Effects of polyphenol compounds on inﬂuenza A virus replication
and deﬁnition of their mechanism of action
, Ignacio Celestino
, Roberta Costi
, Giuliana Cuzzucoli Crucitti
, Luca Pescatori
, Ettore Novellino
, Paola Checconi
, Anna Teresa Palamara
, Lucia Nencioni
Roberto Di Santo
Istituto Pasteur Cenci Bolognetti - Dip. Chimica e Tecnologie del Farmaco, ‘‘Sapienza’’ University of Rome, P.le Aldo Moro 5, 00185 Rome, Italy
Istituto Pasteur Cenci Bolognetti - Dip. Sanità Pubblica e Malattie Infettive, ‘‘Sapienza’’ University of Rome, P.le Aldo Moro 5, 00185 Rome, Italy
Dipartimento di Dip. di Scienze di Base e Applicate per l’Ingegneria, ‘‘Sapienza’’ University of Rome, Via del Castro Laurenziano 7, 00161 Rome, Italy
Dipartimento di Chimica Farmaceutica e Tossicologica, Università di Napoli ‘‘Federico II’’, Via D. Montesano 49, 80131 Napoli, Italy
San Raffaele Pisana Istituto Scientiﬁco di Ricerca e Cura, Via della Pisana 235, 00166 Rome, Italy
Received 21 March 2012
Revised 16 May 2012
Accepted 25 May 2012
Available online 4 June 2012
A set of polyphenol compounds was synthesized and assayed for their ability in inhibiting inﬂuenza A
virus replication. A sub-set of them showed low toxicity. The best compounds within this sub-set were
4and 6g, which inhibited the viral replication in a dose-dependent manner. The antiviral activity of these
molecules was demonstrated to be caused by their interference with intracellular pathways exploited for
viral replication: (1) MAP kinases controlling nuclear-cytoplasmic trafﬁc of viral ribonucleoprotein com-
plex; (2) redox-sensitive pathways, involved in maturation of viral hemagglutinin protein.
Ó2012 Elsevier Ltd. All rights reserved.
Each year inﬂuenza A viruses (IAV) cause thousands of deaths
and hospitalizations. The pandemic caused by the 2009 inﬂuenza
A (H1N1) virus—the ﬁrst of the 21st century—was characterized
mainly by mild-moderate infections similar to those seen with
but several cases of acute respiratory distress
syndrome and pneumonia in previously healthy persons were also
Inﬂuenza A viruses are enveloped, negative strand RNA
viruses belonging to the Orthomyxoviridae family. Their genome
consists of eight single-stranded RNA segments encoding 11 pro-
teins including hemagglutinin (HA), one of the main surface glyco-
The receptor binding site of HA is necessary for the virus
to bind galactose-bound sialic acid on the surface of host-cells, and
16 different subtypes have been isolated thus far from different
Inﬂuenza virus replication has been studied in depth, and
several antiviral agents all targeting viral structures, have been
Among them, amantadine and rimantadine have a spe-
ciﬁc inhibitory effect on type A but not on type B inﬂuenza viruses.
These agents block the ion-channel activity of the viral matrix (M2)
protein, which is mainly required for virus uncoating.
the viral neuraminidase (NA) inhibitors such as oseltamivir, zanam-
ivir, and peramivir are the mainstay of pharmacological protocols to
ﬁght global inﬂuenza pandemics.
However, the long-term efﬁcacy
of these inhibitors is often limited by their toxicity and by the high
incidence in the selection of drug-resistant viral mutants that is al-
Actually, the main effort of the preclinical research
is addressed to block the viral replication by interfering with path-
ogen-exploited host-cell machinery.
This approach could give
important advantages, including the broad-spectrum efﬁcacy, the
antigenic properties, and the reduced probability to select drug-
resistant viral strains.
In particular, a very promising and innova-
tive strategy to develop new antiviral agents could be based on the
inhibition of intracellular pathways that are speciﬁcally activated
by inﬂuenza virus to ensure its replication.
of these pathways are highly sensitive to changes in the intracellu-
lar redox state,
and as a matter of fact their activation is induced
by the oxidative stress caused by infections by DNA or RNA virus,
Dietary polyphenols, such as resveratrol (RV) [5-[(1E)-2-(4-
hydroxyphenyl)ethenyl]-1,3-benzenediol (found in red wine) and
curcumin [diferuloyl methane; 1,7-bis-(4-hydroxy-3-methoxy-
0968-0896/$ - see front matter Ó2012 Elsevier Ltd. All rights reserved.
