Flavonoid Covid 19

Chemico-Biological Interactions 328 (2020) 109211
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Chemico-Biological Interactions
journal homepage: www.elsevier.com/locate/chembioint
Roles of flavonoids against coronavirus infection

Maria Russo 1, Stefania Moccia 1, Carmela Spagnuolo, Idolo Tedesco, Gian Luigi Russo *
National Research Council, Institute of Food Sciences, 83100, Avellino, Italy
A R T I C L E I N F O A B S T R A C T
Keywords: In terms of public health, the 21st century has been characterized by coronavirus pandemics: in 2002-03 the
Coronavirus virus SARS-CoV caused SARS; in 2012 MERS-CoV emerged and in 2019 a new human betacoronavirus strain,
Flavonoids called SARS-CoV-2, caused the unprecedented COVID-19 outbreak. During the course of the current epidemic,
SARS-CoV
medical challenges to save lives and scientific research aimed to reveal the genetic evolution and the
MERS-CoV
SARS-CoV-2
biochemistry of the vital cycle of the new pathogen could lead to new preventive and therapeutic strategies
against SARS-CoV-2. Up to now, there is no cure for COVID-19 and waiting for an efficacious vaccine, the
development of “savage” protocols, based on “old” anti-inflammatory and anti-viral drugs represents a valid and
alternative therapeutic approach. As an alternative or additional therapeutic/preventive option, different in
silico and in vitro studies demonstrated that small natural molecules, belonging to polyphenol family, can
interfere with various stages of coronavirus entry and replication cycle. Here, we reviewed the capacity of well-
known (e.g. quercetin, baicalin, luteolin, hesperetin, gallocatechin gallate, epigallocatechin gallate) and un
common (e.g. scutellarein, amentoflavone, papyriflavonol A) flavonoids, secondary metabolites widely present
in plant tissues with antioxidant and anti-microbial functions, to inhibit key proteins involved in coronavirus
infective cycle, such as PLpro, 3CLpro, NTPase/helicase. Due to their pleiotropic activities and lack of systemic
toxicity, flavonoids and their derivative may represent target compounds to be tested in future clinical trials to
enrich the drug arsenal against coronavirus infections.
certainly lead to the development of new therapeutic protocols.

Recent studies evidenced that SARS-CoV-2 is an efficient killer
1. Introduction compared to its close related SARS-CoV, isolated in 2003 since the
former acquired an array of “strategic adaptations” [3]. During its
The 2nd decade of the 21st century began with an unprecedented evolution in nature, probably decades as meta-genetic analysis are
epidemic in human history: the emergence of a new human betacor revealing the new strain of coronavirus “jumped” from a bat to an un
onavirus strain, first isolated and sequenced in China in early 2020 [1] known mammalian and then to humans. Here, SARS-CoV-2 can target
and called SARS-CoV-2 (Severe Acute Respiratory Syndrome different tissues at multiple levels, starting from the cells of nose and
CoronaVirus-2) as the etiological agent of CoronaVIrus Disease 19 throat down to the lung, invading, perhaps, through vasal endothelium,
(COVID-19) [2]. Globally, at the time of writing (July 11, 2020), 12,322, kidneys and nervous system where it can cause severe illness and death
395 confirmed cases of COVID-19 have been registered, including 556, [4,5].
335 deaths worldwide except for the Antarctic continent, as reported by However, there is still much to learn about SARS-CoV-2 and a deeper
WHO (https://covid19.who.int/COVID-19). Although awaited by some knowledge of its biology, comparing metagenomics analysis and
microbiologists, this pandemic found the health systems of many biochemical characteristics of previous coronaviruses (SARS-CoV and
Western countries (Italy, Spain, France, UK and USA) largely unpre MERS-CoV) will be crucial to define efficient therapeutic and/or pre
pared. However, at the same time, it mobilized the scientific community ventive strategies.
with the production of over 6000 peer-reviewed scientific articles on Besides “old” drugs (essentially antivirals, such as favipiravir and
PubMed in the last 5 months. Waiting for a vaccine, there is currently no ribavirin; anti-HIV protease inhibitors, such as ritonavir and lopinavir,
specific cure against COVID-19. A deeper knowledge of the genetics and and anti-inflammatory agents, such as tociclizumab or dexamethasone)
biochemistry sustaining SARS-CoV-2 vital cycle and infectivity will
* Corresponding author. National Research Council, Institute of Food Sciences, Via Roma, 64, 83100, Avellino, Italy.
E-mail addresses: glrusso@isa.cnr.it, gianluigi.russo@cnr.it (G.L. Russo).
1
Equal contribution.
https://doi.org/10.1016/j.cbi.2020.109211
Received 4 June 2020; Received in revised form 13 July 2020; Accepted 23 July 2020
Available online 28 July 2020
0009-2797/© 2020 Elsevier B.V. All rights reserved.
M. Russo et al. Chemico-Biological Interactions 328 (2020) 109211
Abbreviations ORF Open reading frame

PD N-terminal peptidase domain
3CLpro 3C-like-protease PEDV Porcine epidemic diarrhea virus
ACE2 Angiotensin Receptor Enzyme-2 PLpro Papain-like cysteine protease
CC50 50% cytotoxicity concentration RBD Receptor-binding domain structure
CLD C-terminal collectrin RBM Receptor binding motif
COVID-19 Coronavirus Disease 2019 RdRp RNA-dependent-RNA-polymerase
E Small envelope protein RSV Respiratory syncytial virus
EGCG Epigallocatechin-3-gallate RTC Replication/transcription complex
GCG Gallocatechin gallate S Spike glycoprotein
gRNA, sgRNA Genomic, sub-genomic RNA SAR Structure–activity relationship
HE Hemagglutinin esterase SARS-CoV Severe Acute Respiratory Syndrome CoronaVirus
HR Heptad regions (HR1 and HR2) SARS-CoV-2 Severe Acute Respiratory Syndrome CoronaVirus-2
HSV-1 Herpes simplex virus type 1 SPR Surface Plasmon resonance
M Membrane or matrix protein TCM Traditional Chinese Medicine
MERS-CoV Middle East Respiratory Syndrome CoronaVirus TfR Transferrin receptor
Mpro Main protease TM Transmembrane domain
N Nucleocapsid protein TMPRSS2 Transmembrane peptidase serine 2
NSP Not structural proteins UTR Untranslated region
Fig. 1. Basic skeleton (C6–C3–C6) of flavonoids and representative examples of compounds able to counteract coronavirus infection.
