1
J Virol Methods. 2012 Jun;182(1-2):93-8. Epub 2012 Mar 23. 2
3
February 27, 2012 4
Fisetin and rutin as 3C protease inhibitors of enterovirus A71
56
Ying-Ju Lin1,2, Yi-Chih Chang3
, Nai-Wan Hsiao4, Jing-Ling Hsieh3,4, Ching-Ying 7
Wang5, Szu-Hao Kung6, Fuu-Jen Tsai1,2, Yu-Ching Lan7, and Cheng-Wen Lin3,8,* 8
9 1
Department of Medical Research, China Medical University Hospital, Taichung, 10
Taiwan 11
2
School of Chinese Medicine, China Medical University, Taichung, Taiwan 12
3
Department of Medical Laboratory Science and Biotechnology, China Medical 13
University, Taichung, Taiwan 14
4
Institute of Biotechnology, National Changhua University of Education, Changhua, 15
Taiwan 16
5
School of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, 17
China Medical University, Taichung, Taiwan 18
6
Department of Biotechnology and Laboratory Science in Medicine, National Yang 19
Ming University, Taipei, Taiwan 20
7
Department of Health Risk Management, China Medical University, Taichung 404, 21
Taiwan 22
8
Department of Biotechnology, College of Health Science, Asia University, Wufeng, 23
Taichung, Taiwan 24
25 26
Running title: Fisetin and rutin inhibit EV-A71
27 28 29 30 31
*Corresponding author: Cheng-Wen Lin, PhD. Department of Medical Laboratory 32
Science and Biotechnology, China Medical University, No. 91, Hsueh-Shih Road, 33
Taichung 404, Taiwan 1
Telephone: 886-4-22053366 ext 7210. Fax: 886-4-22057414. 2
E-mail address:[email protected] 3
4
Abstract
5Enterovirus A71 (EV-A71) causes severe complications: encephalitis, 6
pulmonary edema, and death. No effective drug has been approved for clinical use. 7
This studyinvestigated theantiviral effects of flavonoids against EV-A71.An invitro
8
inhibitor screening assay using recombinant EV-A71 3C protease (3Cpro) 9
demonstrated fisetin and rutin inhibiting 3Cpro enzymatic activity in a 10
dose-dependent manner. Cell-based fluorescence resonance energy transfer (FRET) 11
assay with an EV-A71 3Cpro cleavage motif probe also confirmed that fisetin and 12
rutin inhibited the replication of EV-A71 in cells. Virus replication assay indicated 13
that fisetin and rutinreduced significantly the EV-A71-induced cytopathic effect and 14
viral plaque titers in RD cells culture. The IC50 values of plaque reduction against 15
EV-A71 were 85 µM for fisetin and 110 µM for rutin. Therapeutic indices 16
(CC50/IC50 of plaque reduction assays) of fisetin and rutin exceeded 10. The study 17
suggests thatfisetin and rutininhibit the replication of EV-A71. 18
19
20
Keywords: enterovirus A71, 3C protease, flavonoids, fisetin, rutin
1. Introduction
1
Enterovirus A71 (EV-A71), an important pathogen in the Picornaviridae family, 2
causes hand-foot-mouth disease (HFMD), herpangina, aseptic meningitis, or severe 3
neurological complications like encephalitis and poliomyelitis-like paralysis (Singh et 4
al., 2002). EV-A71-induced brainstem encephalitis has a poor prognosis and a high 5
fatality rate (Mizuta et al., 2009). Several EV-A71 outbreaks were reported in Western 6
Pacific countries,in association with severe neurological disease (Mizuta et al., 2009; 7
Lin et al., 2006; Yang et al., 2009; Yang et al., 2009). For example, EV-A71 8
outbreaks in Taiwan caused 78 deaths in 1998 (Lin et al., 2003; Huang et al., 1999; 9
Ho et al., 1999; Chang et al., 1999), 25 deaths in 2000, and 26 deaths in 2001 (Lin et 10
al., 2006). Prevention and/or treatment of EV-A71 infection are very important, but 11
the FDA-approved drugs are not available. Developing effective agents against 12
EV-A71 infectioncouldcontribute to public health. 13
The enterovirus genome consists of a 7.4 kb single-stranded positive-sense RNA 14
genome (Pallansch and Roos, 2007) encoding a single large polyprotein that is 15
cleaved by enteroviral 2A and 3C proteases. After protein synthesis, viral polyproteins 16
cleave into capsid proteins (VP1-VP4), and nonstructural proteins (2A, 2C, 3A-3D). 17
Inhibition of 2A protease (2Apro), 3C protease (3Cpro) or 3D RNA polymerase could 18
block enterovirus replication significantly, indicating potential molecular targets for 19
development of antiviral agents (Chen et al., 2008; Pawlotsky et al., 2007). Moreover, 1
3Cpro is required for the additional cleavage events within viral protein precursors 2
that produce factors critical to protein processing and RNA replication. 3Cpro has also 3
been showed to cleave several important host proteins including poly(A)-binding 4
protein (PABP), cleavage stimulation factor 64 (CstF-64), TIR-domain-containing 5
adapter-inducing interferon-β (TRIF), and interferon regulatory factor 9 (IRF9) 6
(Rivera et al., 2008; Weng et al., 2009; Hung et al., 2011; Lei et al., 2011). EV-A71 7
3Cpro affects mammalian cell Pol I-III transcription and innate immune defense. 8
Comparison of published amino acid sequences from rhinoviruses and enteroviruses 9
shows considerable variability in the 3Cpro-coding region but strict conservation of 10
catalytic triad residues. The viral 3Cpro structure resembles that of well-defined 11
serine protease chymotrypsin (Kuo et al., 2008). Conservation of critical 3Cpro amino 12
acid residues offers to design potent and broad-spectrum anti-enterovirus agents. 13
Antiviral compounds that inhibit the 3Cpro activity have emerged by mimicking of 14
3Cpro substrates (peptide inhibitors) (Dragovich et al., 1998; Patick et al., 1999; 15
Matthews et al., 1999). An inhibitor of rhinoviruses 3Cpro, rupintrivir (formerly 16
AG7088) shows potencyin vitro against many enteroviruses (Binford et al., 2005).
