南方医科大学学报

南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (3): 474-483.doi: 10.12122/j.issn.1673-4254.2024.03.08

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马铃薯三糖甘草次酸衍生物通过抑制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

万 欣，洪崇竣，王进绅，宋高鹏，刘叔文   

  1. 惠州卫生职业技术学院药学与检验学院，广东 惠州 516000；华南农业大学材料与能源学院，广东 广州 510642；南方医科大学药学院，广东 广州 510515
  • 出版日期:2024-03-20 发布日期:2024-04-03

3-O-β-chacotriosyl glycyrrhetinic acid derivatives as potential small-molecule SARS-CoV-2 fusion inhibitors against SARS-CoV-2 entry into host cells

WAN Xin, HONG Chongjun, WANG Jinshen, SONG Gaopeng, LIU Shuwen   

  1. School of Pharmacy and Laboratory Medicine, Huizhou Health Sciences Polytechnic, Huizhou 516000, China; College of Materials and Energy, South China Agricultural University, Guangzhou 510642, China; School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China
  • Online:2024-03-20 Published:2024-04-03

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摘要： 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。

关键词: SARS-CoV-2；小分子新冠病毒融合抑制剂；刺突蛋白；甘草次酸衍生物

Abstract: 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 pseudovirus-infected 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 protein-mediated 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.

Key words: SARS-CoV-2; small-molecule SARS-CoV-2 fusion inhibitors; spike protein; glycyrrhetinic acid derivatives