Exploiting metabolic toxicities to identify compounds that inhibit cholesterol metabolism in M. Tuberculosis

 描述(申请人提供)：结核病(TB)是一种持久的全球流行病，每年夺走约150万人的生命。结核分枝杆菌(Mtb)是结核病的病原体，这种细菌通过在巨噬细胞内存活并操纵宿主免疫反应来建立长期感染。在肺组织内，结核分枝杆菌可以持续数十年，远离宿主免疫反应的压力。在这种持续感染期间，结核分枝杆菌只利用宿主来源的营养物质生存。结核分枝杆菌具有在感染过程中利用宿主来源的胆固醇提供能量产生和/或生物合成途径的能力。因此，抑制胆固醇分解代谢可能是发现新的抗Mtb药物的弱点。对于这个项目，我们将开发一种适用于HTS的基于细胞的检测方法，它可以识别Mtb中胆固醇分解代谢的抑制剂。目的1：我们将使用Mtb Icl1菌株开发一种强大的化学抑制物HTS检测384孔格式。目标2：我们将完成一次HTS试点，并验证屏幕上的点击。目标3：将重点放在 识别HTS Hits的酶靶标。这些研究将产生HTS Ready化验，并提供“原则证据”，证明我们的方法可以识别结核分枝杆菌中胆固醇分解代谢的选择性抑制剂。

 DESCRIPTION (provided by applicant): Tuberculosis (TB) is a lasting global epidemic that claims ~1.5 million human lives annually. Mycobacterium tuberculosis (Mtb) is the causative agent of TB and this bacterium establishes long term infections by surviving within macrophages and manipulating the host immune response. Within lung tissue Mtb can persist for decades sequestered away from the pressures of the host immune response. During this persistent infection Mtb solely utilizes host-derived nutrients for survival. Mtb has the ability t utilize host-derived cholesterol during an infection to supply energy producing and/or biosynthetic pathways. Therefore, inhibition of cholesterol catabolism may be a weakness that can be exploited for discovery of new anti-Mtb drugs. For this project we will develop a cell-based assay suitable for HTS that can identify inhibitors of cholesterol catabolism in Mtb. Aim 1: we will develop a robust chemical-suppressor HTS assay 384-well format using the Mtb Icl1 strain. Aim 2: we will complete a pilot HTS and validate hits from the screen. Aim 3: will focus on identifying the enzyme targets of the HTS hits. These studies will yield a HTS ready assay and provide "proof of principle" that our approach can identify selective inhibitors of cholesterol catabolism in Mtb.