17
Screening 3Cpro inhibitors highlights function-based approaches to develop rapidly 18
antiviral agents against enteroviral infections. 19
Flavonoids occur in vegetables, Citrus herbs in particular (Benavente et al., 1
2008). Researches show their biological properties, including multiple anticancer, 2
cardiovascular antimicrobial and anti-inflammatory activities (Manthey et al., 2001; 3
Orhan et al., 2009). They also exhibit a broad-spectrum antiviral activity, efficiently 4
inhibiting replication of human rhinovirus, Sabin type 2 poliovirus, hepatitis A virus, 5
coxsackievirus B4 and echovirus 6 (Conti et al., 1992; Conti et al., 1990; Genovese et 6
al., 1995; Salvati et al., 2004). However, their antiviral mechanisms are still unclear. 7
Lack of scientific evidence showing the molecular pathways of their action diminishes 8
their clinical utility. 9
This studyinvestigated theinhibitory effects of flavonoids on the3Cpro activity 10
and EV-A71 replication in RD cells.E. coli-expressed EV-A71 3Cpro was purified for
11
in vitro 3Cpro activity assays; fluorescence resonance energy transfer (FRET) probe
12
with a 3Cpro cleavage motif was used for testing the substrate specificity of EV-A71 13
3Cpro in cells. Among flavonoids, fisetin and rutin showed significantly inhibitory 14
effects on 3Cpro activity and EV-A71 replication. 15
16
2. Materials and methods
17
2.1. Viruses and cells
18
EV-A71 isolate CMUH01 was obtained from the Clinical Virology Laboratory, 19
China Medical University Hospital in Taiwan. RD cells (ATCC accession no. 1
CCL-136) weregrown in Dulbecco’s Modified Eagle's Medium (DMEM; HyClone 2
Laboratories, Logan, Utah, USA) with 10% fetal bovine serum (FBS; Biological 3
Industries, Kibbutz Beit Haemek, Israel). HeLa-G3CwtR HeLa cells transfected with 4
the plasmid expressing the recombinant fusion protein (green fluorescent protein-a 5
3Cpro cleavage motif-red fluorescent protein) as a FRET probe were cultured in 6
DMEM with 10% FBS and 20 µg/ml zeocin.All media were supplemented with 100 7
U/mL of penicillin and streptomycin, and 2mML-glutamine. 8
9
2.2. Chemicals
10
Kaempferol, 5-methoxyflavone, myricetin and rutin were purchased from Sigma 11
(St. Louis, MO, USA), chrysin and fisetin from Extrasynthese (Genay, France). 12
13
2.3. Construction, expression and purification of EV-A71 3C protease
14
A full-length gene encoding EV-A71 3Cpro was amplified by PCR with cDNA 15
of EV-A71 isolate CMUH01. Forward primer
16
5’–GCGCGGATCCGGGCCCAGCTTAGACTT-3’ and reverse primer 17
5’-GCGCCTCGAGTTGCTCGCTGGCAAAATA -3’were used, containing BamHI 18
and XhoI restriction enzyme sites (underlined), respectively.ThePCR products were 19
digested with BamHI and XhoI, and then cloned into pET24a (Merck KGaA, 1
Darmstadt, Germany). Plasmid containing EV-A71 3Cpro gene was subsequently 2
transformed into E. coli Origami B (DE3) (Merck KGaA, Darmstadt, Germany). For 3
expression of recombinant 3Cpro fused with a C-terminal His tag as described in a 4
recent report (Lu et al., 2011), a 20 ml overnight culture of a single colony was used 5
to inoculate 2 L of fresh LB medium containing 25µg/ml kanamycin. Cells were 6
grown to an A600 of 0.6 and then induced with 1mM IPTG. Aftera5-h incubation at 7
37 ℃, cells were harvested by centrifugation at 7000g for 15 min. Cell pellets 8
obtained from 2 L cell culture were suspended in 80 mL lysis buffer containing 25 9
mM Tris-HCl, pH 7.5, and 150 mM NaCl. French-press instrument (AIM-AMINCO 10
spectronic Instruments, NY, USA) disrupted cells at 12,000 psi. After the 11
centrifugation, the supernatant of the cell lysates was loaded onto a 20-mL Ni-NTA 12
column equilibrated with 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 5 mM 13
imidazole. The column was washed with 5 mM imidazole followed by a 30 mM 14
imidazole-containing buffer. His-tagged 3Cpro was eluted with 25 mM Tris-HCl, pH 15
7.5, 150 mM NaCl, and 300 mM imidazole.Purified His-tagged 3Cpro recombinant 16
protein was dialyzed against a 2× 2 L buffer containing 12 mM Tris-HCl, pH 7.5, 17
120 mM NaCl, 0.1 mM EDTA, 7.5 mM β-mercaptoethanol, and 1 mM DTT. The 18
concentration of purified 3Cpro protein was determined using the Bio-Rad Protein 19
Assay Kit (catalog number 500-0001). 1
2
2.4. Protease activity assay
3
Horseradish peroxidase (HRP) type VI-A (Sigma, Saint Louis, Missouri, USA) 4
containinga 3Cpro cleavage motif (Gln-Gly pairs at the residues 295-296) was used 5
as the EV-A71 3Cpro substrate for establishing an in vitro enzymatic assay. To 6
examine the cleavage activity of recombinant 3Cpro protein, the 3Cpro at 7
concentrations of 0, 0.005, 0.01, 0.05, and 0.1 µg/ml was incubated with HRP (0.2 8
µg/ml) for 2h at 37℃, and then the remaining HRP activity was determined using a 9
chromogenic substrate ABTS [2,2'-azino-di-(3-ethyl-benzthiazoline-6-sulphonic 10
acid)]/H2O2and the optical intensity of the developed color was measured at 405 nm.
