Identification of pharmacological chaperones for misfolded proteins

描述(由申请人提供)：稳定蛋白质天然折叠的化合物，称为药理伴侣，在治疗淀粉样蛋白疾病、囊性纤维化和癌症等疾病方面前景广阔，这些疾病的病因源于蛋白质的错误折叠。寻找这类化合物的一个主要障碍是缺乏通用的高通量分析来鉴定稳定分子。对于那些表型不容易分析的蛋白质来说，缺乏合适的分析方法往往是一个不可逾越的障碍。这里描述了三种可用于高通量筛选以确定潜在药理伴侣的独立分析方法的发展。其中两种检测方法是通用的：也就是说，它们被设计成与任何蛋白质一起使用，不需要了解蛋白质的功能。这些分析共同构成了一个独特的平台，不仅用于确定候选伴侣，而且还用于剖析它们的行动模式。介绍了测试每种分析能力的工作，首先检查与各种蛋白质发挥作用的可能性，其次对模型错误折叠的疾病蛋白质变体进行化学筛选，最后检查在这些模型研究中获得的HITS的生物学作用。重要的是，预计这些分析将是快速、直接和可转移的，并将为识别与各种蛋白质靶标相互作用的药理伴侣提供基本方法。具体目标是：目的1.建立酵母为基础的错误折叠蛋白的检测方法。(A)互补试验。在这项试验中，人类疾病蛋白补充了酵母菌株，使菌株在蛋白质具有功能的情况下生长旺盛，但在错误折叠的变体中显示出温度敏感型生长。这种分析方法将被开发出来，并在11,000种化合物的高通量筛选中与人类AGT一起进行测试。(B)基于DHFR的蛋白质错误折叠传感器。在这个实验中，一种不稳定的蛋白质被偶联到必需的酶二氢叶酸还原酶(DHFR)上，它起到了报告的作用。这种方法的通用性将在高通量筛选中用包括AGT和1-半乳糖苷酶A在内的几种人类蛋白质进行测试，如(A)所述。目的2.评价一种基于质谱学的配基诱导蛋白质稳定分析方法。这种方法使用氢-氚交换和MALDI质谱学来报告配体诱导的稳定性变化。我们将使用包括AGT、1-半乳糖苷酶A和2-葡萄糖苷酶在内的疾病蛋白来评估该方法用于鉴定药理伴侣的有效性和通用性。疾病变异体将在高通量化学筛选中进行筛选，以稳定配体。目的3.评估HITS与蛋白质错误折叠的相关性。来自基于细胞的屏幕和基于质谱学的屏幕的数据将被比较。细胞和基于质谱学的筛选中常见的命中将接受Kd值测量；基于细胞的分析所独有的命中将被调查其对细胞内一般折叠和/或质量控制途径的潜在影响。错误折叠的蛋白质是许多人类疾病的罪魁祸首，但几乎没有通用的分析方法来有效地识别可能纠正蛋白质折叠缺陷的化合物或分子。在这项提案中，我们描述了三种独立的分析方法的发展，它们可以用于高通量筛选，以增强蛋白质折叠的因素。由于这些分析是可靠的、可推广的，并且可以扩展到任何感兴趣的错误折叠的蛋白质，它们有望极大地加快错误折叠疾病的治疗化合物的鉴定。

DESCRIPTION (provided by applicant): Compounds that stabilize a protein's native fold, termed pharmacological chaperones, hold great promise as therapeutics for treating diseases such as amyloid diseases, cystic fibrosis, and forms of cancer, that have etiological origins in protein misfolding. A major obstacle in the search for such compounds is the lack of generic high-throughput assays to identify stabilizing molecules. The lack of an appropriate assay is often an insurmountable obstacle for proteins that do not have easily assayable phenotypes. Described here is the development of three independent assays that can be used in high-throughput screens to identify potential pharmacological chaperones. Two of these assays are generic: that is, they are designed to be used with any protein and do not require knowledge of the protein's function. Together the assays constitute a unique platform not only for the identification of candidate chaperones, but also for dissecting their mode of action. Work is described to test the capabilities of each assay, first examining potential for functioning with a wide range of proteins, second carrying out chemical screens with model misfolded, disease protein variants, and finally examining the biological roles of hits obtained in these model studies. Importantly, the assays are expected to be rapid, straightforward, and transferable, and will provide basic methodologies for identifying pharmacological chaperones that interact with a wide variety of protein targets. The specific aims are: Aim 1. Establishment of yeast-based assays for rescue of misfolded proteins. (a) Complementation assay. In this assay, human disease protein complements a yeast strain such that the strain grows robustly if the protein is functional, but shows temperature-sensitive growth with a misfolded variant. The assay will be developed and tested with human AGT in high-throughput screens of 11,000 compounds. (b) DHFR-based protein misfolding sensor. In this assay, an unstable protein is coupled to the essential enzyme dihydrofolate reductase (DHFR), which acts as a reporter. The generality of this approach will be tested with several human proteins, including AGT and 1-galactosidase A, in high-throughout screening as in (a). Aim 2. Evaluation of a mass-spectrometry-based SUPREX assay for ligand-induced protein stabilization. This approach uses hydrogen-deuterium exchange and MALDI mass-spectrometry to report ligand-induced stability changes. We will assess the efficacy and generality of this assay for identification of pharmacological chaperones using disease proteins including AGT, 1-galactosidase A, and 2-glucosidase. Disease variants will be screened in high-throughput chemical screens for stabilizing ligands. Aim 3. Evaluation of relevance of hits to protein misfolding. Data from the cell-based and mass- spectrometry based screens will be compared. Hits that are common to the cell- and mass-spectrometry based screens will be subject to Kd value measurements; hits that are exclusive to the cell-based assays will be investigated for potential effects on general folding and/or quality control pathways within the cell. Misfolded proteins are responsible for many human diseases, yet few generic assays exist for efficiently identifying compounds or molecules that may correct defective protein folding. In this proposal, we describe the development of three independent assays that can be used in high-throughput screens for factors that enhance protein folding. As these assays are robust, generalizable, and can be extended to any misfolded protein of interest, they are expected to greatly accelerate identification of therapeutic compounds for misfolding disease.