Aβ聚集诱导发光体系构建及其在细胞内Aβ聚集抑制剂筛选中的应用

Aggregation of amyloid-β protein (Aβ) is the main etiology of Alzheimer’s disease (AD). Thus, the development of some effective inhibitors is the main strategy for the prevention and treatment of AD. In order to overcome the shortcomings (i.e., high false positive, the dramatic difference between in vitro and in vivo environments, etc.) of the existing screening methods, we proposed to construct the fusion system of Aβ modified with the fluorescent probes with aggregation induced emission properties, which was used to develop an in vivo platform for screening high-effective inhibitors against Aβ aggregation. Molecular simulations will be used to study the existing Aβ fibrils and find some residues which play a minor effect on Aβ aggregation. These sites would be selected to introduce the unnatural amino acids with bio-orthogonal functional groups based on the structural similarity. And then, Aβ variants were labeled with tailor-made fluorophores with aggregation induced emission properties through bio-orthogonal reactions. Thereafter, systematical simulation and experimental studies have been performed to study the aggregation properties of Aβ variants and finally obtain the Aβ-APE probes with similar aggregation properties. And then, the fusion systems of Aβ-APE probes will be expressed in E. Coli cells and obtained the cell-based platform for screening novel inhibitors of Aβ aggregation. The platform was validated by the known inhibitors of Aβ aggregation. The in vivo platform would be used to identify the novel efficient inhibitors. Finally, their inhibitory capacity would be explored using systematic dynamics, thermodynamics and biological experiments and molecular simulation analysis. Based on the above knowledge, the mechanism model of small molecular inhibitors was established. In this project, we would develop a novel high precise and efficient screening method of inhibitors of Aβ aggregation. Some high efficient inhibitors would be developed and used to treat AD. The research results would lay a scientific and technical foundation for the development of high efficient inhibitors against Aβ aggregation, which has some important theoretical significance and practical values.

淀粉质β蛋白（Aβ）聚集是引起阿尔茨海默病（AD）的主要原因，开发Aβ聚集抑制剂是防治AD的主要策略。针对现有筛选方法存在的假阳性高和体内外环境差异大等缺点，本项目提出构建Aβ聚集诱导发光体系并将该体系用于细胞内筛选Aβ聚集抑制剂。利用分子模拟探明Aβ纤维结构获得对Aβ聚集行为影响极小的残基，并基于结构相似性原则采用侧链含有生物正交反应基团的非天然氨基酸替换；利用生物正交反应将设计合成的聚集诱导发光分子连接到Aβ上获得Aβ聚集诱导发光体系；通过系统的分子模拟和实验方法筛选出对Aβ聚集性能无影响的体系；然后在大肠杆菌中表达、构建细胞内筛选系统，并利用已知抑制剂验证；最后利用该系统筛选Aβ聚集抑制剂，并通过系统的动力学、热力学和生物学实验和分子模拟分析，建立小分子抑制剂作用的机理模型。本项目成果可为高效Aβ聚集抑制剂的筛选和开发奠定科学和技术基础，具有重要理论意义和实际价值。

淀粉质β蛋白（Aβ）的错误折叠和聚集与阿尔茨海默病（AD）的发生发展密切相关。因此，抑制Aβ错误折叠和聚集是治疗AD的主要方法之一。本项目首先成功构建了体内和体外的聚集抑制剂筛选系统。基于聚集诱导发光（AIE）特性的淀粉样蛋白质聚集抑制剂的体外筛选系统是利用生物正交反应将AIE分子与淀粉样蛋白的位点特异性偶联来实现的。体内筛选系统是基于β-内酰胺酶抗菌性能构建获得了大肠杆菌体内Aβ42聚集抑制剂筛选体系。然后利用上述两种体内和体外筛选方法获得了7类小分子抑制剂，分别为托卡朋及其衍生物、巴西木素衍生物、二氢杨梅素、矢车菊素、固绿FCF、海洋天然产物fascaplysin及其衍生物和9-Methylfascaplysin；开发了多糖类抑制剂（石莼多糖、香菇多糖和松茸多糖等）以及羟基化和羧基化修饰的单壁碳纳米管抑制剂。.利用系统的体外ThT荧光、原子力显微镜和圆二色光谱等方法系统研究了上述抑制剂抑制Aβ聚集和解聚成熟纤维的能力；利用细胞实验进一步验证了它们能够显著降低Aβ聚集体的细胞毒性；利用线虫和小鼠实验进行了体内药效学评价：水迷宫实验和脑组织切片染色等结果发现上述抑制剂可显著改善AD模型鼠的空间记忆能力、减少脑中Aβ聚集体的数量和保护神经细胞。.利用全原子MD模拟探明了依达拉奉、GV971组分和5-hydroxycyclopenicillone抑制Aβ构象转换的分子机制。此外，还开发了帕金森病（PD）的致病蛋白质α-突触核蛋白的小分子抑制剂：巴西木素、VB12、二氢杨梅素、固绿FCF和四氢叶酸等。上述研究成果为深入研究Aβ及相关淀粉样蛋白质的聚集及其抑制理论，开发具有潜在临床价值的抑制剂奠定了坚实的基础。发表学术论文32篇，其中SCI收录论文25篇，申请中国发明专利7项，获授权专利7项。