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