流感病毒神经氨酸酶NA与Ca2+的互作机制及药物靶点研究

Influenza is an infectious disease caused by influenza viruses, and its periodic outbreak posts a great threat to human life. Moreover, novel drug resistant viral strains accumulated due to antigenic drift and antigenic shift. It is necessary to develop and produce more antiviral drugs. Recently, a new motif called 340-cavity was discovered in two subtypes of influenza neuraminidase (NA) at the calcium binding site. Besides, this cavity is closed to the active site of NA. On one hand, NA is one of the most promising influenza drug targets on account of its conserved structure and crucial function in viral life cycle. On the other hand, the activity and thermostability of NA is known to depend on the presence of the calcium ions. The aim of this project is to detect the potential of the 340-cavity being a drug targeting site through exploring the detailed interacting mechanism between the calcium and NA. Firstly, the mechanism of forming a 340-cavity will be explored by simulating all subtypes of NA. Secondly, hotspot in the 340-cavity area will be discovered by ligand binding site prediction and calcium binding pattern exploration. Finally, high-throughput ligand screening, verification through simulations and experiments will be carried out in order to provide promising ligand which can tightly binds in the 340-cavity and influences normal function of NA. Based on above plan, our study will provide new insight in influenza drug development.

由流感病毒引起的流行性感冒作为一种周期性爆发的传染性疾病一直影响着人类的生命财产安全。同时，流感病毒经过抗原漂移和转移不断积累产生新的耐药毒株。因此，抗病毒药物的持续研发极其必要。近期，研究发现在两种流感病毒神经氨酸酶（NA）的Ca2+结合位点处存在区别于其它亚型的新的空腔，340-腔，且该区域紧邻NA活性位点。NA以其保守的结构和在病毒生命周期中的重要功能在新药研发中最被重视，而Ca2+在维持NA活性和热稳定性上也不可或缺。本项目计划以研究Ca2+与NA的互作机制为突破口，通过模拟计算探索340-腔出现的根本原因，并用探针预测法确认340-腔区域内热点氨基酸，最后利用分子对接、分子模拟、体外抗病毒药物筛选实验等手段，筛选出能够稳定结合于340-腔、通过影响Ca2+的功能从而影响天然底物靶定NA过程的配体分子抑制剂，探索340-腔作为流感病毒药物靶点的潜能，为药物研发提供新思路。

流感病毒神经氨酸酶（NA）以其保守的结构和在病毒生命周期中的重要功能在新药研发中最被重视。结晶结构解析发现N6和N7亚型存在区别于其它亚型的新的空腔，340-腔，这一结构的发现为药物设计指出了新方向。本项目利用分子动力学模拟和计算机辅助药物设计等手段针对340-腔进行结构和功能上的探索：（1）首次揭示了340-腔在NA各亚型中形成的可能性，并以340-腔为标志结构将NA各亚型划分为三类；（2）探索了340-腔的形成机制，发现其与第344位氨基酸的侧链伸展方向相关；（3）dPCA分析发现保守的Ca2+对340-环的稳定性具有重要影响，间接证明了340-环/腔对NA蛋白活性及稳定性的重要作用；（4）刻画了天然底物SA靶定到NA蛋白底物结合腔的路径，发现340-环/腔恰好位于SA的结合路径上，这一结论为340-腔能够作为药物靶点进行新药研发提供了强有力的理论支撑。此外，我们通过分子对接、计算模拟筛选以及底物结合能量计算，提供了一款能够稳定结合于340-腔并使天然底物SA脱靶的配体药物（Chembridge ID：6432010），并通过实验证明该药物能够抑制流感病毒H1N1毒株诱导的细胞病变作用。由流感病毒引起的流行性感冒作为一种周期性爆发的传染性疾病严重影响着人类的生命财产安全，本项目的一系列理论计算和实验结果对人类健康具有重大意义，所提供的理论机制和筛选的配体药物具有深远的应用前景。