抗金属离子诱导Aβ聚集的多功能抑制剂设计和作用的基础研究

Aggregation of amyloid-β protein (Aβ) into various kinds of cytotoxic aggregates is induced by metal ions. And the interactions between Aβ and metal ions leads to the generation of reactive oxygen species (ROS). Both cytotoxic aggregates and ROS are closely related to both the occurrence and development of Alzheimer's disease (AD). However, the current study of small molecular inhibitors has ignored the effect of metal ions on the Aβ aggregation, which seriously hinders the development of effective inhibitors. Based on the complex pathogenesis of AD, the synergistic effects of multiple factors with close relationship are considered and used to rational design multifunctional inhibitors, which may bring to better therapeutic effect. In this project, molecular dynamics (MD) simulations are first used to study the interactions between the known inhibitors and Aβ and then their affinity binding models are built. Combined with the existing models of both metal ion chelating and anti-oxidant agents, the multi-functional interacting model is also constructed, which is used to screen the existing library to obtain many candicate compounds. Then, the multi-stage molecular simulation method is used to screen the high-efficiency multi-functional inhibitors against ions-induced Aβ aggregation. And many studies are performed to explore in detail the inhibitory effect of the selected inhibitors against the Aβ aggregation kinetics and thermodynamic properties in the presence of metal ions, which are used to optimize the structure of the multifunctional inhibitors, and construct a novel more-efficiency multi-functional inhibition model. This study can open up a new and effective way to solve the problem of the multi-mechanism and multi-factor characteristics of the AD caused by Aβ aggregation induced by metal ions. Therefore, this project plays an important role in the molecular mechanism of AD and the finding of high efficiency inhibitors of AD.

金属离子能够加速淀粉质β蛋白质（Aβ）聚集并诱导氧化应激的产生，这与阿尔茨海默病（AD）发生发展密切相关。但目前抑制剂的开发均忽略了金属离子对Aβ聚集的影响，严重阻碍了高效AD抑制剂的研发。考虑到AD的复杂作用机制，将密切相关的多因素综合考虑并基于此设计多功能抑制剂应该会起到更好的治疗效果。针对此问题，本项目拟利用MD模拟研究现有典型Aβ抑制剂与Aβ间的相互作用，构建亲和结合作用模型；然后结合现有金属离子螯合剂和抗氧化剂的作用模式构建多功能抑制剂的作用模型；利用多级分子模拟方法从现有数据库中筛选获得高效的多功能抑制剂，并通过系统的动力学、热力学和生物学实验研究它们对金属离子诱导Aβ聚集的抑制效果，并基于此优化多功能抑制剂的作用模型，为高效Aβ聚集抑制剂的开发奠定基础。本项目的实施可对探明金属离子诱导Aβ聚集所引发AD发病机理和研发治疗AD的多功能药物具有重要理论意义和实际应用价值。

金属离子诱导淀粉样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，抑制金属离子诱导的Aβ错误折叠和聚集是治疗AD的主要方法之一。本项目首先利用MD模拟方法研究了现有典型抑制剂GV971（已在中国获批有条件上市）组分、石莼多糖组分和海洋化合物5-羟基环青霉酮抑制Aβ单体的构象转换，探明了它们抑制Aβ构象转换的分子机制和作用力类型。此外，还利用蛋白质-蛋白质分子对接获得了4种Aβ五聚体和Tau五聚体的杂化寡聚体构象。并利用MD模拟探明了Aβ和Tau交叉做种的分子机制。基于上述分子模拟结果获得了Aβ亲和结合模型。结合金属离子螯合剂和抗氧化剂的作用模型，改造获得4种能够抑制金属离子诱导Aβ聚集的巴西木素衍生物。此外还开发了3种小分子多功能抑制剂，分别为托卡朋衍生物、固绿FCF、矢车菊素；获得了多糖类抑制剂石莼多糖和羧基化碳纳米管抑制剂。利用系统的体外硫磺素T荧光、原子力显微镜、圆二色光谱、点印迹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、保护神经细胞。上述研究成果为深入研究金属离子诱导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收录论文9篇，申请中国发明专利2项。