11
For screening EV-A71 3Cpro inhibitors, fisetin, chrysin, myricetin, kaempferol, 12
rutin, puerin, and 5-methoxyflavone were screened for anti-EV-A71 3Cpro activity. 13
Each flavonoid (200 µM concentration), EV-A71 3Cpro and HRP were incubated for 14
2h at 37℃in 96-well plates. The remaining activity of HRP in each reaction was 15
determined with a chromogenic substrate ABTS/H2O2 and the intensity of the
16
developed color was measured at 405 nm. Percentage inhibition of EV-A71 3Cpro 17
activity was calculated as (OD405HRP+drug+3C protease–OD450HRP+3C protease) / (OD450HRP 18
only –OD450HRP+3C protease) x 100%. To determine the IC50 values, fisetin and rutin at 19
concentrationsof 0, 10, 100, 250, 750 and 1000 µM were incubated with 3Cpro and 1
HRP for 2h at 37 ℃ in 96-well plates in vitro. The inhibitory percentage was 2
determined as described above. Concentration for inhibiting the 3Cpro enzymatic 3
activity by 50% (IC50) was then determined, using the ID50 program developed by
4
John L. Spouge (National Center for Biotechnology Information) (Spouge, 1992). 5
6
2.5. Cell viability assay
7
For the cell viability assay, RD cells were cultured overnight on 96-well plates. 8
DMEM medium containing fisetin or rutin atconcentrations of0, 1, 10, 100 and 1000 9
µM was added and incubated for another 48 hours, followed by WST-1 assay. Cell 10
proliferation reagent WST-1 (Roche, Basel, Switzerland) was used for 11
spectrophotometric quantification of cell proliferation, viability, and chemosensitivity 12
in accordance with manufacturer’s directions. After a 1-h incubation of a WST-1 13
reagent (10µL per well) at 37℃, the optical density was measured at 450 nm was 14
measured using a 96-well plate reader. Cell survival rates were calculated as the 15
optical density ratio at 450 nm of treated to untreated cells. Quadruplicate wells were 16
analyzed for each concentration; data represent mean ± SD of three independent 17
experiments. Cytotoxic concentration showing 50% toxic effect (CC50) was 18
determined using the ID50 program. 19
1
2.6. Fluorescence resonance energy transfer (FRET) assay
2
Approximately 80-90% confluent monolayers of the HeLa-G3CwtR cells 3
expressing the FRET probe (described above) were infected with EV-A71 at a 4
multiplicity of infection (MOI) of 0.25, 0.5, or 1. After 90-min adsorption, inoculum 5
was aspirated and 500 µl of medium (Dulbecco’s modified Eagle’s medium containing 6
2% FBS) alone or medium with fisetin or rutin at 200 µM concentration was added to 7
each well.Cells were harvested 48 h post-infection, and lysates were transferred to 8
96-well plates for fluorescence. The fluorescent intensity of the FRET probes was 9
measured by a fluorescent-plate reader with an excitation wavelength at 390/20 nm 10
(for GFP2 at 510/10 nm) and an emission wavelength at 590/14 nm (for DsRed2), in 11
which DsRed2 was excited by the emission of GFP2at 510/10 nm.Data presented are 12
mean values of experiments in triplicate. 13
14
2.7. Cytopathic reduction (CPE) assay
15
RD cells were cultured in DMEM medium containing 2% FBS and infected with 16
EV-A71 at MOI of 0.5 in the absence and presence of fisetin or rutin at 200 µM 17
concentration. After 48-h incubation at 37℃, EV-A71-induced cytopathic effect was 18
observed and photographed. 19
1
2.8. Plaque reduction assay
2
Confluent layers (> 90%) of RD cells in 6-well plates were inoculated with 3
EV-A71 (100 pfu per well) in the absence and presence of fisetin or rutin at 4
concentrations of 10, 100 and 1000 µM. After 1-h incubation at 37°C, confluent layers 5
of RD cells were covered with 3 mL of the overlay medium containing 1.5% agar and 6
incubated in a CO2 incubator for 2 days at 37°C. Finally, cell layers were fixed with
7
formaldehyde and stained with 0.1% Crystal violet, as described previously (Chen et 8
al., 2009). Concentrationfor reducingthe number of plaques by 50% (IC50) was then 9
determined, using ID50 program. 10
11
2.9. Virucidal Activity Assays
12
EV-A71 (104 pfu) was mixed with testcompounds(200μM)ormedium,and 13
then incubated for 60 min at room temperature. Residual infectivity of 1000-fold 14
dilutions of each virus/compound mixture wasdetermined using the plaque assay as 15 describedabove. 16 17 3. Results 18
