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

• 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
• 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

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）是结核病的致病药物，负责巨大的全球苦难。 MTB具有高度适应性的能力，能够在各种微环境中生存，并且在人类宿主中持续数十年，作为潜在的感染。对第一，第二线和第三行药物的抗药性越来越多，促进了对新治疗的需求。为了打击抗药性和持续感染，我们需要开发有效的化合物，以防止非复制MTB。靶向细菌存活必不可少的蛋白质分泌机制是满足这种需求的一种方法。蛋白质分泌是一个关键的细胞过程，它是细菌存活，促进毒力并允许MTB检测和对其环境反应的关键细胞过程。信号肽酶LEPB在此过程中起着不可或缺的作用，对于生存至关重要。但是，很少有研究以表征LEPB活性并探索其在不同生理状态中的作用。在此应用中，我们建议开发和/或运行高通量筛选（HTS）测定法，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”蛋白质，我们将抑制大量对细菌生存和持久性至关重要的途径。我们将接受 创新的，目标驱动的基于细胞的方法以及传统的生化方法，用于开发LEPB小分子抑制剂的高吞吐量测定和筛选。我们将进行筛选，并将它们与其他测定法相结合，以优先考虑化合物。从这项工作中鉴定出的小分子将被用作探针，以更好地了解结核病的生理学，与持久性和毒力有关的分泌组的变化以及治疗前药物发现剂。由于这些早期的药物发现化合物将通过独特的作用机理起作用，因此它们可能有效地抵抗多药耐药性和极度多药物的TB。我们提出的意义在于它具有双重结果。通过提供化学探针来启用HTS并刺激基本生物学研究，使早期药物管道的再生。