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），结核病的病原体，是造成巨大全球痛苦的原因。结核病具有高度的适应性，能够在各种微环境中生存，并作为潜伏感染在人类宿主中持续数十年。对一线、二线和三线药物日益增长的耐药性正在推动对新治疗方法的需求。为了对抗耐药性和持续性感染，我们需要开发针对非复制型结核病的有效化合物。靶向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。蛋白质分泌是一个关键的细胞过程，它支持细菌的生存，促进毒力，并允许结核分枝杆菌检测和响应其环境。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，很少有研究来表征LepB活性并探索其在不同生理状态下的作用。在本申请中，我们提议开发和/或运行高通量筛选（HTS）测定以鉴定Mtb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。我们将采取一个 创新、靶向驱动、基于细胞的方法以及传统的生物化学方法，以开发高通量测定和筛选LepB的小分子抑制剂。我们将进行筛选，并将其与其他检测方法结合联合收割机，以确定化合物的优先级。从这项工作中鉴定的小分子将被用作探针，以更好地了解结核病的生理学，与持久性和毒力相关的分泌组的变化，并作为治疗前药物发现剂。由于这些早期药物发现化合物将通过独特的作用机制发挥作用，因此它们可能对多重耐药和极端多重耐药结核病有效。我们的建议的意义在于它具有双重结果;通过启用HTS和通过提供化学探针刺激基础生物学研究来再生早期药物管道。

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.