Theaflavin 3-gallate inhibits the main protease (Mpro) of SARS-CoV-2 and reduces its count in vitro

Abstract

The main protease (M^pro) of SARS-CoV-2 has been recognized as an attractive drug target because of its central role in viral replication. Our previous preliminary molecular docking studies showed that theaflavin 3-gallate (a natural bioactive molecule derived from theaflavin and found in high abundance in black tea) exhibited better docking scores than repurposed drugs (Atazanavir, Darunavir, Lopinavir). In this study, conventional and steered MD-simulations analyses revealed stronger interactions of theaflavin 3-gallate with the active site residues of M^pro than theaflavin and a standard molecule GC373 (a known inhibitor of M^pro and novel broad-spectrum anti-viral agent). Theaflavin 3-gallate inhibited M^pro protein of SARS-CoV-2 with an IC₅₀ value of 18.48 ± 1.29 μM. Treatment of SARS-CoV-2 (Indian/a3i clade/2020 isolate) with 200 μM of theaflavin 3-gallate in vitro using Vero cells and quantifying viral transcripts demonstrated reduction of viral count by 75% (viral particles reduced from Log10^6.7 to Log10^6.1). Overall, our findings suggest that theaflavin 3-gallate effectively targets the M^pro thus limiting the replication of the SARS-CoV-2 virus in vitro.

Figure 1

Inhibition of M^pro protein of SARS-CoV-2 by theaflavin 3-gallate. The inhibition of M^pro protein by (a) theaflavin (positive control), (b) theaflavin 3-gallate, and (c) GC376 (positive control) was measured in the presence of increasing concentrations of these molecules. The structures of molecules are shown along with the IC₅₀ curves. Dose–response curves for IC₅₀ values were determined by non-linear regression. Data represent mean ± SE, n = 3 independent replicates.

Figure 2

Effect of theaflavin 3-gallate on the inhibition of SARS-CoV-2. Response to theaflavin (positive control), theaflavin 3-gallate, and remdesivir (positive control) in Vero cells at 50, 100, 150, and 200 μM was calculated using quantitative PCR of N and E viral genes. Graphs represent relative viral RNA % (a,b) and log reduction in viral particles (c,d) after treatment with theaflavin, theaflavin 3-gallate, and positive control remdesivir, respectively.

Figure 3

Analysis of docking results. (a) Different domains of M^pro and 3D representations of docking poses for (b) GC373, (c) theaflavin, and (d) theaflavin 3-gallate.

Figure 4

Analysis of MD trajectories. (a) RMSD of backbone Cα atoms and (b–d) number of H-bonds formed between M^pro and ligands during the entire simulation. The color-coding scheme is as follows: GC373 (black), theaflavin (red), and theaflavin 3-gallate (green).

Figure 5

Analysis of SMD results showing: (a) typical external force profiles of GC373 (black), theaflavin (red), and theaflavin 3-gallate (green); the position of (b) GC373, (c) theaflavin, and (d) theaflavin 3-gallate at different time intervals during SMD simulations. The color-coding is as follows: 100 ns (blue), 194 ns (magenta), 242 ns (orange), and 400 ns (cyan).