Antiviral Potential of a Novel Compound CW-33 against Enterovirus A71 via Inhibition of Viral 2A

17 Pages·2015·3.33 MB·English

Viruses2015,7,3155-3171;doi:10.3390/v7062764
OPENACCESS
viruses
ISSN1999-4915
www.mdpi.com/journal/viruses
Article
Antiviral Potential of a Novel Compound CW-33 against
Enterovirus A71 via Inhibition of Viral 2A Protease
Ching-YingWang1,2,†,An-ChengHuang3,†,Mann-JenHour4,Su-HuaHuang5,
Szu-HaoKung6,Chao-HsienChen1,I-ChiehChen1,Yuan-ShiunChang2,Jin-CherngLien4,*
andCheng-WenLin1,5,*
1 DepartmentofMedicalLaboratoryScienceandBiotechnology,ChinaMedicalUniversity,
Taichung40402,Taiwan;E-Mails:[email protected](C.-Y.W.);
[email protected](C.-H.C.);[email protected](I.-C.C.)
2 SchoolofChinesePharmaceuticalSciencesandChineseMedicineResources,
ChinaMedicalUniversity,Taichung40402,Taiwan;E-Mail:[email protected]
3 DepartmentofNursing,St. Mary’sJuniorCollegeofMedicine,NursingandManagement,
YilanCounty266,Taiwan;E-Mail:[email protected]
4 SchoolofPharmacy,ChinaMedicalUniversity,Taichung40402,Taiwan;
E-Mail:[email protected]
5 DepartmentofBiotechnology,AsiaUniversity,Wufeng,Taichung41354,Taiwan;
E-Mail:[email protected]
6 DepartmentofBiotechnologyandLaboratoryScienceinMedicine,NationalYangMingUniversity,
Taipei11221,Taiwan;E-Mail:[email protected]
† Theseauthorscontributedequallytothiswork.
*Authorstowhomcorrespondenceshouldbeaddressed;E-Mails:[email protected](J.-C.L.);
[email protected](C.-W.L.);Tel.:[phone](ext. 5607)(J.-C.L.);
[phone](ext. 7210)(C.-W.L.);Fax:[phone](C.-W.L.).
AcademicEditor: CurtHagedorn
Received: 15April2015/Accepted: 10June2015/Published: 17June2015
Abstract: Enterovirus A71 (EV-A71) in the Picornaviridae family causes
hand-foot-and-mouth disease, aseptic meningitis, severe central nervous system disease,
even death. EV-A71 2A protease cleaves Type I interferon (IFN)-α/β receptor 1 (IFNAR1)
to block IFN-induced Jak/STAT signaling. This study investigated anti-EV-A7l activity and
synergistic mechanism(s) of a novel furoquinoline alkaloid compound CW-33 alone and in
combination with IFN-β. Anti-EV-A71 activities of CW-33 alone and in combination with
Viruses2015,7 3156
IFN-β were evaluated by inhibitory assays of virus-induced apoptosis, plaque formation,
and virus yield. CW-33 showed antiviral activities with an IC of near 200 µM in EV-A71
50
plaque reduction and virus yield inhibition assays. While, anti-EV-A71 activities of
CW-33 combined with 100 U/mL IFN-β exhibited a synergistic potency with an IC of
50
approximate1µMinplaquereductionandvirusyieldinhibitionassays. Moleculardocking
revealed CW-33 binding to EV-A71 2A protease active sites, correlating with an inhibitory
effectofCW33oninvitroenzymaticactivityofrecombinant2Aprotease(IC =53.1µM).
50
WesternblottingdemonstratedCW-33specificallyinhibiting2Aprotease-mediatedcleavage
ofIFNAR1. CW-33alsorecoveredTypeIIFN-inducedTyk2andSTAT1phosphorylationas
well as 2(cid:48),5(cid:48)-OAS upregulation in EV-A71 infected cells. The results demonstrated CW-33
inhibitingviral2AproteaseactivitytoreduceTypeIIFNantagonismofEV-A71. Therefore,
CW-33 combined with a low-dose of Type I IFN could be applied in developing alternative
approachestotreatEV-A71infection.
Keywords: enterovirusA71;2Aprotease;typeIinterferon;antagonism;inhibitor
1. Introduction
Enterovirus A71 (EV-A71) belongs to the “Enterovirus A” species, genus Enterovirus in the
Picornaviridae family, comprising an icosahedral capsid and single positive-strand RNA genome of
approximately 7400 nucleotides [1]. The genus Enterovirus, one of most common genera within the
family Picornaviridae, comprises 72 serotypes: e.g., poliovirus, Coxsackie A virus (CVA), Coxsackie
B virus (CVB), echovirus, EV-A71 [2–4]. EV-A71 was first isolated and characterized from cases
of neurological disease in California as of 1969 [5], usually infecting children by direct contact with
virus shed from the upper respiratory or gastrointestinal tract. EV-A71 causes hand-foot-and-mouth
disease (HFMD), aseptic meningitis, exanthems, acute flaccid paralysis, pericarditis, severe central
nervous system diseases, even death. The genome contains a single long open reading frame (ORF)
anduntranslatedregions(UTR)at5(cid:48) and3(cid:48) ends. TheORFencodesapolyproteinprecursorcleavedinto
manyconsecutivefunctionalpartsbyviral2Aand3Cproteases,suchasP1,P2andP3fragmentsfirstly
cleaved by 2A protease. The P1 fragment yields structural proteins VP1, VP2, VP3 and VP4, while P2
andP3fragmentsdivideintonon-structural2A,2B,2C,3A,3B,3Cand3D.
