Natural Polyphenols, 1,2,3,4,6-O-Pentagalloyglucose and Proanthocyanidins, as Broad-Spectrum Anticoronaviral Inhibitors Targeting Mpro and RdRp of SARS-CoV-2

The natural plant dietary polyphenols 1,2,3,4,6-O-Pentagalloylglucose (PGG) and proanthocyanidin (PAC) have potent antioxidant activity and a variety of pharmacological activities, including antiviral activity. In this study, we examined the inhibitory effect of PGG and PAC on SARS-CoV-2 virus infection, and elucidated its mode of action. PGG and PAC have dose-dependent inhibitory activity against SARS-CoV-2 infection in Vero cells. PGG has a lower IC50 (15.02 ± 0.75 μM) than PAC (25.90 ± 0.81 μM), suggesting that PGG has better inhibitory activity against SARS-CoV-2 than PAC. The PGG and PAC inhibit similar Mpro activities in a protease activity assay, with IC50 values of 25–26 μM. The effects of PGG and PAC on the activity of the other essential SARS-CoV-2 viral protein, RdRp, were analyzed using a cell-based activity assay system. The activity of RdRp is inhibited by PGG and PAC, and PGG has a lower IC50 (5.098 ± 1.089 μM) than PAC (21.022 ± 1.202 μM), which is consistent with their inhibitory capacity of SARS-CoV-2 infection. PGG and PAC also inhibit infection by SARS-CoV and MERS-CoV. These data indicate that PGG and PAC may be candidate broad-spectrum anticoronaviral therapeutic agents, simultaneously targeting the Mpro and RdRp proteins of SARS-CoV-2.

The SARS-CoV-2 genome shares 79% sequence identity with SARS-CoV, and 50% with MERS-CoV. Four structural proteins (spike proteins, envelope proteins, membrane proteins, and nucleocapsid proteins) and sixteen nonstructural proteins are encoded. Among them, the main protease, or 3C-like protease (Mpro or 3CLpro), a papain-like protease (PLpro), and an RNA-dependent RNA polymerase (RdRp) are essential for virus transcription and replication, and for potential targets for antiviral agents [4]. The sequence identity of Mpro in SARS-CoV-2 and SARS-CoV is approximately 96%, and SARS-CoV-2 RdRp has a 96% sequence identity to SARS-CoV RdRp, so resistance to Mpro or RdRp inhibitors in coronavirus variants is likely to be low [5,6]. SARS-CoV-2 Mpro and RdRp have a substrate specificity unique to the virus and are absent in humans, so a low toxicity is expected. The FDA approved the COVID-19 antiviral drugs Veklury (remdesivir) and Lagevrio (molnupiravir), which inhibit viral RdRp activity, and Paxlovid (nirmatrelvir/ritonavir), which inhibits Mpro activity, for the treatment of COVID-19 [7][8][9][10]. Although the therapeutic benefits are clear, these antiviral drugs have been reported to have serious adverse side effects. The side effects of Veklury include respiratory failure and organ dysfunction, and Lagevrio may cause fetal harm. Paxlovid is contraindicated with drugs which interact with CYP3A (e.g., α1-adrenoreceptor antagonists, HMG-CoA reductase inhibitors, antipsychotics, PDE5 inhibitors, sedatives, antimycobacterials, and anticonvulsants), and it may cause serious and life-threatening reactions or the loss of antiviral efficacy. Moreover, the development of more efficient broad-spectrum anticoronaviral therapeutics against new emerging coronavirus variants is still needed to overcome the COVID-19 pandemic and counteract new emerging coronaviral diseases.
In this study, we evaluated the broad-spectrum anticoronaviral activity of PGG and PAC against SARS-CoV-2, SARS-CoV, and MERS-CoV. To elucidate the mode of action of PGG and PAC, we examined their target viral proteins, such as Mpro, PLpro, and RdRp, using Mpro and PLpro protease activity assays, and a cell-based RdRp activity assay. This study identifies PGG and PAC as potentially valuable natural compound therapeutic candidates, which may inhibit emerging coronavirus infections, targeting two main viral proteins, Mpro and RdRp, simultaneously.
Biomedicines 2022, 10, x FOR PEER REVIEW dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA) and stored at −80 °C use. Compounds were prepared to the indicated concentrations of up to 100 μM wit bovine serum-free medium before use. The concentration of DMSO in this experime not exceed 0.5%.