3.1. Preparation of recombinant 3C protease
For preparing recombinant 3Cpro protein,the cDNA fragment of EV-A71 3Cpro 1
gene was cloned into the pET24a expression vector (Supplemental Figs. 1A-1B) and 2
in-frame fused with a C-terminal hexa-His-tag. Recombinant 3Cpro protein was 3
expressed in the transfected E. coli cells after IPTG induction and purified by 4
immobilized-metal affinity chromatography (IMAC) (Supplemental Fig. 2A). 5
Western blot analysis of purified recombinant 3Cpro protein with anti-His-tag 6
antibody revealed an immunoreactive band near 21-kDa as expected molecular weight 7
(Supplemental Fig. 2B). 8
9
3.2. Inhibiting in vitro cleavage of 3C protease by fisetin and rutin
10
To evaluate recombinant 3Cpro activity, HRP type VI-A containing a 3Cpro 11
cleavage motif (the Gln-Gly pair) as a 3Cpro substrate was used for in vitro enzymatic 12
assays. The enzymatic activity of purified 3Cpro protein at the desired concentration 13
was proportional to the amount of the cleaved HRP, being inversely proportional to 14
the remaining HRP activity in the reaction mixture. The optical density of the color 15
developed by the remaining HRP with its chromogenic substrateexhibited a reverse 16
dose-dependent manner as 3Cpro concentration rose (Table 1).This study established 17
a HRP-based enzymatic assay for initially screening potent EV-A71 3Cpro inhibitors 18
according to substrate specificity, and the anti-3Cpro assay method is capable of 19
accurately screening potent inhibitors. 1
Flavonoids show a wide range of anti-enterovirus activity, efficiently inhibiting 2
human rhinovirus, Sabin type 2 poliovirus, hepatitis A virus, coxsackievirus B4 and 3
echovirus 6 infections (Conti et al., 1992; Conti et al., 1990; Genovese et al.,1995; 4
Salvati et al., 2004). This study tested flavonoids including chrysin, fisetin, 5
kaempferol, myricetin, rutin, puerin, and 5-methoxyflavone for in vitro anti-EV-A71 6
3Cpro activity in 96-well plates (Table 2). Data show fisetin and rutin at 200 µM 7
concentration decreasing EV-A71 activity byover 30%. We selected fisetin and rutin 8
(0, 10, 100, 250, 750 and 1000 µM concentrations) for more detailed examination of 9
3Cpro inhibition study. Figure 1 proves both fisetin and rutin exhibited the 3Cpro 10
inhibition activity in a dose-dependent manner. Results highlighted dose-dependently 11
inhibitory effects of fisetin and rutin on in vitro cleavage activity of recombinant 12
3Cpro using HRP-based enzymatic assays. 13
14
3.3. Inhibition of EV-A71 replication in vitro by fisetin and rutin
15
Both fisetin and rutin (less toxic compounds) showed CC50 values above 1000 16
µM (data not shown). HeLa-G3CwtR cells continuously expressing a FRET probe 17
(GFP2-3C cleavage motif-DsRed2 fusion protein) were used to test the substrate 18
specificity and the enzymatic activity of EV-A71 3Cpro in cells, as described in prior 19
study (Tsai et al., 2009).This cell-based FRET assay was alsoused to test inhibitory 1
ability of fisetin and rutin onthe EV-A71 3Cpro activity and viral replication in cells 2
(Fig. 2). To test the proteolytic efficiency of the FRET probe by EV-A71, 3
HeLa-G3CwtR cells were infected with EV-A71 at MOIs of 0.25, 0.5, and 1. Cells 4
were harvested 48 hours post infection, and then added into a fluorescent-plate. 5
Relative fluorescent intensity of the FRET probe was performed using excitation 6
wavelength at 390/20 nm (for GFP2) and emission wavelength at 590/14 nm (for 7
DsRed2) by a fluorescent-plate reader. Compared with mock control, infected cells 8
exhibited declining the emission intensity as well as increasing the cleaved FRET 9
probes in a virus titer-dependent manner (data not shown), indicating that the 10
emission intensity of the cell-based FRET assay inversely correlates with EV-A71 11
3Cpro amount and virus multiplication. 12
To evaluatethe inhibition of fisetin and rutin on the3Cpro activity and EV-A71 13
replication in cells, HeLa-G3CwtR cells were infected with EV-A71 at a MOI of 1 in 14
the absence and presence of fisetin and rutin. After 90 min of virus adsorption, 15
inoculum was aspirated and 500 µl of DMEM medium withor withoutfisetin or rutin 16
