物方案对艾滋病毒合并感染的患者将特别有用，因为药物-药物相互作用和免疫重建可能会使这两种感染的同时管理复杂化。

 DESCRIPTION (provided by applicant) The lengthy and complicated multidrug therapy currently available to treat active tuberculosis (TB) infection has contributed to medical non-adherence and the emerging problems of multidrug-resistant (MDR)- and extensively drug-resistant (XDR)-TB, which are particularly deadly in the setting of HIV co-infection. In addition, persons with HIV are at increased risk for relapse even upon successful completion of anti-TB treatment. The prolonged therapy required to eradicate TB infection is believed to reflect the ability of Mycobacterium tuberculosis (Mtb) to persist within the host in a non-replicating state characterized by antibiotic tolerance to bactericidal drugs, which predominantly target actively dividing tubercle bacilli. Hyperphosphorylated guanosine ((p)ppGpp), which is synthesized by the stringent response enzyme RelA, plays an important role in bacterial growth restriction and antibiotic tolerance. RelA is essential for long-term Mtb survival during various in vitro and in vivo stress conditions. However, whether RelA is required for Mtb persistence in the host lacking cell-mediated immunity represents an important knowledge gap. Recently, we found that relA-deficient Mtb exhibits continued replication and metabolism during stress conditions, ultimately leading to bacillary death, as well as enhanced susceptibility to isoniazid during nutrient starvation and in the lungs of chronically infected mice. In collaboration with GlaxoSmithKline's Tres Cantos Open Lab for Diseases of the Developing World (GSK-DDW), we have screened a library of over 2 million compounds in RelA-inhibition and whole-cell killing assays, and performed preliminary cytotoxicity studies. A total of 178 RelA inhibitor candidates, representing 18 unique scaffolds, were identified and 12 of 39 compounds tested so far exhibit RelA-specific activity and synergy with isoniazid against nutrient-starved Mtb. The central hypothesis of this proposal is that releasing the molecular "brakes" on Mtb growth and replication through inhibition of the stringent response enzyme RelA is a safe and effective strategy to prevent Mtb persistence, decrease drug tolerance, and enhance susceptibility to bactericidal drugs in the CD4 T-cell-deficient host. Specifically, we will screen hits using whole-cell nutrient starvation assays. Target-specific candidates fulfilling physicochemical and toxicity criteria will be prioritized for safety/PK studis in immune-competent C57BL/6J mice. Up to 3 favorable RelA inhibitor candidates will be tested in dose-ranging studies alone and in combination with the first-line regimen in this mouse model, which is the background strain for the Cd4tm1Mak mice, which will be used to validate RelA as a target for Mtb persisters in the CD4 T-cell-deficient host. Our novel "shock and kill" strategy is expected to shorten the duration of treatment for drug-susceptible and drug-resistant TB in the HIV-infected and HIV-uninfected host. In addition to improving medical adherence and reducing the potential for the development of drug resistance, an abbreviated drug regimen to treat active TB would be particularly useful in HIV co-infected patients, since drug- drug interactions and immune reconstitution may complicate the concurrent management of both infections.