Prophylactic Antiviral Activity of Sulfated Glycomimetic Oligomers and Polymers

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Article

Prophylactic antiviral activity of sulfated

glycomimetic oligomers and polymers

Laura Soria-Martinez, Sebastian Bauer, Markus Giesler, Sonja Schelhaas, Jennifer Materlik, Kevin Alexander

Janus, Patrick Pierzyna, Miriam Becker, Nicole Leigh Snyder, Laura Hartmann, and Mario Schelhaas

J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b13484 • Publication Date (Web): 27 Feb 2020

Downloaded from pubs.acs.org on February 27, 2020

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Journal of the American Chemical Society

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Prophylacticꢀantiviralꢀactivityꢀofꢀsulfatedꢀglycomimeticꢀoligo-

mersꢀandꢀpolymers.ꢀ

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1,2

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4,5 ꢀ

LauraꢀSoria-Martinez ,ꢀSebastianꢀBauer ,ꢀMarkusꢀGiesler ,ꢀSonjaꢀSchelhaas , JenniferꢀMater-

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1,2,ꢀ†

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2,3,

lik ,ꢀKevinꢀJanus ,ꢀPatrickꢀPierzyna ,ꢀMiriamꢀBecker ,ꢀNicoleꢀL.ꢀSnyder ,ꢀLauraꢀHartmann *,ꢀ

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,2,5, ꢀ

MarioꢀSchelhaas

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InstituteꢀofꢀCellularꢀVirology,ꢀZMBE,ꢀUniversityꢀofꢀMünster,ꢀMünster,ꢀGermanyꢀ

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ResearchꢀGroupꢀ“ViroCarb:ꢀglycansꢀcontrollingꢀnon-envelopedꢀvirusꢀinfections”ꢀ(FOR2327),ꢀCoordinatingꢀUniversi-

tyꢀofꢀTübingen,ꢀGermanyꢀ

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InstituteꢀofꢀOrganicꢀChemistryꢀandꢀMacromolecularꢀChemistry,ꢀHeinrich-Heine-UniversityꢀDüsseldorf,ꢀDüsseldorf,ꢀ

ꢀ

Germany

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EuropeanꢀInstituteꢀforꢀMolecularꢀImaging,ꢀUniversityꢀofꢀMünster,ꢀMünster,ꢀGermany

CellsꢀinꢀMotionꢀInterfacultyꢀCentreꢀCiMIC,ꢀUniversityꢀofꢀMünster,ꢀMünster,ꢀGermanyꢀ

DepartmentꢀofꢀChemistry,ꢀDavidsonꢀCollege,ꢀDavidson,ꢀNorthꢀCarolina,ꢀUSAꢀ

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*correspondingꢀauthorsꢀ

ꢀ

Antiviralꢀresearch,ꢀvirus,ꢀglycomimetics,ꢀGAGꢀmimetics,ꢀprecisionꢀglycooligomers,ꢀglycopolymersꢀ

ABSTRACT:ꢀInꢀthisꢀwork,ꢀweꢀinvestigateꢀtheꢀpotentialꢀofꢀhighlyꢀsulfatedꢀsyntheticꢀglycomimeticsꢀtoꢀactꢀasꢀinhibitorsꢀofꢀ

viralꢀbinding/infection.ꢀOurꢀresultsꢀindicateꢀthatꢀboth,ꢀlongꢀchainꢀglycopolymersꢀandꢀshortꢀchainꢀglycooligomersꢀareꢀca-

pableꢀofꢀpreventingꢀviralꢀinfection.ꢀNotably,ꢀglycopolymersꢀefficientlyꢀinhibitꢀhumanꢀpapillomavirusꢀ(HPV16)ꢀinfectionꢀinꢀ

vitroꢀandꢀmaintainꢀtheirꢀantiviralꢀactivityꢀinꢀvivo,ꢀwhileꢀtheꢀglycooligomersꢀexertꢀtheirꢀinhibitoryꢀfunctionꢀpostꢀattachmentꢀ

ofꢀvirusesꢀtoꢀcells.ꢀMoreover,ꢀwhenꢀweꢀtestedꢀtheꢀpotentialꢀforꢀbroaderꢀactivityꢀagainstꢀseveralꢀotherꢀhumanꢀpathogenicꢀ

viruses,ꢀ weꢀ observedꢀ broad-spectrumꢀ antiviralꢀ activityꢀ ofꢀ theseꢀ compoundsꢀ beyondꢀ ourꢀ initialꢀ assumptions.ꢀWhileꢀ theꢀ

compoundsꢀtestedꢀdisplayedꢀaꢀrangeꢀofꢀantiviralꢀefficacies,ꢀvirusesꢀwithꢀratherꢀdiverseꢀglycanꢀspecificitiesꢀsuchꢀasꢀHerpesꢀ

SimplexꢀVirusꢀ(HSV),ꢀInfluenzaꢀAꢀVirusꢀ(IAV)ꢀandꢀMerkelꢀCellꢀPolyomavirusꢀ(MCPyV)ꢀcouldꢀbeꢀtargeted.ꢀThisꢀopensꢀupꢀ

newꢀopportunitiesꢀtoꢀdevelopꢀbroadlyꢀactiveꢀglycomimeticꢀinhibitorsꢀofꢀviralꢀentryꢀandꢀinfection.ꢀ

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INTRODUCTIONꢀꢀ

21 sionꢀ betweenꢀ organisms.ꢀ Atꢀ best,ꢀ aꢀ conservedꢀ andꢀ com-

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monꢀ mechanismꢀ forꢀ severalꢀ virusesꢀ wouldꢀ beꢀ targeted.ꢀ

Oneꢀ suchꢀ mechanismꢀ isꢀ theꢀ initialꢀ bindingꢀ toꢀ cellularꢀ

glycansꢀ forꢀ primaryꢀ attachment.ꢀ Manyꢀ virusesꢀ haveꢀ

evolvedꢀtoꢀengageꢀglycansꢀthatꢀareꢀpresentedꢀonꢀcellꢀsur-

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Viralꢀdiseasesꢀareꢀaꢀmajorꢀhealthꢀburdenꢀassociatedꢀwithꢀ

significantꢀ socioeconomicꢀ loss. ꢀ Often,ꢀ preventionꢀ andꢀ

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treatmentꢀ ofꢀ viralꢀ infectionsꢀ remainsꢀ aꢀ majorꢀ challenge.ꢀ

