Abstract
Myricetin (3,5,7,3′,4′,5′ -hexahydroxyflavone), a common dietary flavonoid, has been reported for its roles in improving health due to various pharmacological activities, such as antioxidant, antimicrobial, anti-inflammatory, analgesic, antitumor, hepatoprotective, and antidiabetic. Myricetin has also been shown to have a broad spectrum of antiviral effects against a variety of viruses including Rauscher murine leukemia virus (RLV), human immunodeficiency virus (HIV), Coxsackie virus, Ebolavirus, Zika virus, herpes simplex virus (HSV-1 and HSV-2), dengue virus, murine norovirus, infectious bronchitis virus, African swine fever virus, and both DNA polymerase α and DNA polymerase I. Intensive research suggests that the remarkable potential of myricetin in promoting either the prevention or overcoming of SARS-CoV-2 infection is due to the wide range of its effects on SARS-COV-2 proteases, including modulation of inflammatory processes and immune responses. In silico and in vitro studies demonstrated that myricetin can effectively interfere at various stages of viral infection, including the coronavirus entry and replication cycle due to its high-binding affinity with S-protein, ACE2 receptor, PLpro, Mpro, RdRp, exonuclease, and endoribonuclease. Based on the findings discussed in this review, myricetin, its glycosides, and dihydromyricetin, can be considered as multi-targeted agents having beneficial effects in combatting COVID-19.
Introduction
Myricetin (3,5,7,3′,4′,5′-hexahydroxyflavone, or 5,7,3′,4′,5′-pentahydroxyflavonol or 3,5,7-trihydroxy-2-(3,4,5-trihydroxyphenyl)chromen-4-one. Synonyms: cannabiscetin, myricetol, Figure 1), a dietary polyphenol, abundant in fruits, berries, vegetables, teas, wine, and many medicinal herbs, is well recognized for its nutraceutical and/or health benefits.1‐12 Myricetin (abbreviated as MYR) occurs naturally in free as well as in glycosidic form, having mostly O-linked monosaccharides, such as glucose, galactose, rhamnose, xylose, and arabinose, thus resulting in mono- and di-glycosides; sometimes these sugar moieties are C-linked, thus resulting in O- and C-glycosides. In some cases, the hydroxyl group of the sugar moieties is also esterified with acids such as acetic and gallic to produce acylated glycosides.1‐13
Chemical structure of myricetin (MYR).
Pharmacological Significance
It has been reported that MYR possesses a wide-spectrum of pharmacological properties such as antioxidant, anticancer, antimicrobial, anti-thrombotic, antihypertensive, anti-obesity, anti-osteoarthritic, neuroprotective, hepatoprotective, cardioprotective, immunomodulatory, analgesic, anti-inflammatory, and antihyperglycemic.4‐58 Its roles in bone remodeling, wound healing, non-alcoholic fatty liver disease, cellular autophagy, diabetic eye disorders, gastric injury, protective effects against Alzheimer's disease, as a mitochondria-activating agent, anthelmintic, antivirulence candidate to control Staphylococcus aureus pathogenicity, management of glaucoma, regulation of metabolic inflammation, treatment of thrombotic disorders, preventing diabetes, associated kidney injuries, and cardiotoxicity have been documented recently.59‐85 Several review articles on the pharmacology of MYR have been published in the past few years.4,5,8,11‐13,29,40,45,52,53,56,81,83,86‐90
Antiviral Significance
Emerging literature91‐107 suggests that flavonoids exhibit prominent activities against several viruses, including COVID-19. In general, viruses, whether having a DNA or RNA genome rely on the cellular machinery of the hosts and their surroundings to infect and continue to multiply. There are several mechanisms by which flavonoids inhibit and act on viruses. They can obstruct the internalization of the viruses by preventing either their binding to the receptors or inhibiting the function of the host-receptor itself; hampering the viral replication process by inhibition of viral protease, RNA polymerase, and mRNA and/or new particle assembly; and blocking the release and spread of the virus, modulating the inflammation and immune system, and reducing viral load. Due to the multifaceted biological actions by targeting and modulating the expression of various molecular targets which are involved in viral infectivity, several flavonoids have been considered to have potential for antiviral clinical trials and/or for combined therapy involving flavonoids in combination with antivirals, assuming synergistic effects.
