Target-Based Virtual Screening and LC/MS-Guided Isolation Procedure for Identifying Phloroglucinol-Terpenoid Inhibitors of SARS-CoV‑2

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to more than 5 million deaths worldwide to date. Due to the limited therapeutic options so far available, target-based virtual screening with LC/MS support was applied to identify the novel and high-content compounds 1–4 with inhibitory effects on SARS-CoV-2 in Vero E6 cells from the plant Dryopteris wallichiana. These compounds were also evaluated against SARS-CoV-2 in Calu-3 cells and showed unambiguous inhibitory activity. The inhibition assay of targets showed that compounds 3 and 4 mainly inhibited SARS-CoV-2 3CLpro, with effective Kd values. Through docking and molecular dynamics modeling, the binding site is described, providing a comprehensive understanding of 3CLpro and interactions for 3, including hydrogen bonds, hydrophobic bonds, and the spatial occupation of the B ring. Compounds 3 and 4 represent new, potential lead compounds for the development of anti-SARS-CoV-2 drugs. This study has led to the development of a target-based virtual screening method for exploring the potency of natural products and for identifying natural bioactive compounds for possible COVID-19 treatment.由严重急性呼吸综合征冠状病毒2型（SARS-CoV-2）引发的2019冠状病毒病（COVID-19）大流行，截至目前已在全球造成超500万人死亡。鉴于当前可用治疗手段十分有限，本研究采用液相色谱-质谱联用（LC/MS）辅助的基于靶点的虚拟筛选策略，从植物喜马拉雅鳞毛蕨（Dryopteris wallichiana）中筛选得到4种全新高含量化合物1~4，上述化合物在Vero E6细胞中展现出显著的抗SARS-CoV-2活性。随后，研究团队在Calu-3细胞模型中针对SARS-CoV-2对这些化合物开展了活性验证，结果显示其均具备明确的抑制效果。靶点抑制实验结果表明，化合物3与4主要通过靶向抑制SARS-CoV-2 3CLpro发挥作用，且拥有有效的解离常数（Kd）数值。通过分子对接与分子动力学模拟，本研究阐明了化合物3与3CLpro的结合位点，全面解析了二者的相互作用模式，包括氢键、疏水键相互作用以及B环的空间占位效应。化合物3与4可作为开发抗SARS-CoV-2药物的新型潜在先导化合物。本研究构建了一套基于靶点的虚拟筛选方法，可用于挖掘天然产物的抗新冠病毒活性，并筛选可用于COVID-19潜在治疗的天然生物活性成分。