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

鉴定两种对非复制结核分枝杆菌具有活性的新型化合物的分子靶标，并描述假定靶标 InhA 在非复制状态下的作用

基本信息

批准号：
10538631
负责人：
Kohta Saito
金额：
$ 17.2万
依托单位：
WEILL MEDICAL COLL OF CORNELL UNIV
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-01-25 至 2024-12-31
项目状态：
已结题

项目摘要

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.

项目摘要候选人和职业发展计划：Kohta Saito博士是一种致力于在非复制病原体的抗菌发现和生物学领域的转化研究，特别是结核分枝杆菌（MTB）。他的总体目标是成为一名独立医生 - 科学家追求病原体应激反应和抗生素耐受性的问题，以发现新颖治疗方法。在短期内，他的目标是：1。获得MTB遗传操作的技能，2。学习脂质细胞学技术，并将其应用于病原体对药物和宿主压力的生理学3。为了磨练与MTB免疫细胞相互作用的研究专业知识，4。过渡到独立研究者。环境：Weill Cornell Medicine（纽约，纽约）非常适合发展Saito博士的职业能够完成拟议的研究。三机构资源的大型传染病部门，一个合作的结核病研究部门为适当的赛托博士将接受的适当培训提供了肥沃的基础。研究：拟议的工作扩展了Saito博士在MTB上的经验，这些经验变得容忍了抗生素暴露于导致进入非复制（NR）状态的压力。他将使用两种杀死NR的化合物分枝杆菌并识别NR靶标。具体而言，他将测试inha（inha）是否合成酶，是相关目标。他还将在不复合暴露的情况下测试INHA的重要性。具体目标1。确定两种杀死NR分枝杆菌的新型化合物的NR靶标。我们假设这一点两种化合物AN1和AN2通过直接抑制INHA杀死NR MTB。我们将使用3种方法进行测试：1。 MTB中的重组INHA突变，并测试NR条件中对AN1和AN2的敏感性，2。 MTB中INHA的条件敲低并评估其对AN1和AN2的敏感性，以及3。复合暴露前后NR MTB的轮廓。具体目标2。确定NR MTB不同模型中INHA的本质。我们假设Inha是在NR州对MTB至关重要。我们将创建有条件的INHA敲除菌株，然后评估其在相关的应力条件，包括暴露于人类巨噬细胞，中性粒细胞和血清。代谢组在NR条件下敲低菌株发生的脂质组变化也将被研究。意义：拟议的工作将定义一个对NR州MTB生存至关重要的过程，并描述 INHA在NR州的作用。这将促进该领域对MTB对生理压力的反应的理解，并提供有望缩短结核病治疗的药物靶标。