In a study related to differential inhibitory effects of various flavonoids on the activities of reverse transcriptase (RT) and cellular DNA and RNA polymerases, Ono and coworkers reported MYR to be a strong inhibitor of RT from Rauscher murine leukemia virus (RLV), human immunodeficiency virus (HIV), and both DNA polymerase α and DNA polymerase I.108,109 Complete inhibition of HIV RT was observed by its presence at 2 pg/mL. 109 Pasetto et al reported anti-HIV-1 activity of MYR and found that it can inhibit HIV-1 BaL infection by ≥90%. 110 MYR also showed more than 80% anti-HIV activity in H9 and PBMC cells infected by HIV-1 MN and HIV-1 89.6. 110 MYR can also inhibit (IC50 203 mM) HIV-1 RT, 108 and thus shows promising results against different strains of HIV-1.108‐110
Ortega et al evaluated the anti-HIV-1 activity of myricetin 3-O-rhamnoside and myricetin 3-O-(6-rhamnosylgalactoside) obtained from Marcetia taxifolia and found that both glycosides possess antiviral activity against HIV in vitro, with EC50 values of 120 and 45 µM, respectively. 111 The antiviral effect was exerted through the inhibition of HIV RT with IC50 values of 10.6 µM for the rhamnoside, 13.8 µM for the rhamnosylgalactoside, and 7.6 µM for MYR reflecting MYR to be the most potent. 111 The molecular docking results suggest that MYR and HIV-1 RT residues exhibit 5 hydrogen bond interactions with residues Lys101 (A), Glu138 (B), and Ile180 (A) and electrostatic interactions Pi–Pi, Pi–Alkyl, and Pi–sigma with residues Tyr188, Leu100, Val106, Leu234, and Val179. These authors also suggested that the glycosyl moiety could play an important role in the entry and then, after enzymatic cleavage of the glycosyl moiety, the released MYR was ultimately responsible for the anti-HIV activity. 111 Thus, MYR may modulate important steps in the HIV-1 life cycle, such as entry, RT, integration, and maturation.108‐111
Based on an in vitro test by SRB assay against Coxsackie virus A16 (one of the main causative agents of hand, foot, and mouth diseases in young children), MYR, isolated from the leaves of Ardisia splendens, exhibited activity against Coxsackie virus A16 with an IC50 value of 40.1 µM. 112 In another study, MYR, isolated from Limonium morisianum, was identified as an Ebolavirus inhibitor as it inhibited VP35-viral double-stranded RNA (dsRNA) interactions with an IC50 value of 2.7 µM. 113
MYR moderately inhibited the NS2B-NS3 protease of Zika virus (ZIKV NS2B-NS3pro), and Zika virus replication in a concentration-dependent manner, but the effect was observed immediately after infection, whereas, after 1 h of infection, only moderate inhibition was observed. 114 The IC50 value was 22 ± 0.2 µM with a Ki value of 8.9 ± 1.9 µM. 114 In another study, MYR and quercetin were found to have potent anti-Zika virus activity by targeting the replication process of the virus. The IC50 value for MYR was 0.58 ± 0.17 μM, and the CC50 value for Vero cells was >500 mM. 115
Peng et al 116 investigated the anti-viral activity of MYR against infectious bronchitis virus (IBV) and found that MYR can significantly inhibit IBV replication in primary chicken embryo kidney cells and can upregulate the transcription levels in the nuclear factor kappa-B (NF-kB) and interferon regulatory factor 7 (IRF7) signaling pathways. These studies reflected that MYR could increase the ubiquitin modification level on tumor necrosis factor receptor-associated factors 3 and 6 (TRAF3 and TRAF6) reduced by IBV PLpro, and thus MYR exerts antiviral activity against IBV. 116
Lyu et al carried out anti-herpetic assays on flavonoids using a virus-induced cytopathic effect inhibitory assay, a plaque reduction assay, and a yield reduction assay. When flavonoids were applied at various concentrations to Vero cells infected by herpes simplex virus-1 and HSV-2, MYR showed moderate inhibitory effects against HSV-1. 117 The anti-HSV-1 and HSV-2 inhibiting activity of MYR was due to inhibition of the EGFR/PI3K pathway and reduced activity of Akt through blocking the gD protein in vivo. 118
In a study by Sarwar et al, more than 100 flavonoids were used for docking analysis to investigate their ability against dengue NS2B-NS3 protease; 10 were considered as the best inhibitors on the basis of the docking results. One of these was MYR, having a docking score of −21.987 kcal/mol and, consequently, was considered as an inhibitor of NS2B-NS3. 119 The pharmacological potential of flavonoids against neurotropic viruses has been recently reviewed by Castro e Silva et al. 120
Antiviral effects of 60% aqueous methanol extracts of Zanthoxylum armatum and Hibiscus sabdariffa were demonstrated on murine norovirus. 121 Of the constituents, the phenolic compounds myricetin, quercetin, kaempferol, and luteolin exhibited significant activity, but quercetin showed the most significant logarithmic viral reductions. 121
Jo et al 122 screened several flavonoids to investigate systematically their African swine fever virus (ASFV) protease inhibition by a fluorescence resonance energy transfer (FRET) method. MYR and myricitrin (MOR) were found to have the most prominent anti-ASFV protease activity, the former having an IC50 value of 8.4 µM. The 3´,4´,5´-trihydroxyl-substituted ring-B of MYR seems to be essential for its inhibitory activitiy and the additional rhamnoside, as in MOR, can positively contribute to the interaction with ASFV protease. Thus, these flavonoids can contribute to the development of anti-ASFV agents. 122
Brief Description of SARS-CoV-2 Genome
To identify effective viral inhibitors, it is necessary to have an understanding of the SARS-CoV-2 genome, which consists of 10 open reading frames (ORFs) and among them, ORF1a and ORF1b occupy approximately two-thirds of the whole genome. Apart from ORF1a and ORF1b, the remaining ORFs are distributed in the last third of the SARS-CoV-2 genome, and they encode at least 4 structural proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N), as well as some accessory proteins.123‐125
