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: 8033736
• 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/8033736
• 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（由申请人提供）：基于酵母的HTS分析技术。蛋白质的蛋白水解处理是对广泛生物过程的重要性的不可逆后翻译后修饰。因此，蛋白酶已成为各种人类疾病的药物发现的有希望的靶标，包括炎症，传染病，神经退行性疾病，缺血性疾病和癌症。使用纯化的蛋白酶开发高吞吐量筛选（HTS）测定可能相对简单，也可能非常具有挑战性，尤其是当需要多组分系统以实现蛋白酶激活时。同样，由于某些蛋白酶的活性位点的相似性，如果无法实现，化学抑制剂的选择性可能很难，强调了需要鉴定靶向蛋白酶上游激活剂而不是直接抑制蛋白酶的替代筛查方法的需求。我们建议使用caspases作为原型生成和优化细胞内蛋白酶的HTS系统。为此，我们设计了基于酵母的细胞系统，可以在这些简单的真核生物中重新构造整个哺乳动物途径的组合中易于表达蛋白酶和蛋白酶激活的蛋白质。在集成到酵母系统中的测定方法中，有可裂解的报告基因激活剂，其中蛋白酶介导的裂解激活了转录因子。目的是：（1）设计多组分系统，以重建酵母中的哺乳动物蛋白酶激活途径； （2）调整必要的变量以实现HTS质量测定性能； （3）执行基于酵母菌的蛋白酶测定系统的Pilot化学库筛选，以定义命中率和测试可靠性； （4）制定次要测定策略和方法，用于筛选后击中反卷积和验证。此外，我们将通过将其应用于成熟的HTS广告系列来验证该HTS技术，在该广告系列中，将确定和优化化合物，以选择性地抑制上游CASPASE-1激活剂NLRC4（IPAF1； ClAN），这是天生免疫力和宿主对宿主对内细胞内细胞元元元元元元元合基的宿主免疫和关键调节剂的组成部分。公共卫生相关性：蛋白酶是裂解其他蛋白质的蛋白质。这些酶在许多疾病中起重要作用。因此，蛋白酶已成为有希望的药物发现靶标，但是获得选择性抑制剂通常可能具有挑战性。我们建议设计一种新型技术，用于对大量化学物质的高吞吐量筛查，以鉴定细胞内蛋白酶上游活化剂的化学调节剂。为了获得概念证明，我们专注于对炎症和传染病重要的蛋白酶。