Antiviral Screening of Multiple Compounds against Ebola Virus

17 Pages·2016·0.59 MB·English

viruses
Article
Antiviral Screening of Multiple Compounds against
Ebola Virus
StuartD.Dowall1,6,*,KevinBewley1,RobertJ.Watson1,SeshadriS.Vasan1,2,
ChandradhishGhosh3,MohiniM.Konai3,GroGausdal4,JamesB.Lorens4,JasonLong5,
WendyBarclay5,IsabelGarcia-Dorival6,JulianHiscox6,7,AndrewBosworth1,7,IreneTaylor1,
LindaEasterbrook1,JamesPitman1,SianSummers1,JennyChan-Pensley1,SimonFunnell1,
JuliaVipond1,SueCharlton1,JayantaHaldar3,RogerHewson1,7andMilesW.Carroll1,7
1 PublicHealthEngland,PortonDown,Salisbury,WiltshireSP40JG,UK;[email protected](K.B.);
[email protected](R.J.W.);[email protected](S.S.V.);
[email protected](A.B.);[email protected](I.T.);[email protected](L.E.);
[email protected](J.P.);[email protected](S.S.);[email protected](J.C.-P.);
[email protected](S.F.);[email protected](J.V.);[email protected](S.C.);
[email protected](R.H.);[email protected](M.W.C.)
2 DepartmentofPreventiveandSocialMedicine,JawaharlalInstituteofPostgraduateMedicalEducation&
Research,Puducherry605006,India
3 ChemicalBiologyandMedicinalChemistryLaboratory,NewChemistryUnit,JawaharlalNehruCentrefor
AdvancedScientificResearch,Jakkur,Bengaluru560064,Karnataka,India;[email protected](C.G.);
[email protected](M.M.K.);[email protected](J.H.)
4 BerGenBio,JonasLiesvei91,Bergen5009,Norway;[email protected](G.G.);
[email protected](J.B.L.)
5 ImperialCollegeLondon,StMary’sCampus,LondonW21PG,UK;[email protected](J.L.);
[email protected](W.B.)
6 InstituteofInfectionandGlobalHealth,UniversityofLiverpool,LiverpoolL697BE,UK;
[email protected](I.G.-D.);[email protected](J.H.)
7 NIHRHealthProtectionResearchUnitinEmergingandZoonoticInfections,UK
* Correspondence:[email protected];Tel.:[phone]
AcademicEditor:JensH.Kuhn
Received:19September2016;Accepted:19October2016;Published:27October2016
Abstract:InlightoftherecentoutbreakofEbolavirus(EBOV)diseaseinWestAfrica,therehavebeen
renewedeffortstosearchforeffectiveantiviralcountermeasures. Arangeofcompoundscurrently
availablewithbroadantimicrobialactivityhavebeentestedforactivityagainstEBOV.Usinglive
EBOV,eighteencandidatecompoundswerescreenedforantiviralactivityinvitro. Thecompounds
wereselectedonarationalbasisbecausetheirmechanismsofactionsuggestedthattheyhadthe
potentialtodisruptEBOVentry,replicationorexitfromcellsorbecausetheyhaddisplayedsome
antiviral activity against EBOV in previous tests. Nine compounds caused no reduction in viral
replicationdespitecellsremaininghealthy,sotheywereexcludedfromfurtheranalysis(zidovudine;
didanosine;stavudine;abacavirsulphate;entecavir;JB1a;Aimspro;celgosivir;andcastanospermine).
Asecondscreenoftheremainingcompoundsandthefeasibilityofappropriatenessforinvivotesting
removedsixfurthercompounds(ouabain;omeprazole;esomeprazole;Gleevec;D-LANA-14;and
Tasigna). The three most promising compounds (17-DMAG; BGB324; and NCK-8) were further
screened for invivo activity in the guinea pig model of EBOV disease. Two of the compounds,
BGB324andNCK-8,showedsomeeffectagainstlethalinfectioninvivoattheconcentrationstested,
which warrants further investigation. Further, these data add to the body of knowledge on the
antiviralactivitiesofmultiplecompoundsagainstEBOVandindicatethatthescientificcommunity
shouldinvestmoreeffortintothedevelopmentofnovelandspecificantiviralcompoundstotreat
Ebolavirusdisease.
Viruses2016,8,277;doi:10.3390/v8110277 www.mdpi.com/journal/viruses
Viruses2016,8,277 2of17
Keywords: Ebolavirus;antiviral;downselection;drugrepurposing
1. Introduction
Ebolavirus is a genus of the family Filoviridae and includes five species: Bundibugyo virus
(BDBV),Restonvirus(RESTV),Sudanvirus(SUDV),TaïForestvirus(TAFV)andEbolavirus(EBOV).
Ebolavirusistheprototypespecies[1,2](formallydesignatedZaireebolavirus)andwasresponsible
forthelargeoutbreakofEbolavirusdisease(EVD)inpartsofWestAfricafirstrecognizedinDecember
2013[3]. EBOVisthemostvirulentspeciesofthefamilywithacasemortalityofupto90%,whereas
theRestonspeciesisvirtuallynon-pathogenicinhumans[4].InresponsetotheoutbreakinWestAfrica
andthethreatoffurtheroutbreaksintheabsenceofapprovedandproventherapeuticsorvaccines,
therehasbeenincreasedinternational,political,humanitarianandscientificmomentumtoidentify
treatmentstrategies. Inthiscontext,duringthe2013/2014EBOVoutbreak,PublicHealthEngland
(PHE)wasapproachedbyseveralacademicandcommercialentitiesrequestingrapidevaluationof
repurposeddrugsandexperimentaltherapiesforEBOV,usingitsContainmentLevel4(CL4)facilities.
