Eradicating persistent M. tuberculosis by synthetic lethality of terminal respiratory oxidases

项目摘要 结核分枝杆菌(Mtb)每年导致约180万人死亡，比任何其他传染病都要多 疾病。抗击结核病的两个主要挑战是迅速增加的 耐多药的临床分离株，以及缺乏完全消毒结核分枝杆菌感染的药物。后者是 归因于伴胞细胞的存在，这些细胞不会被抗生素杀死，也可以逃避免疫 回应。结核分枝杆菌持续体是驻留在肺肉芽肿中的代谢休眠、非复制细胞， 致密的免疫细胞聚集体，这是结核病的标志。成熟期血管构筑减少 肉芽肿创造了一个低营养和低氧气的微环境，导致结核分枝杆菌代谢停滞。 转变为静止的确切机制尚不清楚，但氧化还原动态平衡的维持和 通过电子传递链的氧化磷酸化似乎在这一过程中是必不可少的。 下一代抗结核化疗应该是高活性、 能同时杀死活跃的分裂细胞和伴胞细胞的协同药物。分枝杆菌的FDA批准 三磷酸腺苷合成酶抑制剂贝达奎兰已证实结核分枝杆菌的能量产生机制是一种可行的药物 目标。几种抑制细胞色素(Cyt)Bc1：Aa3复合体的新药候选药物(例如Q203)，a 结核分枝杆菌呼吸链的组成部分正在筹备中。然而，所有的bc1抑制剂都是抑菌的。 山地车。这一提议的科学前提是，这类药物缺乏杀灭活性是由于 存在第二种酶，细胞色素BD氧化酶(Cyt-BD)。除了作为终端氧的作用外 还原酶，Cyt-BD在细胞氧化还原缓冲中是必需的，以响应氧化还原应激源。我们假设(1) 联合抑制Cyt-BD和Cyt-Bc1：Aa3将消除结核分枝杆菌的末端氧化，甚至在肉芽肿中也是如此。 (2)抑制Cyt-BD将增强一线和新药根除感染的疗效。我们的预赛 结果显示，Cyt-BD的失活增加了对氧化应激和标准护理抗结核药物的敏感性 毒品。我们还证明了缺乏Cyt-BD的结核分枝杆菌在小鼠肺中被迅速杀死和清除。 Q203(Kalia et al.2017年，PNAS)。 在这里，我们建议采取下一步的新的化疗方法。在我们的第一个目标中，我们 将在一个动物模型中研究末端氧化酶抑制的协同致死性 肉芽肿性病变，类似于人类结核病。在第二个目标中，我们将评估防治结核病之间的协同作用。 杀微生物剂和抑制末端氧化。最后，在目标三中，我们将专注于开发新的、小型的- Cyt-BD的分子抑制剂，与流水线Cyt-Bc1：Aa3抑制剂Q203协同作用。这将包括 构效关系研究、细胞毒性和药代动力学评价及体内外研究 效力测试。成功完成拟议的研究将有助于防治结核病的药物 抗药性和开发一种针对结核病的消毒疗法。 好了！

Project Summary Mycobacterium tuberculosis (Mtb) kills around 1.8 million people a year, more than any other infectious disease. The two main challenges of combating tuberculosis (TB) are the rapidly increasing number of multidrug-resistant clinical isolates, and the lack of drugs that completely sterilize Mtb infection. The latter is ascribed to the presence of persister cells that are not killed by antibiotics, and also evade the immune response. Mtb persisters are metabolically resting, non-replicating cells that reside in lung granulomas, compact aggregates of immune cells that are the hallmark of tuberculosis. The reduced vasculature of mature granulomas creates a microenvironment low in nutrients and oxygen that induces metabolic quiescence in Mtb. The exact mechanism of the transition to quiescence is unclear, but maintenance of redox homeostasis and oxidative phosphorylation via the electron transport chain appear to be essential to this process. The next generation of anti-TB chemotherapy should be a rational combination of highly active, synergistic drugs that kill both actively dividing cells and persister cells. The FDA-approval of the mycobacterial ATP synthase inhibitor bedaquiline has validated the energy generating machinery of Mtb as a viable drug target. Several new drug candidates (e.g. Q203) that inhibit the cytochrome (Cyt) bc1:aa3 complex, a component of the respiratory chain of Mtb, are in the pipeline. However, all bc1 inhibitors are bacteriostatic in Mtb. The scientific premise of this proposal is that the lack of cidal activity by this class of drugs is due to the presence of a second enzyme, the cytochrome bd oxidase (Cyt-bd). In addition to its role as a terminal oxygen reductase, Cyt-bd is required in cellular redox buffering in response to redox stressors. We hypothesize that (1) combined inhibition of Cyt-bd and Cyt-bc1:aa3 will abrogate terminal oxidation in Mtb, even in granulomas, and (2) inhibition of Cyt-bd will enhance efficacy of front-line and novel drugs to eradicate infection. Our preliminary results show that inactivation of Cyt-bd increases sensitivity to oxidative stress and to standard-of-care anti-TB drugs. We also demonstrated that Mtb lacking Cyt-bd is rapidly killed and cleared in mouse lungs treated with Q203 (Kalia et al. 2017, PNAS). Here, we propose to take the next step towards a new chemotherapeutic approach. In our first aim, we will investigate the synergistic lethality of terminal oxidase inhibition in an animal model that develops granulomatous lesions, similar to human tuberculosis. In aim two, we will evaluate synergies between anti-TB microbicides and inhibition of terminal oxidation. Finally, in aim three, we will focus on developing novel, small- molecule inhibitors of Cyt-bd that synergize with Q203, the pipeline Cyt-bc1:aa3 inhibitor. This will include structure-activity relationship studies, cytotoxicity and pharmacokinetics assessment and in vitro and in vivo potency testing. Successful completion of the proposed studies will contribute to combating TB drug resistance and to developing a sterilizing treatment against TB. !