Virologie et Immunologie Moléculaires, UR892, Institut National de la Recherche Agronomique, Domaine de Vilvert, 78350 Jouy en Josas, France.
Antimicrob Agents Chemother. 2013 May;57(5):2231-42. doi: 10.1128/AAC.02335-12. Epub 2013 Mar 4.
The nucleoprotein (NP) binds the viral RNA genome and associates with the polymerase in a ribonucleoprotein complex (RNP) required for transcription and replication of influenza A virus. NP has no cellular counterpart, and the NP sequence is highly conserved, which led to considering NP a hot target in the search for antivirals. We report here that monomeric nucleoprotein can be inhibited by a small molecule binding in its RNA binding groove, resulting in a novel antiviral against influenza A virus. We identified naproxen, an anti-inflammatory drug that targeted the nucleoprotein to inhibit NP-RNA association required for NP function, by virtual screening. Further docking and molecular dynamics (MD) simulations identified in the RNA groove two NP-naproxen complexes of similar levels of interaction energy. The predicted naproxen binding sites were tested using the Y148A, R152A, R355A, and R361A proteins carrying single-point mutations. Surface plasmon resonance, fluorescence, and other in vitro experiments supported the notion that naproxen binds at a site identified by MD simulations and showed that naproxen competed with RNA binding to wild-type (WT) NP and protected active monomers of the nucleoprotein against proteolytic cleavage. Naproxen protected Madin-Darby canine kidney (MDCK) cells against viral challenges with the H1N1 and H3N2 viral strains and was much more effective than other cyclooxygenase inhibitors in decreasing viral titers of MDCK cells. In a mouse model of intranasal infection, naproxen treatment decreased the viral titers in mice lungs. In conclusion, naproxen is a promising lead compound for novel antivirals against influenza A virus that targets the nucleoprotein in its RNA binding groove.
核蛋白（NP）结合病毒 RNA 基因组，并与聚合酶形成核糖核蛋白复合物（RNP），该复合物是流感 A 病毒转录和复制所必需的。NP 没有细胞对应物，其序列高度保守，这使得 NP 成为抗病毒药物研究的热门靶点。我们在此报告，单体核蛋白可以被小分子结合到其 RNA 结合槽中而被抑制，从而产生针对流感 A 病毒的新型抗病毒药物。我们通过虚拟筛选发现，一种抗炎药萘普生（naproxen）靶向核蛋白，抑制 NP 功能所需的 NP-RNA 结合，从而抑制了核蛋白。进一步的对接和分子动力学（MD）模拟确定，在 RNA 槽中存在两种相互作用能量相似的 NP-萘普生复合物。使用携带单点突变的 Y148A、R152A、R355A 和 R361A 蛋白，对预测的萘普生结合位点进行了测试。表面等离子体共振、荧光和其他体外实验支持了萘普生结合 MD 模拟确定的结合位点的观点，并表明萘普生与 RNA 竞争结合野生型（WT）NP，并保护核蛋白的活性单体免受蛋白水解切割。萘普生保护 Madin-Darby 犬肾（MDCK）细胞免受 H1N1 和 H3N2 病毒株的病毒攻击，并且在降低 MDCK 细胞的病毒滴度方面比其他环氧化酶抑制剂更有效。在鼻内感染的小鼠模型中，萘普生治疗降低了小鼠肺部的病毒滴度。总之，萘普生是一种有前途的新型抗流感 A 病毒先导化合物，它靶向核蛋白的 RNA 结合槽。