WithsupportfromtheEbolaresearchfundinginitiativefromtheWellcomeTrust,aprojecttodetermine
theviabledrugcandidatesforfurtherdevelopmentwasdeveloped. Theeighteencandidatesinthis
reportwereselectedfromsixtycredibleleadsbyascientificpanel;theycoveredarangeofpotentially
promisingmechanismsofactionagainstEBOV.Briefdetailsofthecompoundsnominatedforinclusion
areoutlinedbelow:
• Ouabain: Originallyusedforthetreatmentofheartdiseases[5],whichhasbeendemonstratedto
reduceEBOVreplicationbyaroundhalfwhentestinginvitroinastudylookingintotheviral
protein24(VP24)proteinandtheinterruptionofcellularinteractingproteins[6]
• 17-DMAG: An inhibitor of heat shock protein 90 (HSP90), which has been shown to reduce
invitroEBOVreplication[7]
• BGB324: AninhibitorofAxlreceptortyrosinekinase,whichappearstobeinvolvedwithEbola
virusentryintohostcells[8]
• JB1a: An antibody therapy, targeting beta-1 integrins, which have been proposed to facilitate
theentryoffiloviruses;treatmentoftargetcellswiththeJB1aclonereducedinfectionusinga
vesicularstomatitisvirus(VSIV)pseudotypedwithEBOVglycoprotein[9]
• Omeprazoleandesomeprazolemagnesium: Membersofthebenzimidazolesthatmaystopviral
entryviaclathrin-mediatedendocytosisbyraisingtheendosomalpH.Bothcompoundswere
showntoinhibitlentivirus-basedpseudotypesexpressingEBOVglycoprotein[10]
• GleevecandTasigna(marketnamesforimatinibmesylateandnilotinib,respectively): Specific
tyrosinekinaseinhibitorsoriginallydevelopedasanticancercompoundsandproposedtoinhibit
phosphorylation of the VP40 matrix protein which is required for EBOV exit from cells [11].
Duringlarge-scalescreensofantiviralsagainstEBOV,othergroupshaveidentifiedGleevec[12]
andTasigna[13]aspotentialEBOVinhibitors
• Aimspro(anti-inflammatoryimmuno-suppressivedrug): Originallydevelopedforthetreatment
of human immunodeficiency virus (HIV) by the production of hyperimmune serum in goats
injectedwithinactivatedHIVIIIB,theserumhasrevealedthepresenceofarangeofcomponents,
includingthecytokinesinterleukin(IL)-4andIL-10,proopiomelanocortin,argininevasopressin,
β-endorphinandcorticotropin-releasingfactor[14]
• NCK-8andD-LANA-14: Smallmoleculesthatmimicthepropertiesofantimicrobialpeptides,
NCK-8[15,16]andD-LANA-14[17]havedemonstratedpotentactivityagainstdrug-resistant
bacteriaandtheirbiofilms. Theactivityofthisclassofcompoundsisattributedtotheirmembrane
disrupting properties [18–20]. Peptide mimics [21] and several other small molecules have
demonstratedactivityagainstEBOV.Owingtothemembrane-disrupting[22,23]modesofaction
Viruses2016,8,277 3of17
of this class of compounds (e.g., NCK-8 and DLANA-14), they were expected to be active
againstEBOV
• Celgosivir and its prodrug castanospermine: Broad spectrum inhibitors of host glucosidases.
Inhibitorsofendoplasmicreticulum(ER)α-glucosidaseshavebeenshowntoactasantivirals
withseveralhaemorrhagicfeverviruses,includingEBOV[24]
• Zidovudine,didanosine,stavudine,abacavirsulphateandentecavir: Compoundsincludedinthe
studyuponrequestoftheWellcomeTrust
2. MaterialsandMethods
2.1. InVitroScreening
2.1.1. VirusAssay
MRC-5(humanfoetallung)andVeroE6(AfricanGreenmonkeykidney)cellsweresourcedfrom
theEuropeanCollectionofCellCultures(ECACC)andseededinto96-wellplates. MRC-5cellswere
chosen as a host-matched human cell line susceptible to EBOV infection [6] and VeroE6 for being
widelyusedinEBOVstudies[25–27]. Compoundsweresourcedcommercially(SelleckChemicals,
Boston,MA,USA;andDaltonPharmServices,Toronto,ON,Canada)whereverpossibleordirectly
from the supplier if they were not readily available (BGB324, BerGenBio, Bergen, Norway; JB1a,
Avipero,Edinburgh,UK;Aimspro,DavalInternational,Eastbourne,UK).NCK-8andD-LANA-14
weresynthesizedandcharacterizedinJawaharlalNehruCentreforAdvancedScientificResearch,India.
Stocksolutionsofthesecompoundsweremadeatdoublethefinaldilutiontotakeintoaccountanequal
volumeofvirussuspensiontobeadded. Severalstocksweresuppliedindimethylsulfoxide(DMSO)
solution. Afterdilution,thehighestconcentrationofDMSOwas≤0.05%,exceptforomeprazoleand
esomeprazole,wherethehighestconcentrationswere10%and7.5%,respectively.
WithintheCL4laboratory,mediawereremovedfromtheinnerwellsof96-wellplates. Dueto
edge-effects,theouterwellswereleftwithmediaadded. Compoundswereaddedatfivereplicates
per dilution; three for virus addition and two mock-infected. For the first invitro screen, EBOV
suspension(strainME718,recentlyrenamed1976/Yambuku-Ecran[28])wasaddedataTCID (tissue
50
cultureinfectiousdose50percent)concentrationofapproximately500/welltotriplicatewellsper
compounddilution,withtheremainingtwowellshavingmediaaloneadded. Basedontheinhibition
ofEBOV-inducedcytopathiceffectindifferentcelllines,thesupernatantsfromMRC-5andVeroE6
cellswereharvestedondays3and6post-infection,respectively. Cellsweremicroscopicallyassessed
fortheconditionofthemonolayer.