2
that clinicians are currently using against the severe cases of the recent structural proteins (NSP), while the remaining ORFs give rise to acces
COVID-19 outbreak [6–8], natural compounds isolated from the plant sory and structural proteins [22]. In particular, the first ORF (ORF1a/b)
kingdom and belonging to the multiple and heterogeneous class of fla translates two polyproteins: pp1a and pp1ab for the presence of a
vonoids (Fig. 1) may represent an interesting option. In fact, flavonoids frameshift between ORF1a and ORF1b. These polyproteins are pro
lack systemic toxicity, their ability to synergize with conventional drugs cessed by the main protease (Mpro) also known as 3C-like-protease
has been largely demonstrated and, finally, they are “pleiotropic” (3CLpro) and one or two papain–like proteases (PLpro) into 16 NSPs,
compounds, meaning that their functional groups can interact with which produce viral RNA that encodes the four main structural proteins
different cellular targets and intercept multiple pathways [9,10]. These [23] (Fig. 2).
features make flavonoids potential candidates to interfere with the The importance of 3CLpro in the viral cycle and the absence of its
coronavirus life cycle. human homologue makes this enzyme an attractive target for the
Flavonoids include a large number of secondary metabolites, found development of new drugs directed against coronavirus infection. 3CLpro
in vegetables, seeds, fruit, and beverages, such as red wine and tea [11]. is a three domains cysteine protease with an active site highly conserved
There are more than 6000 structurally identified flavonoid molecules. among all coronavirus. Domain I and II are six-stranded antiparallel β
These compounds are synthesized in plants in response to stressful barrels very similar to the architecture of chymotrypsin and picorna
conditions and play an important role in defending plant cells against virus 3C proteinases. The substrate-binding site is located in a cleft be
pathogens and insects [12–14]. From a chemical point of view, flavo tween these two domains. A long loop (residues 184 to 199) connects
noids are hydroxylated phenolic molecules synthesized by the phenyl domain II to the C-terminal domain (domain III, residues 200 to 300).
propanoid pathway and are distinguished by their structural class, This latter domain, a globular cluster of five helices, has been implicated
degree of hydroxylation, and polymerization. The hydroxyl functional in the proteolytic activity of 3CLpro. Anand et al. [24] and Dai et al. [23]
groups of flavonoids are responsible for their antioxidant activity and analyzed substrate-binding pocket of SARS-CoV and SARS-CoV-2,
are formed by two benzene rings (A-and B-rings), connected via a het respectively to design novel inhibitors for this protease and found that
erocyclic pyrene ring (C-ring) (Fig. 1). Flavonoids are divided into the thiol group of a cysteine residue in the S1′ site is important to anchor
different classes, such as anthocyanins, chalcones, dihydrochalcones, molecules by a covalent linkage, obtaining efficacious antiviral activity.
dihydroflavonols, flavanols, flavanones, flavones, flavonols, iso The S1′ site represents one of the four sites (S1’, S1, S2 and S4) highly
flavonoids (see Phenol-Explorer database at http://phenol-explorer.eu). conserved in the catalytic domain of 3CLpro of human coronavirus.
The pharmacological properties of flavonoids include antimicrobial, The four major structural proteins of coronavirus are: 1. the trimeric
antioxidant, anti-inflammatory, and antiviral functions. Flavonoids Spike glycoprotein (S) that localizes on the surface of virus envelope and
have been studied against a wide range of DNA and RNA viruses [15]. essential for virus entry into the host cells; 2. the membrane or matrix
For example, apigenin is active against picornavirus (RNA virus), protein (M); 3. the small envelope protein (E), both essential for the
inhibiting protein synthesis by suppressing IRES viral activity [16]. assembly and release of virions; 4. the nucleocapsid protein (N), that
Epigallocatechin-3-gallate (EGCG), active polyphenolic catechin that binds to RNA genome forming the helically symmetric nucleocapsid
accounts for approximately 59% of the total catechins from the leaves of [19]. In addition, some coronavirus genome encodes for a glycoprotein
the green tea (Camellia sinensis (L.), Kuntze) interferes with the repli of approximately 60–65 kDa called hemagglutinin esterase (HE). The
cation cycle of DNA viruses, such as hepatitis B virus, herpes simplex, role of this protein is still unclear, it possesses an acetyl-esterase enzy
and adenovirus [17]. matic activity able to disrupt sialic acid receptors on the host cells sur
To prepare this review article, especially the PubMed database www. face, helping the invasion and attachment of virion [22].
ncbi.nlm.nih.gov/pubmed/ (https://pubmed.ncbi.nlm.nih.gov/) was Genetic and comparative analysis of different known coronaviruses
consulted up the end of May 2020, to retrieve articles that included the represents a powerful strategy to identify potential drug targets against
following combination of terms: “coronavirus” and “flavonoid”. We the current outbreak and the 3CLpro protease is a good example. The first
selected those papers that convincingly focused on the antiviral activity three SARS-CoV-2 genomes isolated from bronchoalveolar-lavage fluid
of defined flavonoids against human coronaviruses, excluding some very and sequenced in Wuhan (China) showed the typical beta coronavirus
recent preprint articles on SARS-CoV-2 not certified by peer review that, organization (subgenus sarbecovirus): a 5′ untranslated region (UTR),
in our opinion, were of limited quality. We apologize in advance for replicase complex (ORF1a/b), S, E, M and N genes, 3′ UTR and several
possible citations omitted due to space limitations. unidentified non-structural ORF [1]. Comparing RNA sequence of 9
Chinese patients affected by COVID-19 with those of other coronavi
2. Coronavirus biology ruses, Lu et al. (2020) performed a phylogenetic analysis to determine
the evolutionary history of the virus going back to its likely origin [25].
2.1. Morphology and biochemistry The authors of this study found that SARS-CoV-2 RNA sequence shared
96% genetic material with a bat virus in a cave of Yunnan (China), but
Coronavirus is a family of one strand (+) RNA enveloped virus in the was distant from SARS-CoV (79% identity) and MERS-CoV (50% iden
order Nidovirales. They were originally identified in the sixties in the tity). Homology modelling revealed, however, that the new virus had a
United Kingdom and the United States where scientists isolated two similar receptor-binding domain structure (RBD) of spike protein to that
viruses causing common colds in humans [18]. Coronaviruses are of SARS-CoV, despite amino acid variation at few key residues. For this
spherical or pleomorphic, with a diameter of 80–120 nm. In 1968 reason, it was hypothesized that SARS-CoV and SARS-CoV-2 shared the
electron microscopy images revealed the virus crown-like structures same cellular receptor to enter in human cells, the protein Angiotensin
resembling the “solar corona” that give rise to the name of this family Receptor Enzyme-2 (ACE2) widely expressed in lung, heart, kidney,
derived from Latin word: “coronavirus” [19]. Since then and until last testis and gastrointestinal tract [25].
year, two highly pathogenic human strains emerged: SARS-CoV, in 2003
and MERS-CoV (Middle East Respiratory Syndrome coronavirus) in 2.2. Attachment and entry
2012 that caused, according to WHO, severe epidemic outbreaks [20,
21]. They are transmitted to humans from market civets and dromedary, Several steps are necessary to start and complete the coronavirus
respectively and both originated from bats, a natural reserve of hun infective cycle: 1. recognition and binding to the cellular receptor(s)
dreds of still unknown coronavirus [22]. (one or two); 2. changes in the conformation and proteolysis of S pro
The coronavirus RNA genome is bigger than other RNA viruses with tein; 3. fusion to cellular membrane; 4. entry of the virus into the host
size ranges from 26,000 to 32,000 bases including from 6 to 11 open cells by endocytosis [26]. In host cells, the virus uses the endogenous
reading frames (ORF). The first ORF (67% of the genome) encodes not cellular machinery to translate its replicase to transcribe and replicate
3
Fig. 2. A. Coronaviruses form enveloped and spherical particles of 100–160 nm in diameter. They contain a positive-sense, single-stranded RNA (ssRNA) genome
and nucleocapside proteins (N) that bind to RNA genome forming the nucleocapsid. The trimeric Spike glycoprotein (S) localizes on the surface of virus envelope and
is essential for virus entry into the host cells. It recognizes the host receptor protein ACE2 on cell membrane after cleavage and activation by two host serine-
proteases: TMPRSS2 and FURIN. Membrane or matrix protein (M) and small envelope protein (E) are both essential for the assembly and release of virions. B.