EV-A71 outbreaks occur worldwide, especially in the Asia-Pacific region. A 1997 epidemic in
Malaysia caused 31 fatalities. In Taiwan, it caused 788 deaths in 1998 and 51 in 2001–2002 [1]. The
2010 outbreak in China saw over 1 million cases: 15,000 severe, with over 600 fatalities. EV-A71
remains a global menace, with no effective vaccine for clinical use, making EV71 vaccine a primary
candidate for development in the non-clinical stage [6]. Recently, Taiwan’s National Health Research
Institutes (NHRI) completed the first Phase I clinical trial of such a vaccine for children [6]. However,
specific preventive agents against EV-A71 are not available at present. Interferons (IFNs), effectively
antiviralcytokines,areusedincombinationwithantiviraldrugs(ribavirin,boceprevirandtelaprevir)for
hepatitisBorCtreatment[7,8]. BecauseIFNsexhibitlesserpotentialagainstEV-A71[9,10],rarereports
Viruses2015,7 3157
Viruses 2015, 7 3

hinedpiactaitties thBe moar sCcl intriecaatlmtreenatt m[e7n,8t]f.o rBEeVca-Aus7e1 IinFfNecs tieoxnh[i1b1it] .lSeisdsee-re fpfeoctetsntwiaill loagftaeinnsatp pEeVar-—A7e1.g .,[9fe,1v0e]r,,
rcahriell sr,ehpeoardtsa cihned,icmautes ctlheeamch aes/p acilnin,imcaall atirseea—tmaefntet rfIoFrN EiVnj-eAct7i1o ni.nfection [11]. Side-effects will often
appPelaerc—onea.rgi.l,, feaverc,l cinhiicllasl, hceoamdapcohuen,d mutascrglee tainchge/VpaPin1,, msaulcacisees—sfualfltyer IinFhNib iintsjecrthioinno. virus and some
entPerloecvoirnuasreisl,, ab utclninoitcaEl Vc-Aom71pou[1n2d] . tarRgeutpiningt riVviPr1i, s stuhcecemssofustllys ucinchesibsfitusl rpheipntoidvoirmusim aentidc 3soCmpreo
einnhteibroitvoirruasgeasi,n bsuttr hnionto EviVru-As,7C1 V[1B22],. RCuVpBin5t,riEviVr -i6s, thane dmEoVst- 9suicncePshsfausle pIeIpctildinoimcailmtertiiacls 3[C6p,r1o3 i,n1h4i]b.itoAr
asegraiienssto frhBinPoRvOiruZs,i mCiVdaBz2o,l iCdiVnoBn5e, dEeVri-v6a, tiavneds EbaVs-e9d ionn P“hWasien ”IIt ecmlinpilcaatel tsrtiraulcst u[r6e,1s,3,l1ik4e]. WAI Nser5ie1s7 1o1f
B(5P-[R7O-[Z4- (4im,5i-ddaihzyodlirdoi-n2o-noex azdoelryilv)apthiveenso xyb]asheedp tyoln]- 3-“mWeitnh”y listeomxapzloatlee) astrreucretuproerst,e dlitkoe taWrgeINt EV5-1A71711
(V5P-[17,-[i4n-h(i4b,i5ti-ndgihyEdVr-oA-27-1oxraezpolilcyal)tipohneninoxvyit]r ohe[p1t5y]l.]-3L-macetothfyerlrisino,xaazlloolpe)h yacroe cryeapnoirnt,eda ntdo Ctahrgineet sEeVh-eArb7a1l
VcoPm1p, oinuhnidbsit(ienugp aEfVol-iAn,7u1r sroelpiclicaactiido,nc hinry svoitsrpol e[n1e5t]in. ,Lpaecntdoufelerrnitni,n ,aglleonpihpyocsoidcey,aannidn,a aloned- eCmhoidniens)e dhiseprblaayl
cinomviptroouanndtsiv i(reaulpaacftoivliinty, augrasionlsict EaVc-iAd,7 1c[h1r6y,s1o7s]p.leHnoewtinev, erp,eanndtui-lEenVt-iAn,7 1geangiepnotssiadree, stainlldi nadloevee-elompomdeinn)t
dfoisrpcllaiyn icinal vuister.o antiviral activity against EV-A71 [16,17]. However, anti-EV-A71 agents are still in
devFeuloropqmueinnot lfionre clainlkicaalol iudsse. are bioactive compounds in many plants in the Rutaceae family,
sucFhuraosquHinoorltiinae oarlekaadloicida,s aHre. baipoiaccutliavtea ,coTmecploeuandasf zienl iim, aOnyr icpilaanstsu aivne othleen Rs,utaacnedaeB faalmfoiulyro, dseuncdhr oans
Hrieodrteilai aonruemadic[a1,8 H].. apicMuloastat, Tfuecroleqau ianfozelilniie, Oarlikcaialo siudasvepoolesnses,s sandm Banaylfoubrioodloegnidcraoln raicetdiveiltiiaensu:m [e1.8g]..,
