American Study Shows That Plant Flavonols And Dihydroflavonols Show Potential As Antivirals As They Inhibit Mpro Of SARS-Cov-2 In Vitro

As the COVID-19 pandemic fast accelerates into a more catastrophic mode and there are still no effective antivirals to date plus antibody therapies as well as vaccines are beginning to show signs of being defeated by the new emerging variants, there is a dire need for effective therapeutics against the SARS-CoV-2 coronavirus and various study teams around the world are frantically searching for prospective antiviral candidates.


 
Researchers and scientists are now finally turning to herbs and phytochemicals for possible solutions.
 
A team of researchers from the North Carolina State University-USA have in a new study found that certain plant flavonols and dihydroflavonols show potential as antivirals as they inhibit mpro of sars-cov-2 in vitro. Among the identified phytochemicals were (+)-taxifolin dihydroquerectin [(+)-DHQ], (+)-dihydrokaempferol [(+)-DHK], quercetin, kaempferol, myricetin, isoquercetin, rutin, epigallocatechin gallate (EGCG)and epicatechin.
 
The SARS-CoV-2 coronavirus has emerged as virus that is not only linked to respiratory issues but also extending to the damage of various organs and a disruption to the immunity system as well as numerous other critical human cellular pathways. The human coronavirus 229E (HCoV-229E) however only causes the common cold. The main protease (Mpro) is an essential enzyme required for the multiplication of these two viruses in the host cells, and thus is an appropriate candidate to screen potential medicinal compounds.
 
Flavonols and dihydroflavonols are two groups of plant flavonoids.
 
The study team report docking simulation with two Mpro enzymes and five flavonols and three dihydroflavonols, in vitro inhibition of the SARS-CoV-2 Mpro, and in vitro inhibition of the HCoV 229E replication.
 
The docking simulation results predicted that (+)-dihydrokaempferol, (+)-dihydroquercetin, (+)-dihydromyricetin, kaempferol, quercetin, myricentin, isoquercetin, and rutin could bind to at least two subsites (S1, S1’, S2, and S4) in the binding pocket and inhibit the activity of SARS-CoV-2 Mpro. Their affinity scores ranged from -8.8 to -7.4. Likewise, these compounds were predicted to bind and inhibit the HCoV-229E Mpro activity with affinity scores ranging from -7.1 to -7.8.
 
 In vitro inhibition assays showed that seven available compounds effectively inhibited the SARS-CoV-2 Mpro activity and their IC50 values ranged from 0.125 to 12.9 µM. Five compounds inhibited the replication of HCoV-229E in Huh-7 cells. These findings indicate that these antioxidative flavonols and dihydroflavonols are promising candidates for curbing the two viruses.
 
The study findings were published on a preprint server and are currently being peer reviewed. https://www.biorxiv.org/content/10.1101/2021.07.01.450756v1
 
The SARS-CoV-2 coronavirus, as well as other coronaviruses like HCoV-229E, encode for the main protease (Mpro), which is an upstream enzyme that is required for viral replication within the host cell. This cysteine protease has a catalytic Cys-His p air that is targeted by several existing antiviral drugs.
 
It should be noted that the Mpro substrate-binding pocket has four subsites, among which Cys145 is located at the space between three of these subsites. The thiol of this cysteine is essential for the enzymatic activity of Mpro; therefore, its binding will inhibit Mpro.
 
It was recently found that flavan-3-ol gallates, such as (-)-epigallotechin-3-gallate, (-)-catechin-3-gallate, and (-)-epicatechin-3-gallate, and dimeric procyanidins promisingly inhibited the Mpro activity. https://www.frontiersin.org/articles/10.3389/fpls.2020.601316/full
 
Silico docking simulation indicated that their inhibitory activity likely resulted from the formation of hydrogen bonds between these compounds and several amino acids in the binding domain of Mpro.
 
This current study focused on plant flavonoids that form hydrogen bonds with many residues in the Mpro binding region, thus revealing another strategy of enzyme inhibition. These compounds, many of which have strong antioxidant activity, were tested against two different coronaviruses, of which included the SARS-CoV-2 and HCoV-229E.
 
Importantly a common feature shared by these molecules is their ability to deliver their carbonyl group to the thiol of the 145 Cys residue. This results in the formation of a covalent linkage that ultimately inhibits Mpro activity.
Interestingly similar effects have been reported for flavan-3-ol gallates, of which the inhibitory activity is likely due to the formation of hydrogen bonds between these compounds and several amino acids in the Mpro binding domain.
 
In order to investigate the antiviral activity of various phytochemicals, the study team evaluated flavonols and dihydroflavonols, both of which are two main groups of plant flavonoids.
 
