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病（TB）感染。在被感染的人中，约有90％的成功免疫反应隔离了肉芽肿中的细菌。肉芽瘤限制了细菌的扩散和生长，并导致持续感染。在削弱免疫系统的条件下，MTB可以从肉芽肿释放并建立活跃的传染病状态。开发新的疗法，以建立，维持或退出持续状态所需的目标机制可能会缩短当前〜6-9个月的MTB药物方案的过程，并打击耐多药的MTB的出现。 MTB会改变其生理学，以响应宿主的免疫压力，从而使细菌能够在人类中保持生存数十年。 MTB是一种驻留在巨噬细胞（M）内的细胞内病原体，这是一种杀死大多数其他细菌的宿主免疫细胞。感染后，M释放了免疫调节剂，这些免疫调节剂在感染周围的颗粒状瘤的形成。肉芽肿限制了养分和氧气的可用性，并驱动MTB驱动其基因表达和生理学以支持非复制性，持久性，持久性（NRP）状态。 DOSRST两个组件调节器系统（TCS）部分控制了NRP的过渡和维护。 DOSR是一种DNA结合响应调节剂，被传感器激酶DOST磷酸化，以诱导大约50个基因的表达，以响应缺氧等线索。 DOSR诱导的基因最强烈的是三酰基甘油合酶1（TGS1），它负责累积细胞内三酰基甘油（TAG）。诱导DOSR和NRP的体外生长条件会导致MTB积累主要由TAG组成的包含体。在感染期间和来自感染人类的​​临床痰样品中还观察到这些标签包容体，这支持了在慢性感染过程中TAM积累的作用。我们建议利用MTB TAG代谢途径来开发针对MTB持久途径的高吞吐量筛选（HTS）平台。我们将开发荧光记者分析，以测量TAG代谢：i）直接通过使用合成TGS1':: GFP报告基因菌株（特定AIM 1）间接染色标签包含物，或II）间接地。这些HTS平台将以384井格式进行优化和验证，最佳性能平台将用于500,000个小型化合物库的HTS，以用于TAG代谢的抑制剂（特定的AIM 2）。屏幕中确定的点击将得到验证，优先级和最有希望的化合物将经历临床前的特征，包括作用机理，结构活动关系研究以及感染动物模型的功效（特定目标3）。该提案的果阿是确定适合进一步优化的铅化合物，并专门针对结核病感染的慢性阶段。