SARS-CoV-2 PLpro Activity Inhibition Assays
The enzymatic activity of SARS-CoV-2 PLpro was detected using SARS-CoV-2 PLpro assay kits (BPS Bioscience), according to the manufacturer's instructions. The fluorescence intensity was measured at 360 nm/460 nm using a Synergy H1 microplate reader (Bio-Tek). GRL061 (BPS Bioscience) was used as a positive control.

Statistical Analysis
Data were presented as the mean ± standard error of at least two independent experiments. The half-maximal inhibitory concentration (IC 50 ) was calculated by nonlinear regression analysis using GraphPad Prism ® 9 (GraphPad Software Inc., San Diego, CA, USA). Probability (p) values were analyzed by one-way analysis of variance (ANOVA), followed by Bonferroni's multiple comparison test using GraphPad Prism ® 9 (GraphPad Software Inc.), as indicated in the figure legends (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001).

PGG and PAC Inhibited Infection with SARS-CoV-2
To examine the effect of PGG and PAC on SARS-CoV-2 infection, immunofluorescencebased antiviral assays were conducted using SARS-CoV-2-infected Vero cells treated with a serially diluted concentration of PGG or PAC and through detecting the SARS-CoV-2 nucleocapsid antigen after 24 h of infection. The data indicated that the CC 50 values of PGG and PAC were >50 µM. PGG and PAC dose-dependently inhibited SARS-CoV-2 infection. The IC 50 values of PGG and PAC were 15.02 ± 0.75 µM and 25.90 ± 0.81 µM, respectively ( Figure 2). These data suggested that PGG and PAC had anti-SARS-CoV-2 activity, and that PGG had better inhibitory activity against SARS-CoV-2 than PAC.

PGG and PAC Inhibited the Mpro Activity of SARS-CoV-2
To elucidate the mode of action of PGG and PAC, the effect of those compounds on the activity of the viral protease, Mpro, was examined using SARS-CoV-2 Mpro activity assay kits. When the Mpro protein was incubated with concentrations of PGG or PAC serially diluted from 100 μM, these compounds inhibited the Mpro activity in a dose-dependent manner ( Figure 3). The IC50 values of PGG and PAC were 25.26 ± 1.04 μM and 26.00 ± 0.81 μM, respectively. These data showed that PGG and PAC inhibited the SARS-CoV-2 Mpro activity to similar extents. However, 25 μM of PGG and PAC did not inhibit the activity of another SARS-CoV-2 viral protease, PL protease, under our PLpro protein activity assay conditions ( Figure S1).

PGG and PAC Inhibited the Mpro Activity of SARS-CoV-2
To elucidate the mode of action of PGG and PAC, the effect of those compounds on the activity of the viral protease, Mpro, was examined using SARS-CoV-2 Mpro activity assay kits. When the Mpro protein was incubated with concentrations of PGG or PAC serially diluted from 100 µM, these compounds inhibited the Mpro activity in a dose-dependent manner (Figure 3). The IC 50 values of PGG and PAC were 25.26 ± 1.04 µM and 26.00 ± 0.81 µM, respectively. These data showed that PGG and PAC inhibited the SARS-CoV-2 Mpro activity to similar extents. However, 25 µM of PGG and PAC did not inhibit the activity of another SARS-CoV-2 viral protease, PL protease, under our PLpro protein activity assay conditions ( Figure S1).

PGG and PAC Inhibited the Mpro Activity of SARS-CoV-2
To elucidate the mode of action of PGG and PAC, the effect of those compounds on the activity of the viral protease, Mpro, was examined using SARS-CoV-2 Mpro activity assay kits. When the Mpro protein was incubated with concentrations of PGG or PAC serially diluted from 100 μM, these compounds inhibited the Mpro activity in a dose-dependent manner (Figure 3). The IC50 values of PGG and PAC were 25.26 ± 1.04 μM and 26.00 ± 0.81 μM, respectively. These data showed that PGG and PAC inhibited the SARS-CoV-2 Mpro activity to similar extents. However, 25 μM of PGG and PAC did not inhibit the activity of another SARS-CoV-2 viral protease, PL protease, under our PLpro protein activity assay conditions ( Figure S1).

