Diltiazem inhibits the early stage of SARS-CoV-2 infection in vitro.

(A and B) Vero-E6 cells (A) and Calu-3 cells (B) were incubated with vehicle or diltiazem for 1 h, and then infected with HRB25 at a M.O.I. of 5 and a M.O.I. of 10, respectively. At the indicated timepoints post-infection, the viral RNA level in the cell lysates were measured by qPCR. (C) The viability of Calu-3 cells was determined in the presence of diltiazem at the indicated concentrations. (D) Vero-E6 cells were treated with diltiazem and remdesivir for 1 h, respectively, and then infected with HRB25 (M.O.I. = 5). For the virus neutralization assay, HRB25 (M.O.I. = 5) was incubated with neutralizing antibody (20 μg/ml) for 1 h at 4°C, and then the mixture was used to infect Vero-E6 cells. At the indicated timepoints post-infection, the viral RNA level in the cell lysate was measured by qPCR. (E) Vero-E6 cells were infected with HRB25 (M.O.I. = 5), and diltiazem was added at -1 h, 1 h, or 2 h post-infection. The viral RNA level in the cell lysate was determined at 6 h post-infection by qPCR. (F) Vero-E6 cells were infected with HRB25 (M.O.I. = 5), then diltiazem and E64D were added at 6 h post-infection. The supernatants were harvested at 24 h post-infection for plaque assays. The data shown are the means ± SDs of three independent experiments or replicates. The two-tailed unpaired Student’s t-test was used for the statistical analysis. ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001.