A High Throughput Screen for Inhibitors of Aggregation of the Alzheimer's Peptide

描述(申请人提供)：一系列生化和遗传学研究表明，阿尔茨海默氏症多肽A？的聚集在阿尔茨海默病(AD)的分子病因学中起着至关重要的作用。特别是，错误折叠和寡聚化的早期步骤被认为会产生有毒物种，从而引发一系列事件，最终导致神经退化。抑制A？的错误折叠和寡聚可能为AD的治疗提供一种治疗策略；然而，寻找能够在不干扰其他蛋白质折叠的情况下具体实现这一目标的化合物仍然是一个重大挑战。为了克服这一挑战，我们建议开发一种快速、可靠和高通量的筛选A？错误折叠和聚集的特定抑制剂。这一目标将通过以下具体目标实现： (1)构建和优化一种新的Aβ聚集抑制物筛选方法。拟议的筛查将使用A？42与绿色荧光蛋白(GFP)的融合。当与GFP融合时，AB42会导致整个融合蛋白错误折叠和聚集，从而阻止蛋白质的GFP部分的荧光。抑制A？聚集的化合物将使GFP折叠成其自然结构，并将通过产生的荧光信号进行识别。 (2)实施筛选，并测试其区分聚集抑制物和非活性化合物的能力。这将使用荧光来筛选化合物的中试文库来进行演示。 (3)用生化和生物物理方法对筛选结果进行验证。这些研究将证实，通过A？42-GFP筛选分离的化合物增强了纯A？42的溶解性并抑制了聚集。 (4)探索构效关系(SAR)。通过筛选分离的化合物将与文库中的相关化合物进行比较。这些研究将阐明导致抑制的化学特征，并将有助于未来的铅优化。 (5)调整屏幕以用于基于细胞或无细胞的表达系统。完成这些目标将产生一种快速筛选新的和特定的聚集抑制物的方法，从而确定用于开发预防或延缓阿尔茨海默病发病的新药的先导化合物。

DESCRIPTION (provided by applicant): A range of biochemical and genetic studies indicate that aggregation of the Alzheimer's peptide, A¿, plays a crucial role in the molecular etiology of Alzheimer's disease (AD). In particular, early steps of misfolding and oligomerization are thought to produce toxic species, which initiate a cascade of events ultimately leading to neurodegeneration. Inhibition of A¿ misfolding and oligomerization may provide a therapeutic strategy for the treatment of AD; however, finding compounds that specifically accomplish this goal without interfering with the folding of other proteins has remained a major challenge. To overcome this challenge, we propose to develop a rapid, reliable, and high throughput screen for specific inhibitors of A¿ misfolding and aggregation. This goal will be achieved thorough the following specific aims: (1) To construct and optimize a novel screen for inhibitors of A¿ aggregation. The proposed screen will use a fusion of A¿42 to Green Fluorescent Protein (GFP). When fused to GFP, AB42 causes the entire fusion protein to misfold and aggregate, thereby preventing the fluorescence of the GFP portion of the protein. Compounds that inhibit A¿ aggregation will enable GFP to fold into its native structure, and will be identified by the resulting fluorescent signal. (2) To implement the screen and test its ability to distinguish inhibitors of aggregation from inactive compounds. This will be demonstrated using fluorescence to screen a pilot library of compounds. (3) To validate the screen using biochemical and biophysical methods. These studies will verify that compounds isolated by the A¿42-GFP screen enhance solubility and inhibit aggregation of pure A¿42. (4) To probe structure-activity relationships (SAR). Compounds isolated by the screen will be compared to related compounds in the libraries. These studies will elucidate the chemical features responsible for inhibition and will facilitate future lead optimization. (5) To adapt the screen for use in either cell-based or cell-free expression systems. Completion of these aims will produce a rapid method of screening for novel and specific inhibitors of aggregation, thereby identifying lead compounds for the development of new pharmaceuticals that prevent or delay the onset of Alzheimer's disease.