The early stage of infection is primarily driven by the identification, fusion, and entry of SARS-CoV-2 in the host, which is followed by viral replication. The structural proteins of SARS-CoV-2 mainly comprise spike (S), membrane (M), envelope (E), and nucleocapsid (N) proteins. The S protein is associated with the process of virus entry by receptor recognition and fusion mediation. Once the receptor-binding domain (RBD) of the S protein binds with the angiotensin-converting enzyme-2 (ACE2) of the epithelial cells, the virus enters the host cell, and becomes disassembled to release the nucleocapsid and viral genome. Host ribosomes translate the ORF 1a/b into 2 polyproteins (pp1a and pp1ab) that encode various enzymes required for the replication and transcription process, such as papain-like protease (PLPro or nsp3), 3-chymotrypsin-like or main protease (3CLpro, Mpro or nsp5), RNA dependent-RNA polymerase (nsp12), helicase/triphosphatase (nsp13), exoribonuclease (nsp14), and endonuclease (nsp15).126‐129 Mpro and PLpro participate in the cleavage of the polyproteins to produce the nonstructural proteins nsp2−nsp16, responsible for the replication and transcription of the viral genome. This process is followed by assembly of the virion components into the endoplasmic reticulum Golgi intermediate compartment complex and release from the infected cells by exocytosis.130,131 Thus, S-protein is responsible for viral entry, and Mpro/3CLpro, PLpro, and RNA-dependent RNA polymerase are crucial for the viral replication cycle, and inhibiting them may block viral entry and the replication cycle. Therefore, these have been considered as major druggable targets of SARS-CoV-2. 132 In fact, both the proteins of the virus and host factors are essential for the pathogenesis of COVID-19 and targeting any of these enzymes represents an effective way to tackle this disease for antiviral therapy. The later stage of infection progression is driven by a tremendous inflammatory/immune response to viral infection that results in damage to tissues and organs, resulting in COVID-19-associated health complications.126‐129,131,132
SARS-COV-Activity
To the best of our knowledge, the usefulness of MYR for severe acute respiratory syndrome-coronavirus (SARS-COV) was investigated for the first time in 2012, in studies related to the inhibitory effects of a library of naturally occurring compounds against SARS helicase, nsp13, which plays a a crucial role in the replication of this virus133,134 by conducting either a FRET-based double-strand DNA unwinding assay or by using a colorimetry-based ATP hydrolysis assay. MYR and scutellarein (4′,5,6,7-tetrahydroxyflavone) were found to potently inhibit the SARS-COV helicase protein in vitro by affecting ATPase activity. The IC50 value for MYR was determined to be 2.71 ± 0.19 µM. It has also been noticed that MYR did not inhibit to the same extent the ATP hydrolysis activity of the hepatitis C virus helicase, indicating specificity for the SARS-COV enzyme.133,134
Dihydromyricetin (also known as ampelopsin) had only weak inhibitory activity against SARS-CoV 3CLpro (IC50 = 364 ± 8.7 µM) when tested in a proteolytic (FRET-based) assay and in molecular docking studies (docking score, −9.9 kcal/mol). 135 In a recent study, to investigate further nsp13 inhibiting activity of MYR in either the presence or absence of detergent (Tween-20), it was found that MYR showed clear inhibition (IC50: 24 mM), but only in the absence of detergent. 136
Myricetin and its Analogs From Medicinal Plants as Potential Antiviral Agents Against SARS-CoV-2
Chikhale et al 137 carried out computational and network pharmacology studies on Phyllanthus emblica for its antiviral use and found that out of 66 isolated and tested compounds, chlorogenic acid, quercitrin, and MYR showed promising binding energy against 3 protein targets of SARS-CoV-2: nsp15 endoribonuclease, main protease (Mpro), and the receptor binding domain (RBD) of prefusion spike protein. The Myr-RBD complex had higher binding energy (ΔGbind = −17.41) than Remdesivir-RBD (ΔGbind = −0.91), indicating MYR's better affinity for the SARS-CoV-2 RBD than Remdesivir. The network pharmacology analysis study confirmed the importance of these compounds in modulating multiple signaling pathways that could play a significant role in the immune response, inflammatory cascade, and cytokine storm. 137
A phytochemical study
138
on the aerial parts of Limonium tubiflorum growing wild in Egypt led to the isolation of MYR and its acylated glycosides: myricetin 3-O-(2″-galloyl)-β-D-galactopyranoside (
Propolis, a resinous material produced by honeybees from plant exudates, is composed of many chemical constituents, some of which have inhibitory effects on ACE2, TMPRSS2, and other proteases. In addition, propolis promotes immunoregulation and thus reduces the risk of cytokine storm syndrome, a major mortality factor in advanced COVID-19 disease. Phytochemicals that showed promise for the inhibition of coronavirus in humans included quercetin, myricetin, and caffeic acid, all components of propolis.139,140
Black garlic extract and 49 polyphenols were studied by Nguyen et al 141 for their inhibitory effects on Mpro. The IC50 values were 137 µg/mL for black garlic extract and 43 ± 1 µM for MYR. The combinations of tannic acid with MYR enhanced the inhibitory effects on Mpro. A detailed structure–activity relationship revealed that hydroxyl groups at 7, 3´, 4´, and 5´-positions contribute to the inhibitory effects of flavonoids on Mpro. 141
In silico studies were carried out by Pereira et al to evaluate the inhibitory power of phytoconstituents from Anacardium occidentale against Mpro enzyme from SARS-CoV-2. 142 Amentoflavone, agathisflavone, myricetin-3-O-rhamnoside (MOR), and MYR showed values of affinity energy of −8.0 kcal/mol, −8.3 kcal/mol, −7.6 kcal/mol, and −6.7 kcal/mol, respectively. Both MOR and MYR showed hydrogen bond interactions with Lys5A, Lys137A, Glu288A, and Glu290A, but with stronger interactions with Asp197A in the case of MOR and with Asp289A for MYR. The molecular dynamics results indicated that agathisflavone had more stable interactions and a higher interaction potential energy with Mpro in comparison to the other isolated compounds. 142
Interactions With S-Protein and ACE 2
Studies have identified ACE2 as a functional receptor for coronaviruses 143 as the SARS-CoV-2 spike protein (S-protein) plays a vital role in virus invasion due to its strong binding affinity to human ACE2. 144 The spike protein first binds to the host's receptor, and then fuses viral and host membranes, allowing the viruses to enter the host cells. 145 Therefore, S-protein and/or ACE-2 enzyme inhibition can be useful for treatments against these virus infections.