2.1.2. MolecularAssay
Viral replication was measured and compared over a series of time points using a real-time
polymerasechainreaction(PCR)approach. Onehundredfortymicrolitersofsupernatantwereadded
to560µLAVLbufferforRNAextractioninsealabledeep96-wellplatesforremovalfromtheCL4
laboratory. ExtractionofRNAwasperformedusingtheMagnaPure96smallvolumeRNAkit(Roche,
BurgessHill,UK),amagneticbead-basedmethodofRNAseparation. Inbrief,sampleswerevessel
transferredintoMagnaPureplatespriortoloadingontotheMagnaPure96automatedextractionrobot
andRNAelutedin60µLnuclease-freewater. Targetamplificationwasperformedusingprimersto
ZaireebolavirusglycoproteinasdescribedinTrombleyetal.,[29]usingtheFastVirusqRT-PCRKit
(Qiagen,Manchester,UK).AnalysiswasperformedusingtheABi7500(AppliedBiosystems,Paisley,
UK)underthefollowingcyclingconditions: 50◦Cfor10min,95◦Cfor30sfollowedby40cyclesof
95◦Cfor15sand60◦Cfor3s;temperaturecyclingwassettothemaximumrampspeed,anddata
wereacquiredandanalyzedusingtheABi7500on-boardsoftwareversion2.0.6(AppliedBiosystems,
Paisley,UK)withathresholdsetto0.05. Cyclethreshold(Ct)valuesfromthePCRassaywereusedto
giveaconsistentreadingoftheamountofEBOVRNAlevelsinthesamples.
Viruses2016,8,277 4of17
2.1.3. ToxicityAssay
Sixcompoundsofinterestwereassessedfortoxicity,basedonavailabilityandresultsfromthefirst
invitroscreeningandavailability(i.e.,omeprazole,esomeprazole,ouabain,17-DMAG,Gleevecand
BGB324). SerialdilutionsweremadeandincubatedonMRC-5cellsforthreedays. Afterincubation,
cellswerevisuallyassessedforcytotoxicity,andmonolayerswerefixedwithformaldehydesolution
beforestainingwithcrystalvioletforagrossvisualinspectionofcellattachment.
2.1.4. RepeatCompoundScreening
Screening assays were repeated to assess for effects against EBOV using MRC-5 cells.
Thefollowingchangestothepreviousmethodwereemployed: (i)compoundsweretestedindilutions
shownnottoexertacytotoxiceffectonuninfectedMRC-5cells;(ii)a10×higherviralinoculumwas
used(approximately5000TCID /well);and(iii)sampleswereharvestedaftertwodays.
50
2.2. InVivoScreening
Guineapigswereusedforefficacystudies[30]usinggroupsizesofn=6. Animalstudieswere
performed under CL4 conditions with all procedures being undertaken according to the United
Kingdom Animals (Scientific Procedures) Act 1986. Studies were approved by the PHE ethics
committee and the Project Licence approved by a UK Home Office inspector. Vascular catheters
wereinsertedpriortoarrivaltoallowsafeaccesstotheintravenousrouteofdeliveryatCL4. Animals
were challenged via the subcutaneous route with a dose of 103 TCID guinea pig-adapted EBOV
50
thathadbeenpassagedfivetimesinvivo[31]. ThechallengepreparationwasbacktitratedinVeroE6
cellstoconfirmthedose. At6hpost-challenge,treatmentwasinitiatedwiththecompoundsBGB324,
NCK-8and17-DMAG.BGB324wasdissolvedin0.5%(w/w)hydroxypropylmethylcellulose/0.1%
(w/w) Tween-80 to give a concentration that equated to 100 mg/kg in 1 mL. Doses of 1 mL were
givenorallytwicedaily. CompoundNCK-8wasdilutedwithsterilewatertogiveaconcentration
that equated to 5 mg/kg. One milliliter was delivered via the intravenous (i.v.) route twice daily.
17-DMAG was supplied commercially (Selleck, product S1142) and dissolved in 0.05% DMSO to
giveaconcentrationthatequatedto30mg/kgin1mL.Dosesweredeliveredintraperitoneally(i.p.)
and scheduled for every other day. A negative control group consisted of guinea pigs that were
EBOVchallenged,butreceivednotreatment. Eachday,animalswereindividuallyweighedandtheir
temperatureswererecordedbyasubcutaneously-insertedtemperature/identification(ID)chip.
To prevent unnecessary animal suffering, humane clinical endpoints were used, which
standardizedwhenanimalswouldbeculledusingaUKHomeOfficeapprovedSchedule1method.
Theendpointsconsistedof: (a)10%weightlossandamoderateclinicalsymptom(e.g.,lethargy,etc.);
(b)20%weightloss;or(c)showingsignsofdistress,asdeterminedinconsultationwiththenamed
AnimalCareandWelfareOfficer.
3. Results
3.1. SelectionofCompounds
Due to the large number of compounds (about sixty) initially proposed for antiviral testing,
ascoringassessmentwasundertakentotriagecompoundsinrelationtothecapacityavailablefor
testingatCL4. TheassessmentwasbasedonTechnicalReadinessLevel(TRL)(TableA1,adaptedfrom
UnitedStatesDepartmentofDefense,2009[32]),availabilityandpreviousevidenceofefficacyagainst
EBOV(TableA2). Inaddition,companiesandinstitutionswhohadsuggestedthecompoundswere
askedtoscoretheirdrugsthemselvesandtoprovidejustifications. Withthisinformation,together
withinformationfromthepublicly-availableliteratureatthetime,ascientificpanelidentifiedeighteen
compoundsthatweresuitableforscreening(Table1).