Theꢀ mostꢀ importantꢀ andꢀ effectiveꢀ antiviralꢀ strategyꢀ isꢀ

vaccination.ꢀ However,ꢀ vaccinesꢀ areꢀ onlyꢀ availableꢀ forꢀ aꢀ

selectꢀnumberꢀofꢀviralꢀinfections.ꢀInꢀaddition,ꢀonlyꢀaꢀlim-

itedꢀ numberꢀ ofꢀ antiviralꢀ drugsꢀ exist,ꢀ andꢀ thoseꢀ thatꢀ do,ꢀ

typicallyꢀ targetꢀ essentialꢀ viralꢀ functions/proteins.ꢀ Asꢀ aꢀ

consequence,ꢀ thereꢀ isꢀ aꢀ highꢀ riskꢀ ofꢀ quickꢀ emergenceꢀ ofꢀ

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faceꢀ proteinsꢀ orꢀ lipidsꢀ toꢀ adhereꢀ toꢀ cells. ꢀ Theꢀ glycansꢀ

recognizedꢀbyꢀvirusesꢀareꢀdiverse,ꢀmostꢀoftenꢀcontainingꢀ

glycansꢀsuchꢀasꢀsialicꢀacidsꢀ(Neu5Ac)ꢀandꢀglycosaminogly-

cansꢀ (GAGs)ꢀ likeꢀ heparanꢀ sulfatesꢀ (HS),ꢀ whichꢀ areꢀ com-

posedꢀ ofꢀ N-acetylꢀ glucosamineꢀ (GlcNAc)ꢀ andꢀ glucuronicꢀ

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acidꢀ(GlcA). ꢀ

escapeꢀmutationsꢀinꢀtheꢀvirus,ꢀrenderingꢀvirusesꢀresistantꢀ

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toꢀdrugꢀtreatments. ꢀHence,ꢀtheꢀdevelopmentꢀofꢀaddition- 32

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alꢀinterventionsꢀwithꢀaꢀbroaderꢀrangeꢀofꢀtargetedꢀvirusesꢀisꢀ

ofꢀhighestꢀinterest.ꢀ

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Inhibitionꢀofꢀviralꢀinfectionsꢀbyꢀnegativelyꢀchargedꢀnaturalꢀ

polysaccharidesꢀ thatꢀ competeꢀ withꢀ bindingꢀ toꢀ cellularꢀ

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35 glycansꢀ suchꢀ asꢀ HSꢀ isꢀ wellꢀ documented. ꢀ Perhapsꢀ theꢀ

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mostꢀprominentꢀexampleꢀisꢀcarrageenan,ꢀaꢀnaturalꢀsulfat-

edꢀ polysaccharideꢀ fromꢀ redꢀ algaeꢀ whichꢀ interferesꢀ withꢀ

infectionꢀofꢀmanyꢀviruses,ꢀforꢀexampleꢀhumanꢀpapilloma-

virusesꢀ (HPVs),ꢀ herpesꢀ simplexꢀ virusꢀ typeꢀ 1ꢀ (HSV-1)ꢀ andꢀ

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Aꢀpromisingꢀapproachꢀtoꢀfightꢀviralꢀinfectionsꢀisꢀbyꢀtarget-

ingꢀvirusꢀentryꢀintoꢀhostꢀcells,ꢀi.e.ꢀtheꢀdeliveryꢀofꢀtheꢀviralꢀ

genomeꢀtoꢀtheꢀintracellularꢀreplicationꢀsiteꢀduringꢀinitialꢀ

infection.ꢀ Thisꢀ couldꢀ avoidꢀ primaryꢀ infectionꢀ andꢀ addi-

tionallyꢀ limitꢀ spreadꢀ withinꢀ theꢀ organismꢀ andꢀ transmis-

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influenzaꢀAꢀvirusꢀ(IAV). ꢀTheꢀantiviralꢀpotentialꢀofꢀcarra-

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geenanꢀhasꢀalsoꢀbeenꢀrealizedꢀinꢀaꢀclinicalꢀtrial,ꢀwhereꢀitꢀ100 polymerizationꢀ ofꢀ glycomonomers.ꢀ Shortꢀ chainꢀ glycooli-

reducedꢀ contractionꢀ ofꢀ HPV16. ꢀ However,ꢀ naturalꢀ poly- 101 gomersꢀ areꢀ generatedꢀ byꢀ solid-phaseꢀ synthesis,ꢀ whichꢀ

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43 saccharidesꢀmayꢀpresentꢀaꢀnumberꢀofꢀchallengesꢀforꢀsafeꢀ102 allowsꢀforꢀpreciseꢀcontrolꢀofꢀtheꢀdegreeꢀofꢀpolymerizationꢀ

44 clinicalꢀ application.ꢀ Forꢀ one,ꢀ theseꢀ moleculesꢀ areꢀ inher- 103 andꢀtherebyꢀchainꢀlengthꢀandꢀnumberꢀofꢀsulfatedꢀglyco-

45 entlyꢀheterogeneous.ꢀPreparationsꢀmayꢀconsistꢀofꢀaꢀvaria- 104 sideꢀsideꢀchains.ꢀꢀ

46 bleꢀmixtureꢀofꢀheterogeneousꢀglycans,ꢀandꢀmayꢀcontainꢀaꢀ

47 varietyꢀofꢀimpurities.ꢀSecond,ꢀasꢀnaturalꢀglycans,ꢀtheyꢀmayꢀ

48 alsoꢀbeꢀinherentlyꢀbioactiveꢀwithꢀtheꢀriskꢀofꢀbiologicalꢀsideꢀ

49 effectsꢀ uponꢀ administration.ꢀ Inꢀ searchꢀ ofꢀ alternatives,ꢀ

50 syntheticꢀ glycanꢀ analoguesꢀ orꢀ so-calledꢀ glycomimeticsꢀ

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ꢀ

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06 Asꢀaꢀmodelꢀsystemꢀtoꢀtestꢀinhibitoryꢀefficacyꢀtheꢀsulfatedꢀ

07 glycomimetics,ꢀweꢀinitiallyꢀusedꢀanꢀHPVꢀinfectionꢀmodel.ꢀ

08 HPVsꢀareꢀsmall,ꢀnon-envelopedꢀDNAꢀvirusesꢀwithꢀtrans-

09 formingꢀpotentialꢀthatꢀinfectꢀskinꢀorꢀmucosa.ꢀHPVꢀvirionsꢀ

10 consistꢀofꢀtwoꢀstructuralꢀproteins:ꢀtheꢀmajorꢀcapsidꢀpro-

11 teinꢀ L1ꢀ thatꢀ self-assemblesꢀ intoꢀ aꢀ totalꢀ ofꢀ 72ꢀ pentamers,ꢀ

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haveꢀbeenꢀchemicallyꢀproducedꢀtoꢀprovideꢀmoreꢀcontrolꢀ

overꢀtheꢀstructureꢀofꢀtheꢀcompound.ꢀSuchꢀglycomimeticsꢀ

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haveꢀbeenꢀshownꢀtoꢀretainꢀorꢀevenꢀexceedꢀtheꢀactivitiesꢀofꢀ

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theirꢀcorrespondingꢀnaturalꢀpolysaccharidesꢀwhileꢀprovid-

ingꢀ additionalꢀ featuresꢀ suchꢀ asꢀ improvedꢀ stability,ꢀ bioa-

vailabilityꢀandꢀhalf-life. ꢀꢀ

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13 pentamers.