at various concentrations added to each well (Fig. 2).Cells were harvested 2 days post 17
infection and the emission intensity of the FRET probe in each lysate was measured 18
by fluorescent-plate reader with an excitation wavelength at 390/20 nm and an 19
emission wavelength at 590/14 nm (for DsRed2). Relative fluorescent emission 1
intensity at 590/14 nm revealedthatfisetin and rutinexhibited an inhibitory effect on 2
in vitro replication of EV-A71 in a dose-dependent manner. The IC50 values of both 3
flavonoids against EV-A71 in vitro using the cell-based FRET assays were similar: 4
142.8 µM for fisetin and 83 µM for rutin (Table 3). 5
6
3.4. Inhibitory Effect of fisetin and rutin on EV-A71 replication
7
To evaluate antiviral effect of fisetin and rutin on EV-A71 replication, this study 8
analyzed their effects by in vitro virucidal activity, cytopathic effect (CPE) reduction 9
and viral plaque reduction assays (Fig. 3). The results demonstrated that fisetin and 10
rutin at 200 µM had no virucidal activity on EV-A71 (104 pfu) infectivity, but 11
significantly reducedEV-A71-induced cytopathic effect (data not shown).In addition, 12
fisetin and rutin definitely inhibited EV-A71 plaque formation in a dose-dependent 13
manner (Fig. 3): IC50values of 84.48 µM and 109.63 µM, respectively (Table 3). 14
15
4. Discussion
16
This study characterized two in vitro enzymatic assays of EV-A71 3Cpro with 17
the 3Cpro cleavage substrates HRP and recombinant FRET probe (Figs. 1 and 2), 18
indicating the 3Cpro enzymatic assays as the rapid methods for anti-EV-A71 drug 19
discovery. Among flavonoid compounds, fisetin and rutin significantly inhibited in 1
enzymatic activity of recombinant 3Cpro proteins and viral 3Cpro in EV-A71-infected 2
cell while also blocking EV-A71 replication in cytopathic effect (CPE) reduction and 3
viral plaque reduction assays (Fig. 3, Table 3). Taken together, the results suggest 4
fisetin and rutin acting as novel inhibitors of EV-A71 3Cpro, exhibiting moderately 5
potent anti-EV-A71 activities. 6
Soluble recombinant EV-A71 3Cpro was expressed in E. coli, purified with 7
Ni-NTA column, and its proteolysis activity characterized by HRP containing the 8
3Cpro cleavage peptide sequence (Supplemental Fig 2 and Fig. 1). This in vitro 9
proteolysis assay for EV-A71 3Cpro activity proved useful in rapid screening of 10
3Cpro inhibitors; it may be non-specific and non-competitive. In cell-based FRET 11
assay, the FRET probe with 3C cleavage motif was cleaved by EV-A71 3Cpro, but not 12
phylogenetically distant herpes simplex virus (Tsai et al., 2009); the specificity of 13
EV-A71 3Cpro hasbeendemonstrated with rupintrivir (an irreversible 3Cpro inhibitor) 14
(Binford et al., 2007), mutational analysis of FRET probe at 3Cpro cleavage motif and 15
Western blotting (Tsai et al., 2009). Hence, 3Cpro inhibitors identified by this assay 16
were confirmed by cell-based FRET assay of viral 3Cpro activity. Among flavonoids, 17
fisetin and rutin decreased EV-A71 activity by 30%,inhibiting 3Cpro activityin both 18
in vitro and cell-based assays. These belong to the flavonoid family of naturally
occurring polyphenolic compounds with anti-cancer, cardiovascular anti-microbial 1
and anti-inflammatory activities (Manthey et al., 2001; Benaventw et al., 2008; Orhan 2
et al., 2009). They show a broad spectrum of antiviral activity, efficiently inhibiting 3
human rhinovirus, Sabin Type 2 poliovirus, hepatitis A virus, coxsackievirus B4 and 4
echovirus 6 (Conti et al., 1992; Conti et al., 1990; Genovese et al., 1995; Salvati et al., 5
2004). Fisetin exhibited antiviral activity against anti-herpes simplex virus Type 1 6
(HSV-1) and anti-moloney murine leukemia virus (Lyu et al., 2005; Chu et al., 1992). 7
Rutin showed anti-HCV, anti-HIV-1, anti-HSV-1, anti-EMC effects (Orhan et al., 8
2009; Panasiak et al., 1989; Tao et al., 2007; Zuo et al., 2005), yet not in poliovirus 9
infection (Castrillo et al., 1986). These priorstudies prove that fisetin and rutin with 10
similar chemical structures of flavonoids have diverse antiviral activities. Besides 11
inhibition of 3Cpro, fisetin and rutinmay be involvedin other mechanisms inhibiting 12
EV-A71 replication. 13
