细胞膜表面小分子抑制淀粉样β多肽构象转换和聚集的多尺度研究

Alzheimer’s disease (AD) is caused by the aggregation of amyloid-β protein (Aβ). The complex aggregation process were not only affected by the inter- and intra-interactions of Aβ but also influenced greatly by the membrane, which can significantly hinders the development of the inhibitors of the aggregation of Aβ. In order to solve this important problem, molecular simulation and experimental methods are coupled to investigate the molecular mechanism of conformational transition and aggregation of Aβ inhibited by the small molecular inhibitors on the surface of membrane using three levels (e.g., atomic, molecular and coarse-grain level). And, a rational design method for high efficient inhibitors is built. Firstly, molecular mechanisms of conformational transition and aggregation of Aβ on the membrane inhibited by the inhibitors were probed using the above three levels. The molecular interactions among Aβ, inhibitors and membrane were probed. Further, the aggregation process of Aβ on the living cells was explored using the real-time visualization single molecular experiments. The important factors of inhibitors on the aggregation of Aβ on the surface of living cells were explored. The study will be basement on the rational design of the high-efficiency inhibitors of Aβ aggregation. Therefore, this project plays an important role in the molecular mechanism of AD and the finding of high-efficiency inhibitors of AD.

淀粉质β多肽（Aβ）聚集与阿尔茨海默病（AD）发生发展密切相关。Aβ聚集过程非常复杂，除了与Aβ分子内和分子间相互作用之外还涉及细胞膜的影响。但目前小分子抑制剂的开发均忽略了细胞膜对抑制剂抑制Aβ聚集的影响，严重阻碍高效AD抑制剂的研发。针对此问题，本项目提出将分子模拟和实验检测相结合，从原子、分子和细胞尺度系统研究细胞膜表面小分子抑制Aβ构象转换和聚集的分子机制，建立抑制剂的理性设计方法。首先利用分子模拟从原子、分子和粗粒化水平研究细胞膜表面小分子抑制Aβ构象转换和聚集的分子机制，探讨细胞膜、抑制剂和Aβ三者间相互作用关系和作用机理；并进一步利用单分子研究方法原位、实时检测小分子抑制活细胞表面Aβ聚集的动态过程，明确抑制剂抑制细胞膜表面Aβ聚集的关键因素，为细胞膜表面Aβ聚集高效抑制剂的开发奠定基础。该课题的开展对认识AD发病机理和开发高效防治药物具有重要理论意义和实际应用

淀粉样β-蛋白质（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。本项目选择进入III期临床研究的茶多酚EGCG分子，首先结合硫磺素T（ThT）荧光和原子力显微镜（AFM）实验系统研究了膜表面EGCG抑制Aβ聚集的效果。结果表明，EGCG能够抑制脂质体POPC、DOPC和POPE表面Aβ40的聚集并能降低其细胞毒性。为了进一步贴近AD的病理环境，研究了红细胞膜表面EGCG抑制Aβ聚集的效果。结果表明，EGCG同样能够抑制红细胞膜表面Aβ40的纤维化过程，并能减低其细胞毒性。在此基础上筛选获得了8种Aβ聚集的小分子抑制剂，分别为二氢杨梅素、矢车菊素、苏木精、巴西木素、他克林二聚体B3C、岩藻黄素、5-羟基环青霉酮和9-Methylfascaplysin。此外，还开发了帕金森病（PD）的致病蛋白质α-突触核蛋白(α-syn)的抑制剂：巴西木素、VB12和二氢杨梅素。考虑到纳米抑制剂具有大的比表面积、易于透过血脑屏障等优点，本项目也设计获得了羟基、氨基和羧基修饰的单壁碳纳米管，并系统研究了它们对Aβ聚集的抑制效果。利用全原子MD模拟探明了依达拉奉抑制Aβ42构象转换的分子机制，并结合MD模拟和MM-PBSA分析方法解析了ZAβ3和Aβ16-40之间亲和作用的分子机制。利用MD模拟研究了Y220C突变体对p53C蛋白质构象转换的分子机制，并基于此筛选获得了小分子稳定剂；建立了利用大肠杆菌来生物表达淀粉样蛋白质的方法，开发了在C端偶联His6标签的Aβ42的生物表达和纯化技术，并利用麦芽糖结合蛋白为融合标签实现了Aβ42的可溶性表达和纯化；实现了α-syn的高效表达和纯化，并基于此构建了基于β-内酰胺酶抗药性的Aβ42聚集抑制剂的筛选体系。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。已发表SCI收录学术论文18篇，申请国家发明专利15项。