ꢀHigh-riskꢀHPVꢀtypesꢀ(e.g.ꢀ16,ꢀ18ꢀandꢀ31)ꢀareꢀ

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114 responsibleꢀforꢀaꢀvarietyꢀofꢀanogenitalꢀandꢀoropharyngealꢀ

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15 cancers,ꢀ causingꢀ mostꢀ cervicalꢀ cancersꢀ worldwide.ꢀ Theꢀ

16 mostꢀ prevalentꢀ HPVꢀ typeꢀ isꢀ 16,ꢀ whichꢀ hasꢀ beenꢀ widelyꢀ

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58 Glycomimeticsꢀofꢀnegativelyꢀchargedꢀnaturalꢀpolysaccha- 117 studiedꢀ andꢀ servesꢀ asꢀ aꢀ prototypeꢀ forꢀ studyingꢀ HPVꢀ

59 ridesꢀ canꢀ beꢀ differentiatedꢀ intoꢀ polyanionicꢀ systemsꢀ andꢀ118 entry. ꢀ HPV16ꢀ attachesꢀ toꢀ basalꢀ cellsꢀ orꢀ theꢀ basementꢀ

60 glycofunctionalizedꢀ systemsꢀ carryingꢀ eitherꢀ sialicꢀ acidꢀ119 membraneꢀofꢀepitheliaꢀcellsꢀviaꢀtheꢀglycanꢀchainsꢀofꢀhepa-

61 motifsꢀ orꢀ sulfatedꢀ glycanꢀ fragments. ꢀ Polyanionicꢀ sys- 120 ranꢀ sulfateꢀ proteoglycansꢀ (HSPGs).

62 temsꢀ andꢀ sialicꢀ acidꢀ functionalizedꢀ glycomimeticsꢀ haveꢀ121 inducesꢀ aꢀ conformationalꢀ changeꢀ inꢀ L1ꢀ thatꢀ facilitatesꢀ

63 beenꢀ widelyꢀ studiedꢀ forꢀ theirꢀ antiviralꢀ activity. ꢀ Forꢀ ex- 122 furtherꢀstructuralꢀprocessingꢀofꢀtheꢀcapsid. ꢀTheseꢀcrucialꢀ

64 ample,ꢀsialicꢀacidꢀcarryingꢀglycopolymersꢀwereꢀintroducedꢀ123 structuralꢀ changesꢀ involveꢀ aꢀ proteolyticꢀ processingꢀ ofꢀ L1ꢀ

65 andꢀstudiedꢀbyꢀRoyꢀandꢀWhitesidesꢀandꢀhaveꢀbeenꢀshownꢀ124 byꢀ secretedꢀ kallikrein-8,ꢀ asꢀ wellꢀ asꢀ cyclophilin-mediatedꢀ

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ꢀ Thisꢀ interactionꢀ

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byꢀseveralꢀotherꢀgroupsꢀtoꢀbeꢀpotentꢀinhibitorsꢀofꢀinfluen- 125 externalizationꢀandꢀsubsequentꢀfurin-dependentꢀcleavageꢀ

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zaꢀvirusꢀcellꢀentry. ꢀHaag,ꢀAzadꢀandꢀco-workersꢀrecentlyꢀ126 ofꢀtheꢀN-terminusꢀofꢀL2. ꢀTheseꢀchangesꢀresultꢀinꢀtheꢀ

demonstratedꢀ thatꢀ highlyꢀ sulfatedꢀ polyanionicꢀ polyglyc- 127 lossꢀofꢀaffinityꢀforꢀHSPGsꢀandꢀinꢀtheꢀtransferꢀofꢀtheꢀvirusꢀ

erolꢀdendrimersꢀsystemsꢀshowedꢀbroad-spectrumꢀantiviralꢀ128 toꢀ anꢀ elusiveꢀ secondaryꢀ receptorꢀ (complex),ꢀ triggeringꢀ

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activityꢀagainstꢀaꢀnumberꢀofꢀviruses. ꢀOnꢀtheꢀotherꢀhand,ꢀ129 viralꢀinternalizationꢀbyꢀaꢀnovelꢀendocyticꢀmechanism.

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sulfatedꢀ glycopolymersꢀ asꢀ glycomimeticsꢀ ofꢀ GAGsꢀ haveꢀ

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beenꢀmuchꢀlessꢀstudiedꢀforꢀtheirꢀpotentialꢀtoꢀinhibitꢀviralꢀ

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highlyꢀsulfatedꢀ

glycooligomersꢀ

andꢀglycopolymers

infections ,ꢀ butꢀ ratherꢀ asꢀ inhibitorsꢀ ofꢀ proteinꢀ aggrega-

tion.ꢀForꢀexample,ꢀglycopolymerꢀGAGꢀmimeticsꢀasꢀintro-

ducedꢀ byꢀ Hsieh-Wilsonꢀ andꢀ co-workersꢀ wereꢀ studiedꢀ asꢀ

anticoagulantsꢀ showingꢀ activityꢀ comparableꢀ toꢀ commer-

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cialꢀ productsꢀ suchꢀ asꢀ Arixtra. ꢀ Miuraꢀ andꢀ co-workersꢀ

demonstratedꢀanꢀinhibitoryꢀeffectꢀofꢀGAGꢀmimeticꢀglyco-

polymersꢀ onꢀ Alzheimer’sꢀ b-secretase,ꢀ playingꢀ anꢀ im-

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portantꢀ roleꢀ inꢀ theꢀ Alzheimer’sꢀ disease. ꢀ Aꢀ firstꢀ studyꢀ

inhibition/ꢀ

competition

testingꢀforꢀtheꢀantiviralꢀactivityꢀofꢀsulfatedꢀglycopolymersꢀ

wasꢀ reportedꢀ byꢀ Tengdeliusꢀ andꢀ co-workersꢀ whoꢀ intro-

ducedꢀ polymethacrylamidesꢀ withꢀ pendantꢀ sulfatedꢀ α-L-

fucosidesꢀthatꢀexhibitedꢀinhibitoryꢀactivityꢀagainstꢀHSV-1ꢀ

similarꢀtoꢀthatꢀofꢀfucoidan,ꢀaꢀnaturalꢀanionicꢀpolysaccha-

virusꢀadhesionꢀ

andꢀentry

cellꢀsurfaceꢀglycans

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rideꢀproducedꢀinꢀalgae. ꢀꢀ

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Inꢀthisꢀstudy,ꢀweꢀsynthesizeꢀandꢀapplyꢀaꢀseriesꢀofꢀsulfatedꢀ132 Schemeꢀ1.ꢀModelꢀforꢀtheꢀinhibitionꢀofꢀviralꢀentryꢀbyꢀsul-

glycomimeticꢀ oligomersꢀ andꢀ polymersꢀ toꢀ testꢀ forꢀ theirꢀ133 fatedꢀglycomimeticꢀpolymersꢀbyꢀcompetingꢀwithꢀcellularꢀ

antiviralꢀ potential.ꢀ Theꢀ compoundsꢀ testedꢀ consistꢀ ofꢀ aꢀ134 glycans.ꢀ

syntheticꢀ linearꢀ oligo(amidoamine)ꢀ scaffoldꢀ withꢀ carbo-

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ꢀ

hydrateꢀsideꢀchainsꢀthatꢀareꢀfullyꢀsulfated,ꢀtherebyꢀmim-

ickingꢀ naturalꢀ polysaccharidesꢀ withꢀ highꢀ degreesꢀ ofꢀ sul-

fationꢀ suchꢀ asꢀ heparin.ꢀ Weꢀ areꢀ specificallyꢀ interestedꢀ inꢀ

theꢀ roleꢀ ofꢀ theꢀ chainꢀ lengthꢀ orꢀ so-calledꢀ degreeꢀ ofꢀ

polymerizationꢀ (dp),ꢀ asꢀ itꢀ hasꢀ beenꢀ shownꢀ forꢀ naturalꢀ

136 Inꢀ thisꢀ work,ꢀ sulfatedꢀ glycomimeticꢀ oligomersꢀ andꢀ poly-

137 mersꢀwereꢀtestedꢀforꢀtheirꢀantiviralꢀactivityꢀagainstꢀHPV16ꢀ

138 toꢀgainꢀinsightsꢀintoꢀtheꢀpotentialꢀdifferencesꢀinꢀmodeꢀofꢀ

139 actionꢀdependingꢀonꢀtheirꢀchainꢀlengthꢀ(Schemeꢀ1).ꢀAddi-

140 tionally,ꢀtoꢀtestꢀforꢀtheirꢀpotentialꢀasꢀviralꢀinhibitors,ꢀfirstꢀ

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GAGs ꢀ andꢀ GAGꢀ mimeticsꢀ usedꢀ asꢀ anticoagulants ꢀ thatꢀ

