Molecular Interactions of Tannic Acid with Proteins Associated with SARS-CoV-2 Infectivity

Abstract

The overall impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on our society is unprecedented. The identification of small natural ligands that could prevent the entry and/or replication of the coronavirus remains a pertinent approach to fight the coronavirus disease (COVID-19) pandemic. Previously, we showed that the phenolic compounds corilagin and 1,3,6-tri-O-galloyl-β-D-glucose (TGG) inhibit the interaction between the SARS-CoV-2 spike protein receptor binding domain (RBD) and angiotensin-converting enzyme 2 (ACE2), the SARS-CoV-2 target receptor on the cell membrane of the host organism. Building on these promising results, we now assess the effects of these phenolic ligands on two other crucial targets involved in SARS-CoV-2 cell entry and replication, respectively: transmembrane protease serine 2 (TMPRSS2) and 3-chymotrypsin like protease (3CLpro) inhibitors. Since corilagin, TGG, and tannic acid (TA) share many physicochemical and structural properties, we investigate the binding of TA to these targets. In this work, a combination of experimental methods (biochemical inhibition assays, surface plasmon resonance, and quartz crystal microbalance with dissipation monitoring) confirms the potential role of TA in the prevention of SARS-CoV-2 infectivity through the inhibition of extracellular RBD/ACE2 interactions and TMPRSS2 and 3CLpro activity. Moreover, molecular docking prediction followed by dynamic simulation and molecular mechanics Poisson-Boltzmann surface area (MMPBSA) free energy calculation also shows that TA binds to RBD, TMPRSS2, and 3CLpro with higher affinities than TGG and corilagin. Overall, these results suggest that naturally occurring TA is a promising candidate to prevent and inhibit the infectivity of SARS-CoV-2.

Keywords: 3CLpro; COVID-19; RBD; SARS-CoV-2; TMPRSS2; molecular dynamics; polyphenols.

Figure 1

Inhibitory effects of different polyphenols on the interaction between SARS-CoV-2 spike protein receptor binding domain (RBD (N501Y)) and human angiotensin-converting enzyme 2 (ACE2). (A) 10 µM of pelargonidin-3-O-glucoside (Pel-3-O-G), malvidin-3-O-glucoside (Mal-3-O-G), cyanidin-3-O-glucoside (Cya-3-O-G), peonidin, tannic acid (TA), 1,3,6-tri-O-galloyl-β-D-glucose (TGG), and corilagin were tested to evaluate their ability to inhibit the binding of immobilized spike protein (0.5 µg/mL) to human, biotin-labeled ACE2 (0.25 µg/mL) by using an enzyme-linked immunosorbent assay (ELISA). Dose effect inhibition of 0.1, 1, and 5 µM (B) TA, (C) TGG, and (D) corilagin. The absorbance of ACE2 (0.25 µg/mL) at 450 nm was set to 100%. Results are expressed as mean ± SD (n = 3). Statistical analysis was performed using one-way ANOVA followed by the Tukey post hoc test with * p < 0.05, ** p < 0.01, *** p < 0.001 compared to ACE2 (0.25 µg/mL).

Figure 2

Biophysical characterization of the molecular interaction between TA and RBD: (A) Binding of polyphenol TA to immobilized RBD by surface plasmon resonance (SPR). The recombinant protein RBD (N501Y) is immobilized on a carboxymethylated dextran (CM5) sensor chip, and increasing concentrations of TA are injected to evaluate binding kinetics. (B) RBD is adsorbed to a gold quartz crystal microbalance with dissipation monitoring (QCMD) sensor, and various concentrations of TA are flowed over the surface for 30 min. TA adsorption is shown by the dimensionless molar ratio of adsorbed TA from solution to adsorbed RBD. The initial slope is a measure of the affinity of TA to RBD.

Figure 3

Inhibitory effects of TA, TGG, and corilagin on human transmembrane protease serine 2 (TMPRSS2) activity. The effects of different concentrations (0.1 to 100 µM) of (A) TA, (B) TGG, and (C) corilagin are tested on the activity of TMPRSS2. The fluorescence units in control conditions are considered as 100%. Blank values are subtracted from all the readings before the conversion into percentage of activity. Results are expressed as mean ± SD (n = 3). Statistical analysis is performed using one-way ANOVA followed by Tukey post hoc test with *** p < 0.001 compared to positive control wells.

