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