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41 inꢀvivoꢀstudiesꢀwereꢀperformedꢀandꢀbroad-spectrumꢀactiv-

42 ityꢀ wasꢀ testedꢀ forꢀ otherꢀ virusesꢀ suchꢀ asꢀ Herpesꢀ Simplexꢀ

theꢀ chainꢀ lengthꢀ stronglyꢀ affectsꢀ bioactivity.ꢀ Longꢀ chainꢀ

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glycopolymersꢀ areꢀ generatedꢀ viaꢀ controlledꢀ radicalꢀ

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43 Virusꢀ (HSV),ꢀ Influenzaꢀ Aꢀ Virusꢀ (IAV)ꢀ andꢀ Merkelꢀ Cellꢀ187 Cu-mediatedꢀ azido-alkyneꢀ clickꢀ reactionꢀ (Schemeꢀ 2).ꢀ Toꢀ

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144 Polyomavirusꢀ(MCPyV).ꢀ

188 moreꢀ closelyꢀ resembleꢀ theꢀ naturalꢀ ligandꢀ ofꢀ HPV16ꢀ inꢀ

189 cells,ꢀGlcNAcꢀoneꢀofꢀtheꢀmonomersꢀformingꢀHS,ꢀwasꢀusedꢀ

190 asꢀcarbohydrateꢀresidue.ꢀHere,ꢀforꢀtheꢀfirstꢀtime,ꢀglycooli-

191 gomersꢀcarryingꢀ2ꢀ(O1-OH),ꢀ6ꢀ(O2-OH),ꢀ8ꢀ(O3-OH)ꢀorꢀ10ꢀ

192 (O4-OH)ꢀGlcNAcꢀsideꢀchainsꢀwereꢀsynthesizedꢀbyꢀSPPoSꢀ

193 (seeꢀSIꢀforꢀdetailedꢀdescriptionꢀofꢀsynthesisꢀandꢀanalyticalꢀ

194 data)ꢀ(Tableꢀ2).ꢀꢀꢀ

1

45

ꢀ

46 RESULTSꢀ

47 Synthesisꢀofꢀsulfatedꢀglycooligomersꢀandꢀglycopolymersꢀ

148

ꢀ

1

49 Glycopolymersꢀ wereꢀ designedꢀ toꢀ resembleꢀ twoꢀ majorꢀ

50 featuresꢀofꢀheparinꢀandꢀcarrageenanꢀ-ꢀaꢀpolymericꢀdisplayꢀ195

51 ofꢀ saccharidesꢀ andꢀ aꢀ highꢀ degreeꢀ ofꢀ negativeꢀ chargeꢀ

ꢀ

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152 throughꢀsulfation.ꢀGlycomonomersꢀwereꢀfirstꢀsynthesizedꢀ

153 byꢀattachingꢀaꢀmethacrylamideꢀunitꢀtoꢀtheꢀanomericꢀposi-

3

6

154 tionꢀofꢀtheꢀmonosaccharideꢀbasedꢀonꢀworkꢀbyꢀYanꢀetꢀal. ꢀ

37

155 andꢀWuꢀetꢀal. .ꢀTheꢀcorrespondingꢀmonomersꢀwereꢀthenꢀ

156 polymerizedꢀ usingꢀ controlledꢀ reverseꢀ addition-

157 fragmentationꢀ chain-transferꢀ (RAFT)ꢀ polymerizationꢀ

158 usingꢀ aꢀ cyano-2-propylꢀ dodecylꢀ trithiocarbonatꢀ chainꢀ

38

159 transferꢀagentꢀasꢀdescribedꢀbyꢀToyoshimaꢀetꢀal. ꢀ(seeꢀSIꢀ

1

60 forꢀ detailedꢀ descriptionꢀ ofꢀ synthesis)ꢀ (Schemeꢀ 2).ꢀ Twoꢀ

61 differentꢀglycopolymersꢀcarryingꢀeitherꢀGalactoseꢀ(Gal)ꢀorꢀ

62 Mannoseꢀ (Man)ꢀ sideꢀ chainsꢀ wereꢀ generated.ꢀ Galꢀ isꢀ theꢀ

63 constitutingꢀ monomerꢀ ofꢀ carrageenan,ꢀ whileꢀ Manꢀ isꢀ aꢀ

64 commonlyꢀusedꢀmonosaccharideꢀinꢀglycopolymerꢀsynthe-

65 sis;ꢀtheꢀlatterꢀwasꢀusedꢀhereꢀasꢀaꢀcontrolꢀforꢀcomparisonꢀ

66 withꢀ Gal.ꢀ Forꢀ theꢀ Gal-presentingꢀ polymer,ꢀ twoꢀ batchesꢀ

67 wereꢀused:ꢀPG1-OHꢀwithꢀanꢀaverageꢀdpꢀofꢀ40ꢀandꢀ PG2-

68 OHꢀwithꢀdpꢀ46,ꢀshowingꢀtheꢀgoodꢀreproducibilityꢀofꢀsyn-

69 theticꢀ proceduresꢀ (seeꢀ SI).ꢀ Theꢀ Man-presentingꢀ polymerꢀ

70 (PM-OH)ꢀwasꢀisolatedꢀwithꢀanꢀaverageꢀdpꢀofꢀ86ꢀ(seeꢀSI).ꢀ

71 Asꢀ negativeꢀ control,ꢀ PG-OHꢀ withꢀ anꢀ averageꢀ dpꢀ ofꢀ 84,ꢀ

72 wasꢀusedꢀwithoutꢀfurtherꢀsulfationꢀ(seeꢀSI).ꢀꢀ

1

73

ꢀ

174 Glycooligomersꢀwereꢀsynthesizedꢀbyꢀapplyingꢀsolidꢀphaseꢀ

39–41

1

75 polymerꢀ synthesisꢀ (SPPoS).