A screening study of ∼7100 molecules of synthetic and natural origin, including active ingredients present in Ayurvedic medicines, was undertaken with ACE2, Rdrp, and Mpro as the targets. Several natural molecules were identified, including myricitrin (MYR-3-O-α-L-rhamnopyranoside, MOR), which showed strong interactions with the ACE-2 receptor by forming polar interactions with Ala348, Phe390, Arg393, and Asn394, with a binding score of −7.5 kcal/mol. 146
Pandey et al focused their docking studies on flavonoids already reported for their antiviral efficacies against other viral diseases, to study their inhibitory potential against 2019-nCoV spike glycoprotein, and identified baicalin (baicalein 7-O-glucuronide or 5,6-dihydroxyflavone-7-O-β-D-glucuronide), which exhibited the highest binding affinity of −7.26 kcal/mol. 147 The binding affinity for MYR and Prefusion 2019-nCoV spike glycoprotein with a single RBD up (6VSB) was −6.15 kcal/mol, having a number of interactions with Phe970, Gln965, Leu962, Thr961, Gln957, Ala958, Gln954, Arg1014, Gln1011, Tyr1007, Gln1010, Thr1006, Ser1003, and Gln1002. 147
Flavonoids, including MYR, can bind with high affinity to the S-protein. The protease sites on the ACE2 receptor are used by SARS-COV-2 to infect host cells. Based on a docking study, Ngwa et al suggested that MYR binds effectively to the ACE2 receptor, causing a conformational change, exhibiting a binding energy of −8.8 kcal/mol, thereby inhibiting virus entry. 148 Further molecular docking studies were performed to analyze the binding mode of MYR towards various proteins related to COVID-19. 149 Based on QSAR and molecular docking interactions, MYR was predicted to interact with 10 out of 12 potential COVID-19 proteins and could potentially bind to ACE2 with a docking score of −8.8 kcal/mol. 149
Several compounds from natural sources were investigated, using molecular docking, for their inhibitory ability against COVID-19 target proteins, such as ACE2, TMPRSS2, RdRp, 3CLpro, and PLpro. Brazilein and brazilin were found to bond to ACE2 with a lower binding energy value than those of chloroquine, arbidol, remdesivir, ribavirin, and lopinavir. 150 MYR exhibit binding energies of −7.08, −6.25, −3.38, −6.65, and −6.57 kcal/mol with ligands ACE2, TMPRSS2, RdRp, 3CLpro, and PLpro, respectively. 150 In a study related to potential inhibiting properties of ethanolic Anatolian propolis extracts against ACE-2 receptors by molecular docking studies, Guler et al 151 found that rutin, quercetin, caffeic acid phenyl ester, myricetin, and hesperetin effectively inhibit the ACE-2 enzyme. The calculated binding energy and inhibition constant (Ki) for the ACE-2-MYR complex were −7.59 kcal/mol and 2.74 µM, respectively. MYR interacted with Arg273, Phe274, His345, Pro346, Thr347, Ala348, Thr371, His374, Glu375, His378, Glu406, Phe504, His505, Tyr515, and Arg518 in the ACE-2 binding site. 151
Myricetin and Mpro Binding
Among various proteases, the coronavirus Main protease (Mpro), also recognized as 3C like protease (3CLpro), has received considerable attention for its significant role in enzymatic activity leading to its post-translational processing and multiplication of replicase polyproteins. It has been reported that the Mpro substrate-binding pocket includes 4 subsites, S1′, S1, S2, and S4, and a catalytic dyad (Cys-145 and His-41), which are crucial residues located at the space between subsites S1, S1′, and S2.152,153 Several studies have reported that binding of a compound to the thiol of the Cys145 residue can inhibit Mpro activity.152‐154
Zhu et al 155 carried out docking simulation studies for 5 flavonols (including MYR) and 3 dihydroflavonols with Mpro enzymes of SARS-CoV-2 and human coronavirus 229E (HCoV-229E, causing the common cold). The ligand-docking simulation results predicted that MYR could bind to the substrate-binding pocket of Mpro. The A-ring, B-ring, and the heterocyclic C-ring of MYR lodged in the S1 and S4 subsites, and the space between S1 and S2. MYR was predicted to bind and inhibit SARS-CoV-2 Mpro and HCoV-229E Mpro activities with affinity scores of −7.4 and −7.1 kcal/mol, respectively. In vitro inhibition assays showed that (+)-dihydrokaempferol, (+)-dihydroquercetin, (+)-dihydromyricetin, kaempferol, quercetin, MYR, isoquercitrin, and rutin) effectively inhibited the SARS-CoV-2 Mpro activity and their IC50 values ranged from 0.125 to 12.9 μM. Thus, the significance of MYR and other flavonoids as promising candidates for curbing coronavirus was suggested. 155
Among 14 phytochemicals investigated for their binding mechanism with the Mpro of SARS-CoV-2 through combinatorial bioinformatics approaches, MYR was found to be 1 of the 3 compounds showing a more prominent binding profile (binding energy of −8.439 kcal/mol) than the rest of the compounds. These studies suggested that MYR interacted with Mpro through several hydrogen bonds (with Met165, Gln189, Asp187, and Thr26) and hydrophobic bonds with the catalytic residue Cys145 and with Met49, and thus MYR could be suitable for Mpro inhibition. 156 In another study, the docking score for MYR against Mpro (PDB ID: 6LU7) was determined to be −7.311 kcal/mol. 157
