Berbamine prevents SARS-CoV-2 entry and transmission

Abstract

Effective antiviral drugs are essential to combat COVID-19 and future pandemics. Although many compounds show antiviral in vitro activity, only a few retain effectiveness in vivo against SARS-CoV-2. Here, we show that berbamine (Berb) is effective against SARS-CoV, MER-CoV, SARS-CoV-2 and its variants, including the XBB.1.16 variant. In hACE2.Tg mice, Berb suppresses SARS-CoV-2 replication through two distinct mechanisms: inhibiting spike-mediated viral entry and enhancing antiviral gene expression during infection. The administration of Berb, in combination with remdesivir (RDV), clofazimine (Clof) and fangchinoline (Fcn), nearly eliminated viral load and promoted recovery from acute SARS-CoV-2 infection and its variants. Co-housed mice in direct contact with either pre-treated or untreated infected mice exhibited negligible viral loads, reduced lung pathology, and decreased viral shedding, suggesting that Berb may effectively hinder virus transmission. This broad-spectrum activity positions Berb as a promising preventive or therapeutic option against betacoronaviruses.

Keywords: drugs; health sciences; virology.

Graphical abstract

Figure 1

Berb inhibits SARS-CoV-2 and its VOCs entry (A) Chemical structure formula of Berbamine dihydrochloride and its molecular weight (MW). (B) Measurement of relative viral load between RDV and Berb by RT-PCR. ∗∗∗∗p < 0.0001 (One-way ANOVA; Tukey’s multiple comparison test). (C and D) Viral titer in VeroE6, A549 and Caco2 cells in the presence or absence of Berb (C), SARS-CoV-2 and its VOCs Supernatants were collected for quantification of viral titer by plaque assay. Data represent mean ± SEM, n = 3 biological replicates. two-way ANOVA followed by Tukey’s multiple comparison test (∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001); PFU, plaque-forming units (D). (E) Berb inhibited SARS-CoV-2 and its VOCs (multiplicity of infection (MOI) of 0.1) in veroE6 cells. Cell lysates were obtained to assess viral load, measured in relation to beta-actin. Data are mean ± SEM, n = 3 biological replicates. One-way ANOVA followed by Tukey’s multiple comparison test (∗p < 0.05, ∗∗p < 0.005, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001). (F and G) A time-of-addition assay was performed using Berb. VeroE6 cells were treated with Berb at different stages: during inoculation (entry stage), after inoculation (post-entry stage), or during both entry and post-entry stages of SARS-CoV-2 infection at an MOI of 3. The effectiveness of each treatment was assessed by measuring viral titers. The values shown are means ± SEM, of triplicate samples. ∗∗∗∗p < 0.0001 (two-way ANOVA followed by Tukey’s multiple comparison test). (H) Percentage entry inhibition of SARS-CoV-2. (I) VSV-based pseudotyped viral particle assay. Vero E6 cells were pre-treated with Berb and infected with SARS-CoV-2 S pseudotyped particles. Luciferase signals were quantified at 24 h after infection. Data are presented as mean ± SEM, n = 3 independent experiments. two-way ANOVA followed by Tukey’s-test (∗∗∗∗p < 0.0001). E−64D, a known coronavirus entry inhibitor, was used as a positive control. (J–L) Spike (SARS-CoV-2, B.1.617.2, and B.1.1.7) and hACE2 plasmids were co-transfected into BHK-21 cells, forming fusion structures observed through immunofluorescence. Data from three experiments in duplicates were analyzed for statistical significance using two-way ANOVA. Error bar represents mean values with ±SEM. Images were captured using an Olympus fluorescence microscope, and quantification was based on three randomly selected GFP+ areas. This experiment was repeated twice. Nuclei were stained with DAPI (blue), scale bar: 50 μm, magnification 20X. Statistical significance was determined via one-way ANOVA with multiple comparisons, using hACE2 expression as a control. Error bars depict mean values with ±SEM. ∗∗∗∗p < 0.0001. (M) Schematic representation of the SARS-CoV-2 infection experiment in an A549 airway tissue model. Cells were inoculated at the apical surface with SARS-CoV-2. Viral growth was monitored by titration of progeny virus in the mucus layer on the apical surface (two-way ANOVA followed by Tukey’s test; ∗p < 0.05, ∗∗∗∗p < 0.0001). (N) Molecular docking analysis of Berb on Ancestral, B.1.1.7, B.1.617.2, B.1.1.529, BA.2, BA.5 and XBB.1.16 variants. (A) Shows the binding of Berb at the interface of RBD-ACE2. (B–H) Insets show the close-up view of Berb binding at the interface. The stearic clash between Berb and superimposed structure of ACE2 is highlighted in transparent red, showing the hinderance caused by the inhibitor. The respective spike proteins of all strains are shown in Surf; Up conformation: orange and ACE protein is rendered in Lime; New cartoon, while Berb is shown in van der Waals (vDw) ice-blue, atom-type C: ice-blue and O:red. (O) Using flow cytometry, surface-expressed spike protein binding to soluble hACE2 was analyzed in HEK293T cells 36 h post-transfection (t test). The bar graph represents ±SEM. All experiments were performed twice to confirm the results. See also Figures S1 and S2 and Tables S1–S3.