Corresponding author. Tel.:+39 6 49693247.
E-mail address: firstname.lastname@example.org (R. Costi).
Authors to indicate that they contributed equally to the work.
Bioorganic & Medicinal Chemistry 20 (2012) 5046–5052
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phenyl)-1,6-heptadiene-3,5-dione] (found in curry powder), were
reported to posses anticancer,
Curcumin is a colouring compound present in the rhizome of Cur-
cuma longa L. It has been used for thousands of years in Southeast
Asia and Indian folk medicine to treat various diseases and eradicate
It shows many pharmacological properties
antioxidant, iron-chelating, and
some other activities.
For example, the antioxidant activity of cur-
cumin is responsible for its ability to decrease the incidence of colon
cancer, and for its anti-atherogenic property.
It has cytoprotective
effect on PC12 cells against 1-methyl-4-phenylpridinium ions-in-
duced neurotoxicity through the anti-apoptotic and anti-oxidative
properties of the Bcl-2-mitochondria-ROS-iNOS pathway.
cently, curcumin has been proven to exert anti-inﬂuenza activity
by inhibition of the virus-cell attachment. These studies demon-
strated that treatment of cells with curcumin greatly reduced the
yield of IAV at sub-cytotoxic doses. Pre-incubation of virus with cur-
cumin pronouncedly inhibited inﬂuenza virus plaque formation.
Unfortunately, curcumin is water insoluble, poorly dissolves in
organic phase, and shows low bioavailability in vivo after oral
It is unstable at neutral-basic pH values and
in serum-free medium degrading to vanillin, ferulic acid, feruloyl
methane and trans-6-(40-hydroxy-30-methoxy-phenyl)-2,4-di-
RV is a very interesting polyphenol belonging to the stilbene
family. It is found in several fruits, vegetables and beverages
including red wine. It is one of the most important plant polyphe-
nols with proven salutary activity on animal health. The most
important source of polyphenols and in particular RV for human
diet is grape (Vitis vinifera).
In the last two decades the potential protective effects of RV
and neurodegenerative diseases,
as the chemo-preventive properties against cancer,
largely investigated. RV appears to be capable of interfering with
several intracellular signaling pathways, including those activated
by protein kinase C (PKC) and by mitogen-activated protein kinases
RV has been also reported to exert an antiviral activity in differ-
ent experimental systems.
A few years ago, we showed that
RV inhibits inﬂuenza virus replication in vitro and it is also effec-
tive in increasing the survival of inﬂuenza virus-infected mice.
Surprisingly, these effects are not related to its well-known antiox-
idant activity. They are rather related to the inhibition of intracel-
lular pathways JNK (c-Jun N-terminal kinase) and p38MAPK
involved in regulation of viral ribonucleoprotein (vRNP) complex
trafﬁc across the nuclear membrane, a key step of viral replication
cycle that precedes viral assembly and release.
The biological activity of RV is limited by its low bioavailability
and metabolic instability. In fact, stilbene double bond is readily
oxidized by cytochrome P450 monooxygenases into highly reac-
tive epoxides, that may act as carcinogenic metabolites.
ther, under UV irradiation, RV converts to the (Z)-isomer
this was claimed as one of the reason for the presence of small
amounts of (Z)-resveratrol in wines. The conversion from (E)- to
(Z)-conﬁguration makes resveratrol less stable
decreases its biological activity.
Therefore, pursuing our studies on compounds active against
inﬂuenza and taking advantage of our experience in the synthesis
of polyhydroxylated analogues,
we decided to synthesize a num-
ber of curcumin and resveratrol analogues with the aim of obtain-
ing compounds with improved ability in interfering with the cell
pathways exploited by the virus for its replication, such as kinase
pathways and intracellular redox state. These compounds were
evaluated for their antiviral activity and their structures are
reported in Figure 1.
All derivatives tested except 6b, were prepared as reported in
Method of synthesis of compound 6b, chemical
and physical data are reported in Supplementary data.
In a ﬁrst set of experiments the cytotoxicity of the compounds
has been evaluated in a test on conﬂuent monolayers of MDCK cells.
Cells were plated at concentration of 2 10
/ml, treated after 24 h
with various concentrations (range 5–40
g/ml) of the synthesized
compounds ( 1, 2, 3, 4, 5, 6a–m), and incubated for the following
24 h. Microscopical examination, Trypan blue exclusion and cells
counts demonstrated that the compounds 1, 2, 3, 5, 6a, 6b, 6d,
6e, 6h, 6i, 6l and 6m, produced a dose-dependent toxic effect.