Myricetin has been identified by Su et al 158 as a non-peptidomimetic and covalent inhibitor of 3CLpro. The crystal structures of the protease bound with myricetin and its derivatives reflect that the 3´,4´,5´-hydroxyl groups (pyrogallol group) act as an electrophile to bind covalently to the catalytic cysteine (Cys145). In addition, the side chain of His41 forms π–π stacking interactions with the chromone region, demonstrating a pivotal role of His41 in binding. The FRET protease assay was utilized to measure the inhibitory activities of myricetin and dihydromyricetin (3,5,7,3′,4′,5′ -hexahydroxyflavanone, DHM) against 3CLpro and >90% inhibition was observed at a concentration of 10 μM. The half-maximal inhibitory concentrations (IC50) of MYR and DHM were 0.63 and 1.14 μM, respectively, thus identifying them as inhibitors of SARS-CoV-2 3CLpro with sub-micromolar or micromolar potency. 158 These authors further evaluated the antiviral efficacy of MYR against SARS-CoV-2 in Vero E6 cells. The half-maximal cytotoxic concentration (CC50) of MYR was over 200 μM as determined by the CCK8 assay, demonstrating a very low cytotoxicity. MYR showed dose-dependent inhibition of the replication of SARS-CoV-2, and the resulting half-maximal effective concentration (EC50) was 8.00 μM. The resulting selectivity index (SI) value was >25 for MYR. Cell-based antiviral experiments demonstrated that MYR can inhibit viral replication. 158 Using cell-based in vitro methodologies, 7-O-methyl-myricetin was found to be the most effective against SARS-CoV-2 3CLpro activity, with an IC50 of 0.30 ± 0.00 µM. In contrast, myricetin-7-yl diphenyl phosphate was the most effective at inhibiting SARS-CoV-2 replication within cells, with an EC50 of 3.15 ± 0.84 µM.158,159 Based on studies with several myricetin derivatives, these authors illustrated that pyrogallol can serve as an electrophile warhead and a template for the design of non-peptidomimetic covalent inhibitors of 3CLpro. 158 The x-ray crystal structure of the complex of MYR and SARS-Cov-2 3CL-Pro confirms covalent binding between the 2′ position of MYR and the catalytic Cys145 sulfur and, therefore, inhibits its enzymatic activity. 160 The unprecedented sulfur addition to the 2′ position of MYR suggests the presence of a quinoid species. 160
A study by Xiao et al, 161 related to molecular docking and enzymatic screening of 15 natural compounds as Mpro inhibitors, identified MYR as having potent inhibiting activity with an IC50 of 3.684 ± 0.076 μM. In a FRET enzymatic assay at a final concentration of 50 µM, MYR showed effective inhibition of enzymatic activity (inhibition rate reached 97.79%). The molecular docking reflects that the chromone ring of MYR interacts with His41 through π-π stacking, and the 3′-, 4′-, and 7-hydroxyl groups interact with Phe140, Glu166, and Asp187 through hydrogen bonds. 161
In an in silico screening study of 8 plant-derived antivirals against the main protease, Sharma et al 162 illustrated that myricetin is one of the potential candidates against 3CLpro, exhibiting binding affinity (−6.15 kcal/mol) with amino acids (Gly 143, Leu 141, His 41, Thr 26) present in the docking pocket of 3CLpro. Molecular dynamics and simulations of the best docked protein-ligand structures revealed the dynamics information on the stability in the biological system, thus restricting the enzymatic activity in the host cells. 162
In a molecular docking and simulation study related to flavonol glycosides based on their ability to interact with the active site of Mpro, Cherrak et al identified MYR-3-O-rutinoside as one of the glycosides having strong docking scores (binding energy ΔG kcal.mol−1 = −9.3) having interactions with amino acid residues Tyr54, His41, Met49, Met165, Thr26, Cys145, Ser144, Leu141, Gly143, Asn142, His163, and Glu166. The binding energies against the main protease for MYR-3-O-galactoside, MYR-3-O-glucoside, and MYR were −9.3, −7.5, and −7.3 kcal/mol−1, respectively. 163
Using a structure-based virtual screening method to reveal the docking profiles of 3 flavonoids, rutin, luteolin, and myricetin, on one of the COVID-19 main proteases (6W63) of SARS-CoV-2, Gencsoy et al found the binding score for MYR was −7.8 kcal/mol. MYR was observed to exhibit only hydrogen bonding with the surrounding amino acid residues (Phe140, His163, Glu166). 164 The computational studies related to 21 selected flavonoids for their inhibitory potential against SARS-CoV-2 main protease 6YNQ identified rutin as having the highest binding energy (−8.7 kcal/mol), whereas MYR exhibited a binding energy of −7.1 kcal/mol, supporting the hypothesis that flavonoids might be helpful for the treatment of COVID-19. 165
