Kim Woojin, Kim Yunkyoung, Min Jaeki, Kim Dong Jin, Chang Young-Tae, Hecht Michael H
Department of Chemistry, Princeton University, Princeton, New Jersey 08544, USA.
ACS Chem Biol. 2006 Aug 22;1(7):461-9. doi: 10.1021/cb600135w.
Aggregation of the Alzheimer's peptide Abeta produces toxic multimeric species that play a key role in the development of Alzheimer's disease. Compounds that inhibit this aggregation may prove useful as therapeutic agents for the prevention or treatment of Alzheimer's disease. Although aggregation inhibitors may already exist in combinatorial libraries, finding these compounds in a cost-effective high-throughput manner poses an enormous challenge. To meet this challenge, we have developed a novel high-throughput screen capable of isolating inhibitors of Abeta aggregation from large libraries of inactive candidates. The screen uses a fusion of Abeta42 to GFP. In the absence of inhibition, the rapid misfolding and aggregation of Abeta42 causes the entire fusion protein to misfold, thereby preventing fluorescence. Compounds that inhibit Abeta42 aggregation enable GFP to fold into its native structure and be identified by the resulting fluorescent signal. By implementing the screen on a pilot library of triazine derivatives, we have identified several putative inhibitors. One of the selected compounds was studied in detail by a series of biochemical and biophysical methods. These studies confirmed that the selected compound inhibits aggregation of synthetic Abeta42 peptide. The fluorescence-based method described here is rapid and inexpensive and can be used to screen large libraries for inhibitors of Abeta42 aggregation and/or amyloidogenesis.
阿尔茨海默病肽β-淀粉样蛋白（Aβ）的聚集会产生有毒的多聚体，这些多聚体在阿尔茨海默病的发展中起关键作用。抑制这种聚集的化合物可能被证明是预防或治疗阿尔茨海默病的有用治疗剂。尽管组合文库中可能已经存在聚集抑制剂，但以具有成本效益的高通量方式找到这些化合物面临着巨大挑战。为了应对这一挑战，我们开发了一种新型高通量筛选方法，能够从大量无活性候选物文库中分离出Aβ聚集抑制剂。该筛选方法使用Aβ42与绿色荧光蛋白（GFP）的融合蛋白。在没有抑制作用的情况下，Aβ42的快速错误折叠和聚集会导致整个融合蛋白错误折叠，从而阻止荧光产生。抑制Aβ42聚集的化合物能使GFP折叠成其天然结构，并通过产生的荧光信号进行鉴定。通过在三嗪衍生物的先导文库上实施该筛选，我们鉴定出了几种假定的抑制剂。其中一种选定的化合物通过一系列生化和生物物理方法进行了详细研究。这些研究证实，选定的化合物能抑制合成Aβ42肽的聚集。这里描述的基于荧光的方法快速且廉价，可用于筛选大型文库以寻找Aβ42聚集和/或淀粉样蛋白生成的抑制剂。