Indeed, morphological alterations, loss of cells viability and modiﬁ-
cation of cell multiplication rate in treated cells were observed
(data not shown). These substances have been excluded for the fol-
lowing experiments. On the contrary, the treatment with the other
compounds did not induce any toxicity on cell monolayer, even if
the compound 6g caused a slight alteration of cell morphology
starting from 20
g/ml and the compounds 4, 6c, and 6f starting
g/ml. These results were further conﬁrmed using the
Comp. R R1 R
6a H H H H OH OH H
6b H H OH H OH H OH
6c H OH H OH H OH H
6d H H OH H OH OH H
6e H NH2 H H OH OH H
6f OH H H H H Cl H
6g OH H OH H H OH H
6h OH H OH H H OCH3 H
6i OH H OH H H CH3 H
6l OH H OH H OH OH H
6m OH H OCH3 H H F H
HO OH X
Figure 1. Structures of curcumin and resveratrol analogues.
R. Fioravanti et al./ Bioorg. Med. Chem. 20 (2012) 5046–5052 5047
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazoliumbromide (MTT)
proliferation assay (data not shown).
3.1. Compounds 4 and 6g inhibit late phases of inﬂuenza virus
First, to assess whether the compounds that did not cause toxic
effect on cells, could exert an antiviral effect on inﬂuenza virus rep-
lication, conﬂuent monolayers of MDCK cells highly permissive to
inﬂuenza virus replication, were infected with inﬂuenza A/Puerto
Rico/8/34 H1N1 (PR8) virus at low multiplicity of infection (0.01
M.O.I.) to allow multi-cycle replication. One hr after adsorption
the cells were washed and different concentrations of compounds
or DMSO (control infected CI cells) were added to MDCK and main-
tained for the duration of the experiment. As shown in Figure 2A,
24 h post infection (p.i.) virus titer, measured as the amount of virus
released into the cell supernatant by means of hemagglutinating
unit (HAU) assay, was dose-dependently (range 5–20
inhibited by compounds 4and 6g. In particular, viral replication
was markedly reduced by 10
g/ml of 6g (75% vs CI cells) and
g/ml of 4(75% vs CI cells) while the concentration of
g/ml of 6g was able to completely inhibit viral replication.
However, since it induced some alteration of the monolayer, we
chose to not use it for the following experiments. The compounds
6c and 6f were able to inhibit inﬂuenza virus in a dose-dependent
manner (data not shown), however the molecular mechanisms
underlying their anti-inﬂuenza activity are still under investigation.
Next, to identify the step(s) of the inﬂuenza virus life-cycle that
were affected by compounds 4and 6g, we ﬁrst evaluated the effect
of these compounds on the PR8 virus alone. No signiﬁcant antiviral
effect was detected when a stock solution of PR8 virus was incu-
bated for 1 h with 4or 6g and then used for infection. Similarly,
virus production was not affected when compounds were present
in cell culture medium only during the 1 h phase of viral adsorp-
tion or when MDCK cells underwent a overnight pre-infection
treatment with two compounds, with drugs washout right before
viral challenge (data not shown). Then, 4and 6g were added to
MDCK cells immediately after virus challenge and afterwards re-
moved at different time points (Fig. 2B). Post-infection treatment
with both compounds for 2 or 4 h produced no signiﬁcant antiviral
effect, which conﬁrmed that they did not act by preventing virus
entry into the cells. In MDCK exposed to 4or 6g for the ﬁrst 6 h
after infection, slight decreases (about 30%) in viral replication
were noted 24 h p.i. More substantial inhibition was observed
when exposure was extended to the ﬁrst 8 h (55%) or 24 h (75%)
p.i. In other experiments, the treatment was started at different
times after PR8 infection, and the compounds remained in the cell
culture medium through 24 h after infection (Fig. 2C).
The highest inhibition (about 79%) of viral replication was
achieved when treatment began 2 and 4 h after viral challenge.
When treatment was delayed until 6 h after infection, effects were
more limited but still observable and ﬁnally, when two compounds
were added 8 h p.i., no signiﬁcant inhibition was noted. These data
indicate that the antiviral activity of the compounds is largely re-
lated to their inhibition of virus life-cycle steps occurring 2-8 h
p.i., and possibly related to post-transcriptional events.