SARS-CoV-2 genome, genes and proteins. There are 10 open reading frames (ORFs). The first ORF (67% of the genome) encodes not structural proteins (NSP), while
the remaining ORFs give rise to accessory and structural proteins. ORF1a/b translates two polyproteins: pp1a and pp1b for the presence of a frameshift between
ORF1a and ORF1b. These polyproteins are processed by a main protease known as 3C-like-protease (3CLpro) and one or two papain–like proteases (PLpro) into 16
NSPs. NSPs produce replicase complex essential for viral replication: NSP12 encodes RNA dependent RNA Polimerase (RdPd) and NSP13 encodes Helicase. ORFs
2–10 encode viral structural proteins: Spike (S), Envelope (E), Membrane (M), Nucleocapsid (N) and other auxiliary proteins. In particular, Spike protein comprises
two regions: S1 with the receptor-binding domain (RBD) essential for the recognition of host receptor and S2, essential for membrane fusion and entry. Between S1
and S2 subunits there is the polybasic sequence recognized by host endo-proteases Furin. The activation site of S protein, is recognized by serine protease TMPRSS2 in
region S2′ of S2 domain.
viral RNA. The following steps consist of the translation of the structural of the viral phospholipidic membrane, is crucial for coronavirus infec
proteins by sub-genomic RNAs (sgRNA) generated during genome tion and pathogenesis. It includes two functional domains or subunits,
transcription/replication and, finally, the virion assembly and release S1, the globular head containing the RBD at the N-terminal, and the S2
[19]. subunit at the C-terminal responsible for virus-cell membrane fusion,
It is well known that the spike glycoprotein S, located on the surface followed by two heptad regions (HR1 and HR2) and the transmembrane
4
domain (TM). The S protein undergoes several post-translational mod complex (RTC) formed by different viral NSPs. After entry, genomic
ifications: its ectodomain is heavily glycosylated and, probably, the ol RNA (gRNA) is translated by host ribosomes in polyprotein pp1a and
igosaccharides could influence priming by host proteases and determine pp1b, which are auto-cleaved to form NSP. These NSPs induce a rear
antibody recognition [26]. In particular, membrane fusion depends on S rangement of cellular membrane to form double-membrane vesicles
protein cleavage by host cell proteases at S1/S2 and S2’ site responsible where the viral replication complexes are anchored [33]. The core
for S protein activation (Fig. 2). Hoffman et al. (2020) recently component of RTC complex is the catalytic subunit, NSP12 of an
demonstrated that the life cycle of SARS-CoV-2 begins with the RBD of RNA-dependent-RNA-polymerase (RdRp). For optimal function, this
the S protein that makes contact with the ACE2 receptor on the host cells enzyme requires accessory factors: NSP7 and NSP8 that increase RdRp
[27,28]. Two host serine proteases participate in this event: the trans template binding and processivity. NSP3 and NSP5 encode papain
membrane TMPRSS2 and the endo-protease Furin. The S1/S2 site in like-protease, PLpro, and 3CLpro, essential, as described above, for the
SARS-CoV-2 harbors multiple arginine residues not present on cleavage of polyprotein pp1a and pp1b [19]. Using the gRNA as a
SARS-CoV, but common to other human coronaviruses, like MERS-CoV, template, the coronavirus replicase synthesizes full-length negative
that are recognized by Furin protease (Fig. 2). The authors speculate that sense (− ) RNA, which, in turn, serves as a template for the synthesis of
the presence of this multibasic cleavage between S1/S2 sites may new genomic (+) gRNA and a set of different sgRNA, synthetized by
expand SARS-CoV-2 tropism and/or enhance its transmissibility discontinuous transcription. These sgRNAs encode viral structural and
compared to SARS-CoV, due to the ubiquitous presence of Furin-like accessory proteins. Amino acidic residues involved in RNA binding and
proteases in human tissues, especially lung [27]. Inhibition of catalytic active site of RdRp are highly conserved in different RNA virus
TMPRSS2 and Furin protease activities can be considered an interesting justifying the use of broad-spectrum antiviral inhibitors, such as
therapeutic option against coronavirus infection, especially COVID-19, Remdesivir. This drug, that very recently showed its efficacy in a
allowing the block and/or prevention of SARS-CoV-2 infection, as placebo-controlled trial in COVID-19 patients [34], is a prodrug of an
recently reported [28]. adenosine analogue that has been proposed as inhibitor of viral RdRp
An alternative therapeutic/preventive strategy may refer to mole through non-obligate RNA chain termination, a mechanism requiring
cules or antibodies capable of disrupting S protein interaction with the conversion of parent compound from mono-phosphate in a
ACE2. This interaction, due to natural selection (mutation and probably tri-phosphate form. In particular, Yin et al. (2020) studied the structure
a recombination event), is 10-20-fold stronger for SARS-CoV-2 respect to of the nucleotide template in complex with RdRp as a model to under
SARS-CoV and may explain the higher infectivity of former [28]. ACE2 stand how these drug categories can inhibit SARS-CoV-2 replicase ac
is a type I transmembrane protein, its physiological role consists in the tivity. A new compound, EIDD-2801, showed more potent effects than
maturation of the peptide hormone angiotensin, essential for the control Remdesivir in blocking virus replication for the capacity to form two
of vasoconstriction and blood pressure. From a biochemical point of extra hydrogen bonds with the key residue Lys545 within the catalytic
view, ACE2 is a dipeptidyl carboxypeptidase with the N-terminal domain of replicase and N4 hydroxyl group off the cytidine ring, and a
peptidase domain (PD) and the C-terminal collectrin (CLD) domain guanine base in the template strand [35].
ending with a single transmembrane helix and a ~40-residues intra Although genome replication/transcription is mainly mediated by
cellular segment [29]. The peptidase activity of ACE2 is not essential for the viral replicase, other host factors have been involved, as an example,
coronavirus infectivity because virus infecting respiratory tissues use coronavirus N protein, known to act as an RNA chaperone to facilitate
this protein essentially as a receptor, being expressed on the cells of the template switching, and the enzyme glycogen-synthase-kinase-3 (GSK3)
respiratory tract. In the lung, ACE2 is present in alveolar epithelial type [19]. Finally, RNA helicases (NSP13) represent the second most
II cells and bronchial epithelial and it was first recognized as a receptor conserved subunit of the RNA synthesis machinery in (+) RNA coro
for SARS-CoV, MERS-CoV and now for SARS-CoV-2. MERS-CoV also naviruses and are involved in diverse steps of their life cycle. They utilize
recognizes dipeptidyl peptidase 4 (DPP4) or CD26 as entry receptors the energy derived from the hydrolysis of nucleoside triphosphates to
[30]. unwind double-stranded RNA [33].