The IC50 values determined by three different assays were diverse. Amounts of 14
fisetin and rutin required to inhibit purified EV-A71 3Cpro activity in vitro were more 15
than those required in the cell-based system and in blocking EV-A71 replication. This 16
may arise from use of horseradish peroxides (HRP) containing Gln-Gly pairs as 17
substrates for assay and ABTS/H2O2 as coupling assay to measure protease activity. 18
Since flavonoids are known as anti-oxidants due to redox structural feature, inference 19
of flavonoids with peroxidase reaction yields higher IC50 values of in vitro 3Cpro 1
activity by peroxidase-based assay. Antiviral compounds targeting 3Cpro activity 2
have been developed based on mimicking of 3Cpro substrates (peptide inhibitor) 3
(Dragovich et al., 1998; Patick et al., 1999). Other studies searched for inhibitors 4
bound to 3Cpro structure (Patick et al., 1999; Matthews et al., 1999).Further study of 5
the affinity of fisetin and rutin for 3Cpro is warranted to ascertain whether these 6
flavonoids affect other viral or cellular functions crucial for EV-A71 replication. 7
In sum, this studyused EV-A71 3Cpro as a significant target for antiviral drug 8
discovery to demonstrate that fisetin and rutin inhibited 3Cpro activity in vitro and 9
cell-based assays and blocked EV-A71 replication, suggesting these flavonoids as 10
moderately potent anti-EV-A71 agents. Our approach could be crucial to fast, 11
cost-effective development of anti-EV-A71 agents. 12
13
Acknowledgements
14
This project was supported by grants from China Medical University 15
(CMU99-NSC-08, CMU100-S-33, CMU98-CT-22), and the Republic of China 16
National Science Council (NSC 99-2628-B-039-006-MY3). 17
18
References
Benavente, G.O., Castillo, J., 2008. Update on uses and properties of citrus flavonoids: 1
new findings in anticancer, cardiovascular, and anti-inflammatory activity. J. 2
Agric. Food Chem. 56, 6185-6205. 3
Binford, S.L., Weady, P.T., Maldonado, F., Brothers, M.A., Matthews, D.A., Patick, 4
A.K., 2007. In vitro resistance study of rupintrivir, a novel inhibitor of human 5
rhinovirus 3C protease. Antimicrob. Agents Chemother. 51, 4366-4373. 6
Castrillo, J.L., Vanden, B.D., Carrasco, L., 1986. 3-Methylquercetin is a potent and 7
selective inhibitor of poliovirus RNA synthesis. Virology 152, 219-227. 8
Chang, L.Y., Lin, T.Y., Hsu. K.H., Huang, Y.C., Lin, K.L., Hsueh, C., Shih, S.R., Ning, 9
H.C., Hwang, M.S., Wang, H.S., Lee, C.Y., 1999. Clinical features and risk 10
factors of pulmonary oedema after enterovirus-71-related hand, foot, and mouth 11
disease. Lancet 354, 1682-1686. 12
Chen, T.C., Weng, K.F., Chang, S.C., Lin, J.Y., Huang, P.N., Shih, S.R., 2008. 13
Development of antiviral agents for enteroviruses. J. Antimicrob. Chemother. 62, 14
1169-1173. 15
Chen, T.C., Chang, H.Y., Lin, P.F., Chern, J.H., Hsu, J.T., Chang, C.Y., Shih, S.R., 16
2009. Novel antiviral agent DTriP-22 targets RNA-dependent RNA polymerase 17
of enterovirus 71. Antimicrob, Agents Chemother. 53, 2740-2747. 18
Chu, S.C., Hsieh, Y.S., Lin, J.Y., 1992. Inhibitory effects of flavonoids on Moloney 19
murine leukemia virus reverse transcriptase activity. J. Nat. Prod. 55, 179-183. 20
Conti, C., Tomao, P., Genovese, D., Desideri, N., Stein, M.L., Orsi, N., 1992. 21
Mechanism of action of the antirhinovirus flavanoid 4',6-dicyanoflavan. 22
Antimicrob. Agents Chemother. 36, 95-99. 23
Conti, C., Genovese, D., Santoro, R., Stein, M.L., Orsi, N., Fiore, L., 1990. Activities 24
and mechanisms of action of halogen-substituted flavanoids against poliovirus 25
Dragovich, P.S., Webber, S.E., Babine, R.E., Fuhrman, S.A., Patick, A.K., Matthews, 1
D.A., Reich, S.H., Marakovits, J.T., Prins, T.J., Zhou, R., Tikhe, J., Littlefield, 2
E.S., Bleckman, T.M., Wallace, M.B., Little, T.L., Ford, C.E., Meador, J.W. 3rd., 3
Ferre, R.A., Brown, E.L., Binford, S.L., DeLisle, D.M., Worland, S.T., 1998. 4
Structure-based design, synthesis, and biological evaluation of irreversible 5
human rhinovirus 3C protease inhibitors. 2. Peptide structure-activity studies. J. 6
Med. Chem. 41, 2819-2834. 7
Genovese, D., Conti, C., Tomao, P., Desideri, N., Stein, M.L., Catone, S., Fiore, L., 8
1995. Effect of chloro-, cyano-, and amidino-substituted flavanoids on 9
