Screening for inhibitors of M. tuberculosis persistence-related lipid metabolism

结核分枝杆菌持久性相关脂质代谢抑制剂的筛选

• 批准号: 8509336
• 负责人: Robert B Abramovitch
• 金额: $ 19.01万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-08 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8509336
• 关键词: Age; Air; American; Animal Model; Animals; Anti-Inflammatory Agents; Anti-inflammatory; Antibiotics; Bacteria; Biological Assay; Carbon Monoxide; Cessation of life; Chronic; Clinical; Communicable Diseases; Cues; DNA Binding; Development; Drug resistance; Environment; Exhibits; Fluorescence; Gene Expression; Genes; Goals; Granuloma; Growth; HIV; HIV Infections; Health; Human; Hypoxia; Immune; Immune response; Immune system; Immunity; In Vitro; Inclusion Bodies; Infection; Lead; Libraries; Lipid Inclusion; Lipids; Maintenance; Malnutrition; Measures; Metabolism; Mission; Modeling; Multi-Drug Resistance; Mycobacterium tuberculosis; National Institute of Allergy and Infectious Disease; Nitric Oxide; Nutrient; Oxygen; Pathway interactions; Phagosomes; Pharmaceutical Chemistry; Pharmaceutical Preparations; Phase; Phosphotransferases; Physiology; Play; Population; Predisposition; Recycling; Regimen; Reporter; Role; Sampling; Sputum; Staging; Staining method; Stains; Structure-Activity Relationship; System; Triglycerides; Tuberculosis; aging population; cell killing; combat; disease transmission; global health; high throughput screening; inhibitor/antagonist; lipid metabolism; macrophage; nile red; pathogen; pre-clinical; pressure; public health relevance; resistant strain; response; screening; sensor; tuberculosis drugs

DESCRIPTION (provided by applicant): Infection with Mycobacterium tuberculosis (Mtb) often results in a chronic tuberculosis (TB) infection. Of those infected, ~90% mount a successful immune response that sequesters the bacterium in a granuloma. The granuloma limits the spread and growth of the bacterium and results in a persistent infection. Under conditions that weaken the immune system Mtb can be released from the granuloma and establish an active, infectious disease state. Developing new therapies that target mechanisms required for Mtb to establish, maintain or exit a persistent state has the potential to shorten the course of current ~6-9 month Mtb drug regimens as well as combat the emergence of multidrug-resistant Mtb. Mtb alters its physiology in response to host immune pressures thus enabling the bacterium to remain viable in humans for decades. Mtb is an intracellular pathogen that resides within macrophages (M), a host immune cell that kills most other bacteria. Following infection, the M releases immune modulators that orchestrate the formation of a granuloma around the infected M. The granuloma limits the availability of nutrients and oxygen to the bacterium and drives Mtb to realign its gene expression and physiology to support a non-replicative, persistent (NRP) state. Transition into and maintenance of NRP is governed, in part, by the DosRST two component regulator system (TCS). DosR is a DNA binding response regulator that is phosphorylated by the sensor kinase DosT to induce expression of approximately 50 genes in response to cues such as hypoxia. One of the most strongly DosR induced genes is triacylglycerol synthase 1 (tgs1), which is responsible for the accumulation of intracellular triacylglycerol (TAG). In vitro growth conditions that induce DosR and NRP cause Mtb to accumulate inclusion bodies that are primarily composed of TAG. These TAG inclusion bodies are also observed during infections and in clinical sputum samples from infected humans, supporting a role for TAG accumulation in the progression of TB during chronic infection. We propose to exploit Mtb TAG metabolism pathways to develop high throughput screening (HTS) platforms that target Mtb persistence pathways. We will develop fluorescent reporter assays that measure TAG metabolism: i) directly by staining TAG inclusions with the fluorescent stains, or ii) indirectly by using a synthetic tgs1'::GFP reporter strain (Specific Aim 1). These HTS platforms will be optimized and validated in a 384 well format and the best performing platform will be used for a HTS of a 500,000 small compound library for inhibitors of TAG metabolism (Specific Aim 2). Hits identified in the screen will be validated, prioritized and the most promising compounds will undergo preclinical characterization including, mechanism of action, structure activity relationship studies, and efficacy in animal models of infection (Specific aim 3). The goa of this proposal is to identify a lead compound that is suitable for further optimization and that specifically targets the chronic stage of tuberculosis infection.

描述（由申请人提供）：结核分枝杆菌（Mtb）感染通常会导致慢性结核（TB）感染。在感染者中，约 90% 成功地发起了免疫反应，将细菌隔离在肉芽肿中。肉芽肿限制了细菌的传播和生长并导致持续感染。在削弱免疫系统的情况下，结核分枝杆菌可以从肉芽肿中释放出来，并建立一种活跃的传染病状态。开发针对 Mtb 建立、维持或退出持久状态所需机制的新疗法有可能缩短当前约 6-9 个月的 Mtb 药物治疗方案的疗程，并对抗多重耐药 Mtb 的出现。 结核分枝杆菌会改变其生理机能以应对宿主的免疫压力，从而使该细菌能够在人体中存活数十年。 Mtb 是一种存在于巨噬细胞 (M) 内的细胞内病原体，巨噬细胞是一种杀死大多数其他细菌的宿主免疫细胞。感染后，M 会释放免疫调节剂，协调受感染 M 周围肉芽肿的形成。肉芽肿限制了细菌对营养和氧气的利用，并驱使 Mtb 重新调整其基因表达和生理机能，以支持非复制性、持久性。 （NRP）状态。 NRP 的过渡和维护部分由 DosRST 两部分调节器系统 (TCS) 控制。 DosR 是一种 DNA 结合反应调节剂，被传感器激酶 DosT 磷酸化，诱导大约 50 个基因表达，以响应缺氧等提示。 DosR 诱导最强烈的基因之一是三酰甘油合酶 1 (tgs1)，它负责细胞内三酰甘油 (TAG) 的积累。诱导 DosR 和 NRP 的体外生长条件会导致 Mtb 积累主要由 TAG 组成的包涵体。这些 TAG 包涵体也在感染过程中以及感染者的临床痰样本中观察到，支持 TAG 积累在慢性感染期间结核病进展中的作用。我们建议利用 Mtb TAG 代谢途径来开发针对 Mtb 持久性途径的高通量筛选 (HTS) 平台。我们将开发测量 TAG 代谢的荧光报告测定法：i) 通过用荧光染料直接对 TAG 内含物进行染色，或 ii) 通过使用合成的 tgs1'::GFP 报告菌株间接进行（具体目标 1）。这些 HTS 平台将以 384 孔格式进行优化和验证，性能最佳的平台将用于 TAG 代谢抑制剂的 500,000 个小型化合物库的 HTS（具体目标 2）。筛选中确定的命中将得到验证和优先排序，最有前途的化合物将进行临床前表征，包括作用机制、结构活性关系研究和动物感染模型中的功效（具体目标 3）。该提案的目标是确定一种适合进一步优化且专门针对结核病感染慢性阶段的先导化合物。