ꢀ Inꢀ contrastꢀ toꢀ glycopoly-

76 merꢀ synthesis,ꢀ thisꢀ allowsꢀ forꢀ absoluteꢀ controlꢀ overꢀ theꢀ

77 chainꢀlengthꢀandꢀtherebyꢀtheꢀnumberꢀandꢀpositioningꢀofꢀ

78 sugarsꢀ withinꢀ theꢀ glycooligomer.ꢀ Inꢀ short,ꢀ tailor-madeꢀ

79 buildingꢀblocksꢀcarryingꢀaꢀfreeꢀcarboxylicꢀacidꢀgroupꢀandꢀ

80 Fmoc-protectedꢀamineꢀgroupꢀwereꢀcoupledꢀinꢀaꢀstepwiseꢀ

81 fashionꢀonꢀsolidꢀsupportꢀfollowingꢀstandardꢀFmoc-peptideꢀ

82 couplingꢀ protocols.ꢀ Whenꢀ combiningꢀ buildingꢀ blocksꢀ199

83 withꢀalkyneꢀsideꢀchainꢀandꢀhydrophilicꢀmainꢀchainꢀmotifs,ꢀ

84 weꢀ couldꢀ assembleꢀ monodisperse,ꢀ sequence-definedꢀ oli-

85 go(amidoamine)ꢀ scaffoldsꢀ thatꢀ allowedꢀ forꢀ site-selectiveꢀ

86 introductionꢀ ofꢀ azidoꢀ functionalizedꢀ carbohydratesꢀ viaꢀ

1

96

ꢀ

1

97 Schemeꢀ 2.ꢀ Generalꢀ synthesisꢀ ofꢀ sulfatedꢀ glycooligomersꢀ

98 (A)ꢀandꢀglycopolymersꢀ(B).ꢀꢀ

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Tableꢀ1.ꢀStructuresꢀofꢀsulfatedꢀglycopolymersꢀ

Page 4 of 26

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01

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h

#

ꢀ

N(sugars)ꢀ

Degreeꢀofꢀꢀsulfationꢀ[%]ꢀ Dispersity ꢀ Structureꢀ

ꢀ

a

c

PG1ꢀ

40 ꢀ

n.d. ꢀ

2.0ꢀ

ꢀ

ꢀa

46 ꢀ

c,d

PG2ꢀ

PMꢀ

97.6

ꢀ

1.08ꢀ

1.25ꢀ

seeꢀPG1ꢀ

0

1

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9

0

1

ꢀ

a

c

86 ꢀ

n.d. ꢀ

ꢀ

b

e

f

Heparinꢀ

30-34 ꢀ

85% /43% ꢀ

n.a.ꢀ

ꢀ

g

Carageenanꢀ

n.a.ꢀ

25-34 ꢀ

ꢀ

a

2 202

3 203

4 204

5 205

6 206

7 207

8 208

AverageꢀnumberꢀcalculatedꢀfromꢀtheꢀM

ꢀasꢀdeterminedꢀbyꢀaqueousꢀGelꢀPermeationꢀChromatographyꢀ(GPC)ꢀforꢀallꢀpoly-

merꢀsamplesꢀ(seeꢀSI).ꢀ Ofꢀdisaccharideꢀrepeatingꢀunitsꢀasꢀshownꢀinꢀtheꢀtable,ꢀcalculatedꢀfromꢀanꢀaverageꢀMWꢀofꢀ17-19ꢀkDaꢀ

asꢀ providedꢀ byꢀ theꢀ supplier.ꢀ Accordingꢀ toꢀ NMRꢀ analysis,ꢀ quantitativeꢀ sulfationꢀ wasꢀ achievedꢀ forꢀ glycopolymers.ꢀ Forꢀ

sampleꢀPG2,ꢀadditionalꢀanalysisꢀviaꢀelementalꢀanalysisꢀconfirmedꢀhighꢀdegreeꢀofꢀsulfationꢀ(seeꢀSI).ꢀ Correspondsꢀtoꢀ2,4-2,6ꢀ

sulfationsꢀperꢀdisaccharide ,ꢀoutꢀofꢀtheꢀ3ꢀsulfationsꢀpresentꢀinꢀnaturalꢀheparinꢀ(seeꢀstructure).ꢀ Asꢀcalculatedꢀforꢀglycopol-

ymers,ꢀcountingꢀallꢀresiduesꢀsusceptibleꢀforꢀsulfationꢀ Literatureꢀvaluesꢀforꢀfoodꢀgradeꢀcarrageenanꢀ(mainlyꢀiotaꢀtype). ꢀ

OfꢀtheꢀunsulfatedꢀprecursorꢀpolymerꢀasꢀdeterminedꢀbyꢀaqueousꢀGPCꢀ(seeꢀSI).ꢀn.d.ꢀ=ꢀnotꢀdetermined,ꢀn.a.ꢀ=ꢀinformationꢀ

n

b

c

d

e

4

2

f

g

43

h

2

09

notꢀavailable.ꢀ

9

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210 Tableꢀ2.ꢀStructuresꢀofꢀsulfatedꢀglycooligomersꢀ

212

ꢀ

Degreeꢀ

ofꢀ

213 ꢀBoth,ꢀglycopolymersꢀandꢀglycooligomers,ꢀwereꢀthenꢀsul-

214 fatedꢀ usingꢀ sulfurtrioxideꢀ trimethylamineꢀ complexꢀ

15 (TMA*SO )ꢀ (Schemeꢀ 2).ꢀ Theꢀ degreeꢀ ofꢀ sulfationꢀ wasꢀ de-

3

16 terminedꢀviaꢀ H-NMRꢀandꢀadditionallyꢀbyꢀelementalꢀanal-

17 ysisꢀ forꢀ PG2,ꢀ showingꢀ nearlyꢀ fullꢀ sulfationꢀ forꢀ allꢀ struc-

218 tures,ꢀ sulfatedꢀ glycooligomersꢀ (O1-O4)ꢀ andꢀ glycopoly-

219 mersꢀ(PG1,ꢀPG2,ꢀPM)ꢀ(seeꢀSIꢀforꢀanalyticalꢀdata)ꢀ(Tableꢀ1ꢀ

220 andꢀ2).ꢀ

#

ꢀ

N(sugars)ꢀ

a

Structureꢀ

sulfationꢀ[%] ꢀ

2

1

O1ꢀ

2ꢀ

95-98.5ꢀ

2

21

ꢀ

22 SulfatedꢀglycopolymersꢀareꢀstrongꢀantagonistsꢀofꢀHPV16ꢀ

ꢀ

2

23

ꢀ

2

24 Naturalꢀ sulfatedꢀ polysaccharidesꢀ suchꢀ asꢀ heparinꢀ andꢀ

25 carrageenanꢀareꢀgoodꢀinhibitorsꢀofꢀHPV16.

O2ꢀ 6ꢀ

98.7ꢀ

0ꢀ

7,21,44

ꢀHowever,ꢀ

26 theseꢀnaturallyꢀderivedꢀpolysaccharidesꢀexhibitꢀdispersityꢀ

27 withꢀ respectꢀ toꢀ theirꢀ carbohydrateꢀ content,ꢀ length,ꢀ andꢀ

28 degreeꢀ ofꢀ sulfation.ꢀ Forꢀ example,ꢀ heparinꢀ canꢀ occurꢀ inꢀ

29 bothꢀhighꢀandꢀlowꢀmolecularꢀweightꢀformsꢀwithꢀbetweenꢀ

30 40-50%ꢀsulfation,ꢀwhileꢀcarrageenanꢀexhibitsꢀbetweenꢀ25-

31 35%ꢀsulfationꢀ(Tableꢀ1).ꢀThisꢀcanꢀmakeꢀitꢀdifficultꢀtoꢀde-

32 termineꢀpreciseꢀstructure/functionꢀrelationships.ꢀꢀInꢀaddi-

33 tion,ꢀ biologicalꢀ sourcesꢀ ofꢀ heparinꢀ canꢀ causeꢀ unwantedꢀ

34 sideꢀeffectsꢀwithinꢀtheꢀhostꢀe.g.ꢀinterfereꢀwithꢀnormalꢀHSꢀ

35 mediatedꢀ cellꢀ processes.

