Fig 1.
Antiviral activity of Berbamine hydrochloride against SARS-CoV-2 in vitro.
Vero-E6 (a) or Caco2 (b) cells were infected with SARS-CoV-2. After incubation with berbamine hydrochloride at 37°C for 36 h, cell culture fluids were harvested for qRT-PCR assay. Cytotoxicity was examined by CCK-8 assay. The CC50, IC50, and SI values for each inhibitor are shown above the figures. Error bars represents standard deviation. (c) Immunofluorescence assay of SARS-CoV-2-infected Vero E6 cells that received berbamine hydrochloride treatment. Scale bar, 100 μm. Data was expressed as mean ± standard deviation and shown are representative results of two independent experiments with two technical replicas per experiment.
Fig 2.
(a) The schematic diagram of time-of addition. Approximately 8×104 Vero-E6 cells were seeded per well of a 24-well plate. After incubation for 24 h, the cells were infected with the SARS-CoV-2 virus at an MOI of 0.05. Berbamine hydrochloride was added to the infected cells at a final concentration of 10 μM at indicated time points. At 12 h p.i., the cell culture fluids were harvested for qRT-PCR assay (b). Data was expressed as mean ± standard deviation and shown are representative results of two independent experiments with two technical replicas per experiment. Error bars represents standard deviation.
Fig 3.
(a) Transient replicon assay. (b) SARS-CoV-2 pseudovirus assay. Vero E6 cells were treated with the berbamine hydrochloride (10 μM) or DMSO, and then infected with SARS-CoV-2 pseudovirus for 24 h. Luciferase activity in cell lysates was determined and compared with the DMSO control. (c) Impact of berbamine hydrochloride on SARS-CoV-2 attachment. Vero E6 cells were treated with berbamine hydrochloride (10 μM) or DMSO for 1 h prior to SARS-CoV-2 infection at 4°C for 1 h. The cells containing binding virions were collected for quantification of viral RNA copies by real-time RT-PCR assay. Data was expressed as mean ± standard deviation and shown are representative results of two independent experiments with two technical replicas per experiment. An unpaired two-tailed student’s t-test was performed for statistical analysis. n.s. not significant, *p<0.05, **p<0.01 and ***p<0.001. Error bars represents standard deviation.
Fig 4.
Microscopic observation and immunofluorescence analysis the inhibitory activity of bis-benzylisoquinoline alkaloids against SARS-CoV-2 S-mediated cell–cell fusion.
(a) Structure of bis-benzylisoquinoline alkaloids. Vero-E6 cells co-culture with BHK that transfected with the SARS-CoV-2 S expression construct and treated with 10 μM berbamine hydrochloride, liensinine, isoliensinine, tetrandrine, fangchinoline, cepharanthine or DMSO. Cells were harvested at 12 h after treatment. The syncytial phenotype is indicated by the multinucleated giant cells as indicated by the red arrows. (b) Microscopic observation and (c) immunofluorescence images of cells staining with DAPI (blue) or anti-S Ab (green). Scale bar, 100 μm. Experiments were performed at least twice with two technical replicas per experiment.
Fig 5.
Generated the binding mode of berbamine hydrochloride in the post fusion core of SARS-CoV-2 S2 subunit.
The 3D structure of berbamine hydrochloride and proteins were obtained from ZINC (ID: ZINC38139356) and PDB (ID: 6LXT) databases, respectively. The interaction sites between berbamine hydrochloride and S2 were predicted by Autodock vina algorithm. The SARS-CoV-2 S2 residues involved in berbamine hydrochloride binding are shown as green sticks.