The RBD of S protein has a receptor binding motif (RBM) that makes The assembly of viral particles takes place in the ER-Golgi interme
the primary contact with the carboxyl-peptidase domain of ACE2 re diate complex under the control of M protein through homotypic in
ceptor. The amino-acidic sequence of RBM is 50% conserved between teractions. In this phase, M protein acts as a scaffold for virus assembly
SARS-CoV and SARS-CoV-2 and structural studies performed by Yan because the interactions between S and M and M and N proteins allow
et al. demonstrated that the extracellular PD of ACE2 is recognized by the recruitment of structural proteins to the assembly site. E protein
RBD through polar residues [31]. In particular, these researchers found contributes in this phase interacting with M and inducing membrane
that the most prominent alteration is the substitution of Val404 in the curvature [19]. Finally, mature virions are released in smooth-walled
SARS-CoV-RBD with Lys417 in the SARS-CoV-2-RBD. Structural data on vesicles via the secretory pathway and released by exocytosis.
co-crystalized proteins demonstrated that the surface of ACE2 contains In summary, coronavirus replication takes place in a membrane-
two “virus-binding hot-spot”, two Lys residues, essential for SARS-CoV protected and nuclease resistant microenvironment that contains (and
binding ACE2 creating positive charges that need to be neutralized by sequesters) the protein functions required for viral RNA synthesis. This
the coronavirus [32]. Two key residues in the RBM of SARS-CoV-2, strategy is believed to improve duplication/transcription fidelity and, in
Gln493 and Leu455, bind to these hotspots leading to considerable parallel, repress host defenses triggered by the presence of double-
stabilization of binding and a higher affinity for ACE2 than SARS-CoV. In stranded RNA [33].
addition, SARS-CoV-2 RBD shows a significant higher ACE2 binding
affinity due to specific substitutes (residues 482–485: Gly-Val-Glu-Gly) 3. Flavonoids against coronaviruses
that stabilize the interaction between them. Finally, Phe486, present
in the RBM of SARS-CoV-2, is inserted into a hydrophobic pocket of 3.1. Early works and effects of flavonoids on veterinarian coronaviruses
N-terminal α1 helix of ACE2, while a leucine residue present in RBM of
SARS-CoV forms probably a weaker contact owing to its smaller chain The first appearance in PubMed of flavonoids as potential antiviral
[32]. agents is dated back in 1951 [36] and in 1966 quercetin was indicated
among these compounds [37]. In 1977, the viricidal effect of quercetin,
2.3. Genome replication/transcription and virion assembly and release together with other flavonoids (apigenin, pelargonidin, procyanidin),
was demonstrated on parainfluenza virus Type 3 and herpes simplex
Virus replication takes place at the level of the cytoplasmic mem virus, but not on poliovirus Type 1 and adenovirus Type 3. The authors
brane and is mediated by a multi-subunit replication/transcription concluded that flavonoids may possess a potent viricidal activity, but
5
only a moderate inhibitory effect on virus multiplication [38]. However, cytotoxicity. This value was 200-400-fold higher respect to the Cmax of
later studies indicated that quercetin was both effective in reducing the 60 ng/ml (half-life about 3 h), considering that, in humans, a standard
infectivity and intracellular replication of Herpes Simplex Virus type 1 oral dose corresponds to about 1500 mg. This makes hardly practicable
(HSV–I), polio-virus type 1, Parainfluenza virus type 3 (Pf-3), and Res baicalin for antiviral prophylaxis or treatment, although after an intra
piratory Syncytial Virus (RSV) [39]. Hesperetin had no effect on infec venous administration of 360 mg in humans, the molecule reached a
tivity, but reduced intracellular replication, while catechin and naringin peak serum concentration of 74 μg/ml [44]. Two other flavonoids,
had no or limited effects on either virus infectivity or replication [39]. luteolin and quercetin, showed the capacity to block the entry of
The second half of the eighties saw a proliferation of studies on the SARS-CoV into host cells [45]. Luteolin inhibited in a dose-dependent
antiviral activities of flavonoids, due to the boost of antiviral researches manner, SARS-CoV infection of Vero E6 cells with EC50 value of 10.6
following the HIV emergency. A review article published in 1991 and μM (CC50 = 155 μM), while quercetin antagonized HIV-luc/SARS
focused on the capacity of 3-methoxyflavones to inhibit polio- and rhi pseudotyped virus entry with an EC50 of 83.4 μM. The cytotoxicity of
noviruses infection, concluded that natural products can interfere with quercetin was very low (CC50 = 3.32 mM) [45] (Table 1).
several antiviral mechanisms, from “adsorption of the virus to the host The medical herb Cinnamomi Cortex, obtained from the dried bark
cell to release from it” [40]. of Cinnamomum cassia (L.) J. Presl, has been used to prepare several
One of the first paper exploring the antiviral effect of flavonoids on organic fractions enriched in bioactive polyphenols. Among these, the n-
coronaviruses appeared in 1990 [41]. Here, the authors showed that butanol fraction was the most active in inhibiting HIV/SARS-CoV
quercetin reduced infectivity of human and bovine coronaviruses, OC43 pseudovirus infection dose-dependently with an IC50 of 37.3 μg/ml.
and NCDCV, respectively, by 50% at a concentration of 60 μg/ml. Other This result was confirmed also against the wild-type SARS-CoV infection
flavonoids, such as kaempferol, were ineffective, although the latter, at and the measured IC50 for the same fraction was 7.8 μg/ml [46]. The
10 μg/ml, reduced virus replication by 65% in NCDCV and 50% in OC43 authors attributed the observed antiviral activity to the presence in the
[41]. Bovine coronavirus, BCV, was also sensitive to a mixture of thea extract of procyanidin A2, procyanidin B1 and cinnamtannin B1, which
flavins from black tea (theaflavin, theaflavin-3-monogallate, thea showed an IC50 ranging between 30 and 40 μM in the plaque reduction
flavin-3′ -monogallate, and theaflavin-3,3′ digallate) with a mean EC50 of assay on SARS-CoV. However, none of the procyanidins inhibited the
34.7 μg/ml in infectivity assays on HRT-18 cell line [42]. On a different internalization of TfR (transferrin receptor, a marker of
Coronaviridae of veterinarian interest, the Porcine Epidemic Diarrhea clathrin-mediated endocytosis) and they did not affect ACE2 expression
Virus (PEDV), quercetin 7-rhamnoside inhibited PEDV replication in which, as reported above, is a SARS-CoV receptor [46] (Table 1).
