Within the past two decades, three extremely pathogenic and deadly human coronaviruses namely, SARS-CoV, MERS, SARS-CoV-2 have emerged28. They belong to the group β-coronavirus and draw more attention because of their ability to cross animal-human barriers, ultimately becoming major human pathogens29. The timely development of antiviral substances is of utmost importance, which in such a short span is extremely challenging. Moreover, the conventional drug takes years to develop and to get into the market. Drug repurposing is a choice in which the molecules/compounds already known for their therapeutic effects can be screened and tested for inhibition of SARS-CoV-2.
Since time immemorial, natural compounds have been used as therapeutic agents for the treatment of several ailments30. Plant-based natural compounds offer a rich reservoir for novel antiviral drug development. Some natural medicines have been reported to possess antiviral activities against many virus strains including herpes simplex virus, coronavirus, influenza virus, hepatitis B and C viruses, and HIV virus31–33. Our previous in-silico studies showed that tea molecule theaflavin-3-O-gallate has the affinity to bind to the substrate-binding pocket of Mpro of SARS-CoV-234. To quantify the inhibition on a scale, we tested theaflavin 3-gallate against Mpro protein of SARS-CoV-2, and calculated its IC50. The IC50 is a quantitative measure that represents how much of a particular inhibitory substance is needed to inhibit a given biological process or biological component by 50% under in vitro conditions. With our aforesaid method, the IC50 values of theaflavin 3-gallate against Mpro was calculated to be 18.48 ± 1.29 µM. Also, incubation of theaflavin 3-gallate with SARS-CoV-2 led to inhibition of the virus as evident by reduction of viral count by 75% at a concentration of 200 µM.
To understand the mechanism of inhibitory action of theaflavin 3-gallate we performed molecular docking studies which provide a platform to predict the estimated binding affinity and optimal binding pose between receptor and ligand. The binding poses were compared with a standard drug GC373 and theaflavin. This method has been implemented for more than three decades and a significant number of experimental drugs have been identified and developed accordingly14,15,35. Our molecular docking analysis showed that theaflavin 3-gallate interacts strongly with the binding sites on Mpro with a higher docking score than GC373 and theaflavin. Our results showed that Theaflavin 3-gallate acquired the active site of Mpro by interacting with many residues (Asn142, Ser144, His145 His163, and Glu166) crucial for dimerization and biological activity. Many experimental and computational studies have shown potent inhibitor molecules interacting with these residues7,16,17,23,36,37. The stability of binding poses was validated by analysis of different MD-driven time-dependent analyses. The low deviations in RMSD values for all the three structures suggested that the binding of ligands on protein had no impact on stability of the binding pocket. We also compared the number of H-bonds formed by GC373, theaflavin, and theaflavin 3-gallate with the binding site of Mpro throughout the simulation. The analysis of the H-bond profiles of the three molecules showed that theaflavin 3-gallate formed highest number of bonds during the simulation. To get an in-depth insight of the molecular interactions during the simulations, protein-ligand conformations at different time intervals were extracted from the MD trajectories. In comparison to GC373 and theaflavin, theaflavin 3-gallate formed exclusive H-bonds with residues His41, Val186, and His164 at different time intervals during the simulation. These interactions were not observed for GC373 and theaflavin. Also, theaflavin 3-gallate interacted with residue Cys145 by much stronger interactions than theaflavin. The residue Cys145 is involved in the formation of catalytic dyad of the active site of Mpro 7. The strong interactions shown by theaflavin 3-gallate with Mpro were further validated and compared with GC373 and theaflavin by caculating the binding free energy by the MM-PBSA method. The MM-PBSA is an efficient, reliable method for evaluating protein-ligand interactions38,39. The binding free energy results confirmed that theaflavin 3-gallate showed the highest binding affinity for Mpro than GC373 and theaflavin. Our results showed that the van der Waal energy contributed most favorably to the binding of theaflavin 3-gallate with Mpro. Moreover, we also performed SMD simulations to analyze the amount of external force required for unbinding GC373, theaflavin, and theaflavin 3-gallate from the binding pocket. Our SMD results demonstrated that theaflavin 3-gallate required highest amount of external force to unbind it from the binding pocket of the Mpro of SARS-CoV-2. Also, theaflavin 3-gallate remained at the active site for a longer duration than both the GC373 and theaflavin during the pulling simulations, suggesting strong interactions with the residues of the binding site.
In conclusion, in-silico and experimental results suggested theaflavin 3-gallate as a potential candidate molecule that could be rapidly developed as a therapeutic agent to fight COVID-19. Theaflavin 3-gallate performed better than the standard molecule GC373 and theaflavin in both the in-silico and experimental analyses. Theaflavin 3-gallate is a major component of black which is already known for its anti-oxidant properties and is the most consumed beverage in the world. Since theaflavin 3-gallate is already consumed by humans through tea for ages and being edible, crossing cell-cell barriers in the body40 makes it a good potential inhibitor to be used against SARS-CoV-2. Either the molecule alone or in formulations with other such anti-viral compounds as a cocktail can provide an effective first line of defense against diseases associated with coronaviruses.