3.2. Compound 4 inhibits expression of late viral proteins
Nucleoprotein (NP) is the most abundant inﬂuenza A viral pro-
tein that is synthesized immediately after infection, whereas the
major external glycoprotein hemagglutinin (HA), neuraminidase
(NA) and matrix protein 1 (M1) are late gene products.
to determine whether the inhibition of viral replication was related
to the modulation of viral protein synthesis, the cells were infected
with PR8, and compounds 6g (10
g/ml) or 4(20
immediately added after PR8 adsorption. Twenty-four hours after
infection, protein from cell lysates were separated by sodium
dodecyl sulfate-polyacrylammide gel electrophoresis in reducing
conditions and immunoblotted with anti-inﬂuenza Abs. As shown
in Figure 3 (left panel), while the treatment with 6g did not cause
any effect on the expression on viral proteins, except for NA
Figure 2. Compounds 4and 6g inhibit speciﬁc steps of inﬂuenza virus life-cycle. (A) Different concentrations of each compound were added to PR8-infected (M.O.I = 0.01)
MDCK cells. Viral yields 24 h p.i. are expressed as percentages of those recorded for control infected (CI) cells treated with 0.02% dimethyl sulfoxide (DMSO; the concentration
present in culture medium containing the highest dose of compounds). Values shown are means of four experiments, each run in duplicate.
vs CI. Viral yields 24 h p.i. for CI cells and cells infected/treated as follows: (B) 4and 6g (20 and 10
g/ml, respectively) were added to cell cultures immediately after PR8
challenge and removed at different time points (2, 4, 6, 8 or 24 h) after infection; (C) 4and 6g were added 2, 4, 6, or 8 h p.i., and maintained in the culture medium for 24 h
after infection. Values shown are means of two experiments, each run in duplicate.
P<0.01 vs CI.
5048 R. Fioravanti et al./ Bioorg. Med. Chem. 20 (2012) 5046–5052
protein (by 42% vs untreated cells), a signiﬁcant inhibition of some
viral proteins was observable after treatment with 4.
In particular, densitometric analysis (Fig. 3, right panel) re-
vealed decreased expression of HA (by 25% vs untreated), NA (by
56% vs untreated) and M1 (by 62% vs untreated), even if the
expression of NP protein was slight inhibited. These results suggest
that the antiviral activity exerted by 4could be due to an interfer-
ence with viral protein synthesis, while the effect of 6g to the inhi-
bition of other steps of virus life-cycle.
3.3. Compounds 6g and 4 retain viral NP in the nucleus of
During inﬂuenza virus replication, viral RNAs are packaged into
helical ribonucleoprotein (RNP) complexes with polymerase and
NP in the host-cell nucleus and are subsequently exported into
the cytosol to be assembled with the other structural proteins.
Our previous studies demonstrated that RV blocks the nuclear-
cytoplasmic translocation of RNP complexes.
whether the same mechanism was involved in 4and 6g inhibition
of inﬂuenza virus replication, immunoﬂuorescence analysis of
vRNP trafﬁcking was performed. MDCK cells were infected with
PR8 at a high M.O.I. to allow single-cycle replication. Eight hours
after infection, cells were ﬁxed, permeabilized and stained with
-diamidino-2-phenylindole hydrochloride (DAPI), and then
incubated in turn with primary anti-NP antibody and with ﬂuores-
cein isothiocyanate-conjugated secondary antibody. As shown in
Figure 4 in the untreated cells, NP was located predominantly in
the cytosol, and addition of compounds 4and 6g led to the inhibi-
tion of viral RNP export from nucleus to cytoplasm. In particular,
the treatment with 6g caused a strong inhibition of nuclear-cyto-
plasmic trafﬁc of NP, suggesting that this compound could inter-
fere with some pathways that regulate this important step of
inﬂuenza virus replication. These results were conﬁrmed by wes-
tern blot analysis of NP localization in the nuclear and cytoplasmic
extracts after 8 h p.i. As shown in Figure 5 the treatment with the
compounds, especially with 6g, caused an inhibition of NP levels in
the cytosol, while the protein was mainly expressed in the nucleus
of the cells. Several kinase cascades, such as PKC and MAPKs are
activated during inﬂuenza virus infection.
Pleschka et al.
onstrated that ERK (extracellular signal-regulated kinase) phos-
phorylation promotes vRNP trafﬁc and virus production. Our
previous studies demonstrated the role of p38MAPK activity in nu-
clear export of RNP complexes of inﬂuenza virus. In particular, the
inhibition of this kinase decreased vRNP trafﬁc, phosphorylation of
viral NP (a key event for the export in the cytoplasm), and viral
titers in cells supernatants.