Manotisftu fnugraolqu[i1n9o]l,inea natlikmaliocirdosb ipaolss[e2s0s] m, aannyt iboixoildoagnictal[ 2ac1t]i,vitainesd: ea.gn.t,i caannticfeurngaacl t[i1v9it]i,e asnt[i2m2i]c.robiSale v[2e0ra]l,
asynntitohxeisdiaznedt [2c1o]m, apnodu nadnsticbaansceedr aocntiviftuieros q[u2i2n]o. lSineeversakle slyetnotnh,esiszuecdh coamspno-uanlkdys lb-2a,s3e,d4 ,o9n-t efutrraohqyudinroofluinroe
s[2k,e3l-ebto]nq,u sinuochli naes- 3n,-4a-ldkiyoln-2es,3a,4n,d9-Nte-tbreanhzyydlr-o7f-umroe t[h2o,x3y-b-2] ,q3u,4in,9o-ltienter-a3h,y4d-droiofunreos[ 2an,3d- bN]-qbueinnzoyliln-7e--m3,4et-hdoioxnye-
2,3,4,9-tetrahydrofuro[2,3-b]quinoline-3,4-dione (HA-7), exhibit anti-inflammatory, antiallergic and
(HA-7),exhibitanti-inflammatory,antiallergicandantiarrhythmicactivities[23,24]. CompoundCW-33
antiarrhythmic activities [23,24]. Compound CW-33 is a novel synthetic derivative of furoquinoline
is a novel synthetic derivative of furoquinoline alkaloid; flow diagram of CW-33 synthesis appears in
alkaloid; flow diagram of CW-33 synthesis appears in Figure 1A. This study rates anti-EV-A7l activity
Figure 1A. This study rates anti-EV-A7l activity of a novel furoquinoline alkaloid compound CW-33
of a novel furoquinoline alkaloid compound CW-33 alone and in combination with IFNβ. CW-33 shows
alone and in combination with IFNβ. CW-33 shows an inhibitory effect on EV-A71 replication
an inhibitory effect on EV-A71 replication in vitro. Combined treatment with CW-33 and IFN-β exhibits
invitro. Combined treatment with CW-33 and IFN-β exhibits a synergistic antiviral effect on reducing
a synergistic antiviral effect on reducing EV-A71-induced cytopathy (apoptosis), virus yield, and
EV-A71-induced cytopathy (apoptosis), virus yield, and plaque formation. Molecular docking analysis
plaque formation. Molecular docking analysis indicated the binding of CW-33 to EV-A71 2A protease
indicated the binding of CW-33 to EV-A71 2A protease active sites, confirmed by the inhibitory effect
active sites, confirmed by the inhibitory effect of CW-33 on recombinant 2A enzymatic activity as well
of CW-33 on recombinant 2A enzymatic activity as well as recovering the protein levels of IFNAR1 in
as recovering the protein levels of IFNAR1 in EV-A71-infected cells.
EV-A71-infectedcells.

(A)
Figure1. Cont.
Figure 1. Cont.
Viruses2015,7 3158
Viruses 2015, 7 4

(B)

(C)
Figure 1. CW-33 chemical synthesis and NMR characterization (Ethyl
Figure 1. CW-33 chemical synthesis and NMR characterization (Ethyl
2-(3(cid:48),5(cid:48)-dimethylanilino)-4-oxo-4,5-dihydrofuran-3-carboxylate). Flow diagram of
2-(3′,5′-dimethylanilino)-4-oxo-4,5-dihydrofuran-3-carboxylate). Flow diagram of CW-33
CW-33 synthesis appears in (A). 1H-NMR and 13C-NMR spectra of CW-33 are shown in
synthesis appears in A. 1H-NMR and 13C-NMR spectra of CW-33 are shown in B and C,
(B,C),respectively.
respectively.
Viruses2015,7 3159
2. MaterialsandMethods
2.1. VirusesandCells
EV-A71 strain CMUH2005/V978, isolated from throat swab culture of a young child with
encephalitis [25], grew in RD cells. The cells maintained at 37 ◦C, 5% CO in Dulbecco’s Modified
2
Eagle’s Medium (DMEM) with 10% fetal bovine serum (FBS). Titers of EV-A71 were quantified
by plaque assay on RD cell monolayer, stocks stored at −80 ◦C until use, as described in prior
reports[26,27].
