A cell-based screen for inhibitors of intracellular Abeta aggregation

描述(申请人提供)：有一个新的共识，非纤维细胞内的Abeta聚集体，而不是不可溶的纤维，是最有害的Abeta物种，可能在阿尔茨海默病(AD)的发病机制中发挥核心作用。因此，治疗AD的一个有吸引力的方法是通过稳定细胞内单体形式的Abeta或破坏寡聚结构的稳定来选择性地降低潜在突触毒性Abeta聚集体的水平。低分子药物是最具吸引力的抑制Abeta聚集的治疗药物，因为许多小分子能够穿透血脑屏障(BBB)和穿过细胞膜。然而，从历史上看，蛋白质聚集一直是合成类药物分子处理的一个极其困难的目标，部分原因是两个相互作用的蛋白质通常覆盖了很大的表面积，以及蛋白质之间巨大而平坦的结合表面。另一个挑战是，尽管新型有机化合物在实验室中针对孤立的目标进行测试时可能非常有效，但它们可能会与所需目标以外的细胞成分发生交叉反应。自然界中发现的小分子，通常被称为“天然产物”，通常在进化过程中在细胞内停留了一段时间，不太可能以破坏细胞组件(如膜或DMA)的方式相互作用。此外，最近已经表明，许多天然产品在抑制多种蛋白质-蛋白质相互作用方面相当有效。因此，我们正在探索的一个重要问题是，能否分离出既能有效抑制Abeta聚集，又能被活细胞耐受的天然产物或天然产物类分子。这项研究的长期目标是确定具有治疗AD潜力的细胞内Abeta聚集的天然产物样抑制物。为了实现这一目标，我们建立了一种基于细胞的分析方法，用于直接监测细胞内环境中的Abeta折叠。这一特殊的应用旨在：(1)配置我们新颖的基于细胞的折叠实验，用于高通量筛选组合小分子文库；以及(2)从面向多样性的合成文库中分离能够拮抗Abeta聚集的天然产物类化合物。这些化合物将作为AD治疗的先导，并用于生物学研究，阐明Abeta折叠在介导神经毒性中的生理作用。

DESCRIPTION (provided by applicant): There is an emerging consensus that non-fibrillar intracellular Abeta aggregates, rather than insoluble fibrils, are the most deleterious Abeta species and may play a central role in Alzheimer's disease (AD) pathogenesis. Thus, an attractive therapeutic approach to AD would be to seletively reduce the levels of potentially synaptotoxic Abeta aggregates by either stabilizing intracellular Abeta in its monomeric form or destabilizing the oligomeric structure. Low molecular weight drugs represent the most attractive therapeutics for inhibiting Abeta aggregation as many small molecules are capable of permeating the blood-brain barrier (BBB) and crossing cell membranes. Historically, however, protein aggregation has been an extremely difficult target to address with synthetic drug-like molecules, owing in part to the large surface area generally covered by two interacting proteins and to the large, flat binding surfaces between the proteins. Another challenge is that while new types of organic compounds may be extremely potent when tested against isolated targets in the laboratory, they may cross-react with cellular components other than the desired target. Small molecules found in nature, often called 'natural products', typically have spent time inside of a cell during the course of evolution and are less likely to interact in a manner that damages cellular components such as membranes or DMA. In addition, it has been shown recently that many natural products are quite effective at inhibiting a diverse array of protein-protein interactions. Thus, an important question that we are exploring is whether natural products or natural product-like molecules can be isolated that effectively inhibit Abeta aggregation and, at the same time, be tolerated by living cells. The long-term goal of this research is to identify natural product-like inhibitors of intracellular Abeta aggregation that have potential as therapeutic agents for treating AD. Towards this goal, we have generated a cell-based assay for directly monitoring Abeta folding in the intracellular environment. This particular application seeks to: (1) configure our novel cell-based folding assay for high-throughput screening of combinatorial small-molecule libraries; and (2) isolate natural product-like compounds from diversity-oriented synthesis libraries that are capable of antagonizing Abeta aggregation. Such compounds will serve as leads for AD therapy and for biological studies that illuminate the physiological role of Abeta folding in mediating neurotoxicity.