The search for effective antivirals against the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the virus responsible for the coronavirus disease 2019 (COVID-19), remains increasingly important as new mutations of this virus continue to arise.
Study: Flavonols and dihydroflavonols inhibit the main protease activity of SARS-CoV-2 and the replication of human coronavirus 229E. Image Credit: paulista / Shutterstock.com
In a new paper published on the preprint bioRxiv* server, a group of researchers discuss their findings that a group of plant flavonols and dihydroflavonols exhibit antiviral efficacy against both SARS-CoV-2 and the virus responsible for the common cold, which is known as human coronavirus 229E (HCoV-229E).
SARS-CoV-2, 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 pair that is targeted by several existing antiviral drugs. In the current study, the researchers screened existing antiviral medicines or designed chemicals and found that cinanserin, ebselen, and GC376, 11a, and 11b all showed inhibitory effects on Mpro activity.
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.
Ebselen is a small molecule inhibitor with a three-ring structure that effectively inhibits Mpro at a half-maximal inhibitory concentration (IC50) of less than 0.5 micromolar (μM). This three-ring component donates its carbonyl group to the Cys145, thus inactivating the enzyme.
What did the study show?
The 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.
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. 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 an effort to investigate the antiviral activity of more readily available drugs, the researchers evaluated flavonols and dihydroflavonols, both of which are two main groups of plant flavonoids. The researchers were interested in these particular plant compounds as a result of previous studies demonstrating their strong antioxidant properties.
Both the flavonol and dihydroflavonol compounds were first 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.
Docking features at the binding pocket
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.
Ebselen, for example, binds to this cysteine via three-ring structures facing two of these subsites. 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.
The exact site occupied by the various compounds differed. Moreover, glycosylation with rutin and isoquercitin reduced the affinity of binding to SARS-CoV-2 but not to HCoV-229E.
The 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.
Taken together, 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.
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.
Inhibition of HCoV-229E replication
An additional 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.
For instance, 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.
What are the implications?
The researchers of the current study have presented strong evidence to support the further exploration of compounds that inhibit the SARS-CoV-2 Mpro by binding the Cys145 residue at the binding space between the enzyme’s subsites.
The 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.
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.
The experimental inhibition of HCoV-229E is important in demonstrating 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. 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.
“In general, these compounds are safe nutrients sold as supplements or in food products such as onion and common dinner table fruits.”
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.