Objective: To study the inhibitory activities of 3-O-β-chacotriosyl glycyrrhetinic acid derivatives against the entry of SARS-CoV-2 into host cells.
Methods: With pentacyclic triterpene saponin glycyrrhizic acid (a natural SARS-CoV-2 entry inhibitor) as the lead compound, a series of 3-O-β-chacotriosyl glycyrrhetinic acid derivatives were designed and synthesized based on hypridization principle, and their inhibitory activities against virus entry were tested in SARS-CoV-2 pseudovirusinfected cells. The antiviral targets of the lead compound 1b was identified by pseudotyped SARS-CoV-2 infection assay and surface plasmon resonance (SPR) assay, and the S protein-mediated cell-cell fusion assay was used to evaluate the effect of 1b on virus-cell membrane fusion. Molecular docking and single amino acid mutagenesis were carried out to analyze the effect of 1b on binding activitiy of S protein.
Results: The lead compound 1b showed significant inhibitory effect against Omicron pseudovirus with an EC50 value of 3.28 μmol/L (P < 0.05), and had broad-spectrum antiviral activity against other SARS-CoV-2 pseudovirus. Spike-dependent cell-cell fusion assay demonstrated an inhibitory effect of 1b against SARS-CoV-2 S proteinmediated cell-cell fusion. Molecular docking analysis predicted that the lead compound 1b could be well fitted into a cavity between the attachment (S1) and fusion (S2) subunits at the 3-fold axis, where it formed multiple hydrophobic interactions with Glu309, Ser305, Arg765 and Lys964 residues with a KD value of -8.6 kcal/mol. The compound 1b at 10, 5, 2.5 and 1.25 μmol/L showed a significantly reduced inhibitory activity against the pseudovirus with mutated Arg765, Lys964, Glu309 and Leu303 (P < 0.01).
Conclusion: 3-O-β-chacotriosyl glycyrrhetinic acid derivatives are capable of stabilizing spike protein in the pre-fusion step to interfere with the fusion of SARS-CoV-2 with host cell membrane, and can thus serve as potential novel small-molecule SARS-CoV-2 fusion inhibitors.
目的: 研究马铃薯三糖甘草次酸衍生物能否通过抑制SARS-CoV-2进入靶细胞，作为潜在的小分子新冠病毒融合抑制剂。
方法: 以天然SARS-CoV-2进入抑制剂甘草酸为先导化合物，利用活性亚结构的拼合原理等设计并合成了系列马铃薯三糖甘草次酸衍生物。利用SARS-CoV-2假病毒体外细胞感染模型，检测该系列甘草次酸衍生物的体外抗SARS-CoV-2活性；利用表面等离子共振技术及假病毒模型寻找先导化合物1b的抗病毒作用靶点；利用SARS-CoV-2 S蛋白介导的细胞-细胞融合体系，检测先导化合物1b是否作用于SARS-CoV-2病毒入侵宿主的膜融合过程；基于分子对接与定点突变技术，确定先导化合物1b与S蛋白的作用模式等。
结果: 先导化合物1b对SARS-CoV-2奥密克戎假病毒有显著抑制作用，EC50值为3.28 μmol/L（P < 0.05），对其它SARS-CoV-2变异株假病毒有广谱抗病毒活性。细胞-细胞膜融合实验显示1b能够抑制合胞体的形成。分子对接预测先导化合物1b可与S1与S2亚基交界处的空腔中的Glu309、Ser305、Arg765、Lys964等多个保守氨基酸残基产生氢键作用，亲和力为-8.6 kcal/mol。化合物1b在10、5、2.5、1.25 μmol/L时对Arg765、Lys964、Glu309和Leu303突变后的假病毒的抑制活性显著降低（P < 0.01）。
结论: 马铃薯三糖甘草次酸衍生物能够靶向作用于S蛋白，特异性干扰病毒-细胞膜融合阶段，继而发挥抗SARS-CoV-2感染的作用，是一类结构新颖的小分子SARS-CoV-2融合抑制剂。
Keywords: SARS-CoV-2; glycyrrhetinic acid derivatives; small-molecule SARS-CoV-2 fusion inhibitors; spike protein.