Two Distinctive Binding Modes of Endonuclease Inhibitors to the N-Terminal Region of Influenza Virus Polymerase Acidic Subunit
Two Distinctive Binding Modes of Endonuclease Inhibitors to the N-Terminal Region of Influenza Virus Polymerase Acidic Subunit
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DOI：
10.1021/acs.biochem.5b01087
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发表时间：
2016-05-10
期刊：
影响因子：
2.9
通讯作者：
Hoshino, Tyuji
中科院分区：
文献类型：
--
作者：
Fudo, Satoshi;Yamamoto, Norio;Hoshino, Tyuji
流感病毒是对人类的全球性威胁，仍需要开发新的抗病毒药物来为大流行做准备，并克服对当前药物的新耐药性。流感聚合酶酸性蛋白N末端结构域(PA(N))具有核酸内切酶活性，是新型抗病毒药物的合适靶点之一。首先，我们对PA(N)与两种核酸内切酶抑制剂的络合物结构进行了X射线共晶分析。蛋白质结晶和缓蚀剂在pH为5.8的条件下浸泡。这两种抑制物的结合模式不同于以前报道的其他流感病毒核酸内切酶抑制物的共同结合模式。此外，我们还在pH为7.0时阐明了PA(N)与相同的两种核酸内切酶抑制剂的复杂结构。在其中一个晶体结构中，观察到一个额外的抑制剂分子，它与活性中心的两个金属离子发生螯合作用。在pH值为7.0的晶体结构的基础上，我们对这两个配合物进行了100 ns的分子动力学模拟。对模拟结果的分析表明，尽管模拟结构与晶体结构有部分偏离，但各缓蚀剂与PA(N)的结合模式是稳定的。此外，还对PA(N)与缓蚀剂的络合物进行了晶体结构分析和MD模拟，已有报道表明PA(N)具有较高的复配效力以供比较。在PA(N)底物结合口袋周围存在多个结合位点的发现将为提高抑制剂的结合亲和力提供线索。
Influenza viruses are global threat to humans, and the development of new antiviral agents are still demanded to prepare for pandemics and to overcome the emerging resistance to the current drugs. Influenza polymerase acidic protein N-terminal domain (PA(N)) has endonuclease activity and is one of the appropriate targets for novel antiviral agents. First, we performed X-ray cocrystal analysis on the complex structures of PA(N) with two endonuclease inhibitors. The protein crystallization and the inhibitor soaking were done at pH 5.8. The binding modes of the two inhibitors were different from a common binding mode previously reported for the other influenza virus endonuclease inhibitors. We additionally clarified the complex structures of PA(N) with the same two endonuclease inhibitors at pH 7.0. In one of the crystal structures, an additional inhibitor molecule, which chelated to the two metal ions in the active site, was observed. On the basis of the crystal structures at pH 7.0, we carried out 100 ns molecular dynamics (MD) simulations for both of the complexes. The analysis of simulation results suggested that the binding mode of each inhibitor to PA(N) was stable in spite of the partial deviation of the simulation structure from the crystal one. Furthermore, crystal structure analysis and MD simulation were performed for PA(N) in complex with an inhibitor, which was already reported to have a high compound potency for comparison. The findings on the presence of multiple binding sites at around the PA(N) substrate-binding pocket will provide a hint for enhancing the binding affinity of inhibitors.