Fig 1.
Diltiazem inhibits SARS-CoV-2 infection in cells.
(A) Vero-E6 cells were treated with DMSO or inhibitors at the indicated concentrations for 1 h, and then infected with HRB25 (M.O.I. = 0.01). The supernatants were harvested at 24 h post-infection for plaque assays. PFU, plaque-forming units. (B) The viability of Vero-E6 cells was determined in the presence of inhibitors at the indicated concentrations. (C) Vero-E6 cells were treated with diltiazem at different concentrations or vehicle to determine the CC50. CC50: 50% cytotoxic concentration. (D and E) IC50 of diltiazem to HRB25 infectivity. Vero-E6 cells were treated with vehicle or diltiazem at the indicated concentrations for 1 h, and then infected with HRB25 (M.O.I. = 0.01); the supernatants were harvested at 24 h post-infection to determine viral RNA copy numbers (D) and virus titers (E) by use of qPCR and plaque assays, respectively. IC50: 50% inhibitory concentration. (F and G) Vero-E6 cells were treated with diltiazem for 1 h, and then infected with HRB25 at an M.O.I. of 0.01 (F) or 5 (G); the supernatants were harvested at the indicated timepoints for plaque assays. (H and I) BEAS-2B cells (H) and Calu-3 cells (I) were treated with diltiazem for 1 h, and then infected with HRB25 (M.O.I. = 5). At 24 h post-infection, the viral RNA level in the cell lysate was measured by qPCR. 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. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 2.
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.
Fig 3.
Diltiazem inhibits SARS-CoV-2 binding.
(A) Cells were subjected to flow cytometry to detect the surface expression of ACE2. (B) Cells were lysed to determine ACE2 mRNA copy numbers by qPCR. (C) Schematic of the viral binding assay. (D) Diltiazem preincubated cells were incubated with HRB25 (M.O.I. = 10) at 4°C for 1 h. The viral RNA level in the cell lysate was measured by qPCR. (E) Diltiazem preincubated Vero-E6 cells were incubated with HRB25 at an M.O.I. of 10, 1, 0.1, or 0.01. The viral RNA level in the cell lysate was measured by qPCR. (F) Vero-E6 cells were treated and infected as described in (C). The cells were incubated with an anti-SARS-CoV-2 nucleocapsid rabbit monoclonal antibody, and visualized with Alexa Fluor 488-conjugated goat anti-rabbit IgG (green). Cell nuclei were stained with Hoechst 33342. Representative images are shown. The fluorescence intensities of cell-bound HRB25 in at least 110 cells per sample were quantified. (G) HEK293T cells were treated and assessed as described in (E). The data shown represent, or are from, three independent experiments or replicates (means ± SDs). 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.
Fig 4.
Diltiazem decreases the cell surface expression of ACE2.
(A and B) Vero-E6 cells were preincubated with diltiazem, then the expression of ACE2 on the cell surface and in the total cells was detected by western blotting (A) and flow cytometry (B), respectively. (C and D) Diltiazem-treated Vero-E6 cells, Vero-E6-ACE2 cells (C), A549 cells, or A549-ACE2 cells (D) were incubated with HRB25 (M.O.I. = 10) at 4°C for 1 h, then washed with PBS. The viral RNA level in the cell lysate was measured by qPCR. The expression of exogenous ACE2 was confirmed by western blotting. (E) Diltiazem-treated Vero-E6-ACE2 cells were infected with HRB25 (M.O.I. = 0.01), and the supernatants were harvested at 24 h post-infection for plaque assays. The data shown represent, or are from, three independent experiments or replicates (means ± SDs). The two-tailed unpaired Student’s t-test was used for the statistical analysis. ns, not significant, **p < 0.01, ***p < 0.001.
Fig 5.
Diltiazem inhibits the internalization of SARS-CoV-2.
(A) Cells were incubated with HRB25 (M.O.I. = 10) for 1 h at 4°C, then washed with acid buffer/trypsin to remove bound virus and lysed for qPCR to detect SARS-CoV-2. (B and C) Diltiazem-treated cells were incubated with HRB25 (M.O.I. = 1 or 10) at 4°C for 1 h and washed with PBS, and then shifted to 37°C for 1 h; they were then washed with PBS (B) or acid buffer/trypsin (C). The washed cells were lysed for qPCR to detect SARS-CoV-2 binding to cells (B) or internalized into cells (C). (D) HEK293T cells transiently expressing ACE2 and TMPRSS2 were treated with DMSO, E64D, camostat mesylate, or diltiazem, and then infected with HRB25 (M.O.I. = 5). The viral RNA level in the cell lysate was measured by qPCR at 6 h post-infection. (E) Schematic representation of the SARS-CoV-2 S protein-mediated cell-cell fusion assay. (F) Representative images of DMSO-, camostat mesylate-, or diltiazem-treated cell-cell fusion. Scale bar: 4 × field, 1000 μm; 10 × field, 400 μm; and 20 × field, 200 μm. 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 < 0.01, ***p < 0.001.
Fig 6.
Knockdown of Cav1.2 α1c inhibits the attachment and internalization of SARS-CoV-2.