PGG and PAC Inhibited the RdRp Activity of SARS-CoV-2
We also examined whether PGG and PAC could inhibit the activity of the essential viral protein, RdRp. We previously generated a cell-based SARS-CoV-2 RdRp activity assay system [32] and used it for determining the effect of PGG and PAC on the activity of RdRp using a treatment with serially diluted concentrations of PGG and PAC. In this system, the expression level of NLuc represented the activity of SARS-Co-2 RdRp, and the expression level of FLuc was used as the internal control to normalize NLuc activity, as previously described. The expression levels of NLuc were decreased in a dose-dependent manner through treatment with PGG or PAC. The levels of FLuc, the internal control, were maintained, suggesting that PGG and PAC inhibited the SRAS-CoV-2 RdRp activity without cytotoxicity or the inhibition of host transcription (Figure 4). Data showed that the IC 50 values of PGG and PAC were 5.098 ± 1.089 µM and 21.022 ± 1.202 µM, respectively. These data suggest that PGG and PAC had inhibitory activity against the RdRp of SARS-CoV-2, and PGG had stronger inhibitory activity against the RdRp of SARS-CoV-2 than PAC. These findings were consistent with those that PGG had better inhibitory activity against SARS-CoV-2 than PAC, although these compounds had similar inhibitory activity against Mpro. Taken together, these results indicated that PGG and PAC inhibited SARS-CoV-2 infection by targeting the Mpro and RdRp of SARS-CoV-2. and 100 μM GC376 was used as a positive control. The IC50 values are presented by nonlinear regression analysis (right panel). Statistical comparisons were conducted using one-way analysis of variance (ANOVA), followed by Bonferroni's multiple comparison test. * p < 0.05; *** p < 0.001 vs. 0 μM. The data represent triplicate experiments, and are presented as the mean ± SEM.

PGG and PAC Inhibited the RdRp Activity of SARS-CoV-2
We also examined whether PGG and PAC could inhibit the activity of the essential viral protein, RdRp. We previously generated a cell-based SARS-CoV-2 RdRp activity assay system [32] and used it for determining the effect of PGG and PAC on the activity of RdRp using a treatment with serially diluted concentrations of PGG and PAC. In this system, the expression level of NLuc represented the activity of SARS-Co-2 RdRp, and the expression level of FLuc was used as the internal control to normalize NLuc activity, as previously described. The expression levels of NLuc were decreased in a dose-dependent manner through treatment with PGG or PAC. The levels of FLuc, the internal control, were maintained, suggesting that PGG and PAC inhibited the SRAS-CoV-2 RdRp activity without cytotoxicity or the inhibition of host transcription (Figure 4). Data showed that the IC50 values of PGG and PAC were 5.098 ± 1.089 μM and 21.022 ± 1.202 μM, respectively. These data suggest that PGG and PAC had inhibitory activity against the RdRp of SARS-CoV-2, and PGG had stronger inhibitory activity against the RdRp of SARS-CoV-2 than PAC. These findings were consistent with those that PGG had better inhibitory activity against SARS-CoV-2 than PAC, although these compounds had similar inhibitory activity against Mpro. Taken together, these results indicated that PGG and PAC inhibited SARS-CoV-2 infection by targeting the Mpro and RdRp of SARS-CoV-2.  Statistical comparisons were conducted using one-way analysis of variance (ANOVA), followed by Bonferroni's multiple comparison test. * p < 0.05; *** p < 0.001; **** p < 0.0001 vs. 0 µM (left graph). The IC 50 values were determined using nonlinear regression analysis (right graph). The data are representative of three independent experiments, and presented as mean ± SEM.