2.2. SynthesisofCompoundCW-33
Compound CW-33 (ethyl 2-(3(cid:48),5(cid:48)-dimethylanilino)-4-oxo-4,5-dihydrofuran-3-carboxylate was
synthesized (Figure 1A). Briefly, 250 mL of 6 M sodium hydride in tetrahydrofuran (THF) was slowly
added to 250 mL of 6 M diethyl malonate in THF by shaking for 20 min, then bathed in water at
10–12 ◦C. Then, 400 mL of 2 M chloroacetyl chloride in THF was added dropwise to the mixture over
1 h, incubated at 40–50 ◦C for another hour and cooled to 10–12 ◦C; 3,5-dimethylaniline (0.75 mole)
in THF was added dropwise to the reaction solution over 1 h. Finally, reaction mixture left at room
temperature overnight was heated under reflux for 2 h, then cooled and poured into ice water. Solid
precipitate was extracted with chloroform, washed with water, and dried with magnesium. Solvent was
partially evaporated, with concentrated residue refrigerated for six days; precipitate was subsequently
collected and recrystallized from ethanol to form compound CW-33 (21.7 g, yield 79%). After
purification by high-performance liquid chromatography, CW-33 identity and purity were confirmed by
nuclearmagneticresonance(NMR)spectroscopyandmassspectrum. Meltingpoint(m.p.): 144–147◦C;
MS (m/z): 275 (M+); IR (KBr disc, cm−1): 3251 (–NH–), 1708 (C4=O), 1662 (C3-CO–OEt); UVλmax
nm(CHCl ) (log): 283 (4.4); 297 (4.5); 1H-NMR (200 MHz, DMSO-d6) (Figure 1B): 1.24 (3H, t,
3
J = 7.0 Hz, H-2(cid:48)(cid:48)), 2.25 (6H, s, C3(cid:48)-CH , C5(cid:48)-CH ), 4.20 (2H, q, J = 7.0 Hz, H-1(cid:48)(cid:48)), 4.67 (2H, s, H-5),
3 3
6.88 (1H, s, H-4(cid:48)), 7.05 (2H, s, H-2(cid:48), H-6(cid:48)), 10.13 (1H, s, NH); 13C-NMR (200 MHz, DMSO-d6) δ
(Figure1C):14.62(C-2(cid:48)(cid:48)),21.04(3(cid:48)-CH ,5(cid:48)-CH ),59.41(C-1(cid:48)(cid:48)),75.41(C-5),86.85(C-3),120.58(C-2(cid:48),
3 3
C-6(cid:48)),127.73(C-4(cid:48)),135.03(C-1(cid:48)),135.03(C-3(cid:48),C-5(cid:48)),164.32(C-2),177.25(C-3(cid:48)(cid:48)),188.65(C-4).
2.3. CellCytotoxicityAssay
RD cells (3 × 104 per well) were added into 96-well plates, incubated at 37 ◦C, 5% CO overnight,
2
then treated with or without CW-33 (100, 200, 500, 700, or 1000 µM) and DMSO (5%). Survival
rates of mock and treated cells were rated by MTT assay 48 h post-treatment, cells reacted with MTT
solutionfor4h;insolublepurpleformazanconvertedfromMTTbydehydrogenaseenzymesoflivecells
was dissolved by isopropanol/HCl (300:1 (v/v)). Survival rate was derived from ratio of optical density
(OD) oftreatedcellstoOD ofmockcells,asdescribedinpriorreports[26,27].
570−630nm 570−630nm
Viruses2015,7 3160
2.4. CytopathicEffect(CPE)ReductionandVirusYieldAssays
RD cells were cultured in 6-well plates (37 ◦C, 5% CO ) overnight and infected with EV-A71 at
2
multiplicityofinfection(MOI)of0.1andsimultaneouslytreatedwithorwithoutsingleandcombination
of CW-33 (2.5, 25, and 125 µM) and IFN-β (100 or 1000 U/mL) (Hoffmann-La Roche, Basel,
Switzerland). Cytopathic effect was photographed by inverted microscope 24 and 48 h post-infection.
Supernatant harvested from each well quantified virus yield by plaque assay 48 h post-infection: each
serially diluted, then added onto monolayer of RD cells, following overlaying 3% agarose in DMEM
with 2% FBS. Cell monolayer was stained with 0.1% Crystal Violet 48 h post- incubation in 37 ◦C and
5%CO . Plaquenumbercountmeasuredvirusyield.
2
2.5. PlaqueReductionAssayfor50%InhibitoryConcentration(IC )
50
Monolayer of RD cells in each well of 6-well plates was infected with EV-A71 (50 pfu) and
simultaneouslytreatedwithorwithoutsingleandcombinedCW-33(2.5,25,125,or250µM)andIFN-β
(10, 100, or 1000 U/mL). After 48-h incubation (37 ◦C, 5% CO ), plaque was quantified after staining
2
by0.1%CrystalVioletand50%inhibitoryconcentration(IC )fromthreeindependentexperiments,as
50
describedearlier[26,27].
2.6. CellCycleAnalysisofFlowCytometry
RD cells (4 × 105) infected with EV-A71 (MOI 0.1) in the presence and absence of CW-33, IFN-β,
or combination thereof were cultured for 36 h. Cells were washed in PBS, trypsinized, collected, and
centrifugedat2000rpmfor3min,pelletsdissolvedwith490µLofbindingbufferand5µLofpropidium
ioidide (PI)/Annexin V-FITC reagent (Apoptosis Detection Kit, BioVision). After 10-min incubation at
roomtemperatureinthedark,cellswereanalyzedbyflowcytometry(BDFACSAria,BectonDickinson)
with488nmexcitationand633nmemissionwavelength.
2.7. MolecularDocking
To model CW-33 interaction with viral EV-A71 2A protease, crystal structure of 2A proteinase
C110A mutant (PDB: 3w95) deposited in the RCSB Protein Data Bank (Avaliable online:
http://www.rcsb.org/pdb)servedastemplate. Muetal. derivedcrystalstructurebyX-raydiffractionwith
resolution of 1.85 Å, revealing active site as composed of catalytic triads C110A, H21 and D39, where
acidicmemberofD39stabilizesactivesitegeometrybycenteringhydrogenbindingnetworkwithH21,
N19,Y90,andS125[28]. MoleculardockingusedLibDockprogramwithinsoftwarepackageDiscovery
Studio2.5(Accelrys,SanDiego,CA,USA).First,buildmutantsprotocolwasusedtoexchangealanine
residue at position 110 to cysteine in order to return all amino acid residues of 3w95 to original 2A
proteinasesequence. ProteinsitefeaturesdefinedbyLibDockwerelabeledasHotSpotspriortodocking.