(A) The CACNA1C mRNA level in the indicated siRNA-transfected cells was measured by qPCR. siControl, scrambled siRNA. siCACNA1C, siRNA specific for CACNA1C mRNA. (B) CACNA1C-silenced Vero-E6 cells were infected with HRB25 (M.O.I. = 0.01), and the supernatants were harvested at 24 h post-infection for plaque assays. (C) CACNA1C-silenced cells were incubated with HRB25 (M.O.I. = 10) for 1 h at 4°C. The viral RNA level in the cell lysate was measured by qPCR. (D and E) The expression of ACE2 on the cell surface and in total cells was detected by western blotting (D) and flow cytometry (E) in CACNA1C-silenced Vero-E6 cells. (F) The CACNA1C mRNA level in the indicated siRNA-transfected cells was measured by qPCR. (G) CACNA1C-silenced cells were incubated with HRB25 (M.O.I. = 10) for 1 h at 4°C, and washed with PBS. The viral RNA level in the cell lysate was measured by qPCR. (H) CACNA1C-silenced Vero-E6-ACE2 cells were infected with HRB25 (M.O.I. = 0.01), and the supernatants were harvested at 24 h post-infection for plaque assays. (I and J) CACNA1C-silenced cells were incubated with HRB25 (M.O.I. = 10) at 4°C for 1 h and washed with PBS, then shifted to 37°C for 1 h. The cells were then washed with PBS (I) or acid buffer/trypsin (J). The washed cells were lysed for qPCR to detect SARS-CoV-2 binding to cells (I) or internalized into cells (J). (K) DMSO-, E64D-, camostat mesylate-, or diltiazem-treated CACNA1C-silenced HEK293T cells transiently expressing ACE2 and TMPRSS2 were infected with HRB25 (M.O.I. = 5). The viral RNA level in the cell lysate was measured by qPCR at 6 h post-infection. 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.
Fig 7.
Cav1.2 α1c interacts and colocalizes with SARS-CoV-2 S protein and ACE2.
(A) HEK293 cells were co-transfected with Cav1.2 α1c-Flag and SARS-CoV-2 S-Myc, and then subjected to immunoprecipitation (IP) by using anti-Flag agarose beads. Representative western blots of whole-cell lysates and eluates after IP are shown. (B) The co-immunoprecipitation of Cav1.2 α1c-Flag and the SARS-CoV-2 S protein truncation mutant SARS-CoV-2 S1-Myc. (C) The co-immunoprecipitation of Cav1.2 α1c-Flag and ACE2-Flag with the SARS-CoV-2 S protein truncation mutant SARS-CoV-2 RBD-Myc. (D) Purified soluble His-tagged SARS-CoV-2 S1 protein was pooled with the lysate from Cav1.2 α1c-Flag transfected HEK293 cells and then pulled-down by using anti-Flag agarose beads. (E) The co-immunoprecipitation of Cav1.2 α1c-Flag and ACE2-Myc. (F and G) Immunofluorescence assay. Vero-E6 cells were transfected with Cav1.2 α1c-Flag for 24 h, then cells were incubated with SARS-CoV-2 (M.O.I. = 10) at 4°C for 1 h. The images comprising three single fluorescence channels were analysis by using Imaris software. The arrowhead indicates the representative colocalization of Cav1.2 α1c-Flag (green), ACE2 (purple), and SARS-CoV-2 nucleocapsid protein (red) (F). The colocalization of Cav1.2 α1c-Flag, ACE2, and SARS-CoV-2 nucleocapsid protein (indicated by the yellow arrowhead) is shown in three dimensions (G). The data shown are representative of three independent experiments.
Fig 8.
Diltiazem inhibits the replication of SARS-CoV-2 in mouse lung.
(A and B) IC50 of diltiazem to HRB26M infectivity. Vero-E6 cells were treated with diltiazem at different concentrations or with vehicle (water) for 1 h at 37°C, and then infected with HRB26M (M.O.I. = 0.01), the supernatants were harvested at 24 h post-infection to determine viral RNA copy numbers (A) and viral titers (B) by using qPCR and plaque assays, respectively. (C) Protocol for in vivo experiments with BALB/c mice (white) and K18-hACE2 mice (black). (D and E) The mice were intramuscularly administered diltiazem (5 mg/kg) starting 1 h before being infected with HRB26M. The infectious viruses in the lungs of mice (n = 9) were measured by qPCR (D) and plaque assay (E) respectively. (F) Viral antigen in mouse lungs was detected by using an IHC assay. The red arrowhead indicates the representative viral antigen signals. (G and H) The mice were intramuscularly administered diltiazem (5 mg/kg) starting 6 h after being infected with HRB26M. The infectious viruses in the lungs of mice (n = 6) were detected as described in (D) and (E). (I and J) The mice were intranasally administered diltiazem (0.01 mg/kg) starting 1 h before being infected with HRB26M. The infectious viruses in the lungs of mice (n = 6) were detected as described in (D) and (E). (K and L) K18-hACE2 mice were intramuscularly administrated diltiazem (5 mg/kg) starting 1 h before being infected with HRB25. The survival (K) and body weight (L) of the mice (n = 9) were monitoring for 12 days after infection. The horizontal dashed lines indicate the limit of detection. The data shown in panels D and E represent the sum of three independent experiments, those in panels G–L represent the sum of two independent experiments. The two-tailed unpaired Student’s t-test was used for the statistical analysis, mean ± SDs, *p < 0.05, ***p < 0.001.