3.2 Lancemaside A inhibits two main SARS-CoV-2 entry pathways with a similar efficiency
Based on the results showing that LA has better inhibitory activity against SARS-CoV-2 than LB (Figure. 1d), we chose LA for further experiments. First, we evaluated the ability of LA to inhibit pSARS-CoV-2 entry at various concentrations and obtained the IC50 values of 1.77 and 2.85 μM in ACE2+ and ACE2/TMPRRS+ H1299 cells, respectively (Figure 2a and 2b). Notably, LA treatment in the range of concentrations did not induce cytotoxicity in H1299 cells (Figure 2c). We further extended our analysis to examine the effect of LA against the infection of authentic SARS-CoV-2 into Vero cells that express high levels of ACE2 but no detectable TMPRSS2 (Kim et al., 2021). Assessment of SARS-CoV-2 infection based on an immunostaining of SARS-CoV-2 nucleocapsid N protein in Vero cells showed that LA exhibited antiviral activity against ancestral SARS-CoV-2 with IC50 of 2.62 μM (Figure 2d). Importantly, LA was found to be more effective than remdesivir, an FDA-approved anti-SARS-CoV-2 drug (IC50: 7.24 μM) (Figure 2e). We further verified the inhibitory activity of LA against TMPRSS2-dependent cell surface entry pathway of SARS-CoV-2 by measuring the infection of a recombinant SARS-CoV-2 reporter virus expressing the nanoluciferase (Nluc) (SARS-CoV-2-Nluc) into A549 cells that overexpress both ACE2 and TMPRSS2. The result showed that LA effectively blocked the viral entry in the cells with an IC50 of 3.92 µM (Figure 2f). Overall, these results demonstrate that LA restricts SARS-CoV-2 entry both at the endosomal compartments and at the cell surface