Ebola virus (EBOV) causes hemorrhagic fever in humans with high morbidity and fatality. Although over 45 years have passed since the first EBOV outbreak, small molecule drugs are not yet available. Ebola viral protein VP30 is a unique RNA synthesis cofactor, and the VP30/NP interaction plays a critical role in initiating the transcription and propagation of EBOV. Here, we designed a high-throughput screening technique based on a competitive binding assay to bind VP30 between an NP-derived peptide and a chemical compound. By screening a library of 8004 compounds, we obtained two lead compounds, Embelin and Kobe2602. The binding of these compounds to the VP30-NP interface was validated by dose-dependent competitive binding assay, surface plasmon resonance, and thermal shift assay. Moreover, the compounds were confirmed to inhibit the transcription and replication of the Ebola genome by a minigenome assay. Similar results were obtained for their two respective analogs (8-gingerol and Kobe0065). Interestingly, these two structurally different molecules exhibit synergistic binding to the VP30/NP interface. The antiviral efficacy (EC₅₀) increased from 1 μM by Kobe0065 alone to 351 nM when Kobe0065 and Embelin were combined in a 4:1 ratio. The synergistic anti-EBOV effect provides a strong incentive for further developing these lead compounds in future studies.

埃博拉病毒（EBOV）会引起人类出血热，发病率和死亡率很高。尽管自第一次埃博拉病毒爆发以来已经过去了 45 年多，但小分子药物尚未上市。埃博拉病毒蛋白VP30是一种独特的RNA合成辅因子，VP30/NP相互作用在启动EBOV的转录和传播中发挥着关键作用。在这里，我们设计了一种基于竞争性结合测定的高通量筛选技术，以在 NP 衍生肽和化合物之间结合 VP30。通过筛选 8004 个化合物库，我们获得了两种先导化合物：Embelin 和Kobe2602。这些化合物与 VP30-NP 界面的结合通过剂量依赖性竞争结合测定、表面等离子共振和热位移测定进行了验证。此外，通过小基因组测定证实这些化合物能够抑制埃博拉基因组的转录和复制。它们的两种类似物（8-姜酚和Kobe0065）也获得了类似的结果。有趣的是，这两种结构不同的分子表现出与 VP30/NP 界面的协同结合。当Kobe0065和Embelin以4:1的比例组合时，抗病毒功效(EC ₅₀ )从单独使用Kobe0065的1μM增加到351nM。协同抗埃博拉病毒效应为未来研究中进一步开发这些先导化合物提供了强有力的动力。