In one study, 7 flavonoids, representing 1 isoflavone (genistein), flavones (apigenin and luteolin), and flavonols (fisetin, kaempferol, myricetin, and quercetin), were evaluated for COVID-19 treatment using cell-based assays and in silico calculations. The flavonols were found to be better SARS-CoV-2 inhibitors than the isoflavone and flavones. 166
Screening for the capacity to inhibit SARS-CoV-2 replication was performed using cell-based assays. Fisetin (3,3′,4′,7-tetrahydroxyflavone) and MYR presented the lowest EC50 values (2.03 ± 0.10 and 0.91 ± 0.05 µM, respectively), indicating that these flavonols are preferred candidates to inhibit SARS-CoV-2 replication. 166 Both of these flavonols are anti-inflammatory (decreasing TNF-α levels) and inhibit SARS-CoV-2 mainly by targeting the processability of the main protease (Mpro) in a non-competitive manner, with a potency comparable to that of the repurposed drug atazanavir. 166
A study related to the inhibitory effect of 37 food phytochemicals on the main protease of SARS-CoV-2 by determining a cleaved product after chromatographic separation showed epigallocatechin gallate and MYR to be the most active of the compounds evaluated. 167 The IC50 for MYR was estimated as 0.9 μM. The decrease in enzyme activity was dose-dependent. The covalent MYR–Mpro adducts were observed along with the decrease in enzyme activity, and the adduction happened at the Cys of the active site of Mpro. Thus, MYR had a considerable suppressive effect on the protease activity of the enzyme. 167
By performing ∼200 virtual screenings of compound libraries based on consensus chemical space arising from the multiple conformations of the Mpro binding site, Gossen et al were able to identify MYR as a nM-binder (IC50 220 nM) of SARS-CoV-2 Mpro, distinguishing it from micromolar inhibitors and identifying it as a novel nM inhibitor. 168 MYR, having several phenolic groups, is considered promiscuous due to its redox features and high number of close H-bond acceptor/donor sites to satisfy several 3D-pharmacophores. 168
Myricitrin (MYR-3-O-α-L-rhamnopyranoside, MOR) shows strong binding with Mpro, having a binding score of −8.9 kcal/mol. 146 The residues His41 and Cys145 form Pi-alkyl and Pi-sulfur interactions with the aromatic scaffold of MOR. Tyr54, Phe140, Gly143, His163, and Glu166 are involved in the formation of 6 hydrogen bonds with hydroxyl and carbonyl oxygen of MOR, in addition to other interactions like van der Waals’ forces that stabilize the MOR-Mpro binding. 146
The docking studies as carried out by Fadaka et al 169 showed that some flavonoid glycosides exhibited good poses in the binding domain of Mpro. The amino acid residues involved in the binding of the selected ligands correlated well with the residues involved with the mechanism-based inhibitor (N3) and the docking score of quercetin-3-O-neohesperidoside (−16.8 Kcal/mol) ranked efficiently with the N3 inhibitor (−16.5 kcal/mol). In addition, the single-structure molecular mechanics-generalized Born surface area (MM/GBSA) rescoring method showed that quercetin-3-O-neohesperidoside (−87.60 kcal/mol) and myricetin 3-O-rutinoside (−87.50 kcal/mol) are more energetically favored than N3 (−80.88 kcal/mol), whereas myricitrin (MOR) is significantly less favored (−49.22 kcal/mol). 169
Based on the screening analysis of 32 297 anti-viral phytochemicals and their interactions with the 3CLpro sequence, Tahir ul Qamar identified the top 9 hits, among which were myricitrin (MYR-3-O-α-L-rhamnopyranoside
Liu et al 171 found that the ethanol extract of S. baicalensis and its major component, baicalein, inhibit SARS-CoV-2 3CLpro activity in vitro, with IC50 values of 8.52 µg/mL and 0.39 µM, respectively, and also inhibit the replication of SARS-CoV-2 in Vero cells with EC50 values of 0.74 µg/mL and 2.9 µM, respectively. These authors further identified DHM and MYR as prominent flavonoids inhibiting 3CLpro with IC50 values of 1.20 ± 0.09 and 2.86 ± 0.23 µM. 171
Using molecular docking and the FRET-based enzymatic assay, DHM was identified as a potent inhibitor targeting Mpro with an IC50 of 1.716 ± 0.419 μM. In the binding pocket, the dihydrochromone ring of DHM interacts through π-π stacking with His163 and the imidazole side chain. The 1-oxygen of DHM forms a hydrogen bond with the backbone nitrogen of Glu166, whereas rings-A and B hydroxyl groups interact with Gln189, Leu141, Arg188, and Thr190 through hydrogen bonds. Furthermore, DHM prevents BLM-induced pulmonary inflammation and fibrosis. 172
Myricetin and PLpro (NSP3) Binding
