Berberine and Obatoclax Inhibit SARS-Cov-2 Replication in Primary Human Nasal Epithelial Cells In Vitro

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged as a new human pathogen in late 2019 and it has infected over 100 million people in less than a year. There is a clear need for effective antiviral drugs to complement current preventive measures, including vaccines. In this study, we demonstrate that berberine and obatoclax, two broad-spectrum antiviral compounds, are effective against multiple isolates of SARS-CoV-2. Berberine, a plant-derived alkaloid, inhibited SARS-CoV-2 at low micromolar concentrations and obatoclax, which was originally developed as an anti-apoptotic protein antagonist, was effective at sub-micromolar concentrations. Time-of-addition studies indicated that berberine acts on the late stage of the viral life cycle. In agreement, berberine mildly affected viral RNA synthesis, but it strongly reduced infectious viral titers, leading to an increase in the particle-to-pfu ratio. In contrast, obatoclax acted at the early stage of the infection, which is in line with its activity to neutralize the acidic environment in endosomes. We assessed infection of primary human nasal epithelial cells that were cultured on an air-liquid interface and found that SARS-CoV-2 infection induced and repressed expression of specific sets of cytokines and chemokines. Moreover, both obatoclax and berberine inhibited SARS-CoV-2 replication in these primary target cells. We propose berberine and obatoclax as potential antiviral drugs against SARS-CoV-2 that could be considered for further efficacy testing.

Keywords: COVID-19; antiviral compounds; coronavirus.

Figure 1

Berberine and obatoclax are effective antiviral compounds against Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Vero E6 cells were infected with SARS-CoV-2 (BavPat1 isolate) at an MOI of 0.01 for 24 h in the presence of the indicated concentrations of (A) berberine (BBR) or (B) obatoclax (OLX) in a two-fold dilution series or 0.1% DMSO as a control. Infectious viral titers from duplicate cell culture supernatants were assessed by plaque assay and plotted as percentage inhibition compared to the DMSO control. Error bars indicate SD. Dashed lines indicate 50% inhibition. A representative of two independent experiments is shown. Vero E6 cells were treated with the indicated concentrations of (C) berberine or (D) obatoclax or 0.1% DMSO. 24 h post-treatment, ATP content in cells from triplicate wells was measured as indication for cell viability, plotted as percentage compared to the DMSO control. Error bars indicate SD. A representative of two independent experiments is shown. The shaded grey box indicates that different OLX concentrations were used for antiviral (B) and viability assays (D).

Figure 2

Berberine and obatoclax are effective against SARS-CoV-2 Nijmegen1 isolate. Vero E6 cells were infected with the SARS-CoV-2 Nijmegen1 isolate at an MOI of 0.01 for 24 h in the presence of the indicated concentrations of (A–C) berberine (BBR) or (B) obatoclax (OLX) in a two-fold dilution series or 0.1% DMSO as a control. (A,B) Viral RNA from duplicate cell culture supernatants (red line in panel A) was quantified by qRT-PCR and plotted as percentage inhibition compared to the DMSO control. For BBR-treated cells, both extracellular and Intracellular viral RNA was analyzed (red and blue lines, respectively). Intracellular viral RNA levels were normalized to the human β-actin housekeeping gene. (C) Infectious viral titers from duplicate cell culture supernatants were quantified by plaque assay and plotted as percentage inhibition compared to the DMSO control. Error bars indicate SD. Dashed line indicates 50% inhibition. (D) The ratio of relative genome copies to infectious viral particles in cell culture supernatants for the indicated, non-toxic BBR concentrations and DMSO control.

Figure 3

Time-of-addition assay. Vero E6 cells were infected with SARS-CoV-2 (BavPat1 isolate) at an MOI of 1. (A) Schematic layout of the assay. 20 µM berberine (BBR), 0.25 µM obatoclax (OLX), or 0.1% DMSO as a control was added to the infected cells at the indicated time points. (B) Plaque assay titers from cell culture supernatants that were collected at 10 hpi. Bars and error bars represent means and SD of n = 2 replicates.

Figure 4

SARS-CoV-2 infection of primary nasal epithelial cells. Primary nasal epithelial cells, cultured on an air-liquid interface, were infected with SARS-CoV-2 (BavPat1 isolate) at an MOI of 10. At the indicated time points, cells were harvested and viral RNA from (A) cells or (B) apical and basolateral compartment supernatants was quantified by RT-qPCR. Intracellular viral RNA levels were normalized to the human β-actin housekeeping gene. Error bars indicate SD (n = 2). (C) Infectious viral titers from apical supernatants corresponding to the indicated time points were quantified by plaque assay. Error bars indicate SD (n = 2). (D) Immunofluorescence staining of mock (left panel), SARS-CoV-2 infected Vero E6 cells fixed at 24 hpi (MOI = 0.01; middle panel) or SARS-CoV-2 infected primary nasal epithelial cells fixed at 96 hpi (MOI = 10; right panel). The presence of SARS-CoV-2 Spike protein subunit S1 (pink) was assessed in cells stained with DAPI (nucleus, blue) and phalloidin (F-actin, red). Nasal epithelial cells were additionally stained with anti-Muc5AC antibodies (Muc, goblet cells, green). Bar represents 25 µm.

Figure 5

The induction and repression of specific cytokine and chemokines during SARS-CoV-2 infection of primary nasal epithelial cells. Primary nasal epithelial cells cultured on an air-liquid interface were infected with SARS-CoV-2 (BavPat1 isolate) at an MOI of 10 or mock infected. At the indicated time points, medium from the basolateral compartment was harvested and the concentration of the indicated cytokines and chemokines was analyzed by a bead-based immunoassay. Cytokines and chemokines with increased (A) and decreased (B) expression are shown. Means and SEM (shading) of n = 2 replicates are shown.

Figure 6

Berberine and obatoclax inhibit SARS-CoV-2 replication in primary nasal epithelial cells. Primary nasal epithelial cells cultured on an air-liquid interface were infected with SARS-CoV-2 (BavPat1 isolate) at an MOI of 10 in the presence of the indicated concentrations of (A) berberine (BBR) or (B) obatoclax (OLX) in a two-fold dilution series or 0.1% DMSO as a control. Viral RNA from duplicate apical surface supernatants at 72 hpi was quantified by qRT-PCR and plotted as percentage inhibition compared to the DMSO control. Error bars indicate SD (n = 2). Dashed line indicates 50% inhibition. Primary nasal epithelial cells cultured on an air-liquid interface were treated with the indicated concentrations of (C) BBR or (D) OLX or 0.1% DMSO. At 72 h, the ATP content in cells from duplicate samples was measured as a measure of cell viability, plotted as a percentage of the DMSO control. Error bars indicate SD. Dashed lines indicate 50% viability. A representative of two independent experiments is shown. The shaded grey box indicates that different OLX concentrations were used for antiviral (B) and viability assays (D).