Vero cells with an IC50 of 0.014 μg/ml and a CC50 (cytotoxicity con
centration 50%) of 100 μg/ml [43]. Other flavonoids, including quer 3.2.1. Flavonoids interaction with SARS-CoV and MERS-CoV proteases
cetin, apigenin, luteolin, catechin also showed significant anti-PEDV As reported above, SARS-CoV RNA genome encodes for proteinases
activity, but with IC50 values from 10- (apigenin) to 800-fold (catechin) that are required for replication and transcription. NSP3 and NSP5 are
higher than quercetin 7-rhamnoside indicating the importance of the two non-structural regions encoding for PLpro and 3CLpro (also called
o-dihydroxy functional groups at C-3′ and C-4′ and the rhamnoside at Mpro, see Section 2), respectively [47]. The former cleaves the
position 7 [43] (Table 1). NSP1-NSP3 replicase polyproteins [48], while the latter is responsible
for the processing of NSP4-NSP16 replicase products into individual
polypeptides [24]. Therefore, both the SARS-CoV 3CLpro and SARS-CoV
3.2. Flavonoids against SARS-CoV and MERS-CoV PLpro have been soon considered a potential target for the design and
development of antiviral drugs [47]. Using two independent assays
In a formulation of Traditional Chinese Medicine (TMC) for the measuring SARS-CoV 3CLpro cleavage activity, based on a cell-free and a
prevention and treatment of SARS-CoV, one of the components was the cell-based method, and aqueous extract from Isatis indigotica Fortune ex
flavone glycoside baicalin from Scutellaria baicalensis Georgi. This Lindl. (synonym of Isatis tinctoria L.) root showed a dose-dependent
compound was tested on fRhK4 cell line by a neutralization assay using capacity to inhibit the SARS-CoV 3CLpro proteolytic cleavage activity
10 isolated of SARS-CoV coronavirus from 10 different patients. Baicalin with an IC50 of 53.8 μg/ml and 191.6 μg/ml on the cell-free and
showed an EC50 of 12.5–25 μg/ml at 48 h without significant cell-based assay, respectively [49]. In the same experimental models,
several herb-derived flavonoids with accredited antiviral effects were
Table 1 tested (hesperetin, quercetin, and naringenin). Among these, only hes
Studies reporting antiviral activity of natural flavonoids against human and non- peretin dose-dependently inhibited cleavage activity of the 3CLpro in
human coronaviruses. cell-free and cell-based assays (IC50 60 μM and 8.3 μM, respectively)
Coronavirus Compounds Effects Methods Reference [49]. It is of interest that quercetin did not show any inhibitory effect on
Porcine epidemic Quercetin 7- IC50 = Vero cells [43] SARS-CoV 3CLpro, although it was reported to block the entry of
diarrhea virus rhamnoside 0.014 μg/ SARS-CoV into host cells [45]. To support the latter conclusion, using a
(PEDV) ml molecular docking approach, it was established that querce
Bovine coronavirus Theaflavins EC50 = HRT-18 [42] tin-3-β-galactoside bound to SARS-CoV 3CLpro with residue Gln189
(BCV) 34.7 μg/ cells
ml
playing a key role in stabilizing the binding [50]. In an in vitro assay,
HIV/SARS Quercetin EC50 = Vero E6 [45] using His-tag recombinant SARS-CoV 3CLpro, quercetin-3-β-galactoside
pseudotyped virus 83.4 μM cells inhibited the protease activity competitively with IC50 of 42.79 μM.
HIV/SARS Cinnamomi IC50 = Hep-G2 [46] Mutation of Gln189 to alanine did not reduce the enzymatic activity of
pseudotyped virus Cortex extract 37.3 μg/ cells
SARS-CoV 3CLpro, but lowered of about 2-fold the inhibitory potency of
ml
SARS-CoV Baicalin EC50 = fRhK4 [44] quercetin-3-β-galactoside, due to the reduction in binding affinity [50]
12.5-25 cell line (see Section 4). Quercetin was also used as a reference compound in a
μg/ml different study where several metabolites isolated from the leaves of
SARS-CoV Luteolin EC50 = Vero E6 [45] Torreya nucifera (L.) Siebold & Zucc were tested for their capacity to
10.6 μM cells
SARS-CoV Cinnamomi IC50 = 7.8 Vero E6 [46]
inhibit the proteolytic activity of a commercially available form of
Cortex extract μg/ml cells SARS-CoV 3CLpro using an in vitro assay based on FRET method [51].
SARS-CoV Procyanidin A2 IC50 = 30- Vero E6 [46] The most potent inhibitory effect was attributed to the biflavone
Procyanidin B1 40 μM cells amentoflavone with an IC50 of 8.3 μM and a non-competitive inhibition
Cinnamtannin B1
(Ki = 13.8 μM). In comparison, quercetin and luteolin showed an IC50
6
about 3-fold higher, apigenin was the less efficient with an IC50 about capacity to inhibit the proteolytic activity of the two virus proteases
34-fold higher respect to amentoflavone. An in silico docking simulation 3CLpro and PLpro from both SARS-CoV and MERS-CoV viruses, only the
demonstrated that the biflavone nicely fitted into the SARS-CoV 3CLpro prenylated quercetin derivative, papyriflavonol A, showed a potent,
binding pocket [51] (see section 4 below). A different series of flavo non-competitive inhibitory effect on SARS-CoV PLpro with an IC50 value
noids belonging to the four major groups, e.g. flavonol, flavanonol, of 3.7 μM. All the other compounds, presented IC50 values in the tens
isoflavone, and flavan-3-ol were tested in a different work [52]. Here and/or hundreds micromolar ranges, on all four proteases, although the
quercetin, gallocatechin gallate (GCG), EGCG resulted the most effective inhibition was dose-dependent [55]. Surface plasmon resonance (SPR)
in inhibiting the activity of recombinant SARS-CoV 3CLpro expressed in indicated that the interaction between papyriflavonol A and SARS-CoV
Pichia pastoris, a methylotrophic yeast, with an IC50 in the range 47–73 PLpro was due to a specific binding event with a KD of 212 μM, stimu
μM. Others, such as ampelopsin, puerarin, daidzein showed an IC50 lating the design of more effective coronavirus inhibitors [55]. A more
higher than 350 μM. A more detailed characterization of GCG inhibitory specific study on the inhibition of MERS-CoV 3CLpro by flavonoids was
capacity indicated a competitive inhibition mechanism (Ki = 25 μM). In published later [56]. Here, the authors demonstrated that among 40
addition, a computational docking analysis and hydrophobic and flavonoids tested ach at the concentration of 20 μM, four of them,
hydrogen bond interactions displayed binding energy of − 14 kcal/mol namely herbacetin, isobavachalcone, quercetin 3-β-D-glucoside and
of GCG to the active site of SARS-CoV 3CLpro highlighting the impor helichrysetin were the most effective with and IC50 of 40.59, 35.85,
tance of the galloyl moiety at 3-OH position for the inhibitory activity 37.03 and 67.04 μM, respectively. Docking analysis showed the S1 and
[52] (see section 4). The ethanol extract of Psoralea corylifolia L. (syn S2 sites of the protease play a key role in interaction with flavonoids. In
onym of Cullen corylifolium (L.) Medik) seeds led to the isolation of six fact, the 4-hydroxyl group of helichrysetin forms a hydrogen bond with
flavonoids, namely, bavachinin, neobavaisoflavone, isobavachalcone, the hydroxyl group of Tyr54 of the protease MERS-CoV 3CLpro; this
4′ -O-methylbavachalcone, psoralidin and corylifol A, all capable to seems to indicate a better affinity of helichrysetin since Tyr54 is located
inhibit in vitro SARS-CoV PLpro proteolytic activity using a fluorogenic deep inside of the hydrophobic S2 site [56]. Recently, the same research
peptide (Z-Arg-Leu-Arg-Gly-Gly-7-amido-4-methylcoumarin; Z-RLRG group tested the 64-flavonoid library in search of potential inhibitors of
G-AMC). All of them inhibited the protease in a dose-dependent manner SARS-CoV 3CLpro [57]. The screening ended up with the identification of
in the IC50 range of 4.2–38.4 μM with isobavachalcone and psoralidin herbacetin, rhoifolin and pectolinarin as the most prominent inhibitors
being the most active [53]. It is of interest that the mode of inhibition is with IC50 values of 33.17, 27.45 and 37.78 μM, respectively. Also in this
mixed, being the inhibition constants referred to type I, with the in case, docking studies demonstrated that the better affinity of rhoifolin
hibitor bound to the free enzyme, or type II, where the enzyme-substrate could be due to the coordinated binding through the polar S1 site, the
complex is the target of the inhibitor [53]. Using a similar approach, five hydrophobic S2 and the S3′ site with no strong tendency [57] (Table 2).