Papain-like protease (PLpro) encoded by nsp3 results in the maturation of nsp1-3, thus supporting viral replication. In addition, it suppresses the innate immune response of the host, which makes PLpro an attractive target to treat COVID-19. Inhibition of PLpro could not only prevent viral replication but also restore the interferon related antiviral immunity of the host.173,174 In a study related to 16 natural products, myricetin showed the strongest antiviral effect on IBV PLpro. The results showed that myricetin can significantly inhibit IBV replication and upregulate the transcription levels in the NF-kB and IRF7 signaling pathways. 116 Bioactive compounds from natural sources were investigated, using molecular docking, for their inhibitory ability against COVID-19 target proteins; MYR exhibited poor binding (binding energy −6.57 kcal/mol) with PLpro (PDB: 4OW0). 150
Myricetin and Rdrp (NSP12) Binding
Another potential target for SARS-CoV-2 is RNA-dependent RNA polymerase (RdRp), which is a key component of the replication machinery of the virus to make multiple copies of the RNA genome. 175 RdRp of SARS-CoV exhibits ∼97% sequence similarity with that of SARS-CoV-2. More importantly, there is no human polymerase counterpart that resembles the sequence/structural homology with RdRp from coronaviruses, and hence, the development of RdRp inhibitors could be a potential therapeutic strategy without risk of crosstalk with human polymerases. 176
In an in silico analysis of a library of bioactive polyphenols against RdRp, MYR was identified as 1 of the 8 most prominent compounds to be docked in the active site of RdRp of SARS-CoV-2. The amino acids, W617, W800, D760, E811, K798, D618, Y619, C622, D761, and F812 of the RdRp binding pocket are involved in interactions with MYR (binding energy: −7.2 kcal/mol). 177
Molecular docking and simulation studies related to more than 100 different flavonoids led to the identification of MYR as 1 of the 3 top hits as an inhibitor of the allosteric site of SARS-CoV-RdRp, exhibiting a good binding energy (S-score) of −18.17 kcal/mol. Interactions between 1 arene-cation and 4 hydrogen bonds with Met755, Gly584, Thr604, Arg583, Ser592 were observed between the allosteric site and MYR. In silico ADME and evaluation of the toxic properties of MYR with the admetSAR online server reflect that MYR had the highest enhanced human intestinal absorption score. 178
In another study, Joshi et al 146 found that MOR exhibited strong interaction with RdRp, with a binding score of −7.9 kcal/mol. The carbonyl group of MOR forms hydrogen bonds with Arg553, Arg555, and Thr556, along with Pi-anion interactions between the aromatic rings of MOR and the active site residue Asp623. 146
Myricetin and Helicase (NSP13) Binding
Nsp13 is a critical component for viral replication and shares the highest sequence conservation across the CoV family, 179 and provides the RNA helicase and 5′-triphosphatase activities. 180 To the best of our knowledge, the first report about anti-helicase activity of MYR was related to the FRET-based double-strand DNA unwinding assay and a colorimetry-based adenosine triphosphate (ATP) hydrolysis assay. This study revealed that MYR inhibits nsp13 in vitro (IC50 2.71 ± 0.19 µM) by affecting ATPase, but not the unwinding activity. 133
In a recent study, molecular docking simulations were performed for flavonoids with 2 binding sites of nsp13, the nucleotide and 5′-RNA pockets. MYR was shown to exhibit mild binding and DHM low binding with these targets reflecting possible inhibition of enzymatic activities. 181 MYR inhibited the nsp13- associated unwinding activity, showing an IC50 value of 0.41 ± 0.11 µM, whereas an IC50 value of >30 (87%) was observed for the nsp13-associated ATPase activity. 181 In molecular docking studies, MYR exhibited 2 strong interactions with Glu540 and Ser289 in the nsp13 binding sites. Based on these studies, MYR was found to inhibit the SARS-CoV-2 nps13 unwinding activity at nanomolar concentrations. 181
Myricetin and 3′-5′ Exonuclease or ExoN (nsp14) Binding
SARS-CoV-2 possesses a 3′–5′ exonuclease for proofreading to maintain the integrity of the genome. 182 The replication complex of coronaviruses consists of several viral proteins, including RdRp, its 2 accessory proteins (nsp7 and nsp8), and the exonuclease (nsp14) with its accessory protein (nsp10). 183 Generally, the SARS-CoV-2 ExoN has 2 Mg (II) ions in the catalytic site; however, the absence of the second Mg(II) ion has already been reported. 184 Therefore, using in silico models, Chaves et al evaluated the binding of flavonoids for both possibilities. 166 In both, MYR exhibited hydrogen bond interactions with Val91, Asn104, and Glu191, and Van der Waals’ interactions with Met58, Asp90, Glu92, Ala187, Phe190, and Glu191. The presence of 2 Mg (II) ions improved the docking score and binding profile of MYR, suggesting that MYR might interact with ExoN. 166
Myricetin and NSP15 Endoribonuclease Binding