Rigid ligand poses were placed into the active site, HotSpots matched as triplets. In the structure of
EV-A71 2A protease, Asn19, His21, Asp39, Tyr 90, Ala110, and Ser125 amino acids were defined as
active site (sphere radius: 11.5015 Å) [28]. Poses were pruned, final optimization step performed, and
thebestscoringposessubsequentlyreported.
Viruses2015,7 3161
2.8. InVitroEnzymaticAssayofRecombinant2AProtease
Recombinant EV-A71 2A protease was synthesized in E. coli, as detailed in prior study [26].
Briefly, expression vector pET24a containing protease gene was transformed into E. coli BL21
(DE3), with 10 mL overnight culture of a single colony injected into 400 mL of fresh LB medium
containing 25 µg/mL kanamycin for 3 h, induced with 1 mM IPTG for 4 h, harvested by centrifuge
at 6000 rpm for 30 min, then resuspended in denaturing buffer (10 mM imidazole, 8 M urea and
1 mM β-mercaptoethanol) before subjecting to sonication. Recombinant 2A (r2A) protease was
purified with Ni-NTA column by gradient elution with 25 mM Tris-HCl, pH 7.5, 150 mM NaCl
and 300 mM imidazole. Horseradish peroxidase (10 µg/mL) containing Leu-Gly pairs at residues
122–123 served as substrate, incubated 2 h with or without 5 µg/mL of r2A protease and indicated
CW-33 concentrations at 37 ◦C in 96-well plates invitro. Remaining substrate in each reaction was
derived with chromogenic substrate ABTS/H O ; intensity of the developed color was gauged at
2 2
405 nm. Inhibition of r2A protease enzymatic activity was determined as (OD405
subtrsate+CW−33+r2Ao
−OD405 )/(OD405 −OD405 )×100%.
subtrsate+r2A subtrsate subtrsate+r2Apro
2.9. WesternBlotAnalysis
Lysates from un-infected, un-infected/treated, virus-infected/untreated, and virus-infected/treated
cells were dissolved in SDS-PAGE sample buffer containing 2-mercaptoethanol, boiled for 10 min,
then applied to run 8% SDS-PAGE gels. After electronically transferring to nictrocellulose
membranes, blots were blocked with 5% skim milk in TBST, then incubated with specific antibodies
including anti-phospho-STAT1, anti-phospho-ERK1/2, anti-phospho-p38 MAPK, anti-phospho-Tyk2,
anti-IFNAR1, or anti-β-actin antibodies (Cell Signaling Technology, Danvers, MA, USA), respectively.
After reaction with horseradish peroxidase-conjugated secondary antibodies against mouse or rabbit
IgG, immunoreactive bands were developed, using enhanced chemiluminescent substrates (Amersham
PharmaciaBiotech,Piscataway,NJ,USA).
2.10. Real-TimeReverseTranscription-PolymeraseChainReaction(RT-PCR)
Total RNAs isolated from EV-A71 infected RD cells treated with CW-33 alone or combined
with IFN-β, using total RNA purification system (Invitrogen), were reverse-transcribed with oligo
dT primer and SuperScript III reverse transcriptase kit (Invitrogen). To analyze gene expression
in response to CW-33 alone or combined with IFNβ, quantitative PCR used cDNAs, primer pairs,
and SYBR Green I PCR Master Mix. Pairs were forward 5(cid:48)-GATGTGCTGCCTGCCTTT-3(cid:48) and
reverse primer 5(cid:48)-TTGGGGGTTAGGTTTATAGCTG-3(cid:48) for human 2(cid:48),5(cid:48)-OAS (2(cid:48),5(cid:48)-oligoadenylate
synthetase), forward 5(cid:48)-CATGGGCTGGGACCTGA CGGTGAAG-3(cid:48) and reverse primer
5(cid:48)-CTGCTGCGGCCCTTGTTATT-3(cid:48) for IFNAR1 (interferon-α/β receptor 1), or forward
5(cid:48)-AGCCACATCGCTCAGACAC-3(cid:48) and reverse primer 5(cid:48)-GCCCCAATACGACCAAATCC-3(cid:48) for
glyceraldehyde-3-phosphatedehydrogenase(GAPDH).Real-timePCRwascompletedbyamplification
protocol consisting of 1 cycle at 50 ◦C for 2 min, 1 cycle at 95 ◦C for 10 min, 45 cycles at 95 ◦C for
15 s, and 60 ◦C for 1 min. Products were detected in ABI PRISM 7700 sequence detection system (PE
Applied Biosystems); relative change in mRNA levels of indicated genes were normalized by mRNA
levelofhousekeepinggeneGAPDH.
Viruses2015,7 3162
2.11. StatisticalAnalysis
Data from three independent experiments, representing mean ± standard deviation (S.D.), were
Vstiartuissetisc a2l0ly15a, n7a lyzed by ANOVA, SPSS program (Version 10.1, SPSS Inc.; Chicago, IL, USA), an8d

Scheffetest,withpvalue<0.05asstatisticallysignificant.
