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

描述（由申请人提供）：结核分枝杆菌（Mtb）感染通常导致慢性结核（TB）感染。在这些感染者中，约90%的人产生了成功的免疫反应，将细菌隔离在肉芽肿中。肉芽肿限制了细菌的扩散和生长，导致持续感染。在削弱免疫系统的条件下，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。开发针对结核分枝杆菌建立、维持或退出持续状态所需机制的新疗法，有可能缩短目前约6-9个月的结核分枝杆菌药物治疗方案的疗程，并与耐多药结核分枝杆菌的出现作斗争。结核分枝杆菌会改变其生理机能以应对宿主的免疫压力，从而使这种细菌在人体内存活数十年。结核分枝杆菌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，结核分枝杆菌释放免疫调节剂，在被感染结核分枝杆菌周围形成肉芽肿。肉芽肿限制了细菌获得营养和氧气的机会，并促使结核分枝杆菌重新调整其基因表达和生理，以支持非复制、持续（NRP）状态。NRP的过渡和维持部分由DosRST双组分调节系统（TCS）控制。DosR是一种DNA结合反应调节剂，可被传感激酶DosT磷酸化，诱导约50个基因在缺氧等信号下的表达。其中最强烈的DosR诱导基因是三酰基甘油合成酶1 (tgs1)，它负责细胞内三酰基甘油（TAG）的积累。诱导DosR和NRP的体外生长条件导致Mtb积累主要由TAG组成的包涵体。在感染期间和感染者的临床痰样本中也观察到这些TAG包涵体，支持TAG积累在慢性感染期间结核病进展中的作用。我们建议利用结核分枝杆菌TAG代谢途径来开发针对结核分枝杆菌持续途径的高通量筛选（HTS）平台。我们将开发荧光报告基因检测来测量TAG代谢：i)直接用荧光染色染色TAG内含物，或ii)间接使用合成的tgs1'::GFP报告菌株（Specific Aim 1）。这些HTS平台将在384孔格式中进行优化和验证，性能最好的平台将用于50万个小化合物库的HTS，用于TAG代谢抑制剂（Specific Aim 2）。筛选中确定的靶点将被验证、优先排序，最有希望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的疗效（Specific aim 3）。本提案的目的是确定一种适合进一步优化的先导化合物，并专门针对慢性结核感染。

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.