Nsp15, also known as uridylate-specific endoribonuclease, is encoded by the coronavirus as an RNA-processing enzyme and hence plays an essential role in viral replication and survival in the host cell. Thus, inhibition of nsp15 can slow viral replication. 185 The active site of nsp15 is shaped by 6 critical amino acids (His235, His250, Lys290, Thr341, Tyr343, and Ser294). An in silico approach was used for 8 plant-derived antiviral compounds for their inhibition of nsp15. The docking results suggested that MYR showed significant binding with Lys290, His235, His250, and Glu340 (binding energy −6.52 kcal/mol) of nsp15 (PDB ID: 6VWW). 162 In a study to identify phytochemicals as potential agents that bind to nsp15, Kumar et al identified that MYR can bind (−7.0 kcal/mol) with nsp15 (PDB ID: 6VWW) via H-bonds with Ser294, His250, His235, and Gly248. 186
In a detailed investigation, bioactive constituents of tea were docked onto the active site of nsp15 (PDB ID: 6W01). Based on their docking scores, MYR was 1 of the 3 top compounds, and its conformational behavior was analyzed via molecular dynamics simulations by Sharma et al 187 The results indicated that MYR shows strong MYR-NSP-15 binding (binding energy, −11.9 kcal/mol) via H-bonds, C-H bonds and π-π stacking interactions with all 5 critical amino acids (His235, His250, Lys290, Thr341, and Tyr343). MYR also interacts with Val292, forming 1 H bond and 1 π-alkyl bond, thus reflecting a strong MYR-nsp15 association. 187
Myricetin Reduced the Inflammatory Response
COVID-19, from an asymptomatic state, frequently leads to fatal inflammatory responses including mild to moderate pneumonia, acute respiratory distress syndrome, and associated complications and mortality. 188 The viral infection can be associated with uncontrolled release of pro-inflammatory cytokine mediators such as IL-6 and lactate dehydrogenase, as well as other inflammatory cytokines (IL-1, IL-1α, TNF-α, and IFN-γ), which can cause inflammatory cascade and cytokine storms and subsequent organ failure.149,165,189,190 Due to the excessive immune responses that trigger cytokine release and which can result in the overproduction of proinflammatory cytokines, the reversion of hyper-inflammation can play an important role in the treatment of COVID-19. 191 The anti-inflammatoy and immunomodulatory potential of flavonoids, including MYR, has been well-described.22,24,26,35,46,78,84,85,107,165,170,192‐195 Thus, flavonoids could potentially be useful in the modulation of COVID-19-related inflammatory processes and immune responses.
Li et al have shown that myricetin may be able to attenuate viral pneumonia and encephalitis symptoms in HSV infected mice. 118 MYR shows effective interactions with the GP130 protein and with NF-κB. 149 The effect of MYR on pulmonary inflammation with bleomycin treated mice was investigated by Xiao et al, who showed that MYR was effective in inhibiting the infiltration of inflammatory cells and the secretion of inflammatory factors such as IL-6, TNF-α, IFN-γ, and IL-1α in the bronchoalveolar lavage fluid. 161 MYR exhibits reasonable effects in the modulation of inflammatory mediators and signaling cascades, including NLRP3 (NLR family, pyrin domain-containing 3 protein) inflammasomes. Chen et al have shown the effect of MYR on NLRP3-driven inflammatory diseases by promotion of reactive oxygen species (ROS)-independent ubiquitination of NLRP3, thus resulting in inhibition of the NLRP3 inflammasome assembly. 85 The significance of DHM in vivo on BLM-induced pulmonary inflammatory and fibrosis was evaluated by Xiao et al and the results indicated that it inhibits the migration and activation of myofibroblasts and extracellular matrix production by transforming growth factor (TGF)-β1/Smad signaling pathways. 170 Hence, MYR and DHM can be potential modulators for COVID-19-related inflammatory and immune deregulations.
Concluding Remarks
Taken together from various in silico and in vitro studies, it can be concluded that myricetin possesses multifaceted biological potential which can interfere with various stages of coronavirus entry and its replication cycle due its interaction with s-protein and ACE-2, Mpro, PLpro, helicase, exonuclease, and endoribonuclease. A unique mode of covalent bonding of MYR in targeting the Mpro responsible for suppression of enzyme activity suggests its therapeutic potential in the treatment of COVID-19. Myricetin also exhibits significant beneficial effects in the modulation of inflammatory and immune processes. The FDA has listed it as Generally Recognized as Safe. 148 Thus, available evidence supports the multifaceted biological and/or prophylactic potential of MYR for further optimization for COVID-19 therapeutic treatment.
Footnotes
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) received no financial support for the research, authorship, and/or publication of this article.
