Yeast-based HTS Assay Technologies for Proteases

基于酵母的蛋白酶高温超导检测技术

• 批准号: 8212269
• 负责人: JOHN C REED
• 金额: $ 46.8万
• 依托单位: SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-02-01 至 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8212269
• 关键词: Active Sites; Animals; Apoptotic; Atherosclerosis; Autoimmunity; Biological Assay; Biological Process; Caspase; Caspase-1; Cell Death; Chemicals; Chronic; Cleaved cell; Collection; Communicable Diseases; Complex; Cysteine Protease; Development; Disease; Endopeptidases; Enzymes; Eukaryota; Event; Family; Genes; Health; Human Genome; Immune response; In Vitro; Inflammation; Inflammatory; Libraries; Malignant Neoplasms; Mediating; Methods; Natural Immunity; Nerve Degeneration; Noise; Pathway interactions; Peptide Hydrolases; Performance; Play; Post-Translational Protein Processing; Proteins; Proteolytic Processing; Reporter Genes; Research; Role; Screening procedure; Signal Transduction; System; Technology; Testing; Validation; Yeasts; base; cytokine; drug discovery; high throughput screening; human disease; inhibitor/antagonist; interest; member; new technology; novel; pathogen; protein complex; prototype; reconstitution; small molecule libraries; tool; transcription factor

DESCRIPTION (provided by applicant): Yeast-based HTS Assay Technologies for Proteases. Proteolytic processing of proteins is an irreversible post-translational modification of importance for a wide-variety of biological processes. Consequently, proteases have emerged as promising targets for drug discovery for a wide variety of human diseases, including inflammation, infectious diseases, neurodegeneration, ischemic diseases, and cancer. Development of high throughput screening (HTS) assays using purified proteases can be relatively straightforward or it can be quite challenging, particularly when multi-component systems are required to achieve protease activation. Also, due to similarity of the active sites of some groups of proteases, selectivity of chemical inhibitors can be difficult if not impossible to achieve, highlighting the need for alternative screening methods for identifying compounds that target upstream activators of proteases rather than directly inhibiting the protease of interest. We propose to generate and optimize HTS systems for intracellular proteases, using Caspases as a prototype. For this purpose, we have devised yeast-based cellular systems that permit facile expression of proteases and protease-activating proteins in combinations that reconstitute entire mammalian pathways in these simple eukaryotes. Among the assay methods integrated into the yeast system are cleavable reporter gene activators, in which protease-mediated cleavage activates a transcription factor. The Aims are to: (1) Devise multi-component systems that reconstitute mammalian protease activation pathways in yeast; (2) Adjust the necessary variables to achieve HTS-quality assay performance; (3) Perform pilot chemical library screens of multi-component yeast-based protease assay systems to define hit-rates and test reliability; and (4) Develop secondary assay strategies and methods for post- screening hit deconvolution and validation. In addition, we will validate this HTS technology by applying it for a full-fledged HTS campaign in which compounds will be identified and optimized that selectively inhibit the upstream Caspase-1 activator NLRC4 (Ipaf1; CLAN), a component of innate immunity and critical regulator of host responses to intracellular bacterial pathogens. PUBLIC HEALTH RELEVANCE: Proteases are proteins that cleave other proteins. These enzymes play important roles in many diseases. Consequently, proteases have emerged as promising targets for drug discovery, but it can often be challenging to obtain selective inhibitors. We propose to devise a novel technology for high- throughput screening of large collections of chemicals for identifying chemical modulators of the upstream activators of intracellular proteases. For proof of concept, we focus on proteases important for inflammatory and infectious diseases.

描述(申请人提供)：酵母型高温超导蛋白酶检测技术。蛋白质的蛋白质水解性加工是一种不可逆的翻译后修饰，对多种生物过程具有重要意义。因此，蛋白酶已成为治疗多种人类疾病，包括炎症、感染性疾病、神经退行性疾病、缺血性疾病和癌症的药物开发的有希望的靶点。使用纯化的蛋白酶开发高通量筛选(HTS)分析可能相对简单，也可能非常具有挑战性，特别是当需要多组分系统来实现蛋白酶激活时。此外，由于某些组蛋白水解酶活性部位的相似性，化学抑制剂的选择性即使不是不可能实现，也是困难的，这突显了有必要采用替代筛选方法来确定针对蛋白水解酶上游激活剂的化合物，而不是直接抑制所需的蛋白水解酶。我们建议以Caspase为原型，生成和优化细胞内蛋白水解酶的HTS系统。为此，我们设计了基于酵母的细胞系统，允许在这些简单的真核生物中以组合的方式方便地表达蛋白酶和蛋白水解酶激活蛋白，从而重建整个哺乳动物的通路。在酵母系统中集成的检测方法有可切割的报告基因激活剂，在这种方法中，蛋白酶介导的切割激活了转录因子。其目的是：(1)设计在酵母中重组哺乳动物蛋白酶激活途径的多组分系统；(2)调整必要的变量以实现HTS质量的检测性能；(3)对基于酵母的多组分蛋白酶检测系统进行中试化学文库筛选，以确定命中率和测试可靠性；以及(4)开发二次检测策略和方法，用于筛选后HIT去卷积和验证。此外，我们将通过将这项HTS技术应用于全面的HTS活动来验证这项技术，在HTS活动中，将识别和优化选择性抑制上游Caspase-1激活物NLRC4(Ipaf1；Clan)的化合物，NLRC4是先天性免疫的组成部分，也是宿主对细胞内细菌病原体反应的关键调节因子。与公共健康相关：蛋白水解酶是一种能分解其他蛋白质的蛋白质。这些酶在许多疾病中起着重要作用。因此，蛋白酶已成为药物发现的有希望的目标，但获得选择性抑制剂往往是具有挑战性的。我们建议设计一种新的技术，用于高通量筛选大量化学物质，以确定细胞内蛋白水解酶上游激活剂的化学调节剂。为了验证概念，我们将重点放在对炎症性疾病和感染性疾病至关重要的蛋白酶上。