寨卡病毒（Zika virus，ZIKV）属于黄病毒家族成员，主要传播方式为蚊虫传播、性传播和母婴传播。2015至2016年，ZIKV疫情在美洲大规模爆发，70多万人被感染，造成了大量成人神经系统免疫性疾病和孕妇感染导致的胎儿小头畸形病例。鉴于此次疫情的严重性，世界卫生组织宣布将其列为“国际关注的突发公共卫生事件”。迄今为止，暂时没有针对ZIKV感染的疫苗或药物批准上市。

ZIKV的遗传物质为单股正链RNA，RNA编码的多聚蛋白包含三个结构蛋白（E、C和pr M）和七个非结构蛋白（NS1、NS2A、NS2B、NS3、NS4A、NS4B和NS5），其中位于NS5的C端的RNA依赖的RNA聚合酶（RdRp）在病毒的复制和转录中发挥了重要作用，且人体中不含有类似结构，因此RdRp成为一个有潜力的抗ZIKV药物研究靶点。

本研究以ZIKV NS5 RdRp为靶点，利用LibDock对抗感染化合物库和天然产物库中的935个化合物进行虚拟筛选，根据LibDock得分选取排名靠前的11个化合物进行研究。依次通过酶活抑制实验、细胞毒性实验和亲和力实验对化合物进行检测，最终选取Posaconazole（POS）进行后续研究。体外检测结果显示，POS对RdRp具有良好的抑制活性，其IC₅₀值为4.29 μM，且没有明显的细胞毒性（CC₅₀ = 101 μM）。表面等离子共振结果表明POS能够直接与RdRp结合且亲和力较强（K_D = 2.76´10^-5 M）。利用分子对接和定点突变技术，分析得出化合物POS与RdRp蛋白作用的关键位点为第666位天冬氨酸。在抗ZIKV测定中发现，POS对ZIKV具有良好的抑制活性，其EC₅₀值为0.59 μM。

综上所述，本研究通过虚拟筛选技术和一系列生物学实验，得到ZIKV NS5 RdRp抑制剂 POS。进一步实验证明，POS通过与RdRp蛋白的第666位天冬氨酸结合而发挥抑制作用，同时具有良好的抗ZIKV活性，为抗ZIKV药物研究提供了先导化合物。此外，虚拟筛选技术与生物学实验相结合的方式可以节约筛选成本和时间，提高候选化合物的命中率，是抗感染药物研发中的重要手段。

Zika virus (ZIKV) is a member of the Flaviviridae family, transmitting through mosquitoes, sex intercourse or vertical transmission from mother to fetus. The pandemic of ZIKV in Americas infected more than 700,000 people between 2015 and 2016, leading to numerous cases of neuroimmune disease in adults and fetal microcephaly in pregnant women. Concerning a global health crisis, WHO declared ZIKV a Public Health Emergency of International Concern. There are currently no approved vaccines or drugs for ZIKV infection.

ZIKV is a single-stranded positive-sense RNA, encoding a polyprotein of three structural proteins (E, C and prM) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5). Since NS5 RdRp plays an important role in replication and transcription of ZIKV and is absent in human beings, it is proposed a potential drug target for anti-ZIKV agents.

In this work, we screened 935 compounds against RdRp via LibDcok from an anti-infection compound library and a natural product library, and 11 compounds showing the top 11 LibDock scores were selected. These were further tested by RdRp enzymatic inhibition, cytotoxicity evaluation and affinity analysis. Finally, we found that Posaconazole (POS) had a good inhibitory effect on RdRp with an IC₅₀ of 4.29 μM and no significant toxic effect with CC₅₀ = 101 μM. The results of surface plasmon resonance reaction showed that POS was able to bind directly to RdRp with good affinity (K_D = 2.76´10^-5 M). D666 was identified as the key amino in the reaction between POS and RdRp by molecular docking and surface plasmon resonance assay. Finally, POS showed good anti-ZIKV activity in antiviral assays with an EC₅₀ value of 0.59 μM.

In conclusion, POS, an inhibitor of ZIKV NS5 RdRp, was identified by virtual screening and biological assays. Further experiments revealed that POS inhibited RdRp activity by binding with the key amino acid D666 and exhibited potent anti-ZIKV activity. Our reaserch might provide the lead compound to anti-ZIKV drugs. Moreover, the combination of virtual screening and biological assays is an important tool in anti-infective drugs development, which can improve the hit rate of candidate compounds and save cost and time of screening.