Viruses2016,8,277 5of17
Table1.ScoringofselectedcompoundsforEbolavirus(EBOV)screening.TRL,TechnicalReadinessLevel.
Name TRLScore1 Availability2 Efficacy3 Total
Ouabain 4 2 1 7
17-DMAG 4 2 1 7
BGB324 4 2 1 7
Zidovudine 4 2 1 7
Didanosine 4 2 1 7
Stavudine 4 2 1 7
Abacavirsulphate 4 2 1 7
Entecavir 4 2 1 7
JB1a 3 2 1 6
Omeprazole 3 2 1 6
Esomeprazolemagnesium 3 2 1 6
Gleevec 3 2 0.5 5.5
Aimspro 3 2 0 5
NCK-8 3 2 0 5
D-LANA-14 3 2 0 5
Tasigna 3 1 0.5 4.5
Celgosivir 2 2 0 4
Castanospermine 2 2 0 4
1Scoredfrom1to9(TLRtable;AppendixA).2Availabilityforuseintheclinic.3Previousdataonefficacy
againstEBOV.
3.2. EffectsofCompoundsagainstInVitroEBOVReplication
3.2.1. InitialScreenatRecommendedConcentration
Therecommendeduseconcentrationforeachcompoundwassoughtfromthesuppliersand/or
informationinthepublicdomain. Usingtheseconcentrations,thelevelsofEBOVRNAafterinfection
oftwocelllines,MRC-5andVeroE6,wereassessedtogiveareadoutofviralreplication. Inadditionto
viralRNAlevels,thecytopathiceffects(CPEs)wereassessedfortoxiceffects(Table2).Resultsidentified
nine compounds that had no reduction in viral replication despite cells remaining healthy, and
these (zidovudine, didanosine, stavudine, abacavir sulphate, entecavir, JB1a, Aimspro, celgosivir,
andcastanospermine)wereremovedfromfurtherevaluation. Whileseveralcompoundsshoweda
reductioninviralRNAlevels,thesedrugs(ouabain,17-DMAG,omeprazole,esomeprazolemagnesium,
Gleevec,andTasigna)alsoexhibitedsignificantCPEincellmonolayers. However,threecompounds
gavereductionsintheCtvaluewithcellsremainingattached: BGB324,NCK-8andD-LANA-14;albeit
thatthelattertwocompoundsdidaffectthemorphologyofthecellstosomeextent.
Table2.ChangesinEBOVRNAlevelsandcellhealthinMRC-5andVeroE6cellstreatedafterinfection
withcompoundsattherecommendedconcentrations.
MRC-5 VeroE6
Name Concentration
CtDifference1 CellAppearance2 CtDifference CellAppearance
Ouabain 20nM 3.48±0.21 x −3.73±4.88 x
17-DMAG 5µM 3.72±0.18 x −0.63±1.39 x
BGB324 3µM 3.05±0.75 (cid:51) −1.83±1.13 (cid:51)
Zidovudine 5µM −3.12±0.27 (cid:51) −7.91±2.67 (cid:51)
Didanosine 5µM −0.43±3.87 (cid:51) −2.52±1.27 (cid:51)
Stavudine 5µM −2.87±0.22 (cid:51) −3.93±0.25 (cid:51)
Abacavirsulphate 5µM −1.54±3.26 (cid:51) −3.95±2.69 (cid:51)
Entecavir 5µM −3.08±0.20 (cid:51) −4.44±1.11 (cid:51)
JB1a 2µg/mL −4.02±0.13 (cid:51) −5.48±0.50 (cid:51)
Omeprazole 100µM 1.35±1.35 x 2.21±1.08 x
Esomeprazole
75µM 1.05±0.79 x 1.62±0.36 x
magnesium
Gleevec 20µM 3.60±0.63 x 3.49±0.54 x
Viruses2016,8,277 6of17
Table2.Cont.
MRC-5 VeroE6
Name Concentration
CtDifference1 CellAppearance2 CtDifference CellAppearance
Aimspro Neat −2.03±0.95 (cid:51) −4.60±1.15 (cid:51)
NCK-8 1mg/mL >10 * >10 *
D-LANA-14 1mg/mL >10 * >10 *
Tasigna 20µM 3.59±0.57 x −0.13±0.33 (cid:51)
Celgosivir 200µM −2.52±0.21 (cid:51) −2.41±0.12 (cid:51)
Castanospermine 200µM −1.58±3.23 (cid:51) −0.26±4.11 (cid:51)
1Differencebetweenmeanvalueofuntreatedcells(n=3)versustreatedcells(n=3).Apositivevalueindicatesa
reductioninviralRNAlevels.Valuesshownaremeanoftriplicates±standarddeviation.2Presenceofhealthy
andadherentcells.Asterisksindicatethatcellswereattached,butwithachangedmorphologicalappearance.
3.2.2. SecondaryScreeningwithaHighVirusInoculation
Arepeatscreeningassayofcompoundsthatshowedanti-EBOVactivitywasconductedusing
MRC-5cellsonly,asintheinitialscreen,similarresponseswereobservedbetweenMRC-5andVeroE6
cells,withahigherconcentrationofvirusinoculumandincubationfortwodays. Additionally,for
thecompoundsthatwerepreviouslytestedattherecommendedconcentration, buthadexerteda
cytotoxiceffect,theconcentrationsusedwereadjustedtodeterminetheoptimumconcentrationthat
causedtolerabletoxicityafterincubationonuninfectedMRC-5cellsfortwodays. Resultsfromthis
experimentconfirmedtheinvitroantiviralactivityagainstEBOVforallcompoundstested(Table3).
