Bromhexine inhibits SARS-CoV-2 Omicron and variant pseudovirus infection via ACE2-targeted mechanisms

Figures

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  FIGURE 1. Immunofluorescence and Western blot analysis of ACE2 in HEK-293 and HEK-293/ACE2 cell lines. (A,C) Immunofluorescence localization of ACE2 proteins (red fluorescence). (B,D) Immunostaining images with primary antibodies omitted (control). All images show cell nuclei stained with DAPI (blue fluorescence). The scale bar represents 20 µm. (E) Representative immunoblots for ACE2 (90 kDa) and Actin (42 kDa) as a loading control are shown for HEK-293 cells and the stable cell line overexpressing human ACE2 (HEK-293/ACE2). (F) The relative abundance of ACE2 protein levels is expressed as the ratio of ACE2 to Actin band intensities. Data are shown as mean ± SEM from three independent experiments.

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  FIGURE 2. Effect of bromhexine on SARS-CoV-2 Omicron pseudovirus infectivity in HEK-293/ACE2 cells. (A,C,E) Representative fluorescence microscopy images of cells infected with Omicron pseudovirus treated with 1, 10, and 100 µM bromhexine, respectively. (B) Positive control (Omicron pseudovirus infection without bromhexine). (D) Negative control (cells without Omicron pseudovirus infection or bromhexine). (F) Quantitative analysis of Omicron pseudovirus infection in HEK-293/ACE2 cells, based on the percentage of GFP-positive cells after infection. GFP expression indicates successful pseudovirus entry. Data are presented as means ± SEM (n = 4) from at least three different experiments. An asterisk (*) indicates statistically significant differences (p < 0.05) compared to the positive control, determined by one-way ANOVA with post hoc Tukey HSD test.

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  FIGURE 3. Luciferase activity and IC50 determination on HEK-293/ACE2 cells. (A) Infectivity of Omicron pseudoviruses was assessed by measuring luciferase activity in relative luminescence units (RLUs) after infecting cells with the viruses. Data are shown as means ± SEM (n = 4). An asterisk (*) indicates statistically significant differences (p < 0.05) determined by one-way ANOVA with post hoc Tukey HSD test. (B) Dose-response curve used to determine the half-maximal inhibitory concentration (IC50) of bromhexine in HEK-293/ACE2 cells infected with Omicron pseudovirus, with an IC50 of 17.3 ± 0.9 µM. Results are presented as means ± SEM (n = 4). Curves are fitted to a 4-parameter logistic model and generated using the average of fitted parameters from individual experiments.

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  FIGURE 4. Reduction in infectivity of SARS-CoV-2 pseudovirus variants in HEK-293/ACE2 cells treated with bromhexine. Infectivity of pseudoviruses representing Alpha, Beta, and Delta SARS-CoV-2 variants was measured by luciferase activity, expressed in relative luminescence units (RLUs), 48 h after treatment with 40 μM bromhexine or a vehicle (control). The cells were infected with pseudoviruses engineered to express each variant. Data are shown as means ± SEM (n = 4). An asterisk (*) indicates statistically significant differences (p < 0.05) compared to the control group, determined by two-way ANOVA followed by post hoc Tukey HSD test.

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  FIGURE 5. Illustration of the root mean square deviation (RMSD) profiles of the hACE2-bromhexine complex over a 500 ns molecular dynamics simulation. The plot shows the RMSD progress of the hACE2 backbone (gray, left Y-axis) and bromhexine (light blue, right Y-axis) relative to their initial positions during the simulation. The protein backbone remains in a stable conformation with minimal fluctuations throughout the simulation. Bromhexine maintained a sustained binding within the pocket, without significant displacements over the simulation.

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  FIGURE 6. Molecular interactions between bromhexine and key hACE2 residues. (A) Interaction frequency plot of Phe40, Phe390, and Asn394 residues over 500 ns of MD simulations. Frequencies indicate the proportion of simulation time during which each interaction type was maintained (π–cation, hydrophobic, or hydrogen bond). Only residues with an interaction frequency of at least 0.2 during the 500 ns simulation are shown. (B) Structural representation of bromhexine in complex with hACE2 at the 500 ns snapshot. The spike RBD (blue) and hACE2 (white) are depicted as ribbons, with bromhexine shown in stick format. Dashed lines indicate π–π-cation (green), hydrophobic (magenta), and hydrogen bond (yellow) interactions with the specified residues.