Antiviral targeting of virus envelope proteins is an effective strategy for therapeutic intervention of viral infections. Here, we took a computer-guided approach with the aim of identifying new antivirals against the envelope protein E2 of bovine viral diarrhea virus (BVDV). BVDV is an enveloped virus with an RNA genome responsible for major economic losses of the cattle industry worldwide. Based on the crystal structure of the envelope protein E2, we defined a binding site at the interface of the two most distal domains from the virus membrane and pursued a hierarchical docking-based virtual screening search to identify small-molecule ligands of E2. Phenyl thiophene carboxamide derivative 12 (PTC12) emerged as a specific inhibitor of BVDV replication from in vitro antiviral activity screening of candidate molecules, displaying an IC₅₀ of 0.30 μM against the reference NADL strain of the virus. Using reverse genetics we constructed a recombinant BVDV expressing GFP that served as a sensitive reporter for the study of the mechanism of action of antiviral compounds. Time of drug addition assays showed that PTC12 inhibited an early step of infection. The mechanism of action was further dissected to find that the compound specifically acted at the internalization step of virus entry. Interestingly, we demonstrated that similar to PTC12, the benzimidazole derivative 03 (BI03) selected in the virtual screen also inhibited internalization of BVDV. Furthermore, docking analysis of PTC12 and BI03 into the binding site revealed common interactions with amino acid residues in E2 suggesting that both compounds could share the same molecular target. In conclusion, starting from a targeted design strategy of antivirals against E2 we identified PTC12 as a potent inhibitor of BVDV entry. The compound can be valuable in the design of antiviral strategies in combination with already well-characterized polymerase inhibitors of BVDV.

病毒包膜蛋白的抗病毒靶向是治疗病毒感染的有效策略。在这里，我们采用了计算机指导的方法，目的是确定针对牛病毒性腹泻病毒（BVDV）的包膜蛋白E2的新型抗病毒药。BVDV是一种带有RNA基因组的包膜病毒，可造成全球养牛业的重大经济损失。基于包膜蛋白E2的晶体结构，我们在病毒膜的两个最远端结构域的界面处定义了一个结合位点，并进行了基于分层对接的虚拟筛选搜索，以鉴定E2的小分子配体。苯基噻吩羧酰胺衍生物12（PTC12）作为BVDV复制的特异性抑制剂从体外出现候选分子的抗病毒活性筛选，显示IC ₅₀相对于病毒的参考NADL株为0.30μM。使用反向遗传学，我们构建了表达GFP的重组BVDV，用作研究抗病毒化合物作用机理的敏感报告基因。药物添加分析的时间表明PTC12抑制了感染的早期步骤。进一步剖析了作用机理，以发现该化合物在病毒进入的内在化步骤中起特定作用。有趣的是，我们证明了与PTC12类似，在虚拟屏幕中选择的苯并咪唑衍生物03（BI03）也抑制了BVDV的内在化。此外，对PTC12和BI03的结合位点的对接分析显示与E2中氨基酸残基的共同相互作用表明这两种化合物可以共享相同的分子靶标。综上所述，从针对E2的抗病毒药物的针对性设计策略开始，我们确定PTC12是BVDV进入的有效抑制剂。该化合物与已经充分表征的BVDV聚合酶抑制剂结合在一起，在抗病毒策略设计中可能很有价值。