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，称为药理学分子伴侣，作为治疗疾病如淀粉样蛋白病、囊性纤维化和癌症形式的治疗剂，具有很大的希望，这些疾病的病因起源于蛋白质错误折叠。寻找这种化合物的主要障碍是缺乏通用的高通量测定来鉴定稳定分子。缺乏适当的测定通常是不具有容易测定的表型的蛋白质不可逾越的障碍。这里描述的是三个独立的检测，可用于高通量筛选，以确定潜在的药理学伴侣的发展。其中两种检测方法是通用的：也就是说，它们被设计用于任何蛋白质，不需要了解蛋白质的功能。这些试验共同构成了一个独特的平台，不仅用于鉴定候选分子伴侣，而且用于剖析其作用模式。工作描述测试的能力，每个检测，首先检查功能与广泛的蛋白质的潜力，第二次进行化学筛选与模型错误折叠，疾病蛋白变体，最后检查在这些模型研究中获得的命中的生物学作用。重要的是，预计该测定是快速的，直接的，和可转移的，并将提供基本的方法，用于识别与各种蛋白质靶点相互作用的药理学分子伴侣。具体目标是：目标1。基于酵母的错误折叠蛋白质拯救分析的建立。(a)互补测定。在该测定中，人疾病蛋白补充酵母菌株，使得如果蛋白质是功能性的，则该菌株稳健地生长，但显示具有错误折叠变体的温度敏感性生长。该检测方法将在11，000种化合物的高通量筛选中使用人AGT进行开发和测试。(b)基于DHFR的蛋白质错误折叠传感器。在该测定中，将不稳定的蛋白质偶联到作为报告基因的必需酶二氢叶酸还原酶（DHFR）。在高通量筛选中，将用几种人蛋白质（包括AGT和1-半乳糖苷酶A）测试该方法的通用性，如（a）中所述。目标2.用于配体诱导的蛋白质稳定化的基于质谱的SUPREX测定的评价。该方法使用氢-氘交换和MALDI质谱法来报告配体诱导的稳定性变化。我们将评估该测定法用于使用疾病蛋白（包括AGT、1-半乳糖苷酶A和2-葡萄糖苷酶）鉴定药理学伴侣的有效性和一般性。将在高通量化学筛选中筛选疾病变体以稳定配体。目标3。评价命中与蛋白质错误折叠的相关性。将比较来自基于细胞和基于质谱的筛选的数据。将对基于细胞和质谱的筛选中常见的命中进行Kd值测量;将研究基于细胞的测定中独有的命中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导致许多人类疾病的原因，但很少有通用的检测方法可以有效地鉴定可以纠正缺陷蛋白质折叠的化合物或分子。在这个建议中，我们描述了三个独立的检测，可用于高通量筛选增强蛋白质折叠的因素的发展。由于这些测定法是稳健的、可推广的，并且可以扩展到任何错误折叠的目标蛋白质，因此预计它们将大大加速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.