new and rare geranylated flavonoids, tomentin A, tomentin B, tomentin Chalcones also represented an interesting flavonoid group with sig
C, tomentin D, tomentin E inhibited SARS-CoV PLpro with IC50 raging nificant inhibitory activity against the two main coronavirus proteases.
between 5.0 and 14.4 μM [54]. Among 10 new polyphenols derived In a study based on nine alkylated chalcones isolated from the Japanese
from Broussonetia papyrifera (L.) L’Hér. ex Vent and assayed for their plant Angelica keiskei (Miq.) Koidz, it was demonstrated a significant
Table 2
Studies reporting inhibitory activity of natural flavonoids against human coronavirus proteins.
Viral proteins Compounds Effects Methods Reference
SARS-CoV PLpro Geranylated flavonoids (tomentin A- IC50 = 5.0–14.4 μM fluorogenic peptide Z-RLRGG-AMC [54]
E)
SARS-CoV PLpro Bavachinin IC50 = 4.2–38.4 μM fluorogenic peptide Z-RLRGG-AMC [53]
Corylifol A Isobavachalcone
4′ -O-methylbavachalcone
Neobavaisoflavone
Psoralidin
SARS-CoV PLpro Papyriflavonol A IC50 = 3.7 μM fluorogenic peptide Z-RLRGG-AMC [55]
SARS-CoV PLpro Xanthoangelol E IC50 = 1.2 μM cell-free method [58]
SARS-CoV 3CLpro Xanthoangelol E IC50 = 11.4 μM cell-free method [58]
IC50 = 7.1 μM cell-based method
SARS-CoV 3CLpro Herbacetin IC50 = 33.17 recombinant protein; FRET method [57]
Pectolinarin IC50 = 27.45
Rhoifolin IC50 = 37.78 μM
SARS-CoV 3CLpro Isatis indigotica extract IC50 = 53.8 μg/ml cell-free method [49]
Hesperetin IC50 = 60 μM
SARS-CoV 3CLpro Quercetin-3-β-galactoside IC50 = 42.79 μM recombinant protein [50]
SARS-CoV 3CLpro Amentoflavone IC50 = 8.3 μM recombinant protein; FRET method [51]
SARS-CoV 3CLpro Epigallocatechin gallate IC50 = 47–73 μM. recombinant protein; FRET method [52]
Gallocatechin gallate
Quercetin
MERS-CoV 3CLpro Helichrysetin IC50 = 40.59 μM recombinant protein FRET method [56]
Herbacetin IC50 = 35.85 μM
Isobavachalcone IC50 = 37.03 μM
Quercetin 3-β-D-glucoside IC50 = 67.04 μM
SARS-CoV NTPase/ Quercetin IC50 = 8.1 μM recombinant protein FRET-based dsDNA unwinding assay [62]
helicase
SARS-CoV NTPase/ 7-O-arylmethylquercetin derivatives IC50 = 2.7–5.2 μM recombinant protein FRET-based dsDNA unwinding assay [63]
helicase
SARS-CoV NTPase/ Myricetin IC50 = 2.71 μM recombinant protein FRET-based dsDNA unwinding assay [64]
helicase
ATPase activity Scutellarein IC50 = 0.86 μM ATP hydrolysis colorimetric assay
N protein Catechin gallate 0.05 μg/ml (40% quantum dots (QDs)-conjugated RNA oligonucleotide on [60]
Gallocatechin gallate inhibition) biochip
7
capacity to inhibit both SARS-CoV 3CLpro and SARS-CoV PLpro protease Paidu Decoction (QFPD), consisting of 21 components (herbs and min
activity in cell-based and cell-free assays. For the former protease, the erals drugs), showed an effectiveness of 92% in patients at all stages
cell-free assay indicated a range of IC50 of 11.4–39.4 μM for 7 out of 9 including subjects cured and discharged, cases where clinical symptoms
chalcones. The same compounds inhibited SARS-CoV PLpro with an IC50 were disappeared, remained stable without aggravation or significantly
of 1.2–26.0 μM. The most active resulted xanthoangelol E, containing improved [66]. The beneficial effects of QFPD were evident after 6 days
the perhydroxyl group, that showed an IC50 of 11.4 and 1.2 μM for SARS- of treatment with chest CT results that ameliorated, tracheobronchial
CoV 3CLpro and SARS-CoV PLpro, respectively. In the cell-based cleavage, shadow was normal, and inflammation was also absorbed accordingly
this chalcone resulted to inhibit SARS-CoV 3CLpro with an IC50 of 7.1 μM with the theory of TMC that identifies in the lung the primary target of
and a CC50 of 65.6 μM [58] (Table 2). QFPD against COVID-19 [65]. In the attempt to identify the major
constituents of QFPD and to investigate its pharmacological mechanism
3.2.2. Flavonoids interaction with SARS-CoV N protein against COVID-19 infection, Yang et al. [66] applied an integrated
As discussed in the previous paragraphs, the genome of SARS-CoV multidisciplinary approach (in silico technology that included pharma
encodes structural proteins, including the N protein, an alkaline pro cological network and molecular networking of LC-MS data). They
tein with a lysine-rich region that suggests a nuclear localization signal. identified 129 compounds clustered in 14 groups, where flavonoids
The N factor plays a key role in virion assembly through its interactions represented 45% of the total. Finally, a recent paper, using a computa
with the viral genome and M protein. It also participates in viral RNA tion approach, demonstrated that narcissoside, an iso
synthesis [59]. An interesting approach was established for the rhamnetin-3-O-rutinoside flavonoid (glycosyloxyflavone) present in
screening of potential inhibitors of SARS-CoV N protein using a several wild plants, is a potent inhibitor of 6W63, the term that indicates
mimicking on glass chip the direct binding of viral RNA to N protein. The the experimental structure of SARS-CoV-2 3CLpro protease (https://co
screening showed that among the 23 polyphenolic compounds tested, vid-19.uniprot.org/uniprotkb/P0DTD1). The narcissoside showed a
only (− )-catechin gallate and (− )-GCG were able to displace the binding higher affinity than the standard inhibitor X77. The two inhibitors
of N protein to the RNA oligonucleotide. Starting from 0.005 μg/ml, shared the same complex, but narcissoside interacted with three amino
both compounds in a concentration-dependent manner attenuated the acids (Leu167, Pro52 and Pro168) different than those interacting with
binding affinity on the designed biochip and at the concentration of X77 (His172, Leu27 and Thr26). Finally, the better fit of the glyco
0.05 μg/ml, they showed up to 40% inhibition activity [60] (Table 2). syloxyflavone into the active site of the 6W63 was also reinforced by
thirteen hydrogen bonds compared to the four established by X77 [67].