3. Results
3. Results
3.1. Antiviral Activity of CW-33 against EV-A71
3.1. AntiviralActivityofCW-33againstEV-A71
Cytotoxicity of CW-33 to RD cells was initially assessed using MTT assay (Figure 2A). Survival rate
excCeeydtoetdo x5i0c%ity wohfeCnW c-e3ll3s ttoreRatDedc ewllisthw haisghin citoianlclyenatrsasteisosne dofu sCinWg-M33T Tat a1s0sa0y0 (μFMig,u rpero2vAin)g. SCuWrvi-v3a3l
draetfeineixtecleye dleesds 5c0y%totwoxhiecn. Acenltlisvtirreaal taecdtiwviittyh ohfig ChWco-n3c3e natgraaitniosnt EoVf C-AW7-13 3waats 1la0t0e0r µteMste,dp rboyv icnygtoCpWat-h3ic3
edfeffiecnti tienlhyiblietsiosnc yantodt opxlaicq.ueA rnedtiuvcirtiaolna acstisvaiyty (FoifguCrWes -23B3,Cag aaninds t3AEV,B-)A. 7C1Ww-3a3s claotnecretnetsrtaetdionb-ydecpyetnodpeanthtliyc
seuffpepcrteisnsheidb iEtiVon-Aan7d1-pinladquuceerde dcyutcotipoanthaiscs aeyff(eFcitg iunr eRsD2B c,eCllas n(dFi3gAu,rBe )2.BC)W, a-3s 3wceolnl caesn trreadtiuocni-ndge paepnodpetonttilcy
rsautpep roefs sveidruEsV-i-nAfe7c1t-eidn dcueclelds c(yFtiogpuarteh ic2Cef)f e(cpt in< R0D.0c0e1l)ls. (CFiWgu-r3e32 Blik),eawsiwsee lslhaoswreeddu cpinlagqaupeo pretodtuicctriaotne
aocftivviirtuys -winifthec ItCed50c oefll s19(F3i.8g uμrMe 2. CT)h(ep r<es0u.l0ts0 1in).diCcaWte-d3 3ClWike-3w3i sdeissphloawyiendg pal amquoedereradtuec taionntivaicrtailv iatcytiwviittyh
aICgainostf E19V3-.A87µ1M. .TheresultsindicatedCW-33displayingamoderateantiviralactivityagainstEV-A71.
50
B.
A.
140
120
)
%
100
(
la 80 EV71(MOI=0.1) - + -
v CW-33(μM) - - 125
i 60
v
r
u 40
S
20
0
0 1002003004005006007008009001000 EV71(MOI=0.1) + + +
CW-33 (μM) CW-33(μM) 2.5 25 125
C. 80
70 *
s
llec60
)+
I-(50
P
/)+40 ***
V-(
n30
ix
en20
n
A
10
0
EV71(MOI=0.1) -1 2+ 3- 4+ +5 +6
CW-33(μM) - - 125 2.5 25 125
Figure 2. Reducing EV-A71-infected cytopathic effects by CW-33. Firstly, cytotoxicity
Figure 2. Reducing EV-A71-infected cytopathic effects by CW-33. Firstly, cytotoxicity of
of CW-33 was performed using MTT assays (A). Survival rates of cells were calculated as
CW-33 was performed using MTT assays (A). Survival rates of cells were calculated as the
the ratio of OD of treated cells to OD of untreated cells. Next, antiviral
570−630nm 570−630nm
ratio of OD of treated cells to OD of untreated cells. Next, antiviral activity
activity of C57W0–6-3303 nmwas evaluated using the570r–e6d3u0 cnmtion of virus-induced cytopathic effect and
of CW-33 was evaluated using the reduction of virus-induced cytopathic effect and
apoptosis. Virus-induced cytopathicity was photographed post-infection by phase-contrast
apoptosis. Virus-induced cytopathicity was photographed post-infection by phase-contrast
microscopy (B), while apoptosis of infected and/or treated cells were analyzed by flow
microscopy (B), while apoptosis of infected and/or treated cells were analyzed by flow
cytometrywithAnnexin-V/PIstain(C).*pvalue<0.05;***pvalue<0.001byScheffetest.
cytometry with Annexin-V/PI stain (C). * p value < 0.05; *** p value < 0.001 by Scheffe test.
Viruses2015,7 3163
Viruses 2015, 7 9

A. B. 60
80
)70 )50 ***
u % **
f60
p( r50 ( no40
eti40 itib30
t su30 ihnI20
riV20
10 10
0
0
1 2 3 4 5
EV71 + + + + +
0 50 100 150 200 250
CW-33(μM) - 2.5 25 125 250 CW-33 (μM)
C.
120 D.
60
)100 50
u )
f %
p( r 80 ( n40
e o
tit s 60 itib30
u 40 ih20
riV nI
20 10
0 0
EV71 +1 2+ 3+ 4+ 0 200 400 600 800 1000
IFN-β (U/mL)
IFNβ(U/mL) - 10 100 1000
Figure 3. Inhibition of EV-A71 plaque formation by CW-33 or IFN-β alone. Monolayer
Figure 3. Inhibition of EV-A71 plaque formation by CW-33 or IFN-β alone. Monolayer of
of RD cells in 6-well plates were infected with EV-A71 (100 pfu), and then immediately
RD cells in 6-well plates were infected with EV-A71 (100 pfu), and then immediately
treated with indicated concentrations of CW-33 (A) or IFN-β (C). After 1-h absorption,
treated with indicated concentrations of CW-33 (A) or IFN-β (C). After 1-h absorption, cell
cell monolayer was washed with PBS, then overlaid with medium containing 1.5% agar.
monolayer was washed with PBS, then overlaid with medium containing 1.5% agar. After
After 48-h incubation, plaque number was counted after staining by 0.1% Crystal Violet,
48-h incubation, plaque number was counted after staining by 0.1% Crystal Violet, Inhibitory
Inhibitory activities of CW-33 (B) or IFN-β (D) were calculated from ratio of experimental
activities of CW-33 (B) or IFN-β (D) were calculated from ratio of experimental data to
datatomockcontrol. ***pvalue<0.001byScheffetest.
mock control. *** p value < 0.001 by Scheffe test.
