Yeast-based HTS Assay Technologies for Proteases

描述（由申请人提供）：基于酵母的蛋白酶HTS测定技术。蛋白质的蛋白水解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，蛋白酶已经成为用于多种人类疾病（包括炎症、感染性疾病、神经变性、缺血性疾病和癌症）的药物发现的有希望的靶标。使用纯化的蛋白酶开发高通量筛选（HTS）测定可以是相对简单的，或者它可以是相当具有挑战性的，特别是当需要多组分系统来实现蛋白酶活化时。此外，由于某些蛋白酶组的活性位点的相似性，化学抑制剂的选择性如果不是不可能实现的话也是困难的，这突出了对用于鉴定靶向蛋白酶的上游活化剂而不是直接抑制感兴趣的蛋白酶的化合物的替代筛选方法的需要。我们建议使用半胱天冬酶作为原型，生成和优化细胞内蛋白酶的HTS系统。为此目的，我们设计了基于酵母的细胞系统，允许蛋白酶和蛋白酶激活蛋白的组合，在这些简单的真核生物中重建整个哺乳动物的途径容易表达。整合到酵母系统中的测定方法是可裂解的报告基因激活剂，其中蛋白酶介导的裂解激活转录因子。其目的是：（1）设计在酵母中重建哺乳动物蛋白酶活化途径的多组分系统;（2）调整必要的变量以实现HTS质量测定性能;（3）进行基于多组分酵母的蛋白酶测定系统的中试化学文库筛选以定义命中率和测试可靠性;以及（4）开发用于筛选后命中解卷积和验证的次级测定策略和方法。此外，我们将通过将其应用于全面的HTS活动来验证这种HTS技术，其中将鉴定和优化选择性抑制上游Caspase-1激活剂NLRC 4（Ipaf 1; CLAN）的化合物，NLRC 4是先天免疫的一个组成部分，也是宿主对细胞内细菌病原体反应的关键调节剂。公共卫生相关性：蛋白酶是切割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，蛋白酶已成为药物发现的有希望的靶点，但获得选择性抑制剂通常具有挑战性。我们建议设计一种新的技术，用于高通量筛选大量的化学品，以鉴定细胞内蛋白酶上游激活剂的化学调节剂。为了证明概念，我们专注于对炎症和感染性疾病重要的蛋白酶。

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.