Table3. ChangesinEBOVRNAlevelsinMRC-5cellstreatedwithcompoundsatthreedilutions
determinednottocausecytotoxicityinnon-infectedtreatedcells.
Name Concentration CtDifference1
20nM 0.06±0.10
Ouabain 6.7nM 0.09±0.23
2.2nM 0.33±0.35
63.3nM 0.30±0.20
17-DMAG 21.1nM 0.26±0.57
7.0nM 0.44±0.06
1µM 0.90±0.15
BGB324 0.3µM 0.67±0.09
0.1µM 0.34±0.05
100µM 0.70±0.10
Omeprazole 33.3µM 0.77±0.31
11.1µM 0.86±0.22
25µM 0.78±0.25
Esomeprazole 8.3µM 0.50±1.06
0.93µM 0.17±0.16
6.7µM 1.55±0.20
Gleevec 2.2µM 1.03±0.42
0.74µM 0.64±0.03
150µg/mL 1.54±0.44
NCK-8 50µg/mL 1.33±0.09
16.7µg/mL 1.09±0.17
60µg/mL 0.96±0.19
D-LANA-14 20µg/mL 0.37±0.11
6.7µg/mL 0.40±0.32
1Differencebetweenthemeanvalueofuntreatedcells(n=3)versustreatedcells(n=3). Apositivevalue
indicatesareductioninviralRNAlevels.Valuesshownarethemeanoftriplicates±standarddeviation.
Viruses2016,8,277 7of17
3.3. ScreeningofCompoundsforEffectsagainstDiseaseinEBOV-InfectedGuineaPigs
Three of the compounds that had demonstrated invitro activity against EBOV were screened
fVoirruseesf f2e0c1t6s, 8,a 2g7a7 inst EBOV disease in the guinea pig model: 17-DMAG, BGB324, and NC7 Kof- 188. 
ThesecompoundswerechosenbecausetheyhadcausedthehighestlevelsofviralRNAreduction,
rreepprreesseenntteeddd dififfefreernetnmt modoedseosf oafc taivcittiiveistiaensd awnder ewseuriet asbuleitafobrleu sfeorin utshee icnu rtrheen tciunrrveivnot imno vdievlow mithooduetl 
fwuritthhoeurtm fuodrtihfiecra tmioondoifriccahtaionng eosr tcoheatnhgiceasl tloic eetnhsiicnagl .licensing.  
33..33..11.. TTeessttiinngg ooff BBGGBB332244 iinn EEBBOOVV-‐IInnffeecctteeddG GuuiinneeaaP Piiggss 
BBGGBB332244 wwaassd edleivlievreerdedor aolrlayllwy itwhittwh otwdoos edsopseers dpaeyr sdtaaryti nsgtaartti6nhg paot s6t- chh aplolesnt‐gceh.aRlleesnugltes. sRheoswueltds 
tshhaotwBeGdB th3a2t4 BtGreBa3tm24e tnrteaftamileednt tfoaileexde rtot aexneyrts atantyis stticaatilsltyi-csailglyn‐isfiicgannifticparnott epcrtoitveecteivffee cetfsfeccotsm copmarpedaretod 
uton turneatrteeadteadn iamniamlsa(lns (=n 6= p6e pregrr goruopu;pp; p= =0 .03.5385,8l,o lgog-r‐arnankks usurvrvivivaalla annaalylyssisis))( F(Figiguurree1 1aa)).. WWeeiigghhtt aanndd 
tteemmppeerraattuurreed  idffifefreernecnecseps opsto-EstB‐EOBVOcVh alclhenaglleenshgeo wsehdowtheadt anthimata lasnthimatamls etthhautm  maneet chliunmicaalneen dcplioniinctasl 
eexnhdipboitiendts wexehigibhittelods ws aenigdhta llolsasn aimnda lasllh aandimaarliss ehiand tae mripsee riant uterme p(Feirgauturere1 (bF,icg).urHe o1wb,ec)v. eHr,owweeivgehrt, 
lwosesigwhta slonsos twoabss enrovte dobisnerovneeda inni monael farnoimmathl efrBomGB t3h2e4 B-tGreBa3te2d4‐gtrreoautepd, wgrhoiuchp,s wurhviicvhe dsuurvnitviledda uyn1t8il 
pdoasyt -1c8h aplloesnt‐gceh,atlhleensgceh,e tdhuel esdcheendduloefdt heendst uodf yt.hTe hsitsuidnyd. icTahtiess itnhdaitciantetsh itshaant iimn athl,iBs GanBi3m24ale, xBeGrtBe3d2a4 
perxoetretectdiv ae perfofetcetc.tive effect. 

BGB324 treated
Untreated
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Time (days post-challenge)
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Figure 1. Survival and clinical parameters of guinea pigs treated with 100 mg/kg BGB324 twice daily 
Figure1.Survivalandclinicalparametersofguineapigstreatedwith100mg/kgBGB324twicedaily
ccoommppaarreedd ttoo uunnttrreeaatteeddc coonnttrroolsls( n(n= = 66 ppeerr ggrroouupp)).. ((aa)) SSuurrvviivvaalla annaalylyssisisa affteterrc chhaalllelennggeew witihth1 10033T TCCIIDD50 
50
EBOV; (b) Weight changes as a percentage compared to the day of challenge; (c) Temperature changes 
EBOV;(b)Weightchangesasapercentagecomparedtothedayofchallenge;(c)Temperaturechanges
aass ◦°CC ddiiffffeerreennccee ccoommppaarreedd ttoo tthhee ddaayy ooff cchhaalllleennggee.. 