33..22.. SSyynneerrggiissttiicc AAnnttiivviirraall AAccttiivviittyy ooff CCWW--3333i ninC Coommbbininaatitoionnw witihthI Innteterrfeferroonn( I(FIFNN)-)β-β
IIFFNN--ββ ata at loaw lcoowncecnotrnactieonntr (a1ti0o0n U/(m10L0) slUig/hmtlLy) inhsliibgithetdly EVin-Ahi7b1it-eindduEceVd- Acy7t1o-pinatdhuyc e(Fdigucyret o3pCa,tDhy).
(PFliagquuree re3dCu,cDti)o.n aPslsaaqyu eindriceadtuecdt ioIFnN-aβs seaxyhibiintidnigc alteesds anIFtiNvi-rβal aecxtihviibtyit i(nIgC l e=s s966a.n2t iUvi/rmalL) aacgtiaviintyst
50
(EICV-A=719 6i6n.f2ecUti/omnL. )CaogmabininstedE Vtr-eAat7m1enint foefc tisoenr.ial Ccoonmcebnintreadtiotrnesa tomf eCntWo-f33s ewriiatlh c1o0n0c eUn/tmraLtio InFsNo-fβ
50
CshWo-w3e3d wa imthor1e0 p0otUen/mt iLnhIiFbNiti-oβn oshf ovwirueds-iandmucoered cpyottoepnatthinich iebfifteicotn anodf avpiorupst-oisnidsu ccoemdpcayretodp taot hCicWe-3ff3e cotr
aInFdN-aβp oaplotonsei s(Fciogmurpe a4r evds. tFoigCuWre- 323). FororI FdNet-eβrmailnoinneg a(Fdidgiutirvee 4orv ssy.nFeriggiusrteic2 a)n.tiFvoirrald aectetirvmitiyn ionfg CaWdd-3it3iv ien
ocromsybnineragtiiosnti cwaitnht iIvFiNra-lβ,a ccotimvibtyineodf tCreWat-m3e3nti nofc soemriabli ncaotniocenntwraittihonIsF oNf- CβW, c-o3m3 bwiinthe d10tr0e Uat/mmeLn tIFoNf-sβe wriaals
ceovnacluenatteradt iobnys EoVf -CAW7-13 3plwaqiuthe 1a0n0d Uy/imelLd IrFedNu-cβtiowna sasesvaaylsu a(tFeidgubryesE V5- Aan7d1 6p)l.a qCuoemabnidneydie ltdreraetmduecntti oonf
aCssWay-3s3( Fwigituhr e1s005 Uan/mdL6 )I.FCNo-βm ebxinheibdittereda stymneenrtgiosftiCc Wan-t3iv3irwali tahct1i0vi0tiUes/ magLaiInFsNt E-βV-eAxh7i1b i(tIeCd s yonf e0r.g9i sμtiMc
50
afnotri vpilraaqluaec trivedituiecstiaogna ainnsdt EICV-A o7f1 1(.I4C μMo ffo0r. 9viµruMs yfoierlpdl areqduuecrteiodnu)c.t iConWa-n3d3 IiCn coomfb1i.n4atµioMn fwoirthv i1ru0s0
50 50 50
yUie/mldLr eIdFuNct-iβo ne)x.ceCeWde-d3 310in0-cfoomldb lionwateior nICwit hva1l0u0esU a/mgaLinIsFt NE-Vβ-Aex7c1e erdepedlic1a0ti0o-nfo ilnd vloitwroe rcIoCmpavraeldu etso
50 50
aCgWain-3st3 EalVo-nAe.7 1Rerseuplltisc dateimononinstrvaitterod ac osmynpearrgeidstitco aCntWiv-ir3a3l aaclotinveit.y oRfe CsuWlt-s33d eimn oconmstrbaitneadtioans wynitehr gai slotiwc
acnotnivciernatlraactitoivni toyf oIfFCNW-β- a3g3aiinnscto EmVb-iAna7t1io. nwithalowconcentrationofIFN-βagainstEV-A71.
Viruses2015,7 3164
Viruses 2015, 7 10
Viruses 2015, 7 10

A. C.
100
A. C. 80
100
)%( lavivruS)%( lavivruS 46824680000000 V nsilxlIeePcn/ ))++-(-(V nsilxlIeePcn/ n))++A-(-(2345678123456700000000000000 ** ****** ******
20 nA100
0 EV71(MO0I=0.1) 1- +2 3- 4- +5 +6 7+
0 0 200 400 600 800 1000 EIFVN71ß(M(10O0IU=/0m.1l)) 1-- +2- 3-- 4+- +5+ +6+ 7 ++
0 C20W0-33 (μ40M0) plus6 0IF0N-β(810000U/m1l)000 ICFWNß-3(31 0(μ0UM/)m l ) -- -- 1-25 + - 2+.5 2+5 1+25
CW-33 (μM) plus IFN-β(100U/ml) CW-33 (μM) - - 125 - 2.5 25 125
B.