236 whetherꢀ syntheticꢀ glycomimeticꢀ compoundsꢀ mayꢀ beꢀ al-

237 ternativesꢀtoꢀnaturalꢀcompounds.ꢀ

O2-

6

ꢀ

OHꢀ

ꢀ

O3ꢀ 8ꢀ

89.9ꢀ

85.2ꢀ

45–47

ꢀ Here,ꢀ weꢀ aimedꢀ toꢀ analyzeꢀ

O4ꢀ 10ꢀ

ꢀ

a

1

2

11 Degreeꢀofꢀsulfationꢀasꢀdeterminedꢀbyꢀ H-NMRꢀ(seeꢀSI).ꢀ 238

ꢀ

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2

39 Toꢀcompareꢀtheꢀantiviralꢀefficacyꢀofꢀtheꢀsynthesizedꢀgly- 265 toꢀheparinꢀandꢀcarrageenanꢀ(Fig.ꢀ1A).ꢀPG1ꢀandꢀPG2ꢀpro-

40 comimeticꢀ compounds,ꢀ glycopolymersꢀ wereꢀ mixedꢀ inꢀ266 videdꢀ highlyꢀ similarꢀ resultsꢀ (dataꢀ notꢀ shown),ꢀ showingꢀ

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241 differentꢀconcentrationsꢀwithꢀHPV16ꢀcapsidsꢀcontainingꢀaꢀ267 excellentꢀ reproducibilityꢀ fromꢀ batchꢀ toꢀ batch.ꢀ However,ꢀ

242 reporterꢀ plasmidꢀ encodingꢀ forꢀ eGFPꢀ (pseudovirusesꢀ orꢀ268 noꢀ significantꢀ differenceꢀ wasꢀ observedꢀ forꢀ theꢀ inhibitoryꢀ

4

8

243 PsV) ꢀ priorꢀ toꢀ infection.ꢀ Despiteꢀ HPV16ꢀ beingꢀ aꢀ BSL2ꢀ269 effectsꢀofꢀPMꢀandꢀPG1.ꢀThisꢀconfirmedꢀthatꢀaꢀdpꢀofꢀ40ꢀisꢀ

244 pathogen,ꢀtheꢀuseꢀofꢀPsVꢀallowsꢀworkꢀunderꢀBSL1ꢀcondi- 270 sufficientꢀtoꢀyieldꢀmaximalꢀblockingꢀefficacy.ꢀItꢀmayꢀalsoꢀ

245 tionsꢀdueꢀtoꢀtheꢀincorporationꢀofꢀaꢀpseudogenomeꢀinsteadꢀ271 provideꢀ aꢀ hintꢀ thatꢀ theꢀ identityꢀ ofꢀ theꢀ sugar,ꢀ changingꢀ

246 ofꢀtheꢀviralꢀgenome.ꢀAfterꢀbindingꢀtoꢀHeLaꢀcells,ꢀtheꢀinoc- 272 fromꢀgalactoseꢀ(PG)ꢀtoꢀmannoseꢀ(PM),ꢀmayꢀnotꢀdramati-

247 ulumꢀwasꢀremoved,ꢀandꢀtheꢀinfectionꢀwasꢀscoredꢀ48hꢀpostꢀ273 callyꢀaffectꢀtheꢀinhibitionꢀpotential.ꢀAsꢀmentionedꢀabove,ꢀ

248 infectionꢀ (p.i.).ꢀ Heparinꢀ andꢀ carrageenanꢀ wereꢀ usedꢀ asꢀ274 weꢀchoseꢀonlyꢀoneꢀtypeꢀofꢀunsulfatedꢀcontrolꢀpolymerꢀ(i.e.ꢀ

2

49 positiveꢀ controlsꢀ toꢀ evaluateꢀ glycopolymerꢀ binding.ꢀ Asꢀ275 PG-OH),ꢀsinceꢀitꢀisꢀalreadyꢀwellꢀestablishedꢀthatꢀbindingꢀ

50 expected,ꢀ HPV16ꢀ infectionꢀ wasꢀ blockedꢀ byꢀ heparinꢀ andꢀ276 ofꢀ glycansꢀ toꢀ HPV16ꢀ requiresꢀ aꢀ negativeꢀ chargeꢀ broughtꢀ

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21,26

51 carrageenanꢀinꢀaꢀdose-dependentꢀfashionꢀ(Fig.ꢀ1A,ꢀ ).ꢀBothꢀ277 aboutꢀbyꢀsulfation. ꢀSimilarly,ꢀmannose-basedꢀglycopol-

4

9

52 heparinꢀandꢀcarrageenanꢀabrogatedꢀHPV16ꢀinfectionꢀatꢀaꢀ278 ymersꢀ doꢀ notꢀ inhibitꢀ HPV16ꢀ infection. ꢀ Therefore,ꢀ weꢀ

53 concentrationꢀ ofꢀ 0.01mg/ml,ꢀ althoughꢀ carrageenanꢀ wasꢀ279 omittedꢀ theꢀ additionꢀ ofꢀ PM-OHꢀ asꢀ control.ꢀ Inꢀ lineꢀ withꢀ

54 moreꢀefficientꢀthanꢀheparinꢀatꢀlowerꢀconcentrations.ꢀTheꢀ280 theꢀ previousꢀ data,ꢀ PG-OHꢀ didꢀ notꢀ significantlyꢀ inhibitꢀ

55 half-maximalꢀ inhibitoryꢀ concentrationꢀ (IC50)ꢀ forꢀ heparinꢀ281 HPV16ꢀinfectionꢀ(Fig.ꢀ1A).ꢀTheꢀIC50ꢀofꢀPMꢀandꢀPG1ꢀwereꢀ

56 andꢀ carrageenanꢀ wereꢀ 3·10-3ꢀ mg/mlꢀ andꢀ 4·10-5ꢀ mg/ml,ꢀ282 7·10-5ꢀmg/mlꢀandꢀ2·10-4ꢀmg/ml,ꢀrespectivelyꢀ(Fig.ꢀ2C,ꢀD).ꢀ