PGG and PAC Inhibited Infection with SARS-CoV and MERS-CoV
We further examined whether PGG and PAC could inhibit other coronaviruses, SARS-CoV and MERS-CoV, to identify whether these compounds have broad-spectrum anticoronaviral effects. As in the SARS-CoV-2 experiment, immunofluorescence-based antiviral assays were conducted in 0.05 MOI SARS-CoV-infected Vero cells, treated with a serially diluted concentration of PGG or PAC, and the expression of the SARS-CoV spike protein was detected using an anti-SARS-CoV spike protein antibody, 24 h postinfection ( Figure 5). The data showed that PGG and PAC dose-dependently inhibited SARS-CoV infection, with IC 50 values of 15.67 ± 1.02 µM and IC 50 15.19 ± 0.28 µM, respectively, with a CC 50 > 50 µM. These results suggested that PGG and PAC could inhibit the SARS-CoV infection in Vero cells to a similar degree.
The IC50 values were determined using nonlinear regression analysis (right graph). The data are representative of three independent experiments, and presented as mean ± SEM.

PGG and PAC Inhibited Infection with SARS-CoV and MERS-CoV
We further examined whether PGG and PAC could inhibit other coronaviruses, SARS-CoV and MERS-CoV, to identify whether these compounds have broad-spectrum anticoronaviral effects. As in the SARS-CoV-2 experiment, immunofluorescence-based antiviral assays were conducted in 0.05 MOI SARS-CoV-infected Vero cells, treated with a serially diluted concentration of PGG or PAC, and the expression of the SARS-CoV spike protein was detected using an anti-SARS-CoV spike protein antibody, 24 h postinfection ( Figure 5). The data showed that PGG and PAC dose-dependently inhibited SARS-CoV infection, with IC50 values of 15.67 ± 1.02 μM and IC50 15.19 ± 0.28 μM, respectively, with a CC50 > 50 μM. These results suggested that PGG and PAC could inhibit the SARS-CoV infection in Vero cells to a similar degree. To assess the effect of PGG and PAC on MERS-CoV infection, immunofluorescencebased antiviral assays performed with 0.0625 MOI MERS-CoV-infected Vero cells were detected using anti-MERS-CoV spike protein, 24 h postinfection, and then analyzed. The data showed that PGG and PAC dose-dependently inhibited MERS-CoV infection, with IC50 values of 3.466 ± 0.231 μM and 4.949 ± 0.181 μM, respectively ( Figure 6). Therefore, these data suggested that PGG and PAC could inhibit the coronaviruses SARS-CoV and MERS-CoV, as well as SARS-CoV-2.

Discussion
The polyphenolic natural products PGG and PAC are found in fruits, and exhibit strong antioxidant activity and a variety of pharmacological activities. The inhibitory effects of PGG and PAC on coronavirus infections were examined, and we found that PGG and PAC exhibited anticoronaviral activity against SARS-CoV and MERS-CoV, as well as SARS-CoV-2 in Vero cells, by simultaneously inhibiting the activity of Mpro and RdRp in SARS-CoV-2.
It has previously been reported that PGG blocks the entry of HCV [22] and human respiratory syncytial virus cell entry by using the pseudotyped lentiviral system [33]. Recently, the binding between PGG and the SARS-CoV-2 RBD protein has been shown using molecular docking and biolayer interferometry binding assays, and the blocking of SARS-CoV-2-RBD binding to hACE2 was shown by ELISA, immunocytochemistry assays, and spike protein RBD-pseudotyped lentivirus infection experiments [34]. PGG has also been reported to inhibit viral protein activity, such as HIV-1 integrase activity [19] and the activity of the neuraminidase of the influenza A virus [35]. Hydrolyzed tannins, including PGG, were shown to bind to Mpro proteins using surface plasmon resonance and molecular docking, as well as to inhibit the activity of Mpro in protein activity assays [36]. PAC was reported to inhibit SARS-CoV-2 replication by directly binding to the SARS-CoV-2-E channel [37]. The derivatives of flavan-3-ols, similar to PAC, procyanidin A2 (PA2), and procyanidin B2 (PB2), were shown to bind in the binding pocket of Mpro in docking simulation data. However, only PB2, not PA2, inhibited Mpro activity in Mpro protein activity assays [38]. In our data, PAC and PGG were compared and shown to inhibit Mpro activity. Overall, these data suggest that the presence of more hydroxy groups, galloylation, and the oligomerization of polyphenols such as PGG and PAC induce stronger inhibitory activity of Mpro [36], as it was previously discovered that higher numbers of hydroxyl groups of polyphenols produced enhanced antioxidant activity [11].
In this study, we demonstrated an inhibitory effect of PGG and PAC on SARS-CoV-