Moreover, it is known that RV inter-
feres with several intracellular pathways including those activated
by PKC and MAPKs.
Therefore to determine the underlying mechanism for the
inhibitory effect of 4and 6g on nuclear translocation of vRNP com-
plexes, the possible involvement of the PKC pathway in the antivi-
ral activity of the compounds was investigated in human NCI-H292
infected cells. As shown in Figure 6, the phosphorylation of PKD,
the PKC downstream effector,
was observed in infected cells
6 h p.i., and this event was markedly reduced by compounds 4
and 6g. Then, the phosphorylation of p38MAPK, JNK, and ERK path-
ways was analyzed. As shown in Figure 6, the compound 6g
strongly diminished p38MAPK and JNK phosphorylation, but had
no effect on that of ERK 1 and 2. Similar results were obtained in
cells treated with 4, although the inhibition of p38MAPK phos-
phorylation was less pronounced. These results indicate that the
strong inhibition of p38MAPK phosphorylation by 6g leads to vRNP
retaining in the nucleus of infected cells.
3.4. Compound 4 restores the intracellular redox balance during
viral infection and affects viral HA localization
Inﬂuenza virus infection is associated with redox changes char-
acteristic of oxidative stress, including depletion of GSH levels and
a general oxidative stress in both in vivo and in vitro experimental
Our recent studies have demonstrated a key role of GSH
levels in the regulation of viral HA maturation. Indeed, the addition
of a GSH derivative was able to restore the reduced environment in
infected cells and, as a consequence, to interfere with intracellular
redox-regulated pathways responsible for the folding of viral pro-
teins. The ﬁnal effect on viral replication was an impairment of vir-
al propagation by impeding HA plasma-membrane insertion.
Therefore we evaluated the potential antioxidant activity of these
compounds. Twenty-four hours after infection, as expected inﬂu-
enza virus-induced a signiﬁcant depletion of intracellular GSH con-
tent (Fig. 7A). On the contrary, in infected cells treated with 4, GSH
levels did not diminish during viral infection, and GSH content was
similar to that measured in control (mock-infected) cells. The
treatment with 6g was also able to restore the intracellular GSH
content, even if in less extent. A conﬁrmation of this difference
was obtained by measuring the redox potentials of 4and 6g. Com-
pound 4showed Epa1 = 0.75 V compared to Epa1 = 1,35 V found
for 6g. It is evident the higher reducing property of 4 compared
to that of 6g.
Next, we evaluated whether these compounds could interfere
with HA maturation, especially with HA localization on the plas-
ma-membrane. Immunoﬂuorescence studies of HA localization
were performed 8 h after PR8 infection with high M.O.I. Cells were
ﬁxed and stained with anti-HA antibody and with secondary anti-
body conjugated with phycoerythrin. As shown in Figure 7B, viral
protein HA was highly expressed, diffused into the cells and local-
ized predominantly on the plasma-membrane of infected cells.
Figure 3. Compound 4inhibits expression of late viral proteins. (Left panel) expression of viral proteins in inﬂuenza A/PR8/34 H1N1 infected cells untreated or treated with
6g or 4, respectively. After 24 h, cells were lysed and cell homogenates were separated in reducing conditions by 12% gel, transferred to nitrocellulose membrane and
immunostained with goat polyclonal anti-inﬂuenza Abs. PR8 virus protein molecular weights are indicated to the right of the ﬁgure. HA, hemagglutinin; NP, nucleoprotein;
NA, neuraminidase; M1, matrix protein 1. Results are shown for one representative experiment of three performed. (Right panel) densitometric analysis of viral proteins
expression shown in (left panel). Results are expressed as ratio of each viral protein to actin.
R. Fioravanti et al./ Bioorg. Med. Chem. 20 (2012) 5046–5052 5049
On the contrary, the treatment with the compounds, especially
with 4, inhibited HA expression, and viral protein localization on
the plasma-membrane was impaired. In particular, in these latter
cells HA was localized on the peri-nuclear zone (see white arrows,
Fig. 7B). These results suggest that 4could exert its antiviral activ-
ity through regulation of some redox-sensitive pathways involved
in HA maturation. Further studies are in progress to better charac-
terize the molecular mechanism involved in this inhibition.