enterovirus infection in vitro. Antiviral Res. 27, 123-136. 10
Ho, M., Chen, E.R., Hsu, K.H., Twu, S.J., Chen, K.T., Tsai, S.F., Wang, J.R., Shih, 11
S.R., 1999. An epidemic of enterovirus 71 infection in Taiwan. Taiwan 12
Enterovirus Epidemic Working Group. N. Engl. J. Med. 341, 929-935. 13
Huang, C.C., Liu, C.C., Chang, Y.C., Chen, C.Y., Wang, S.T., Yeh, T.F., 1999. 14
Neurologic complications in children with enterovirus 71 infection. N. Engl. J. 15
Med. 341, 936-942. 16
Hung, H.C., Wang, H.C., Shih, S.R., Teng, I.F., Tseng, C.P., Hsu, J.T., 2011. 17
Synergistic inhibition of enterovirus 71 replication by interferon and rupintrivir. 18
J. Infect. Dis. 203, 1784-1790 19
Kuo, C.J., Shie, J.J., Fang, J.M., Yen, G.R., Hsu, J.T., Liu, H.G., Tseng, S.N., Chang, 20
S.C., Lee, C.Y., Shih, S.R., Liang, P.H., 2008. Design, synthesis, and evaluation 21
of 3C protease inhibitors as anti-enterovirus 71 agents. Bioorg. Med. Chem. 16, 22
7388-7398. 23
Lei, X., Sun, Z., Liu, X., Jin, Q., He, B., Wang, J., 2011. Cleavage of the adaptor 24
protein TRIF by enterovirus 71 3C inhibits antiviral responses mediated by 25
Toll-like receptor 3. J. Virol. 85, 8811-8818. 26
Lin, T.Y., Twu, S.J., Ho, M.S., Chang, L.Y., Lee, C.Y., 2003. Enterovirus 71 outbreaks, 1
Taiwan: occurrence and recognition. Emerg. Infect. Dis. 9, 291-293. 2
Lin, K.H., Hwang, K.P., Ke, G.M., Wang, C.F., Ke, L.Y., Hsu, Y.T., Tung, Y.C., Chu, 3
P.Y., Chen, B.H., Chen, H.L., Kao, C.L., Wang, J.R., Eng, H.L., Wang, S.Y., 4
Hsu, L.C., Chen, H.Y., 2006. Evolution of EV-A71 genogroup in Taiwan from 5
1998 to 2005: an emerging of subgenogroup C4 of EV-A71. J. Med. Virol. 78, 6
254-262. 7
Lu, G., Qi, J., Chen, Z., Xu, X., Gao, F., Lin, D., Qian, W., Liu, H., Jiang, H., Yan, J., 8
Gao, G.F., 2011. Enterovirus 71 and coxsackievirus A16 3C proteases: binding 9
to rupintrivir and their substrates and anti-hand, foot, and mouth disease virus 10
drug design. J. Virol. 85, 10319-10331. 11
Lyu, S.Y., Rhim, J.Y., Park, W.B., 2005. Antiherpetic activities of flavonoids against 12
herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) in vitro. Arch Pharm. 13
Res. 28, 1293-1301. 14
Manthey, J.A., Grohmann, K., Guthrie, N., 2001. Biological properties of citrus 15
flavonoids pertaining to cancer and inflammation. Curr. Med. Chem. 8, 16
135-153. 17
Matthews, D.A., Dragovich, P.S., Webber, S.E., Fuhrman, S.A., Patick, A.K., Zalman, 18
L.S., Hendrickson, T.F., Love, R.A., Prins, T.J., Marakovits, 19
J.T., Zhou, .R, Tikhe, J., Ford, C.E., Meador, J.W., Ferre, R.A., Brown, E.L., 20
Binford, S.L., Brothers, M.A., DeLisle, D.M., Worland, S.T., 1999. 21
Structure-assisted design of mechanism-based irreversible inhibitors of human 22
rhinovirus 3C protease with potent antiviral activity against multiple rhinovirus 23
serotypes. Proc. Natl. Acad. Sci. US 96, 11000-11007. 24
Mizuta, K., Aoki, Y., Suto, A., Ootani, K., Katsushima, N., Itagaki, T., Ohmi, A., 25
H., Ahiko, T., 2009. Cross-antigenicity among EV-A71 strains from different 1
genogroups isolated in Yamagata, Japan, between 1990 and 2007. Vaccine 27, 2
3153-3158. 3
Orhan, D.D., Ozcelik, B., Ozgen, S., Ergun, F., 2010. Antibacterial, antifungal, and 4
antiviral activities of some flavonoids. Microbiol. Res. 165, 496-504. 5
Panasiak, W., Wleklik, M., Oraczewska, A., Luczak, M., 1989. Influence of 6
flavonoids on combined experimental infections with EMC virus and 7
Staphylococcus aureus in mice. Acta Microbiol. Pol. 38, 185-188. 8
Patick, A.K., Binford, S.L., Brothers, M.A., Jackson, R.L., Ford, C.E., Diem, M.D., 9
Maldonado, F., Dragovich, P.S., Zhou, R., Prins, T.J., Fuhrman, S.A., Meador, 10
J.W., Zalman, L.S., Matthews, D.A., Worland, S.T., 1999. In vitro antiviral 11
activity of AG7088, a potent inhibitor of human rhinovirus 3C protease. 12
Antimicrob. Agents Chemother. 43, 2444-2450. 13
Pallansch, M.A., Roos, R., 2007. Enteroviruses: polioviruses, coxsackieviruses, 14
echoviruses, and newer enteroviruses. In Fields Virology, 5th edn, pp. 839–893. 15
Edited by D. M. Knipe & P. M. Howley. Philadelphia: Lippincott Williams & 16
Wilkins. 17
Pawlotsky, J.M., Chevaliez, S., McHutchison, J.G., 2007. The hepatitis C virus life 18