Figure 2

Prophylactic treatment of Berb controls viral burden and disease in SARS-CoV-2 infected mice (A) Experimental design for prophylactic treatment in hACE2.Tg mice: Mice received intranasal inoculation of SARS-CoV-2 and Delta variant (1 × 10⁴ PFU). Prophylactic treatment was performed 12 h before and immediately after infection (100 mg/kg; oral/day) till 6 dpi. RDV was used as a comparator drug. At 6 dpi, a group of mice (n = 6) was euthanized for tissue collection. Survival was monitored in another subset (n = 7). (B–E) Body weight change (two-way ANOVA followed by Tukey’s multiple comparison test) and percent survival (Mantel-Cox test) in uninfected (n = 7), SARS-CoV-2, and B.1.617.2-infected mice treated with Berb (100 mg/kg/day; oral), vehicle or RDV (25 mg/kg; i.p.) are shown (n = 7 for each group); ∗p = 0.059. (F) Image shows gross morphological changes in lungs. Black arrows represent hemolytic areas of the lungs. (G) Lung hemorrhage was scored on a 0–5 scale, where 0 represents a healthy pink lung, and 5 indicate a uniformly discolored dark red lung. The bar graph illustrates the mean ± SEM (n = 6 mice per group) and statistical analysis was conducted using one-way ANOVA followed by Tukey’s multiple comparison test; ∗∗∗∗p < 0.0001. (H and I) Viral load and viral titer was measured in control, vehicle, and Berb-treated mice. Error bar represents ±SEM (two-way ANOVA followed by tukey’s multiple comparison test). (J) Immunostaining for SARS-CoV-N antigen (brown) in lung tissue sections (n = 5 mice per group; experiment was performed once; 100 μm scale bar). Statistical significance was determined by two-way ANOVA followed by Tukey’s multiple comparison test. (K) Lung sections were subjected to hematoxylin and eosin staining, and the bar graph illustrates histology scores for various parameters, including hyaline membrane, septal wall thickness, septal degradation, neutrophil infiltration, and protein debris. The images were captured at 40× magnification, with a scale bar of 100 μm. The study included n = 5 mice per group, and statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison test. Bar graph represents ±SEM. (L–N) the experimental design is illustrated: four mice were infected with 1 × 10⁴ PFU of SARS-CoV-2 (infected mice), and another group (contact mice) was cohoused with them (L). Prophylactic Berb treatment was performed 12 h before and immediately after infection (100 mg/kg; oral/day). Each group comprised four infected and four contact mice. Measurements of percentage body weight change (M) and viral load (N) were measured at 6 dpi in infected mice and contact mice. Infected mouse groups received either vehicle or 100 mg/kg of Berb (oral). Values represent means ± SEM. Statistical analysis used two-way ANOVA followed by Tukey’s multiple comparison test, where ∗p < 0.05 and ∗∗∗∗p < 0.0001 indicate significance. (O) The experimental design is illustrated: four mice were infected with 1 × 10⁴ PFU of SARS-CoV-2 (infected mice), and another group (pre-treated contact mice; treated 12 h before cohousing till 6 dpi) was cohoused with them. Each group comprised four infected and four contact mice. Viral load was measured at 6 dpi in infected and contact mice. Contact mice groups received 100 mg/kg of Berb. ∗∗∗∗p < 0.0001; values represent means ± SEM. Statistical analysis used two-way ANOVA followed by Tukey’s multiple comparison test, where ∗∗∗∗p < 0.0001 indicates significance. All experiments were performed twice to confirm the results.

