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

高通量测定检测关键结核分枝杆菌蛋白酶的抑制

基本信息

批准号：
8438134
负责人：
Tanya Parish
金额：
$ 36.43万
依托单位：
ACCESS TO ADVANCED HEALTH INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-07-16 至 2016-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8438134
关键词：
Anti-Bacterial Agents Bacteria Bacterial Infections Biochemical Biological Biological Assay Biology Cause of Death Cell physiology Cells Chemicals Communicable Diseases Critical Pathways Disease Drug resistance Environment Enzyme Inhibition Evaluation Gatekeeping Genetic Growth Human In Vitro Infection Kinetics Laboratories Lead Libraries Measures Metabolic Pathway Multi-Drug Resistance Mycobacterium tuberculosis Natural regeneration Organism Outcome Pathogenicity Peptide Hydrolases Peptides Pharmaceutical Preparations Physiological Physiology Play Process Protein Secretion Proteins Reading Resistance Role Running Testing Therapeutic Therapeutic Agents Tuberculosis United States National Institutes of Health Virulence Work assay development base combat drug development drug discovery drugged driving high throughput screening improved in vivo inhibitor/antagonist innovation insight interest killings latent infection meetings novel therapeutics pathogen public health relevance response screening signal peptidase small molecule stressor tool

项目摘要

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.

描述（由申请人提供）：结核分枝杆菌（Mtb）是结核病的病原体，给全球带来了巨大的痛苦。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感染，我们需要开发针对非复制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。蛋白质分泌是一个关键的细胞过程，它支持细菌生存、增强毒力并允许结核分枝杆菌检测并对其环境做出反应。信号肽酶 LepB 在此过程中发挥着不可或缺的作用，对于生存至关重要。然而，很少有研究来表征 LepB 活性并探讨其在不同生理状态下的作用。在此应用中，我们建议开发和/或运行高通量筛选 (HTS) 测定法来鉴定 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。我们将采取创新、目标驱动、基于细胞的方法以及传统的生化方法，开发高通量检测方法并筛选 LepB 小分子抑制剂。我们将进行筛选并将其与其他测定结合起来以确定化合物的优先顺序。从这项工作中鉴定出的小分子将用作探针，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，并用作治疗前药物发现剂。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，因此它们可能会有效对抗多重耐药性和极度多重耐药性结核病。我们建议的意义在于它具有双重结果：通过启用 HTS 来再生早期药物管道，并通过提供化学探针来刺激基础生物学研究。