Figure 4

Biophysical characterization of the molecular interactions between TA and TMPRSS2. (A) The recombinant protein TMPRSS2 is immobilized on a CM5 sensor chip, and increasing concentrations of TA are injected to evaluate binding kinetics by SPR. (B) TMPRSS2 is adsorbed to a gold QCMD sensor, and various concentrations of TA are flowed over the surface for 30 min. TA adsorption is expressed by the dimensionless molar ratio of adsorbed TA to adsorbed TMPRSS2.

Figure 5

Inhibitory effects of TA, TGG, and corilagin on SARS-CoV-2 3-chymotrypsin like protease (3CLpro) activity. Different concentrations (0.1 to 100 µM) of (A) TA, (B) TGG, and (C) corilagin are tested on the activity of 3CLpro. The fluorescence units in control conditions are considered as 100%. Blank values are subtracted from all the readings before the conversion into percentage of activity. Results are expressed as mean ± SD (n = 3). Statistical analysis is performed using one-way ANOVA followed by the Tukey post hoc test with *** p < 0.001 compared to positive control wells.

Figure 6

Biophysical characterization of the molecular interactions between the polyphenol TA on immobilized 3CLpro: (A) The recombinant protein 3CLpro is immobilized on a CM5 sensor chip, and increasing concentrations of TA are injected to evaluate binding kinetics by SPR. (B) 3CLpro is adsorbed to a gold QCMD sensor, and various concentrations of TA are flowed over the surface for 30 min. TA adsorption is expressed by the dimensionless molar ratio of adsorbed TA to adsorbed 3CLpro.

Figure 7

Molecular structures and docking of TA/RBD complex: one possible structure of (A) TA, (B) RBD (N501Y) (molecular dynamics (MD) 100 ns), and (C) TA/RBD (N501Y) complex (pose 1; highest docking binding affinity of −6.8 kcal/mol). The N501Y mutation is yellow.

Figure 8

Molecular structures (pose 1) of: (A) TA/RBD, (B) TA/TMPRSS2, and (C) TA/3CLpro complexes, before (green) and after (turquoise) 1000-ns MD simulations.

Figure 9

Molecular structures after 1000 ns of MD: (A) TA/RBD complex (pose 1; molecular mechanics Poisson–Boltzmann surface area (MMPBSA) binding free energy of −66 kcal/mol) and (B) ligand interaction map. The interaction map of TA with RBD (N501Y) is shown from the center of the biggest cluster computed on the convergence interval using the protein backbone atoms and ligand non-hydrogen atoms. The other contacts, defined by a distance smaller than 0.40 nm between the ligand and the protein, are shown as red arcs. H-bonds and their donor/acceptor distances are shown in green. The interaction map is generated using LigPlot [50,51].

Figure 10

Molecular structures after 1000 ns of MD: (A) TA/TMPRSS2 complex (pose 1; MMPBSA binding free energy of −68 kcal/mol) and (B) ligand interaction map. The interaction map of TA with TMPRSS2 is shown from the center of the biggest cluster computed on the convergence interval using the protein backbone atoms and ligand non-hydrogen atoms. The other contacts, defined by a distance smaller than 0.40 nm between the ligand and the protein, are shown as red arcs. H-bonds and their donor/acceptor distances are shown in green. The interaction map is generated using LigPlot [50,51].

Figure 11

Molecular structures after 1000 ns of MD: (A) TA/3CLpro complex (pose 1, MMPBSA binding free energy of −65 kcal/mol); (B) ligand interaction map. The contact map of TA with 3CLpro is shown from the center of the biggest cluster computed on the convergence interval using the protein backbone atoms and ligand non-hydrogen atoms. The contacts, defined by a distance smaller than 0.40 nm between the ligand and the protein, are shown as red arcs. The interaction map is generated using LigPlot [50,51].