The scientists were interested in these particular plant compounds as a result of previous studies demonstrating their strong antioxidant properties.
 
Initially both the flavonol and dihydroflavonol compounds were studied by docking simulations that included three main steps of protein preparation, ligand preparation, and protein-ligand docking. These studies demonstrated that several of the flavonols and dihydroflavonols showed high-affinity binding to Mpro, with affinity scores that were better than those reported for ebselen, both in its glycosylated and aglycone forms.
 
It should be noted that the Mpro enzyme is 43% conserved between the two coronaviruses in this study, with the binding site being almost identical. This similarity remains true with respect to the conformation and binding pocket of the Mpro enzyme in both viruses.
 
The docking simulation demonstrated the binding of several plant compounds; namely, three aglycone flavonols, two glycosylated flavonols, and three dihydroflavonols, to this cysteine residue via two subsites.
 
However the exact site occupied by the various compounds differed.
 
Glycosylation with rutin and isoquercitin reduced the affinity of binding to SARS-CoV-2 but not to HCoV-229E.
 
All the identified seven compounds that were tested for their in vitro inhibition of the two Mpro enzymes included (+)-taxifolin dihydroquerectin [(+)-DHQ], (+)-dihydrokaempferol [(+)-DHK], quercetin, kaempferol, myricetin, isoquercetin, and rutin.
 
These compounds were found to inhibit the SARS-CoV-2 Mpro at IC50s within the range of 0.125-12.94 µM. The lowest IC50 value was associated with rutin, thereby indicating its high efficacy against SARS-CoV-2. Comparatively, among the seven tested compounds, (+)-DHQ had the highest IC50 value.
 
Interestingly when all compounds were compared at a concentration of 100 μM, rutin was again found to be the most effective. Interestingly, (+)-catechin and (-)-epicatechin failed to inhibit the SARS-CoV-2 Mpro enzyme at any concentration up to 200 μM.
 
A further detailed experiment was conducted to explore the inhibitory effects of five of these compounds on HCoV-229E replication in cells. These included quercetin, isoquercetin, taxifolin, epigallocatechin gallate (EGCG), and epicatechin. The researchers demonstrated that all five compounds were able to inhibit the replication of this virus at low concentrations.
 
Taxifolin and EGCG were found to inhibit viral replication at 2.5 μM, with further inhibition occurring in proportion to its concentration. Whereas quercetin had an IC50 value below 5 μM, isoquercitrin and epicatechin also showed strong inhibition at 5 µM and 20 μM, respectively.
 
The study of the current study have presented strong evidence to support the further exploration of plant compounds that inhibit the SARS-CoV-2 Mpro by binding the Cys145 residue at the binding space between the enzyme’s subsites.
 
The detailed docking simulation study discussed here shows that these flavonols and dihydroflavonols bind to the Mpro substrate-binding pocket. As a result, these compounds occupy the region between S1 and S2 subsites with their C ring while, in many cases, the A- and B-ring was predicted to bind to the region between two subsites.
 
It should also be known that quercetin is one of multiple flavonoids that are used as nutraceuticals and has also been observed to exhibit antiviral activity against viruses like influenza, Zika virus, Ebola, and hepatitis B virus.
 
The current study suggests that this compound also exhibits antiviral activity against SARS-CoV-2, which is enhanced by glycosylation, since this increases the capacity to occupy the binding site. The glycoside rutin is therefore predicted to occupy all four of the subsites, as shown by the increased affinity of binding- shared also with isoquercitrin.
 
Importantly the experimental inhibition of HCoV-229E demonstrates the ability of these five compounds to block SARS-CoV-2 replication within the host cell, as the Mpro enzymes in both viruses show high identity of the binding site.
 
Also this approach was selected to obviate the necessity for biosafety level 3 (BSL3) conditions that would arise with the use of SARS-CoV-2. Taken together, the data discussed here could be useful in designing new drugs that could help treat both COVID-19 and HCoV-229E infection.
 
At present there are only two commercial products that also contain all these listed phytochemicals in their preparations against the SARS-CoV-2 virus including the Delta variant. One is a therapeutic tea developed by Thailand Medical News itself. https://www.thailandmedical.news/news/new-therapeutic-teas-
 
And the other is a therapeutic suspension developed by a German-Indian biotech company called Vedicinals. https://www.thailandmedical.news/news/vedicinals-an-indian-approved-nutraceutical-shows-efficacy-as-an-adjuvant-to-treat-covid-19-and-long-covid-in-who-registered-clinical-trial
 
For more about herbs and phytochemicals for COVID-19, refer to: https://www.thailandmedical.news/articles/covid-19-herbs