Figure 3

Berb retains antiviral activity against Alpha, BA.1, BA.2, BA.5, and XBB.1.16 in vivo Mice were intranasally inoculated with 1 × 10⁴ PFU of Alpha, and Omicron variants (BA.1, BA.2, and BA.5). The mice were treated with Berb (100 mg/kg) or vehicle (0 mg/kg) b.i.d. from the time of inoculation (0 hpi) to 5 dpi (n = 5 for each group). (A and B) Percentage change in weight (n = 5) and survival (n = 5) was measured in Alpha infected and Berb-treated mice (two-way ANOVA followed by Tukey’s multiple comparison test; ∗∗∗∗p < 0.0001). (C–F) Percentage change in weight (BA.1, BA.2, and BA.5) and survival (Wilcoxon test) was measured in BA.5 infected and Berb-treated mice. (G) Percentage change in weight was measured in XBB.1.16 infected and treated mice. The bar represents ±SEM. two-way ANOVA followed by Tukey’s multiple comparison test (body weight); ∗∗∗∗p < 0.0001. (H–K) The image shows gross lung morphological changes, and the bar graph represents lung hemorrhage score. 0–5, where 0 is a normal pink healthy lung, and 5 is a diffusely discolored dark red lung (black arrow indicates color of the lungs). Bar graph represents ±SEM. One-way ANOVA followed by Tukey’s multiple comparison test; ∗p < 0.05, ∗∗p < 0.005, ∗∗∗p < 0.0005, ∗∗∗∗p < 0.0001. (L and M) Virus titers in the lungs were measured by plaque assay. The values shown are means ± SEM. Statistical significance was measured by one-way ANOVA followed by Tukey’s multiple comparison test; ∗p < 0.05, ∗∗p < 0.005, ∗∗∗p < 0.0002. (N) Lung sections were subjected to hematoxylin and eosin staining, and the bar graph illustrates histology scores as mentioned previously. The images were captured at 40× magnification, with a scale bar of 100 μm. Two-way ANOVA followed by Tukey’s multiple comparison test. Bar graph represents ±SEM. See also Figure S3.

Figure 4

Therapeutic treatment with Berb decreased the viral load of SARS-CoV-2 and reduces disease severity in mice (A) The pictorial diagram shows the therapeutic treatment in a mouse model. Mice were intranasally inoculated with 10⁴ PFU of SARS-CoV-2 Delta variant. For therapeutic treatment, the mice were administration with Berb (100 mg/kg, oral; daily) or vehicle (1% DMSO in PBS) from 12 hpi to 6 dpi. RDV (15 mg/kg; i.p.) was used as a positive comparator drug. (B and C) Percentage change in body weight (n = 6 mice per group; two-way ANOVA; Tukey’s test) and percent survival (n = 7 mice per group; Wilcoxon test) were measured in delta variant infected and treated mice. (D) Image represents gross lung morphological changes in infected and Berb-treated mice (n = 5). The bar graph represents lung hemorrhage score. Values represents +SEM; One-way ANOVA followed by Tukey’s test. (E) Relative viral load was determined by RT-PCR and normalized to Actin-β. Bar graph represents +SEM (One-way ANOVA followed by Tukey’s multiple comparison test). (F and G) Relative mRNA expression analysis of CXCL10 and IL-10 was determined by RT-PCR; ∗p < 0.05. (H and I) Images represent H&E staining (H) and IHC (I) of the lung tissues, respectively. One-way ANOVA followed by Tukey’s multiple comparison test. Bar graph represents +SEM; n = 5 mice per group.

Figure 5

Berb promotes host mediated protective response RNA was isolated from infected (SARS-CoV-2 and B.1.617.2) and Berb-treated mice. cDNA was synthesized using applied biosystem c-DNA kit. (A and B) Relative mRNA expression of ISGs Irf3, Tbk1, IFN-β, and IFITM (one-way ANOVA; Tukey’s multiple comparison test). Bar graph represents ±SEM (ns = non-significant). (C) relative mRNA expression of chemokine CXCL10, measured by qPCR from lung samples (n = 4) (one-way ANOVA followed by Tukey’s, multiple comparison test); bar graph represents as a mean ± SEM. (D and E) antiviral genes (Oas1g and MX2) profile by RT-PCR from lung samples (n = 4) (one-way ANOVA followed by Tukey’s, multiple comparison test). (F and G) The effect of Berb on SARS-CoV and MESR-CoV virus inhibition was determined by neutralization assay. Table represents IC₅₀ values. See also Figure S5.