57 respectivelyꢀ(Fig.ꢀ2A,ꢀB).ꢀ

283 Thus,ꢀtheꢀsulfatedꢀglycopolymersꢀinhibitedꢀHPV16ꢀinfec-

2

84 tionꢀ asꢀ efficientlyꢀ asꢀ carrageenanꢀ andꢀ 10-100ꢀ foldꢀ betterꢀ

85 thanꢀ heparin,ꢀ indicatingꢀ thatꢀ sulfatedꢀ glycopolymersꢀ areꢀ

86 effectiveꢀ inhibitorsꢀ ofꢀ HPV16ꢀ infection.ꢀ Notably,ꢀ incuba-

87 tionꢀ withꢀ theꢀ naturalꢀ glycansꢀ orꢀ theꢀ glycopolymersꢀ dis-

88 playedꢀ noꢀ cytotoxicꢀ effectsꢀ inꢀ cellꢀ cultureꢀ inꢀ theꢀ highestꢀ

89 concentrationsꢀusedꢀ(Fig.ꢀ1B).ꢀ

2

58

ꢀ

2

259 ꢀNext,ꢀ theꢀ glycopolymersꢀ PMꢀ andꢀ PG1ꢀ wereꢀ testedꢀ forꢀ

260 theirꢀinhibitoryꢀeffectꢀonꢀHPV16ꢀinfection.ꢀBasedꢀonꢀourꢀ

261 unpublishedꢀ workꢀ onꢀ theꢀ lengthꢀ ofꢀ naturalꢀ glycans,ꢀ weꢀ

262 expectedꢀthatꢀglycopolymersꢀ≥dp40ꢀwouldꢀdisplayꢀsimilarꢀ

263 efficaciesꢀinꢀengagingꢀtheꢀvirusꢀandꢀpreventingꢀinfection.ꢀ

2

90

ꢀ

64 Indeed,ꢀbothꢀabrogatedꢀinfectionꢀatꢀ0.01mg/ml,ꢀsimilarlyꢀ

2

91

ꢀ

3 292

4 293

5 294

6 295

7 296

8 297

9 298

0 299

ꢀ

Figureꢀ1.ꢀNaturalꢀpolysaccharidesꢀandꢀsulfatedꢀglycopolymersꢀblockꢀinfectionꢀofꢀHPV16.ꢀ(A)ꢀHPV16ꢀwasꢀpreincubatedꢀwithꢀ

theꢀindicatedꢀamountsꢀofꢀheparin,carrageenan,ꢀglycopolymersꢀPMꢀandꢀPG1ꢀandꢀtheꢀunsulfatedꢀcontrolꢀPG-OHꢀforꢀ1hꢀatꢀ

RT.ꢀHeLaꢀcellsꢀwereꢀinfectedꢀandꢀinfectionꢀwasꢀscoredꢀ48hꢀp.i.ꢀbyꢀmicroscopyꢀ(GFPꢀsignal).ꢀResultsꢀareꢀshownꢀinꢀ%ꢀrelativeꢀ

toꢀmock-incubatedꢀHPV16.ꢀAllꢀinfectionꢀvaluesꢀareꢀtheꢀmeanꢀ±ꢀSDꢀofꢀatꢀleastꢀ3ꢀindependentꢀexperiments.ꢀ(B)ꢀRelativeꢀcellꢀ

countꢀofꢀcellsꢀtreatedꢀasꢀinꢀA).ꢀTheꢀrelativeꢀcellꢀnumbersꢀafterꢀinfectionꢀandꢀtreatmentꢀwithꢀglycopolymersꢀorꢀnaturalꢀpoly-

saccharidesꢀareꢀshownꢀrelativeꢀ(inꢀ%)ꢀtoꢀtheꢀuntreatedꢀmockꢀ(uninfected)ꢀconditionꢀofꢀeachꢀexperimentꢀandꢀindicateꢀtheꢀ

absenceꢀofꢀtoxicityꢀbyꢀallꢀtreatmentsꢀatꢀ1mg/ml.ꢀ

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13 HPV16ꢀinfectionꢀwasꢀreducedꢀbyꢀsulfatedꢀglycooligomersꢀ

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314 inꢀvitroꢀ

3

15 Ourꢀ previousꢀ resultsꢀ indicatedꢀ thatꢀ theꢀ antiviralꢀ activityꢀ

16 cannotꢀbeꢀfurtherꢀincreasedꢀaboveꢀaꢀcriticalꢀdpꢀofꢀglyco-

17 polymersꢀandꢀweꢀdidꢀnotꢀseeꢀaꢀnoticeableꢀdependenceꢀonꢀ

18 theꢀtypeꢀofꢀsugarꢀpresentedꢀinꢀtheꢀsideꢀchains.ꢀTherefore,ꢀ

19 weꢀnextꢀlookedꢀatꢀinhibitionꢀemployingꢀshorterꢀglycooli-

20 gomersꢀ ofꢀ definedꢀ chainꢀ lengthꢀ goingꢀ fromꢀ 2ꢀ upꢀ toꢀ 10ꢀ

21 sugars.ꢀAdditionally,ꢀweꢀdecidedꢀtoꢀnowꢀincludeꢀGlcNAcꢀ

322 sideꢀchainsꢀinꢀorderꢀtoꢀmoreꢀcloselyꢀmimicꢀtheꢀnaturalꢀꢀHSꢀ

323 fragments.

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ꢀ

26 Figureꢀ3.ꢀAntiviralꢀactivityꢀbyꢀsulfatedꢀglycooligomersꢀinꢀ

27 HPV16ꢀ infection.ꢀ (A)ꢀ HPV16ꢀ infectionꢀ assayꢀ wasꢀ per-

3

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01 Figureꢀ2.ꢀInhibitoryꢀefficiencyꢀofꢀglycopolymersꢀPMꢀandꢀ328 formedꢀasꢀdescribedꢀinꢀFig.ꢀ1ꢀwithꢀglycooligomersꢀwithꢀ2,ꢀ

02 PG1ꢀandꢀnaturalꢀpolysaccharides.ꢀHPV16ꢀPsVsꢀwereꢀprein- 329 6,ꢀ8ꢀorꢀ10ꢀGlcNAcꢀresiduesꢀ(O1-O4,ꢀrespectively)ꢀandꢀun-

03 cubatedꢀwithꢀtheꢀindicatedꢀcompoundsꢀforꢀ1hꢀatꢀincreas- 330 sulfatedꢀglycooligomerꢀO2-OH.ꢀResultsꢀareꢀshownꢀrelativeꢀ

04 ingꢀ logꢀ concentrations.ꢀ Theꢀ inoculumsꢀ wereꢀ addedꢀ toꢀ331 (inꢀ%)ꢀtoꢀtheꢀmock-infectedꢀsamples.ꢀAllꢀinfectionꢀvaluesꢀ

05 HeLaꢀcellsꢀforꢀ2hꢀandꢀinfectionꢀwasꢀscoredꢀ48hꢀp.i.ꢀInhibi- 332 areꢀtheꢀmeanꢀ±ꢀSDꢀofꢀatꢀleastꢀ3ꢀindependentꢀexperiments.ꢀ

306 tionꢀ curvesꢀ andꢀ IC50ꢀ valuesꢀ forꢀ carrageenanꢀ (A),ꢀ heparinꢀ333 (B)ꢀRelativeꢀcellꢀcountꢀofꢀcellsꢀtreatedꢀasꢀinꢀA).ꢀTheꢀrela-

307 (B),ꢀPG1ꢀ(C)ꢀandꢀPMꢀ(D)ꢀwereꢀcalculatedꢀusingꢀGraphPad.ꢀ334 tiveꢀcellꢀnumbersꢀafterꢀinfectionꢀandꢀtreatmentꢀwithꢀgly-

308 Resultsꢀareꢀshownꢀrelativeꢀ(inꢀ%)ꢀtoꢀmock-infectedꢀsam- 335 cooligomersꢀorꢀnaturalꢀpolysaccharidesꢀareꢀshownꢀrelativeꢀ

309 ples.ꢀAllꢀinfectionꢀvaluesꢀareꢀmeanꢀ±ꢀSDꢀofꢀatꢀleastꢀ3ꢀinde- 336 (inꢀ %)ꢀ toꢀ theꢀ untreatedꢀ mockꢀ (uninfected)ꢀ conditionꢀ ofꢀ

