天然小分子化合物抑制固有无序蛋白聚集的微观机理研究

Intrinsically disordered proteins (IDPs) do not have unique well-defined three-dimensional structures under normal physiological conditions. Aggregation of ordered proteins requires initial unfolding, while IDPs exist as dynamic ensembles of interconverting monomers, and they do not have to unfold prior to aggregation. Therefore, IDPs are particularly prone to aggregation which is usually associated with various amyloid diseases such as Alzheimer’s and Parkinson’s diseases. Researchers have made great efforts for many years to develop drugs against amyloid diseases. However, no effective drugs have been found so far. Recent experimental studies showed that short peptides, nanoparticles and small molecule natural products can interfere with the aggregation process of IDPs and reduce the amount of cytotoxic oligomers. Among them, small molecule natural compounds, such as green tea extract, have been received considerable attention due to their low toxicity, good druggability and easy modification. However, due to the complexity of small-molecule—protein interactions, the molecular mechanism underlying the toxic oligomer inhibition by small molecules is not well understood. In this proposal, we mainly employ all-atom molecular dynamics simulations, replica-exchange molecular dynamics simulations, Markov State Model, and binding free energy calculations, in combination with atomic force microscopy technique, to investigate the self- and cross-aggregation of IDPs in the absence and presence of several different small molecule natural compounds. By analyzing the influences of small molecules with different sizes and geometries on the structures and the free energy landscapes of protein aggregates and by calculating the binding free energies, we will identify the dominant binding sites and binding modes, and find out the dominant factors that inhibit the aggregation of IDPs. The main purpose of this proposal is to elucidate the inhibition mechanism of small molecules against the self- and cross-aggregation of IDPs. This study will provide theoretical guidance for the design of potential drug candidates against amyloidosis.

固有无序蛋白是一类在生理环境下无确定三维结构的蛋白质。此类蛋白因柔性大而比折叠蛋白更易发生病理性聚集并导致包括阿兹海默症和帕金森症等一系列淀粉样变性病。虽然人们长期致力于蛋白质聚集及其抑制的研究，但至今未找到有效的治疗药物。实验研究发现，短肽、纳米颗粒和天然小分子化合物能够有效抑制蛋白聚集并降低细胞毒性。其中，天然小分子因具有低毒、成药性好和易于修饰等特点而备受关注。然而，由于小分子与淀粉样蛋白相互作用的复杂性，其抑制蛋白质聚集的微观机理尚不清楚。本项目拟主要采用分子动力学模拟、副本交换分子动力学模拟、马可夫态模型和结合能计算等方法，并辅以原子力显微成像等实验手段，研究有小分子和无小分子存在情况下几种典型固有无序蛋白的聚集和交叉聚集，通过分析大小和结构不同的天然小分子对固有无序蛋白聚集体结构和自由能面的影响及二者的结合自由能，揭示小分子与固有无序蛋白作用的关键氨基酸位点和结合模式，找出小分子影响无序蛋白聚集的主要因素，进而从原子层次阐明天然小分子化合物抑制固有无序蛋白聚集和交叉聚集的微观机理，为药物设计提供结构基础和理论指导。

蛋白质聚集及其淀粉样沉淀与一系列淀粉样变性病（如：阿尔兹海默症、二型糖尿病和帕金森病）密切相关，固有无序蛋白比折叠蛋白更易发生病理性聚集并形成淀粉样纤维。但由于聚集体结构的多样性、低聚体的不稳定性和高度动态变化等原因，至今未找到有效的治疗药物。实验研究发现，天然小分子化合物能够有效抑制蛋白聚集和纤维化并降低细胞毒性。然而，由于天然小分子与淀粉样蛋白相互作用的复杂性，其抑制蛋白质聚集的微观机理尚不清楚。本项目主要采用分子动力学模拟、副本交换分子动力学模拟、和结合能计算等方法，主要开展了以下四个方面的工作。1）研究了固有无序蛋白--人胰岛淀粉样蛋白（hIAPP）和β-淀粉样蛋白（Aβ）的自聚集和共聚集及其低聚体构象分布和结构特征，发现Aβ和hIAPP的自聚集和共聚集均具有非常相似的分子识别位点和β-sheet区域；2）研究了无小分子情况下，结构不同的Aβ原纤维在磷脂双层膜表面的稳定性及其对生物膜的影响、tau蛋白不同组合的bi-repeat 原纤维（PHF）结构倾向性和肝素（heparin）分子对原纤维结构稳定性的影响。3）研究了天然小分子（如EGCG、多巴胺和去甲肾上腺素）对hIAPP、Aβ低聚体结构和聚集自由能面的影响，揭示了天然小分子抑制这些固有无序蛋白聚集的微观机理；4）研究了天然小分子（如EGCG、EGC、二氢查尔酮（DHC）、白藜芦醇、和多巴胺）对hIAPP、Aβ、和α-突触核蛋白（α-synuclein）原纤维结构或纤维核的影响，分析了不同小分子与同一纤维结构的作用机理，找出了关键氨基酸结合位点和结合模式，阐明了破坏纤维核的关键物理相互作用。研究结果从原子层次揭示了小分子抑制固有无序蛋白聚集和纤维化的微观物理机制，为淀粉样变性病相关药物的设计和开发提供理论参考和指导。