In the present study we have demonstrated the antiviral activ-
ity of compounds 4and 6g. These compounds inhibited the inﬂu-
enza A virus replication in a dose-dependent manner without
inducing any cytotoxic effect. The antiviral activity of these mole-
cules is due to inhibition of two key steps of inﬂuenza virus life-
cycle: (1) nuclear-cytoplasmic trafﬁc of vRNP; (2) maturation of
viral HA protein.
These fundamental steps are ﬁnely regulated by redox-sensitive
intracellular pathways that are activated during viral infection. In
particular, vRNP trafﬁc is regulated by several intracellular kinases,
including PKC and MAPKs.
In our previous paper we have dem-
onstrated that inﬂuenza virus-activated p38MAPK phosphorylated
viral NP, an event needed for vRNP nuclear export. Inhibition of
p38MAP led to retention of NP in the nucleus of infected cells.
Here, we demonstrate that the two compounds were able to inhibit
p38MAPK and, as consequence, to impede the export of vRNP from
the nucleus to the cytoplasm, conﬁrming the role of p38MAPK in
the regulation of this step of viral replication.
Viral infection is often associated with redox changes character-
istic of oxidative stress, and these alterations are mainly due to
virus-induced depletion of intracellular GSH levels.
in GSH content and general oxidative stress have been also demon-
strated during inﬂuenza virus infection in both in vivo and in vitro
We have recently demonstrated a key
role of GSH levels in the regulation of viral HA maturation. Indeed
the addition of a GSH derivative impairs viral propagation by
Figure 4. Compounds 6g and 4block the nuclear-cytoplasmic vRNP trafﬁc. MDCK cells were infected with 1 M.O.I. to allow single-cycle replication. Cells were ﬁxed,
permeabilized and stained with monoclonal anti-NP Ab and analyzed by ﬂuorescence microscopy. Nuclei were stained with DAPI. Merged images are shown in the third
Figure 5. Compounds 6g and 4retain the NP in the nucleus of infected cells.
Cytosolic and nuclear extracts from 4or 6g treated and untreated cells were
subjected to SDS-PAGE and immunoblotted with anti-NP Abs. The same nitrocel-
lulose ﬁlters were then stripped and restained with anti-
-tubulin or anti-lamin A/
C. Above each blot, densitometric analysis expressed as the ratio of viral NP/tubulin
or lamin is reported. Results are shown for one representative experiment of three
Figure 6. Compounds 4and 6g interfere with PKC pathway. NCI-H292 cells were
mock- or PR8-infected (1 M.O.I.), treated with 4or 6g and lysed 6 h p.i. Proteins
were separated by 10% SDS–PAGE and gels were blotted onto nitrocellulose
membranes and immunostained with rabbit anti-phospho-PKD, anti-phospho-ERK
1/2, anti-phospho-p38MAPK or anti-phospho-JNK Abs. The same nitrocellulose
ﬁlters were then stripped and restained with anti-PKD, anti-ERK1/2, anti-p38MAPK,
or anti-JNK Abs, respectively. Results are shown for one representative experiment
of three performed.
5050 R. Fioravanti et al./ Bioorg. Med. Chem. 20 (2012) 5046–5052
impeding HA plasma-membrane insertion.
The antioxidant activ-
ity of RV has been well demonstrated in numerous studies.
ever, in our previous paper, RV was not able to restore intracellular
levels of GSH in inﬂuenza virus-infected cells.
Here, the addition
of the new compounds, in particular of 4, to infected cells was able
to restore virus-induced depletion of GSH. Moreover, we have ob-
served that the compounds, especially 4, were able to interfere
with HA localization on plasma-membrane, an event that occurs
when HA redox-regulated maturation process is completed.
These results suggest that the compounds could interfere with
some redox-sensitive intracellular pathways involved in matura-
tion of viral HA. Further studies are in progress to evaluate the
molecular mechanisms underlying this process.
Overall the data demonstrated that the compounds 4and 6g
exert their anti-inﬂuenza activity by inhibiting intracellular meta-
bolic pathways rather than viral proteins. Inactivation of host-cell
functions that are essential for the virus replication offers two
important advantages: not only it is more difﬁcult for the virus
to adapt to, but it can also expected to affect viral replication
independently from virus type or strain.
This work was partially supported by the Italian Ministry of
Instruction, Universities, and Research (Progetto PON), and PRIN
n°2008 CE75SA the Italian Ministry of Health, and Fondazione
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.05.062.
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