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: 10083173
• 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/10083173
• 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/antagonist; 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.

项目总结