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，比其他任何传染病都多。 疾病防治结核病的两个主要挑战是， 多重耐药临床分离株，以及缺乏完全杀灭结核分枝杆菌感染的药物。后者是 这归因于存在不被抗生素杀死的持续细胞，并且也逃避了免疫系统。 反应Mtb存留者是代谢静止的，非复制的细胞，存在于肺肉芽肿中， 免疫细胞的紧密聚集是结核病的标志。成熟的血管减少 肉芽肿产生了营养物和氧含量低的微环境，其诱导Mtb的代谢静止。 过渡到静止的确切机制尚不清楚，但维持氧化还原稳态和 通过电子传递链的氧化磷酸化似乎是该过程所必需的。 下一代抗结核化疗应该是高效、安全、有效的合理组合， 协同作用的药物，杀死活跃分裂的细胞和持续存在的细胞。FDA批准的分枝杆菌 ATP合成酶抑制剂贝达喹啉已经验证了结核分枝杆菌作为一种可行药物的能量产生机制 目标几种新的候选药物（如Q203），抑制细胞色素（Cyt）bc 1：aa 3复合物， 结核分枝杆菌呼吸链的组成部分，正在管道中。然而，所有的bc 1抑制剂都是抑菌的， Mtb.这一建议的科学前提是，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， 在对氧化还原应激的反应中，细胞氧化还原缓冲需要Cyt-bd。我们假设（1） Cyt-bd和Cyt-bc 1：aa 3的联合抑制将消除Mtb中的末端氧化，甚至在肉芽肿中， (2)抑制Cyt-bd将增强一线和新型药物根除感染功效。我们的初步 结果表明，Cyt-bd的失活增加了对氧化应激和标准治疗抗TB的敏感性， 毒品我们还证明了缺乏Cyt-bd的Mtb在用抗结核药物治疗的小鼠肺中被快速杀死和清除。 Q203（卡利亚等人，2017，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。在目标二中，我们将评估抗结核药物 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颖的，小型的- Cyt-bd的分子抑制剂，与Q203（管道Cyt-bc 1：aa 3抑制剂）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估以及体外和体内研究 效能测试成功完成拟议的研究将有助于防治结核病药物 耐药性和开发针对结核病的消毒治疗。 !

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. !