3.2.3. Flavonoids interaction with SARS-CoV NTPase/helicase
SARS-CoV NTPase/helicase (also called NSP13) represents an 4. Flavonoids as potential inhibitors of SARS-CoV proteins: In
attractive target for anti-SARS therapy since it plays crucial role in the silico studies
viral life cycle [61]. Quercetin and its derivatives have been proposed as
a possible inhibitor of the helicase. Firstly, using the recombinant pro From the previous sections, we learned that the bioinformatics
tein and a FRET-based assay, it was reported that the IC50 of quercetin approach represents an essential tool to identify antagonist compounds
towards the Duplex DNA-unwinding activity of the SARS-CoV NTPa that can specifically target the binding sites of SARS-CoV viral proteins
se/helicase was of 8.1 μM, highlighting the importance of the presence through complex molecular interactions responsible for viral attach
of a diketo acid core and a free catechol unit [62]. The same group later ment and replication. This goal can be achieved by targeting structurally
published the synthesis of several 7-O-arylmethylquercetin derivatives. important binding sites, highly conserved regions in non-structural
Three of them, with 3′′ -Cl, 3′′ -CN, and 4′′ -Cl substituent on arylmethyl proteins including NSP12 RdRp, NSP13 helicase, and the proteases
group, tested in this study showed inhibitory activity against SARS-CoV 3CLpro and PLpro [68]. 3CLpro is highly conserved in all coronaviruses, it
NTPase/helicase ranging between 2.7 and 5.2 μM, the same order of is essential for the polyprotein cleavage and, as reported above, repre
magnitude of the parental compound [63]. The NSP13 helicase pos sents an important target for the development of new inhibitory agents
sesses a dsDNA unwinding activity as well as an ATPase activity [24].
allowing the helicase to translocate along with the nucleic acids by The analysis of SARS-CoV-2 3CLpro crystal structure indicated that
hydrolyzing ATP. A screening of about 64 natural compounds, including the enzyme is a homodimer (chains A and B), composed of 306 amino
several flavonoids (strangely quercetin was not included in this study), acids and each monomer consists of three different domains [69,70].
all tested at the concentration of 10 μM, did not evidence any compound Two catalytic domains, I (residues 8–101) and II (residues 102–184),
able to significantly inhibit the dsDNA unwinding activity of the NSP13 have an antiparallel β-barrel structure, arranged perpendicular to each
helicase in a FRET-based assay [64]. However, when the same com other. The active site is located in the cleft between domain I and
pounds were tested for their capacity to inhibit the ATPase activity of the domain II with a catalytic dyad (His41 and Cys145), connecting to the
helicase, two of them, myricetin and scutellarein, emerged as a potent helical domain III by a long loop region [71]. Domain III (residues
inhibitor of NSP13 ATPase with IC50 values of 2.71 and 0.86 μM, 201–303), a globular cluster of five α-helices, is involved in the dimer
respectively. It is of interest that these compounds did not inhibit to the ization of the 3CLpro, essential for the catalytic activity. N-finger of one
same extent the ATP hydrolysis activity of the hepatitis C virus helicase, subunit is involved in the arrangement of the substrate binding-pocket
indicating specificity for the SARS-CoV enzyme [64] (Table 2). through a salt-bridge interaction between Glu290 and Arg4 of each
protomer. It has also been shown that S1, S2, and S4 subsites are
3.3. Flavonoids against SARS-CoV-2 involved in substrate recognition. S1 subsite is the polar site of 3CLpro
containing a small aliphatic residue (Ser, Gly, Ala) in the P1 position of
Following the COVID-19 pandemic, the interest of the potential the proteolytic site. The hydrophobic S2 subsite contains a large hy
therapeutic use of flavonoids against coronavirus infection focused on drophobic residue in P2, whereas the side chain of Val and Ala are at P3
SARS-CoV-2 virus. Only few papers have been published up to now, but and P4 side, respectively, forming a small hydrophobic pocket [69].
it is easy to predict that this number will exponentially increase in the Due to the COVID-19 pandemic, compelling efforts concentrated in
next months or weeks. Considering the impact of the COVID-19 identifying and designing new inhibitory compounds targeting the
outbreak in China, it was expected that TCM could have a primary SARS-CoV-2 proteases and, among these, flavonoids attracted scientists’
role in the therapy of the SARS-CoV-2 infection or, at least, in the alle interest being them promising agents against SARS-CoV infection (see
viation of its symptoms. In fact, on February 2020, the rate of TCM the section above and these reviews [57,72]). Accumulating evidence
treatment for COVID-19 in China was of about 90% with only 5% of are going in this direction. The inhibitory activity of flavonoids against
patients that manifested worse clinical signs [65]. The formula Qingfei SARS-CoV cysteine proteases in the low micromolar range was higher if
8
compared to the effects of peptide-derived inhibitors [55]. hydrogen bonds with the flavonoid. Moreover, querce
The structural features of flavonoids were responsible for their tin-3-β-galactoside formed hydrophobic interactions with residues
selectivity, suggesting that the two phenyl groups were associated with Leu141, Asn142, Met165, and Glu166 of SARS-CoV 3CLpro (Fig. 4B).
inhibitory potency, as resulting by IC50 values of the studies cited above According to this structure, it is possible to speculate that the four hy
for kaempferol, quercetin, and quercetin-β-galactoside [55]. Docking droxyl groups of quercetin are strongly responsible for its inhibitory role
stimulation screening was performed to predict the binding affinity of and the spatial conformation based on sugar moiety and 7-hydroxy site
flavonoids, showing that apigenin, luteolin, quercetin, daidzein, epi allowed structural changes through hydrogen bonding interactions [50].
gallocatechin, and kaempferol were able to inhibit the proteolytic ac Two other examples can be taken by the works of Nguyen et al. [52]
tivity of SARS-CoV 3CLpro [57] (Fig. 3). In particular, the molecular and Ryu et al. [51] commented in Section 3 above. In the former, the
dynamics simulations allow to predict that His41, Gly143, and Glu166 GCG interaction with the substrate-binding pocket of 3CLpro involved 7
formed interactions with common functional groups of flavonoids that hydrogen bonds with residues in the catalytic binding pocket and hy
showed inhibitory activities [73]. For example, Fig. 4A shows the pre drophobic interactions of carbon atoms with His41, Cys145, Met165,
dicted interaction between the catalytic site of SARS-CoV 3CLpro and the Glu166, Asp187, Arg188, and Gln189 of 3CLpro [52]. Interestingly, the
phenyl group of kaempferol, which locates in the hydrophilic task of methylation of hydroxyl groups at C-7 reduced the inhibitory activity,
SARS-CoV through a hydrogen bond with Glu166. Other hydrogen revealing that the biflavone amentoflavone showed the highest inhibi
bonds are formed between the hydroxyl groups and Ile188, Asp142, tory activity with an IC50 value of 8.3 μM (Table 2). To support the
while the chromen-4-one scaffold was in the hydrophobic S2 site [57]. A effective inhibitor role of this molecule, computer-docking analysis
similar association between inhibitory effect and molecular interaction indicated the interaction with the S1 site of 3CLpro, forming two
established by docking analysis was described for querce hydrogen bonds between the C5 hydroxyl group and the nitrogen atom
tin-3-β-galactoside against SARS-CoV 3CLpro [50]. The analysis of of the imidazole group of His163 and the -OH of Leu141. An additional
structure–activity relationship highlighted crucial pharmacophore fea hydrogen bond occurred between the hydroxyl group in the B ring with
tures, showing that the specific interactions between querce Gln189, involving the S2 site of 3CLpro [51].