3.3.2. Testing of NCK‐8 in EBOV‐Infected Guinea Pigs 
NCK‐8 was delivered intravenously with two doses per day starting at 6 h post‐challenge. 
Although an increase in time to death was observed, this was not statistically significant (n = 6 per 
group; p = 0.076, log‐rank survival analysis) (Figure 2a). All animals exhibited weight loss and had a 
rise in temperature (Figure 2b,c). An animal in the NCK‐8‐treated group began to increase its weight 
nine days post‐challenge, indicating a recovery from EBOV infection. This animal survived to the 
scheduled end of the study.
Viruses2016,8,277 8of17
3.3.2. TestingofNCK-8inEBOV-InfectedGuineaPigs
NCK-8 was delivered intravenously with two doses per day starting at 6 h post-challenge.
Althoughanincreaseintimetodeathwasobserved,thiswasnotstatisticallysignificant(n=6per
group;p=0.076,log-ranksurvivalanalysis)(Figure2a). Allanimalsexhibitedweightlossandhada
riseintemperature(Figure2b,c). AnanimalintheNCK-8-treatedgroupbegantoincreaseitsweight
ninedayspost-challenge,indicatingarecoveryfromEBOVinfection. Thisanimalsurvivedtothe
scheduledendofthestudy.
Viruses 2016, 8, 277  8 of 18 
NCK-8 treated
Untreated
(a) 100
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0 2 4 6 8 10 12 14
Time (days post-challenge)
(b) ge) 120 (c) ge) 4
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hangeay of c 100 e chan ay of c 2
Weight cared to d 90 mperatur ared to d -20
mp 80 Te mp
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( 0 2 4 6 8 10 12 14 ( 0 2 4 6 8 10 12 14
Time (days post-challenge) Time (days post-challenge)

FFiigguurree 22.. SSuurrvviivvaall aanndd cclliinniiccaall ppaarraammeetteerrss ooff gguuiinneeaa ppiiggss ttrreeaatteedd wwitithh5 5m mgg//kkgg NNCCKK-‐88 ttwwiiccee ddaaiillyy 
ccoommppaarreeddt toou unnttrreeaateteddc coonntrtorolsls( n(n= =6 6p peerrg grroouupp).).( (aa))S Suurrvvivivaalla annaalylyssisisa aftfeterrc chhaalllelennggeew witihth1 10033T TCCIDID5500 
EEBBOOVV;; ((bb)) WWeeiigghhttc chhaannggeessa ass aa ppeerrcceennttaaggee ccoommppaarreedd ttoo tthhee ddaayy ooff cchhaalllleennggee;; ((cc)) TTeemmppeerraattuurree cchhaannggeess 
aass ◦°CC ddiiffffeerreennccee ccoommppaarreedd ttoo tthhee ddaayy ooff cchhaalllleennggee.. 
33..33..33.. TTeessttiinngg ooff 1177-‐DDMMAAGG iinn EEBBOOVV-‐IInnffeecctteeddG GuuinineeaaP Pigigss 
1177-‐DDMMAAGG wwaass sscchheedduulleedd ttoo bbee ddeelliivveerreedd vviiaa tthhee iinnttrraappeerriittoonneeaall rroouuttee eevveerryy ttwwoo ddaayyss.. HHoowweevveerr,, 
aallll ttrreeaatteedd aanniimmaallss mmeett hhuummaannee eennddppooiinnttss wwiitthhiinn 2244 hh ddeelliivveerryy ooff tthhee fifirrsstt ddoossee.. TThheerreeffoorree,, tthhee eeffffeeccttss 
ooff tthhiiss ccoommppoouunndd aaggaaiinnsstt EEBBOOVV ccoouulldd nnoott bbee aasscceerrttaaiinneedd iinn tthhiisse exxppeerriimmeenntt.. 
44.. DDiissccuussssiioonn 
TThhee rreecceenntt oouuttbbrreeaakk ooff EEBBOOVV ddiisseeaassee iinn WWeesstt AAffrriiccaa [[33]] hhaass hhiigghhlliigghhtteedd tthhee uurrggeenntt nneeeedd ffoorr 
tthheerraappeeuuttiiccss.. TThheesese neneedesd ms mighigth bte bmeemt metomst oqsuticqkulyic aknlyd aenffdicieefnfitclyie inf talyn eifxiasntinegx idsrtiungg wdirtuhg a kwniothwan 
ksnafoewtyn  psarfoeftiyle pcrooufillde cboeu ldrebpeurreppouserdp ostoe dttroeattr eEatBEOBVO Veffeefcfeticvteivlye.l y.OOuru rsstutuddyy  ssccrreeeenneedd eeiigghhtteeeenn 
tthheeoorreettiiccaallllyy-‐pprroommiissiinngga annttiivviirraallt thheerraappiieessa aggaaiinnssttE EBBOOVVu ussiinngg iinn vviittrroo aanndd iinn vviivvoo eexxppeerriimmeennttss wwiitthh 
lliivvee vviirruuss aatt CCLL44.. GGiivveenn tthhee sseerriioouussnneessss aanndd lloonngg rruunnnniinngg eexxtteenntt ooff tthhee WWeesstt AAffrriiccaann EEVVDD oouuttbbrreeaakk,, 
we developed an experimental workflow to rapidly screen compounds for antiviral activity in order 
to focus efforts and resources on only the most promising therapies. In the first in vitro screen using 
500 TCID50/well (multiplicity of infection (MOI) approximately 0.01), half of the compounds failed to 
show any activity, so these compounds were excluded from further study. For a second screen using 
5000 TCID50/well (MOI approximately 0.1), eight of the remaining nine compounds continued to 
demonstrate a mean reduction in viral replication. In this screen, the drug Tasigna was not retested 
due to compound unavailability, but additionally, the drug Gleevec was included since it was 
reported to operate via the same mechanism and demonstrated better in vitro efficacy. The in vitro 
screens used viral RNA levels as a readout based on previous antiviral testing work [33] and 
infectious doses of MOI 0.01–0.1, similar to levels used in other anti‐EBOV testing studies [6,34,35].
