淀粉质β多肽聚集的亲和纳米抑制剂的设计和作用机理研究

The aggregation and fibrillation of amyloid β-peptide (Aβ) are the main cause of Alzheimer's disease. This project is proposed to develop novel inhibitors for the aggregation and fibrillation of Aβ and to study the working mechanisms of the inhibitors. First of all, molecular interactions in the affinity complex of Aβ-ZAβ3 will be investigated by molecular dynamics (MD) simulations to find the key amino acid residues (hot spots) in ZAβ3 and their distribution. Based on the distribution of the hot spots, an affinity molecular model will be constructed, leading to the formation of a biomimetic short peptide library. Then, a multi-stage MD simulation strategy is established to efficiently screen the peptides and determine the high-ranking peptide ligands for further experimental identification. From the high-ranking peptides determined by MD simulations, experiments of binding thermodynamics will be carried out to determine a few high-affinity peptide ligands. The affinity ligands are coupled to biocompatible nanoparticles to construct affinity nanoparticles. By investigating the thermodynamic interactions between the affinity nanoparticles and Aβ as well as the inhibition kinetics on Aβ aggregation, the affinity nano-inhibitors can be optimized. Then cell toxicity assay and dot blot assay will be performed to study the detoxication effects of the nano-inhibitors and their binding characteristics with Aβ. Finally, MD simulations of the interactions between Aβ and the nano-inhibitor surface will be conducted to elucidate the molecular details involved in the inhibition. Based on the comprehensive kinetics, thermodynamics, biological assays and MD simulations, a mechanistic model for the inhibition effects of the nano-inhibitors on Aβ aggregation can be proposed. Therefore, this project is of importance because the outcomes of this project will form the scientific and technological bases for the further design and development of Aβ aggregation inhibitors.

淀粉质β多肽（Aβ）的聚集和纤维化是阿尔茨海默症形成的重要过程。本项目旨在建立新型Aβ聚集抑制的设计方法并研究其作用机理。首先研究Aβ与其多肽抑制剂ZAβ3的亲和结合作用，发现ZAβ3分子中的关键氨基酸残基和分布规律，构建Aβ的亲和结合分子模型和仿生短肽配基库，进而建立多级分子模拟筛选方法，获得备选短肽配基；然后利用热力学实验分析，鉴定和发现高选择性短肽配基，构筑生物相容性亲和纳米粒子；研究亲和纳米粒子与Aβ作用的热力学特性和抑制Aβ聚集的动力学规律，优化设计亲和纳米抑制剂；进行细胞毒性实验和抗体斑点印迹实验，研究纳米抑制剂对Aβ聚集的解毒作用和Aβ结合特性；最后开展Aβ与亲和纳米抑制剂表面作用的分子模拟研究。通过系统的动力学、热力学和生物学实验研究以及分子模拟分析，建立亲和纳米抑制剂作用的机理模型。本项目成果可为新型纳米抑制剂的设计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。

淀粉样-β多肽（Aβ）聚集形成的寡聚体和纤维具有神经毒性，是阿尔兹海默症（AD）的主要病理特征。因此，抑制Aβ聚集是AD治疗的主要策略之一。本项目提出设计针对Aβ聚集的亲和纳米抑制剂的研究构想，取得了重要研究成果。（1）设计了一条新型亲和多肽序列（Ac-LVFFARK-NH2，简称LK7），将其键合到聚乳酸-羟基乙酸（PLGA）纳米粒子表面，构建了纳米抑制剂LK7@PLGA。LK7@PLGA有效消除了LK7的自聚现象，使LK7对Aβ的亲和性得到充分发挥，抑制功效显著增强，等效抑制浓度仅相当于游离肽的1/69。基于LK7序列，进一步扩展设计3条新肽，显示了包括金属离子螯合作用在内的多功能抑制作用；（2）提出利用不同抑制剂协同作用的理论，设计了两种肽抑制剂VVIA和LPFFD的嵌合肽（VVIACLPFFD，简称VCD10），将其键合到金纳米粒子表面，获得的VCD10@AuNP可在极低浓度下（40 nmol/L）显著降低Aβ的细胞毒性，使细胞存活率提高70%，等效抑制浓度低于游离肽的1/800；（3）合成了3种纳米抑制剂，通过调控纳米粒子表面疏水基团和负电荷基团，或者修饰小分子抑制剂，获得了新型作用功能的纳米抑制剂，包括具有抑制聚集和螯合金属离子双重功效的双功能抑制剂；（4）基于血清白蛋白表面修饰，开发了酸酐酸化的白蛋白（A-HSA）和亚氨基二乙酸修饰的IDA-HSA，两种蛋白类抑制剂均具有包括高容量金属离子螯合作用在内的多功能性。基于A-HSA抑制作用的解析，提出了“疏水结合-静电排斥作用”理论；（5）从小分子库虚拟筛选获得了天然小分子抑制剂巴西木素和苏木精，具有抑制Aβ聚集和解聚Aβ纤维的作用。系统研究解析了上述各抑制剂作用的机理，研究成果为深入研究Aβ聚集及其抑制理论，开发具有临床价值的抑制剂奠定了基础。已发表SCI收录学术论文20篇，申请国家发明专利7项。