Small-Molecule Inhibitors of Dengue-Virus Entry

Flavivirus envelope protein (E) mediates membrane fusion and viral entry from endosomes. A low-pH induced, dimer-to-trimer rearrangement and reconfiguration of the membrane-proximal “stem" of the E ectodomain draw together the viral and cellular membranes. We found stem-derived peptides from dengue virus (DV) bind stem-less E trimer and mimic the stem-reconfiguration step in the fusion pathway. We adapted this experiment as a high-throughput screen for small molecules that block peptide binding and thus may inhibit viral entry. A compound identified in this screen, 1662G07, and a number of its analogs reversibly inhibit DV infectivity. They do so by binding the prefusion, dimeric E on the virion surface, before adsorption to a cell. They also block viral fusion with liposomes. Structure-activity relationship studies have led to analogs with submicromolar IC90s against DV2, and certain analogs are active against DV serotypes 1,2, and 4. The compounds do not inhibit the closely related Kunjin virus. We propose that they bind in a previously identified, E-protein pocket, exposed on the virion surface and although this pocket is closed in the postfusion trimer, its mouth is fully accessible. Examination of the E-trimer coordinates (PDB 1OK8) shows that conformational fluctuations around the hinge could open the pocket without dissociating the trimer or otherwise generating molecular collisions. We propose that compounds such as 1662G07 trap the sE trimer in a “pocket-open" state, which has lost affinity for the stem peptide and cannot support the final “zipping up" of the stem.

黄病毒包膜蛋白（Flavivirus envelope protein, E）介导膜融合与病毒从内体侵入宿主细胞。低pH诱导下，E蛋白胞外域的膜近端“茎区”发生二聚体向三聚体的重排与构象重构，使病毒膜与细胞膜相互靠拢。我们发现，登革病毒（Dengue Virus, DV）的茎源肽可结合无茎区E三聚体，模拟融合通路中的茎区重构步骤。我们将该实验改造为高通量筛选模型，用于筛选可阻断肽结合、从而抑制病毒侵入的小分子化合物。该筛选中发现的化合物1662G07及其多款类似物可可逆抑制登革病毒的感染性，其作用机制是在病毒吸附宿主细胞前，结合病毒粒子表面处于融合前状态的二聚体E蛋白；同时它们还可阻断病毒与脂质体的膜融合过程。构效关系研究已获得对登革病毒2型（DV2）的IC90处于亚微摩尔级的类似物，且部分类似物对登革病毒血清型1、2、4均具有抗病毒活性。该类化合物对亲缘关系相近的昆津病毒无抑制活性。我们推测此类化合物结合于此前已发现的、暴露于病毒粒子表面的E蛋白口袋中；尽管该口袋在融合后三聚体构象中处于闭合状态，但其开口完全暴露。对E三聚体坐标（PDB 1OK8）的分析显示，铰链区周围的构象波动可在不解离三聚体或引发分子碰撞的前提下打开该口袋。我们推测，1662G07这类化合物可将可溶性E（soluble E, sE）三聚体捕获于“口袋开放”状态，该状态下茎区肽的结合亲和力丧失，无法支撑茎区最终的“拉链式”闭合过程。