Viruses2016,8,277 9of17
wedevelopedanexperimentalworkflowtorapidlyscreencompoundsforantiviralactivityinorder
tofocuseffortsandresourcesononlythemostpromisingtherapies. Inthefirstinvitroscreenusing
500TCID /well(multiplicityofinfection(MOI)approximately0.01),halfofthecompoundsfailedto
50
showanyactivity,sothesecompoundswereexcludedfromfurtherstudy. Forasecondscreenusing
5000TCID /well(MOIapproximately0.1), eightoftheremainingninecompoundscontinuedto
50
demonstrateameanreductioninviralreplication. Inthisscreen,thedrugTasignawasnotretested
duetocompoundunavailability,butadditionally,thedrugGleevecwasincludedsinceitwasreported
to operate via the same mechanism and demonstrated better invitro efficacy. The invitro screens
usedviralRNAlevelsasareadoutbasedonpreviousantiviraltestingwork[33]andinfectiousdoses
ofMOI0.01–0.1, similartolevelsusedinotheranti-EBOVtestingstudies[6,34,35]. Giventhatthe
compounds tested in our study were chosen due to their perceived potential effectiveness for use
againstEBOV,thenegativeresultsdemonstratetheimportanceoftestingtherapiesusingtheactual
livepathogentodetermineeffects. Theyalsohighlighttheuncertaintiesinherentinextrapolatingthe
mechanismofactiondatatodrugrepurposing,nomatterhowrational.
OftheeightcompoundsthatshowedrepeatableantiviralactivityagainstEBOVinvitro,three
were selected for invivo studies using the guinea pig model of infection (i.e., 17-DMAG, BGB324
andNCK-8). WhereasomeprazoleandesomeprazoledemonstratedinvitroactivityagainstEBOV,
theresultswereinlinewithapreviousreportusingpseudotypedviruseswherethevaluesofdrug
concentration causing 50% inhibition (IC ) were in the region of 50 µM [10]. This suggested that
50
dosesrequiredforpotentinhibitionwouldbedifficulttoachievewithoutconcomitantandsignificant
toxicity(thelicenceddosingfor40mgesomeprazole,20mgesomeprazoleand20mgomeprazole
generatesmedianmaximumplasmaconcentrationsof1.59–9.61µM,0.51–4.78µMand0.15–3.51µM,
respectively[36]). Gleevec(brandnameforimatinibmesylate)wasnotfollowedthroughforinvivo
testingsincetheoptimaldosingrequirement(i.e.,continuousdosing)wasnotfeasibleforthecurrent
set-upoftheanimalmodelatCL4. Therequirementforcontinuousdosingisduetotheshorthalf-life
inratsofasimilarcompound[37]. Additionally,theplasmaconcentrationsofGleevecusuallyreach
2–3 µM at normal dosages [38]. Others have suggested that the concentration for effective EBOV
inhibitionis20µM[11],whichwouldnotbepossible. However,thedatafromtheinvitrostudies
withliveEBOVreportedhereshoweffectsevenwhen0.74µMwasused. NCK-8andD-LANA-14are
membrane-activesmallmoleculesthatmimicthepropertiesofnaturalantimicrobialpeptidesandmay
negativelyimpacttheviralenvelopeandcellularlipidbilayer. Outofthetwomolecules,D-LANA-14,
waslessactive;henceitsefficacywasnotassessedintheanimalmodel. TheguineapigmodelofEBOV
diseasewasusedinthisworksinceitpresentsarobust,accessibleandbroadlyreflectiverodentmodel
forthescreeningofcountermeasures[39]. Importantly,themodelalsoallowscatheterizedanimals
tobeused,allowingdirectaccesstotheintravenousroute. Thisisthepreferredrouteofdeliveryfor
manyantiviralcompoundsandiscompatibleforworkatthehighestmicrobiologicalcontainment[30].
NopositiveeffectsofBGB324againstEBOVinfectioninguineapigswereobservedinrelationto
survival. BothBGB324-treatedanduntreatedcontrolsexhibited83%mortality. However,thesurvivor
intheBGB324-treatedgroupdidnotshowthesignificantlossinweightthatistypicalofEBOV-infected
guineapigs[31]andthatwasexhibitedbythesurvivorintheuntreatedgroup. Bothsurvivingguinea
pigsexhibitedanincreaseintemperaturetypicalofEBOVinfectioninthismodel[31]. Itispossible
thattheseobservationsareaconsequenceofbroadguineapigresponses,sincetheseanimalswere
outbredindividualsandthereweresubtledifferencesbetweentheirresponses. TheeffectsofBGB324
wereproposedtobeduetotheinhibitionoftyrosinekinaseinhibitors[40].Thus,itcouldbespeculated
thatothertyrosinekinaseinhibitors,includingthetwointhisstudy(GleevecandTasigna),wouldalso
nothavedemonstratedprotectiveeffects.
