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. 2022 Dec:208:105428.
doi: 10.1016/j.antiviral.2022.105428. Epub 2022 Oct 15.

Astersaponin I from Aster koraiensis is a natural viral fusion blocker that inhibits the infection of SARS-CoV-2 variants and syncytium formation

Tai Young Kim  1 , Ji-Young Kim  2 , Hak Cheol Kwon  3 , Sangeun Jeon  4 , Sol Ji Lee  5 , Haejin Jung  6 , Seungtaek Kim  4 , Dae Sik Jang  7 , C Justin Lee  8
Affiliations

Astersaponin I from Aster koraiensis is a natural viral fusion blocker that inhibits the infection of SARS-CoV-2 variants and syncytium formation

Tai Young Kim et al. Antiviral Res. 2022 Dec.

Abstract

The continuous emergence of SARS-CoV-2 variants prolongs COVID-19 pandemic. Although SARS-CoV-2 vaccines and therapeutics are currently available, there is still a need for development of safe and effective drugs against SARS-CoV-2 and also for preparedness for the next pandemic. Here, we discover that astersaponin I (AI), a triterpenoid saponin in Aster koraiensis inhibits SARS-CoV-2 entry pathways at the plasma membrane and within the endosomal compartments mainly by increasing cholesterol content in the plasma membrane and interfering with the fusion of SARS-CoV-2 envelope with the host cell membrane. Moreover, we find that this functional property of AI as a fusion blocker enables it to inhibit the infection with SARS-CoV-2 variants including the Alpha, Beta, Delta, and Omicron with a similar efficacy, and the formation of syncytium, a multinucleated cells driven by SARS-CoV-2 spike protein-mediated cell-to-cell fusion. Finally, we claim that the triterpene backbone as well as the attached hydrophilic sugar moieties of AI are structurally important for its inhibitory activity against the membrane fusion event. Overall, this study demonstrates that AI is a natural viral fusion inhibitor and proposes that it can be a broad-spectrum antiviral agent against current COVID-19 pandemic and future outbreaks of novel viral pathogens.

Keywords: Aster koraiensis; Astersaponin I; COVID-19; Membrane fusion; SARS-CoV-2 variants; Syncytium.

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Conflict of interest statement

Declaration of competing interest The authors declare no conflict of interests.