tin-3-β-galactoside and the active site of the protease occurred by six The structure-activity relationship analysis of flavones namely api
hydrogen bonds with residues of the catalytic binding pocket. Indeed, genin, luteolin and quercetin evidenced that the substitution of C-30
His41, Gly143, Ser144, Cys145, Glu166, and Gln189 residues were hydroxyl group, as in luteolin, and the hydroxyl group at the C-30 po
located near the pharmacophore spheres. In detail, the inhibitory effect sition of quercetin, may play a pivotal role in SARS-CoV 3CLpro inhibi
of quercetin-3-β-galactoside was strongly associated to the bind with tion [51]. This observation was supported by a recent study, which
Gln189 suggesting the formation of four hydrogen bonds with O and N explains by molecular docking study that luteolin forms 5 hydrogen
atoms of the Gln189 side chain (Fig. 4B). In addition, the N atom of the bonds with Gln189, Leu4, Asn142, Thr26 and hydrophobic interaction
main chain of Glu166 was involved in the formation of the other two with Met49 and Val3, in agreement with its lower binding energy [68].
Fig. 3. Graphical representation of computer docking screening indicating the interaction of flavonoids and the binding pocket of SARS-CoV 3CLpro.
9
Fig. 4. A. Predicted interaction of kaempferol with the catalytic site of SARS-CoV 3CLpro by hydrogen bond formation. B. Hydrogen bond and hydrophobic in
teractions between quercetin-3-β-galactoside and the active site of SARS-CoV 3CLpro.
Based on these observations, it is not surprising that molecular docking infections, including SARS-CoV-2. Similarly, it has been hypothesized
approach, summarized in Fig. 3, supports the role of flavonoids in the that treating patients affected by COVID-19 with senolytics and other
inhibition of SARS-CoV 3CLpro by binding His41 and Cys145 of the anti-aging drugs may represent a prominent approach to prevent viral
catalytic site and other active site residues (e.g., Met49, Gly143, His163, transmission and quercetin has been included among these potential
His164, Glu166, Pro168, and Gln89), stimulating their validation by in agents to be tested in clinical trials [77].
vitro and in vivo studies. A recent paper confirmed that luteolin can also We express caution on these interpretations too optimist suggesting
bind and inhibit the SARS-CoV 3CLpro [68]. that coronavirus viral infections can be ameliorated or prevented by
Another interesting field of investigation is represented by inhibition diet. However, although difficult to assess from an experimental and
of RNA viral replication, proposing RdRp as a candidate for targeted clinical point of view, the concept that strengthening the immune sys
drug development. To this purpose, a molecular screening evidenced tem, reducing inflammation and oxidative stress during pandemic can
that theaflavin can interfere with the catalytic pocket of SARS-CoV-2 be achieved by increasing the consumption of food groups and key nu
RdRp, showing binding energy of − 8.8 kcal/mol. By means of molecular trients remains an attractive hypothesis [78,79]. Obstacles to this
docking, it has been demonstrated that hydrophobic interactions are assumption are represented by the well-known and largely accepted
involved in binding of theaflavin to RdRp. In addition, hydrogen bonds limits of natural compounds, including flavonoids, in foods and/or in
were established between functional moieties of theaflavin and residues nutraceutical formulations. The extremely low bioavailability, their
Asp452, Arg553 and Arg624 of RdRp [74]. high bio-transformation due to the intestinal adsorption and complexity
of gut microbiota make unlikely that flavonoids and their metabolites
5. Conclusions and future directions can reach blood concentrations in the micromolar range. These limits
not only make nutritional supplementation ineffective in humans, but
From the data reported in the previous sections, the pleiotropic na are in clear contradiction with the effective concentrations of flavonoids
ture of polyphenols and, among them, the flavonoid class, is emerging as tested against coronaviruses and viral proteins cited in the previous
a promising and powerful cornucopia of natural compounds with po sections, all in the micromolar range [80] (Tables 1 and 2).
tential antiviral capacity. We reported and commented on the results of We and others [72] are more prone to believe that the real appli
several studies largely based on docking simulations suggesting the cability of flavonoids as antiviral drugs resides in the therapy, more than
possibility that specific flavonoids can interact with and inhibit key the prevention. In fact, the large part of the works commented above
factors responsible for the virus life cycle. In different biological fields, refers to the direct binding of specific flavonoids to viral targets,
the therapeutic applications of flavonoids are usually accompanied by although with IC50 of tens of micromolar. This suggests that, based on in
strengthens and weaknesses, generally shared by other bioactive phy vitro assays and docking models, it will be possible to design and
tochemicals. As an example, being flavonoids present in our diet, it is chemically synthesize more efficient compounds based on the flavonoid
easy to associate their beneficial effects with the consumption of foods/ structures, where key active residues are conserved, while others un
dietary patterns enriched in this class of compounds. Focusing our dergo modifications accordingly to the SAR analysis. Of course, both
analysis on their antiviral properties, recent reviews support this view. natural and synthetic compounds will need functional validations that
In fact, Messina et al. (2020) suggested that dietary intervention may cannot be limited to in vitro assays based on recombinant proteins
ameliorate COVID-19 outcomes and polyphenols/flavonoids can (Table 2). To this aim, the recent COVID-19 pandemic is speeding up
contribute to these effects reducing inflammation and blocking nuclear enormously the commercialization of new reagents and kits to identify
NF-κB translocation [75]. Others suggested that “nutraceuticals and SARS-CoV-2 inhibitors that will probably accelerate the development of
functional foods have broad potential for preventing the mechanisms of innovative methods to assess how coronaviruses infect cells and how to
viral infection and modulating immune responses” [76], and hypothe block the infection. The therapeutic application of flavonoids, or their
size a link between the senolytic activity of some flavonoids (e.g. derivatives, as antiviral agents, as pure compounds or in combination
quercetin) and the higher susceptibility of older people to viral with canonical antiviral drugs, presents also the advantage to mitigate
10
the critical issue of their scarce bioavailability. In fact, pharmacological composition and divergence of the novel coronavirus (2019-nCoV) originating in
China, Cell Host Microbe 27 (2020) 325–328.
(not nutritional) doses, given for a limited amount of time can be
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Chemico-Biological Interactions 328 (2020) 109211

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Roles of flavonoids against coronavirus infection

certainly lead to the development of new therapeutic protocols.

Abbreviations ORF Open reading frame

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