TreatmentofEBOV-challengedguineapigswithNCK-8wastheonlyinstancethatshowedan
effect on disease progression, with a demonstrable increased mean time to death; albeit, this was
notstatisticallysignificant. AllanimalstreatedwithNCK-8exhibitedweightlossandtemperature
increases,inlinewiththelevelsofuntreatedanimals. Interestingly,thisclassofcompoundshasbeen
Viruses2016,8,277 10of17
testedagainstbacterialpathogens[15,19,41]andhasbeenfoundtobequiteeffective. Otherpeptide
mimicshavedemonstratedantiviralactivityagainstEBOV,aswell[21]. However,itshouldbenoted
thatalthoughNCK-8showedhighlevelsofantiviralactivity,thecellmonolayerswerephenotypically
different for both VeroE6 and MRC-5 cell lines. This observation warrants further investigation,
especiallythedependenceofactivityontheconcentrationofthecompounds.Nevertheless,NCK-8was
well-toleratedwhentestedinguineapigsandledtotherecoveryofoneanimalfromEBOVinfection.
Whentestedinguineapigs,theeffectsof17-DMAGcouldnotbeclearlyascertainedsincethese
animalsmethumaneendpointspriortodiseasesymptomsofEBOVdisease. Thedoseusedinour
studywas30mg/kg;whereastherearenoreportsof17-DMAGbeingusedinguineapigs,othershave
shownthat75mg/kgdeliveredorallywaswelltoleratedinmiceandrats[42]. Therefore,itislikely
thatthereisanon-compatibleinteractionwithacomponentofguineapigphysiologythatresultsin
atoxiceffect. Tofurthertesttheeffectivenessof17-DMAGagainstEBOV,ananimalsystemwhere
thecompoundhasshowntobenon-toxicshouldbeused,suchasthemouse. However,duetothe
mousemodelnotbeingasrelevanttohumandiseaseastheguineapig[43],thevalueofconducting
suchstudiesmaybecounterproductive.
Insummary,ourresultsprovidedetailsontheantiviralpropertiesofeighteenpotentialtherapies.
Whereastheexperimentswerelimitedandcouldhavebeenextendedtoincludeviableviralloadsby
plaqueassayormodernmethodsfortheassessmentofcytotoxicity,thestudieswereconductedto
providerapidresultsduringanactiveEBOVoutbreakandwithinthelimitationsofCL4facilitieswhere
allhandlingofliveEBOVwaswithinaspecializedcabinet-linesystem. Candidatesforthisstudywere
chosenviaaproject-specificselectioncommittee(comprisingofmembersfromPHE,academiaand
theWellcomeTrust),notbyscreeningapproachesofalargenumberofcompounds[12,44,45],sowere
moreselectivebasedonperceivedactivityagainstEBOV.However,thevalueofthisworkistheuse
ofliveEBOV,providingvaluableinsightintocompoundsthatwarrantfurtherinvestigationand,as
important,thosethatshowednoantiviraleffectsagainstEBOV.
5. Conclusions
Inconclusion,thedatainthisreportwillhelptoinformdecisionsonwhichcompoundsshould
beinvestigatedfurtherand,equallyimportantly,whichonesshouldnot. Giventhelimitedfacilities
andrestrictionsofworkingwithliveHazardGroup4viruses,thefocusshouldnowbeontreatments
thathaveshownpromisewithliveEBOVusinginvitroandinvivomodels.
Acknowledgments: This work was financially supported by the Ebola research funding initiative from the
Wellcome Trust to Public Health England (PHE) scientists Miles W. Carroll, Roger Hewson, Julia Vipond,
Seshadri Vasan and Simon Funnell (Grant Number 106722/Z/15/Z). The authors would like to thank
KeithSpencerfromtheWellcomeTrustforvaluablecontributionsanddiscussionsinassessingcompoundsforthe
suitabilityfortesting.Wewouldalsoliketothankthoseorganizationsthatproposedcompoundsforevaluation,
including:UniversityofLiverpool(ouabainand17-DMAG);BerGenBio(BGB324);Avipero(JB1a);ImperialCollege
London(omeprazoleandesomeprazole);EmoryUniversity(GleevecandTasigna);DavalInternational(Aimspro);
JawaharlalNehruCentreforAdvancedScientificResearch(NCK-8andD-LANA-14);and60◦Pharmaceuticals
(celgosivirandcastanospermine).Theviewsexpressedinthisreportarethoseoftheauthorsandnotnecessarily
thoseoftheemployinginstitutionsorthefundingbody.
Author Contributions: Stuart D. Dowall, Seshadri S. Vasan, Simon Funnell, Julia Vipond, Sue Charlton,
RogerHewsonandMilesW.Carrollconceivedofanddesignedtheexperiments.StuartD.Dowall,KevinBewley,
RobertWatson,AndrewBosworth,IreneTaylor,LindaEasterbrookandJamesPitmanperformedtheexperiments.
StuartD.Dowall,KevinBewley,RobertWatson,AndrewBosworth,JennyChan-Pensely,RogerHewsonand
MilesW.Carrollanalyzedthedata. ChandradhishGhosh, MohiniM.Konai, GroGausdal, JamesB.Lorens,
JasonLong,WendyBarclay,IsabelGarcia-Dorival,JulianHiscox,SianSummersandJayantaHaldarcontributed
reagents,materials,andanalysistools.StuartD.Dowall,RogerHewson,SueCharltonandMilesW.Carrollwrote
thepaper.
ConflictsofInterest: PHEauthorsclaimnoconflictsofinterest. Theremainingauthorscontributedreagents,
whichformedpartoftheirinstitution/companyworkprograms; however, theseauthorshadnoroleinthe
collection,analysesorinterpretationofdata,norinthewritingofthemanuscript.