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

• 批准号: 10320910
• 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
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-01-25 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10320910
• 关键词: Agar; Anaerobic Bacteria; Biological Assay; Biology; Boron; Cell Communication; Cell Wall; Clinical; Collaborations; Communicable Diseases; DNA Sequence Alteration; Data; Development; Development Plans; Dose; Drug Targeting; Environment; Enzymes; Exposure to; Face; Foundations; Future; Gene Mutation; Genetic Transcription; Genus Mycobacterium; Goals; Growth; Heterogeneity; Human; Human body; Immune; In Vitro; Incubated; Isoniazid resistance; Kinetics; Knock-out; Learning; Link; Lipids; Literature; Medicine; Methods; Modeling; Modification; Molecular Target; Mutation; Mycobacterium smegmatis; Mycobacterium tuberculosis; Mycolic Acid; NADH; New York; Nutrient; Outcome; Phagosomes; Pharmaceutical Preparations; Phenotype; Physicians; Physiology; Predisposition; Process; Property; Publishing; Research; Research Personnel; Resistance; Resources; Role; Scientist; Secondary to; Serum; Starvation; Stress; Stress Tests; Techniques; Testing; Therapeutic; Training; Translational Research; Tuberculosis; Work; antibiotic tolerance; antimicrobial; base; biological adaptation to stress; career; career development; chemical property; design; experience; genetic manipulation; improved; in vivo; inhibitor; isoniazid; knock-down; lipidome; lipidomics; macrophage; metabolome; metabolomics; mutant; neutrophil; novel; pathogen; physiologic stressor; resilience; response; skills; therapeutic development; tuberculosis drugs; tuberculosis treatment

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博士是一名传染病医生，致力于 在抗菌剂发现和非复制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- 科学家追求病原体应激反应和抗生素耐受性的问题，以发现新的 治疗方法在短期内，他的目标是：1。为了获得结核分枝杆菌基因操作的技能，2。到 学习脂质组学技术，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; 为了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.转变为独立调查员。 环境：威尔康奈尔医学（纽约，纽约州）非常适合发展博士。 准备完成所提出的研究。一个大的传染病部门，三个机构的资源， 和一个合作的结核病研究单位为齐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Saito博士对结核分枝杆菌的经验，这种结核分枝杆菌对抗生素具有耐受性， 暴露于导致进入非复制（NR）状态的应力。他将使用两种化合物杀死NR 分枝杆菌和确定NR目标。具体来说，他将测试InhA，一种细胞壁合成酶， 是相关的目标他还将测试在没有化合物暴露的NR条件下InhA的必要性。 具体目标1。确定两种新型化合物的NR靶点，杀死NR分枝杆菌。我们假设 两种化合物AN 1和AN 2通过直接抑制InhA杀死NR Mtb。我们将用三种方法来测试：1。 在Mtb中检测InhA突变并在NR条件下测试对AN 1和AN 2的易感性，2.创建 Mtb中InhA的条件性敲低并评估其对AN 1和AN 2的易感性，以及3.比较脂质 在化合物暴露之前和之后的NR Mtb曲线。 具体目标2。确定InhA在不同NR Mtb模型中的重要性。我们假设InhA是 对NR州的Mtb至关重要。我们将创建一个条件性InhA敲除菌株，然后评估其在 相关应激条件，包括暴露于人巨噬细胞、中性粒细胞和血清。代谢组 还将研究NR条件下敲低菌株发生的脂质组学变化。 意义：拟议的工作将定义一个对NR州的Mtb生存至关重要的过程，并描述 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应激反应的理解， 并提供有望缩短结核病治疗时间的药物靶点。