Instituto de Investigaciones Biotecnológicas, CONICET, Universidad Nacional de San Martín, Argentina.
Centro de Investigaciones en Bionanociencias, CONICET, Argentina.
Antiviral Res. 2018 Jan;149:179-190. doi: 10.1016/j.antiviral.2017.10.010. Epub 2017 Oct 12.
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 IC 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.
抗病毒靶向病毒包膜蛋白是治疗病毒感染的有效策略。在这里，我们采用计算机指导的方法，旨在鉴定针对牛病毒性腹泻病毒（BVDV）包膜蛋白 E2 的新型抗病毒药物。BVDV 是一种带有 RNA 基因组的包膜病毒，是全球牛养殖业遭受重大经济损失的罪魁祸首。基于包膜蛋白 E2 的晶体结构，我们在距离病毒膜最远的两个结构域的界面上定义了一个结合位点，并采用分级对接虚拟筛选搜索来鉴定 E2 的小分子配体。苯噻吩甲酰胺衍生物 12（PTC12）从候选分子的体外抗病毒活性筛选中脱颖而出，成为 BVDV 复制的特异性抑制剂，对 NADL 参考株的 IC 为 0.30μM。我们使用反向遗传学构建了一种表达 GFP 的重组 BVDV，作为研究抗病毒化合物作用机制的敏感报告分子。加药时间测定表明，PTC12 抑制了感染的早期阶段。作用机制进一步被剖析，发现该化合物特异性作用于病毒进入的内化步骤。有趣的是，我们证明，与 PTC12 类似，虚拟筛选中选择的苯并咪唑衍生物 03（BI03）也抑制了 BVDV 的内化。此外，将 PTC12 和 BI03 对接分析到结合位点表明，两者与 E2 中的氨基酸残基存在共同相互作用，提示这两种化合物可能具有相同的分子靶标。总之，从针对 E2 的抗病毒靶向设计策略开始，我们鉴定出 PTC12 是一种有效的 BVDV 进入抑制剂。该化合物可与 BVDV 已充分表征的聚合酶抑制剂联合用于设计抗病毒策略。
