Identifying the Molecular Target of Two Novel Compounds Active Against Non-replicating Mycobacterium tuberculosis and Delineating the Role of the Putative Target, InhA, in Non-replicating States

项目摘要 候选人和职业发展计划：Kohta Saito博士是一名传染病内科医生，致力于 在抗菌药物发现和非复制型病原体生物学领域的转化研究， 特别是结核分枝杆菌(Mtb)。他的首要目标是成为一名独立的医生- 科学家探索病原体应激反应和抗生素耐受性问题以发现新的 治疗方法。在短期内，他的目标是：1.获得结核分枝杆菌基因操作的技能；2.到 学习脂肪组学技术，并将其应用于病原体对药物和宿主应激反应的生理学研究。 磨练免疫细胞与结核分枝杆菌相互作用研究的专业知识，4.过渡到独立的研究人员。 环境：威尔·康奈尔医学院(纽约州纽约)非常适合发展斋藤博士的职业生涯，并且已经 具备完成拟议研究的能力。一个大型传染病部门，三个机构资源， 和一个合作的结核病研究单位为斋藤博士将进行的适当培训提供了肥沃的土壤。 研究：这项拟议的工作扩展了齐藤博士治疗结核分枝杆菌的经验，这种结核杆菌对抗生素产生了耐受性 暴露于导致进入非复制(NR)状态的压力。他将使用两种化合物杀死NR 分枝杆菌，并确定NR靶标。具体地说，他将测试InHa，一种细胞壁合成酶， 是相关的目标。他还将在没有接触化合物的NR条件下测试Inha的重要性。 具体目的1.鉴定两个新化合物的NR靶点。我们假设 两个化合物AN1和AN2通过直接抑制INHA而杀死NR Mtb。我们将用3种方法对此进行测试：1. 重组结核分枝杆菌inhA突变和检测NR条件下对AN1和AN2的敏感性，2.创造 结核分枝杆菌inha的条件性敲除及其对AN1和AN2的易感性；3.比较血脂 接触复合制剂前后NR、Mtb的变化 具体目的2.确定INHA在不同的NR Mtb模型中的重要性。我们假设英哈是 对NR州的结核分枝杆菌来说是必不可少的。我们将创造一种有条件的inha基因敲除株，然后评估其在 相关的应激条件，包括暴露在人类巨噬细胞、中性粒细胞和血清中。代谢组学 此外，还将研究在NR条件下发生的基因敲除菌株的脂体变化。 意义：拟议的工作将定义对结核分枝杆菌在NR州的生存至关重要的过程，并描绘出 Inha在NR状态中的作用。这将促进该领域对结核分枝杆菌对生理压力的反应的理解， 并提供有望缩短结核病治疗时间的药物靶点。

Project Summary Candidate and career development plan: Dr. Kohta Saito is an Infectious Diseases physician committed to translational research in the fields of antimicrobial discovery and biology of non-replicating pathogens, specifically Mycobacterium tuberculosis (Mtb). His overarching goal is to become an independent physician- scientist pursuing questions of pathogen stress-response and antibiotic tolerance in order to discover novel treatment methods. In the short term, his goals are: 1. To gain skills in the genetic manipulation of Mtb, 2. To learn lipidomic techniques and apply them to the physiology of pathogen response to drugs and host stress, 3. To hone expertise in the study of immune cell interaction with Mtb, 4. To transition to an independent investigator. Environment: Weill Cornell Medicine (New York, NY) is well suited to develop Dr. Saito's career and already equipped to accomplish the proposed research. A large infectious diseases division, tri-institutional resources, and a collaborative TB Research Unit provide fertile ground for the appropriate training Dr. Saito will undertake. Research: The proposed work extends Dr. Saito's experience with Mtb that become tolerant to antibiotics upon exposure to stresses that cause entry into a non-replicative (NR) state. He will use two compounds that kill NR mycobacteria and identify the NR target. Specifically, he will test whether InhA, a cell wall synthesizing enzyme, is the relevant target. He will also test the essentiality of InhA in NR conditions without compound exposure. Specific Aim 1. Identify the NR target of two novel compounds that kill NR mycobacteria. We hypothesize that two compounds, AN1 and AN2, kill NR Mtb via direct inhibition of InhA. We will test this with 3 methods: 1. Recombineering InhA mutations in Mtb and testing susceptibility to AN1 and AN2 in NR condtions, 2. creating conditional knockdowns of InhA in Mtb and assessing their susceptibility to AN1 and AN2, and 3. comparing lipid profiles of NR Mtb before and after compound exposure. Specific Aim 2. Determine the essentiality of InhA in varied models of NR Mtb. We hypothesize that InhA is essential to Mtb in NR states. We will create a conditional InhA knockout strain, and then assess its viability in relevant stress conditions, including exposure to human macrophages, neutrophils, and serum. Metabolomic and lipidomic changes that occur with knockdown strains in NR conditions will also be investigated. Significance: The proposed work will define a process vital to survival of Mtb in NR states, and delineate the role of InhA in NR states. This will advance the field's understanding of Mtb response to physiologic stresses, and provide drug targets that promise to shorten tuberculosis therapy.