cycle as a target for new antiviral therapies. Gastroenterology 132, 1979-1998. 19
Rivera, C.I., Lloyd, R.E., 2008 Modulation of enteroviral proteinase cleavage of 20
poly(A)-binding protein (PABP) by conformation and PABP-associated factors. 21
Virology 375, 59-72. 22
Salvati, A.L., Dominicis, D.A., Tait, S., Canitano, A., Lahm, A., Fiore, L., 2004. 23
Mechanism of action at the molecular level of the antiviral drug 24
3(2H)-isoflavene against type 2 poliovirus. Antimicrob. Agents Chemother. 48, 25
2233-2243. 26
Singh, S., Poh, C.L., Chow, V.T., 2002. Complete sequence analyses of enterovirus 71 1
strains from fatal and non-fatal cases of the hand, foot and mouth disease 2
outbreak in Singapore. Microbiol. Immunol. 46, 801-808. 3
Spouge, J.L., 1992. Statistical analysis of sparse infection data and its implications for 4
retroviral treatment trials in primates. Proc. Natl. Acad. Sci. USA 89, 7581 5
-7585. 6
Tao, J., Hu, Q., Yang, J., Li, R., Li, X., Lu, C., Chen, C., Wang, L., Shattock, R., Ben, 7
K., 2007. In vitro anti-HIV and -HSV activity and safety of sodium rutin sulfate 8
as a microbicide candidate. Antiviral Res. 75, 227-233. 9
Tsai, M.T., Cheng, Y.H., Liu, Y.N., Liao, N.C., Lu, W.W., Kung, S.H., 2009. Real-time 10
monitoring of human enterovirus (HEV)-infected cells and anti-HEV 3C 11
protease potency by fluorescence resonance energy transfer. Antimicrob. Agents 12
Chemother. 53, 748-755. 13
Weng, K.F., Li, M.L., Hung, C.T., Shih, S.R., 2009. Enterovirus 71 3C protease 14
cleaves a novel target CstF-64 and inhibits cellular polyadenylation. PLoS 15
Pathog. 5, e1000593. 16
Yang, F., Ren, L., Xiong, Z., Li, J., Xiao, Y., Zhao, R., He, Y., Bu, G., Zhou, S., 17
Wang, J., Qi, J., 2009. Enterovirus 71 outbreak in the People's Republic of 18
China in 2008. J. Clin. Microbiol. 47, 2351-2352. 19
Zuo, G.Y., Li, Z.Q., Chen, L.R., Xu, X.J., 2005. In vitro anti-HCV activities of 20
Saxifraga melanocentra and its related polyphenolic compounds. Antivir. Chem. 21
Chemother. 16, 393-398. 22
Table and Figure Legends
1Table 1. Characterization of EV-A71 3Cpro activity with the substrate
2
horseradish peroxidase.
3
Table 2. Screening of EV-A71 3Cpro inhibitors from flavonoid derivatives.
4
Table 3. Antiviral activities of fisetin and rutin against of EV-A71 by two
5
different methods.
6
Fig. 1. Dose-dependent effects of fisetin and rutin on inhibition of in vitro EV-A71
7
3Cpro activity. A horseradish peroxidase (HRP) containing Gln-Gly pairs
8
corresponding to the cleavage site by 3Cpro was designed as substrate for 3Cpro 9
enzyme. Rutin and fisetin at concentrations of 0, 10, 100, 250, 750 and 1000 µM 10
were incubated with 3C protease (0.1μM)and HRP (0.2 μg/ml) for 2h at 37℃in 11
96-well plates in vitro. Mixtures were then developed with ABTS/H2O2 and 12
measured at OD405. Percentage inhibition of EV-A71 3Cpro activity was 13
calculated as (OD405HRP+drug+3C protease – OD450HRP+3C protease) / (OD450HRP 14
only–OD450HRP+3C protease) x 100%. 15
Fig. 2. Inhibitory effects of fisetin and rutin on intracellular EV-A71 3Cpro
16
activity with a FRET probe. HeLa-G3CwtR cells expressed a FRET probe with
17
an in-frame fusion product of GFP2, the 3Cpro cleavage linker, and DsRed2. 18
HeLa-G3CwtR cells were infected with EV-A71 at MOI= 1 for 90-min of 19
adsorption; the inoculum was aspirated and 500 µl of Dulbecco’s modified Eagle’s 20
medium containing 2% FBS with rutin or fisetin at various concentrations―0 µM, 1
1 µM, 10 µM, 100 µM, 200 µM and 400 µM―was added to each well. After 48-h 2
incubation, cells were harvested and subjected to measurement by a 3
fluorescent-plate reader. Relative intensity of fluorescent emission at 590/14 nm 4
(for DsRed2) was detected using an excitation wavelength at 390/20 nm (for 5
GFP2). 6
Fig. 3. EV-A71 plaque reduction by rutin and fisetin. RD cells were infected with
7
EV-A71 at a titer of 100 pfu per well of cells with of medium alone or medium 8
containing fisetin or rutin at concentrations of 10, 100 and 1000 µM. After 1 h 9
incubation, each cell monolayer was covered with 3 mL of agar overlay medium. 10
At the end of 2-day incubation, the cells were fixed with formaldehyde and stained 11
with 0.1% Crystal Violet. The number of plaques in each well was counted. 12
