High throughput assays to detect inhibition of a key M. tuberculosis protease

描述（由申请人提供）：结核分枝杆菌（Mtb）是结核病的病原体，是造成全球巨大痛苦的罪魁祸首。结核分枝杆菌具有高度适应性，能够在各种微环境中生存，并作为潜伏感染在人类宿主体内持续数十年。对一线、二线和三线药物日益增长的耐药性推动了对新疗法的需求。为了对抗耐药性和持续性感染，我们需要开发针对非复制性结核分枝杆菌的有效化合物。针对细菌生存所必需的蛋白质分泌机制是满足这一需求的一种方法。蛋白质分泌是支撑细菌存活的关键细胞过程，促进毒力并使结核分枝杆菌能够检测其环境并对其作出反应。信号肽酶LepB在这一过程中起着不可或缺的作用，对生存至关重要；然而，很少有研究对LepB活性进行表征，并探讨其在不同生理状态下的作用。在本申请中，我们建议开发和/或运行高通量筛选（HTS）试验，以鉴定结核分枝杆菌信号肽酶LepB的化学抑制剂。我们认为，通过抑制这种“看门人”蛋白，我们将抑制大量对细菌生存和持久性至关重要的途径。我们将采取行动。

DESCRIPTION (provided by applicant): Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is responsible for huge global suffering. Mtb is highly adaptive being able to survive in a wide variety of microenvironments as well as persist for decades in the human host as a latent infection. Growing resistance to first, second and third line drugs are driving a need for new treatments. To combat drug-resistance and persistent infections, we need to develop effective compounds against non-replicating Mtb. Targeting protein secretion mechanisms essential for bacterial survival is one way to meet this need. Protein secretion is a key cellular process which underpins bacterial survival, promoting virulence and allowing Mtb to detect and respond to its environment. The signal peptidase, LepB, plays an integral role in this process and is essential for survival; however few studies have been conducted to characterize LepB activity and explore its role in different physiological states. In this application we propos to develop and/or run high-throughput screening (HTS) assays to identify chemical inhibitors of the Mtb signal peptidase, LepB. We think that by inhibiting this "gatekeeper" protein, we will inhibit a large number of pathways critical for bacterial survival and persistence. We will take an innovative, target-driven, cell-based approach, as well as a traditional biochemical approach, to develop high throughput assays and screen for small molecule inhibitors of LepB. We will conduct screens and combine these with other assays to prioritize compounds. Small molecules identified from this work will be used as probes better to understand the physiology of tuberculosis, the changes in the secretome related to persistence and virulence, and as pre-therapeutic drug discovery agents. Because these early drug discovery compounds will act through a unique mechanism of action, they will likely be effective against multi-drug resistant and extremely multi-drug resistant TB. The significance of our proposal is that it has a dual outcome; regeneration of the early drug pipeline by enabling HTS and stimulation of basic biological studies by providing chemical probes.