Figures

Fig. 1
Fig. 1
Astersaponin I from Aster koraiensis inhibits SARS-CoV-2 infection. (A) The effects of various plant extracts on the entry of SARS-CoV-2 pseudovirus (pSARS-CoV-2) into ACE2+ and ACE2/TMPRSS2+ H1299 cells. (B, C) pSARS-CoV-2 entry and cell viability assays for the 70% EtOH extract of Aster koraiensis (B) and triterpenoid saponins isolated from A. koraiensis (C) in ACE2+ and ACE2/TMPRSS2+ H1299 cells. The data from pSARS-CoV-2 entry assay were representative of three independent experiments. The error bars indicate the SEM (n > 3). P values were determined by the unpaired, two-tailed Student's t-test. *P < 0.05; **P < 0.01, ****P < 0.0001.
Fig. 2
Fig. 2
Steroidal saponins and triterpenoid saponins with a phenyl moiety have a limited inhibitory activity against SARS-CoV-2 infection. (A) Chemical structures and the effects of the steroidal saponins obtained from A. cochinchinensis on the entry of pSARS-CoV-2 into ACE2+ H1299 cells. (B) Chemical structures and the effects of onjisaponins obtained from P. tenuifolia on the entry of pSARS-CoV-2 into ACE2+ H1299 cells. The data from pSARS-CoV-2 entry assay were representative of two or three independent experiments. The error bars indicate the SEM (n > 3).
Fig. 3
Fig. 3
Astersaponin I blocks viral membrane fusion with the host cell membrane. (A) Schematic illustration of the binding of SARS-CoV-2 spike receptor binding domain (RBD) fused to GFP (RBD-GFP) to ACE2 protein overexpressed in H1299 cells. (B) Examination of the effect of AK extracts and AI on the interaction between RBD-GFP and ACE2 on the surface of H1299 cells by flow cytometry after treatment with indicated concentration of AK extracts and AI. The grey peaks indicate the control experiments without RBD-GFP addition. (C) ACE2+ H1299 cells were treated with 1 or 5 μM AI for 1 h and washed with fresh media (Wash) or maintained (No wash), followed by the addition of pSARS-CoV-2. The data from pSARS-CoV-2 entry assay were representative of two independent experiments. The error bars indicate the SEM (n > 3). P values were determined by the unpaired, two-tailed Student's t-test (NS, not significant). (D) Schematic illustration of the SARS-CoV-2 S protein-mediated cell fusion. Addition of the cell suspension of H1299 cells stably expressing S protein and GFP (Spike-H1299) to a monolayer of ACE2/TMPRSS2+ H1299 cells with mRuby fluorescence (ACE2/TMPRSS2-H1299) leads to cell-to-cell fusion. (E) Still images at different time points from time-lapse imaging of S-mediated cell fusion. (F) The fusion between Spike-H1299 and ACE2/TMPRSS2+ H1299 was determined by counting the number of cells double-positive for GFP and mRuby by flow cytometry. H1299 cells expressing only GFP (no spike) were used for control experiment. All compounds were used at the concentration of 10 μM. The data were representative of three independent experiments. (G, H) Filipin cholesterol staining of ACE2+ H1299 cells after treatment with DMSO, AI (5 μM), and other indicated compounds (10 μM) for 1 h (G) and quantification of the intensity of membrane filipin staining using Image J software (n = 20 for each group). Error bars in the graphs indicate the SEM. P values were determined by one-way ANOVA followed by Tukey's post hoc test. ****P < 0.0001; NS not significant (H).
Fig. 4
Fig. 4
Astersaponin I prevents SARS-CoV-2 S-induced syncytia formation. (A) Schematic illustration of Split-GFP assay. Ectopic expression of SARS-CoV-2 S protein into cultures of the mixture of ACE2/TMPRSS2+ H1299 cells expressing GFP1-10 or GFP11 leads to cell-to-cell fusion, generating GFP fluorescence. (B) Images of the S-mediated cell-to-cell fusion using Split-GFP. GFP and blue fluorescence indicate the cell-to-cell fusion and DAPI-stained nuclei, respectively. The nuclei are automatically pseudocolored in white and overlapped with GFP fluorescence by CellReporterXpress software. (C) Examination of effect of AI on the S-mediated cell-to-cell fusion using Split-GFP assay. Representative images of GFP-positive cell-to-cell fusion. The GFP signal and DAPI nuclei staining are automatically pseudocolored in green and red by CellReporterXpress software. (D) Quantitative evaluation for cell-to-cell fusion using Split-GFP assay. Images of GFP and nuclei stained with DAPI were obtained in five random fields per well. The percentage of fusion cells were calculated by dividing number of nuclei in GFP-positive cells by total number of nuclei. The data were representative of three independent experiments. The error bars indicate the SEM (n > 3). P values were determined by one-way ANOVA followed by Tukey's post hoc test. *P < 0.05; ****P < 0.0001; NS not significant. (E) Protein lysates from co-culture experiments were assessed by Western blot. GAPDH was used as a loading control.
Fig. 5
Fig. 5
Astersaponin I equally inhibits the infection of SARS-CoV-2 WT and D614G mutant. (A) pSARS-CoV-2 entry assay in ACE2+ and ACE2/TMPRSS2+ H1299 cells. The data were representative of two independent experiments. The error bars indicate the SEM (n > 3). P values were determined by the unpaired, two-tailed Student's t-test (****P < 0.0001). (B) The effects of AI on the infection of WT and D614G mutant of pSARS-CoV-2 in ACE2+ and ACE2/TMPRSS2+ H1299 cells. The data were representative of three independent experiments. The error bars indicate the SEM (n > 3). P values were determined by the unpaired, two-tailed Student's t-test (****P < 0.0001).
Fig. 6
Fig. 6
Astersaponin I inhibits the infection of SARS-CoV-2 and its variants with a similar efficiency. (A, C) Dose-response curves for AI (A) and chloroquine (C) against ancestral SARS-CoV-2 (A, black circle) and SARS-CoV-2 variants Alpha (B.1.1.7, red square), Beta (B.1.351, orange triangle), Delta (B.1.617.2, pink inverted triangle), and Omicron (B.1.1.529, green diamond) in Vero cells. The blue diamond represents cell viability. The mean ± SEM was calculated from duplicate experiments. (B, D) Confocal images of SARS-CoV-2 N protein (green) and cell nuclei at concentrations near the IC50 of AI (B) and chloroquine (D) in Vero cells. (E) Dose-response curve for AI against replicable SARS-CoV-2 recombinant viruses expressing Nanoluciferase (Nluc) into A549 cells that overexpress ACE2 and TMPRSS2. The blue diamond represents cell viability.
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