B.
EV71 (MOI=0.1) - + - -
IFNß(100U/ml) - - - +
ECVW7-13 (3M (μOMI=) 0 . 1 ) -- +- 1-25 --
IFNß(100U/ml) - - - +
CW-33 (μM) - - 125 -
EV71 (MOI=0.1) + + + +
IFNß(100U/ml) + + + +
ECVW7-13 (3M (μOMI=) 0 . 1 ) +- 2+. 5 2+5 1+2 5
IFNß(100U/ml) + + + +
CW-33 (μM) - 2.5 25 125
Figure 4. Inhibitory effect of CW-33 in combination with IFN-β on EV-A71-infected
Figure 4. Inhibitory effect of CW-33 in combination with IFN-β on EV-A71-infected
Figure 4. Inhibitory effect of CW-33 in combination with IFN-β on EV-A71-infected
cytopcayttohpicatheifcf eecftf.ecFt. iFrsirtslytl,y,c ycytotototoxxiicciittyy ooff CCWW-3-333 ini ncocmombibnaintiaotnio wnitwh itIhFNIF-βN t-oβ RtDo RceDlls cells
cytopathic effect. Firstly, cytotoxicity of CW-33 in combination with IFN-β to RD cells
was wpaesr fpoermrfoerdmeuds iunsginMg MTTTTa sassasyayss ((AA)).. RRDD cceellllss ininfefcetcetde dwiwthi thEVE-AV7-A1 7w1erwe efroerthfworitthh with
was performed using MTT assays (A). RD cells infected with EV-A71 were forthwith
treatetrdeawteidth wCithW C-3W3-3a3lo anloenaen adndin inc ocmombbininaatitioonn wwiitthh IIFFNN--ββ. V. Viruirsu-isn-dinudceudc ecdytocpyatothpiacitthyi cwiatys was
treated with CW-33 alone and in combination with IFN-β. Virus-induced cytopathicity was
photophgoratopghreadphpeods pt-oisnt-fiencfteicotniobn ybyp hphasaese--ccoonnttrraasstt mmiiccrroossccooppyy (B(B), )a,paoppotopstios soifs ionffeicntfeedc atnedd/oarn d/or
photographed post-infection by phase-contrast microscopy (B), apoptosis of infected and/or
treated cells analyzed by flow cytometry with Annexin-V/PI staining (C). * p value < 0.05; ***
treated cells analyzed by flow cytometry with Annexin-V/PI staining (C). * p value < 0.05;
treated cells analyzed by flow cytometry with Annexin-V/PI staining (C). * p value < 0.05; ***
p value < 0.001 by Scheffe test.
***pp vvaalluuee << 0.00.0010 b1yb SychSecfhfee ftfeest.t e st.
A. B.
A. 120 B.
90
120 90 ** ***
100 80 ** ***
80
100 70 **
80 70 **
( retit suurifVp)( retit suurifVp)4684600000 **** **** )%( noitibihnI)%( noitibihnI3456345600000000
** 20
20 ** ** 20
20 ** 10
10
0 0
1 1 2 3 4
EV71 0 + + + + + EV701 + + + +
EIFVN7ß1( 1 0 0 U / m l) + - ++ +1+ ++ ++ EIFVN7ß1( 1 0 0 U / m l1 ) ++ 2 ++ 3 ++ 4 ++
ICFNWß-(3130(0μUM/)m l ) -- +- 2+. 5 +2 5 +12 5 ICFNWß-(3130(0μUM/)m l ) +- 2+. 5 + 2 5 +125
CW-33(μM) - - 2.5 25 125 CW-33(μM) - 2.5 25 125
FiguFriegu5r.e I5n.h Iinbhiitbioitniono foEf EVV-A-A7711p pllaaqquuee ffoorrmmaattioionn bby ycocmombinbeind etdreatrtmeaetnmt eonft CoWf C-3W3 w-3i3th with
Figure 5. Inhibition of EV-A71 plaque formation by combined treatment of CW-33 with
IFN-β. Monolayer of RD cells in 6-well plates infected with EV-A71 (100 pfu) was
IFN-β. Monolayer of RD cells in 6-well plates infected with EV-A71 (100 pfu) was
IFN-β. Monolayer of RD cells in 6-well plates infected with EV-A71 (100 pfu) was
immediately treated with CW-33 alone or in combination with IFN-β. After 1-h absorption,
immediatelytreatedwithCW-33aloneorincombinationwithIFN-β. After1-habsorption,
immediately treated with CW-33 alone or in combination with IFN-β. After 1-h absorption,
cell monolayer was washed with PBS, then overlaid with medium containing 1.5% agar.
cell cmelol nmoolanyoelarywera wsaws awsahsehdedw witihth PPBBSS,, tthheenn oovveerrlalaidid wwithit hmemdeiudmiu mconctoainntianign i1n.5g%1 .a5g%ar. agar.
After 48-hour incubation, plaque number was counted after staining by 0.1% Crystal Violet
(A),50%inhibitoryconcentration(IC )calculatedfromratioofexperimentaldatatomock
50
control(B).**pvalue<0.01;***pvalue<0.001byScheffetest.