Discussion
The polyphenolic natural products PGG and PAC are found in fruits, and exhibit strong antioxidant activity and a variety of pharmacological activities. The inhibitory effects of PGG and PAC on coronavirus infections were examined, and we found that PGG and PAC exhibited anticoronaviral activity against SARS-CoV and MERS-CoV, as well as SARS-CoV-2 in Vero cells, by simultaneously inhibiting the activity of Mpro and RdRp in SARS-CoV-2.
It has previously been reported that PGG blocks the entry of HCV [22] and human respiratory syncytial virus cell entry by using the pseudotyped lentiviral system [33]. Recently, the binding between PGG and the SARS-CoV-2 RBD protein has been shown using molecular docking and biolayer interferometry binding assays, and the blocking of SARS-CoV-2-RBD binding to hACE2 was shown by ELISA, immunocytochemistry assays, and spike protein RBD-pseudotyped lentivirus infection experiments [34]. PGG has also been reported to inhibit viral protein activity, such as HIV-1 integrase activity [19] and the activity of the neuraminidase of the influenza A virus [35]. Hydrolyzed tannins, including PGG, were shown to bind to Mpro proteins using surface plasmon resonance and molecular docking, as well as to inhibit the activity of Mpro in protein activity assays [36]. PAC was reported to inhibit SARS-CoV-2 replication by directly binding to the SARS-CoV-2-E channel [37]. The derivatives of flavan-3-ols, similar to PAC, procyanidin A2 (PA2), and procyanidin B2 (PB2), were shown to bind in the binding pocket of Mpro in docking simulation data. However, only PB2, not PA2, inhibited Mpro activity in Mpro protein activity assays [38]. In our data, PAC and PGG were compared and shown to inhibit Mpro activity. Overall, these data suggest that the presence of more hydroxy groups, galloylation, and the oligomerization of polyphenols such as PGG and PAC induce stronger inhibitory activity of Mpro [36], as it was previously discovered that higher numbers of hydroxyl groups of polyphenols produced enhanced antioxidant activity [11].
In this study, we demonstrated an inhibitory effect of PGG and PAC on SARS-CoV-2 infection in vitro. PGG had better inhibitory activity against SARS-CoV-2 infection than PAC. We also showed that PGG and PAC inhibited Mpro activity in protein activity assays to a similar extent, and that the activity of the other viral protease, PLpro, of SARS-CoV-2 was not inhibited by PGG or PAC under these experimental conditions. It was first reported that the activity of another essential viral protein, RdRp, was also dose-dependently inhibited by PGG and PAC, and PGG had a stronger inhibitory effect on SARS-CoV-2 RdRp activity than PAC. These observations suggested that the better inhibitory activity of PGG against SARS-CoV-2 was due to the better inhibition of the RdRp activity, despite its similar inhibitory activity against Mpro. We also demonstrated that PGG and PAC inhibited the other coronaviruses, SARS-CoV and MERS-CoV, suggesting that PGG and PAC have broadspectrum anticoronaviral activity, which is particularly important in light of the ongoing emergence of coronavirus variants. However, the IC 50 of PGG and PAC was much higher than those of the FDA-approved COVID-19 antiviral drug. Toxicity studies of PGG and PAC should be performed with the effective concentrations to exert an antiviral effect in the blood. Furthermore, we need to evaluate the proof of concept of PGG and PAC in in vivo experiments, pharmacokinetic properties (microsomal stability, hERG inhibition, CYP450 inhibition, etc.), and the potential transition to human trials, or the antiviral hygienic applications of PGG and PAC.

Conclusions
These findings suggest that PGG and PAC might be effective broad-spectrum anticoronaviral therapeutic candidates. The mode of action of PGG and PAC by targeting the Mpro and RdRp viral proteins of SARS-CoV-2 could provide valuable information for the development of therapeutic candidates against emerging coronaviral infections.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/biomedicines10051170/s1, Figure S1: The effect of PGG and PAC on the SARS-CoV-2 PLpro activity. PGG and PAC (25 µM) did not affect the PLpro activity of SARS-CoV-2. GRL061 was used as a positive control.