310 pendentꢀexperiments.ꢀ

337 eachꢀ experimentꢀ andꢀ indicateꢀ theꢀ absenceꢀ ofꢀ toxicityꢀ byꢀ

338 allꢀtreatmentsꢀatꢀ1mg/ml.ꢀ

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387 ducedꢀbyꢀ90%ꢀatꢀtheꢀsameꢀconcentrationꢀ(Fig.ꢀ4;ꢀcompareꢀ

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88 Fig.ꢀ 3).ꢀ Whileꢀ theꢀ shorterꢀ O1ꢀ andꢀ O2ꢀ didꢀ notꢀ affectꢀ theꢀ

389 distributionꢀofꢀvirusꢀsignal,ꢀtheꢀlongerꢀO3ꢀandꢀO4ꢀledꢀtoꢀ

390 virusꢀclusteringꢀ(Fig.ꢀ4;ꢀbrighter,ꢀbiggerꢀdots).ꢀNoneꢀofꢀtheꢀ

391 unsulfatedꢀ controlsꢀ (PG-OH,ꢀ O2-OH)ꢀ significantlyꢀ af-

392 fectedꢀ HPV16ꢀ bindingꢀ toꢀ cellsꢀ (Fig.ꢀ 4).ꢀ Inꢀ conclusion,ꢀ

393 whileꢀ glycopolymersꢀ blockedꢀ bindingꢀ asꢀ expected,ꢀ gly-

394 cooligomersꢀallowedꢀbindingꢀbutꢀnotꢀinfection,ꢀindicatingꢀ

395 thatꢀinfectionꢀoccuredꢀatꢀaꢀpost-bindingꢀstep,ꢀthusꢀrepre-

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41 Sinceꢀ theseꢀ glycooligomersꢀ areꢀ shortꢀ inꢀ comparisonꢀ toꢀ

42 GAGs,ꢀweꢀexpectedꢀthatꢀtheyꢀwouldꢀexhibitꢀlowerꢀinhibi-

43 toryꢀ potentialꢀ thanꢀ theꢀ glycopolymers.ꢀ HPV16ꢀ infectionꢀ

44 wasꢀalsoꢀblockedꢀbyꢀglycooligomersꢀO1-O4,ꢀalbeitꢀatꢀrela-

45 tivelyꢀhighꢀconcentrationsꢀ(Fig.ꢀ3A).ꢀWeꢀobservedꢀaꢀreduc-

46 tionꢀ ofꢀ 50%ꢀ infectionꢀ atꢀ aꢀ concentrationꢀ ofꢀ 0.1ꢀ mg/mlꢀ

47 similarꢀ forꢀ allꢀ glycooligomers.ꢀ Increasingꢀ theꢀ concentra-

48 tionꢀofꢀglycooligomersꢀfurtherꢀreducedꢀinfectionꢀbyꢀaboutꢀ

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96 sentingꢀaꢀnovelꢀmodeꢀofꢀinterferenceꢀwithꢀviralꢀinfection.ꢀꢀ

349 90%ꢀforꢀO1ꢀandꢀO2,ꢀbutꢀsurprisinglyꢀwasꢀlessꢀefficientꢀforꢀ

350 theꢀ longerꢀ glycooligomers,ꢀ O3ꢀ andꢀ O4.ꢀ Thus,ꢀ atꢀ higherꢀ397

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351 concentrationsꢀ theꢀ inhibitoryꢀ effectsꢀ ofꢀ glycooligomersꢀ

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98 GlycopolymersꢀdisplayꢀantiviralꢀactivityꢀagainstꢀHPV16ꢀinꢀ

99 vivoꢀ

352 didꢀnotꢀcorrelateꢀwithꢀtheirꢀlength.ꢀThisꢀwasꢀunexpected,ꢀ

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353 asꢀlongerꢀsulfatedꢀpolysaccharidesꢀdisplayꢀaꢀhigherꢀnum-

354 berꢀ ofꢀ bindingꢀ moieties,ꢀ whichꢀ weꢀ reasonedꢀ wouldꢀ in-

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01 Asꢀ theꢀ inꢀ vitroꢀ experimentsꢀ showedꢀ promisingꢀ antiviralꢀ

02 activityꢀ forꢀ bothꢀ glycopolymersꢀ andꢀ glycooligomers,ꢀ weꢀ

03 testedꢀwhetherꢀtheyꢀwouldꢀalsoꢀbeꢀeffectiveꢀinꢀanꢀinꢀvivoꢀ

04 infectionꢀ scenario.ꢀ Forꢀ this,ꢀ theꢀ HPV16ꢀ mouseꢀ vaginalꢀ

355 creaseꢀtheꢀaffinity,ꢀavidityꢀandꢀthereforeꢀtheꢀprobabilityꢀofꢀ

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356 bindingꢀ toꢀ theꢀ virus,ꢀ thusꢀ inhibitingꢀ infection.ꢀ Thisꢀ wasꢀ

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57 notꢀdueꢀtoꢀcytotoxicꢀeffects,ꢀsince,ꢀsimilarꢀtoꢀtheꢀglycopol-

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58 ymers,ꢀ theꢀ glycooligomersꢀ didꢀ notꢀ exhibitꢀ anyꢀ reducedꢀ

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05 challengeꢀ modelꢀ wasꢀ used. ꢀ Glycomymeticsꢀ wereꢀ incu-

06 batedꢀ withꢀ HPV16-luciferaseꢀ asꢀ forꢀ inꢀ vitroꢀ experimentsꢀ

07 andꢀinoculumsꢀwereꢀappliedꢀinꢀtheꢀmiceꢀvaginaꢀafterꢀme-

08 chanicalꢀ disruptionꢀ ofꢀ theꢀ epithelia.ꢀ Usingꢀ non-invasiveꢀ

09 bioluminescenceꢀimaging,ꢀinfectionꢀwasꢀscoredꢀbyꢀsignalꢀ

10 intensityꢀafterꢀapplyingꢀluciferin,ꢀtheꢀsubstrateꢀofꢀlucifer-

11 ase.ꢀ

59 cellꢀ numbersꢀ evenꢀ uponꢀ highꢀ concentrationsꢀ (Fig.ꢀ 3B).ꢀ

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60 Potentially,ꢀ aggregationꢀ behaviorꢀ ofꢀ sulfatedꢀ glycooligo-

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61 mersꢀ increasesꢀ withꢀ increasingꢀ chainꢀ length,ꢀ butꢀ furtherꢀ

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62 studiesꢀ areꢀ requiredꢀ toꢀ investigateꢀ thisꢀ inꢀ depth.ꢀ Never-

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63 theless,ꢀthisꢀfirstꢀgenerationꢀofꢀglycooligomersꢀwereꢀableꢀ

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64 toꢀ interfereꢀ withꢀ HPV16ꢀ infection,ꢀ suggestingꢀ thatꢀ theyꢀ

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65 mayꢀbeꢀfeasibleꢀantiviralꢀcandidates.ꢀꢀꢀ

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13 Allꢀmiceꢀinꢀtheꢀuntreatedꢀcontrolꢀgroupꢀexhibitedꢀlucifer-

14 aseꢀ activity,ꢀ indicatingꢀ successfulꢀ infectionꢀ (2.0x105ꢀ ±ꢀ

15 2.8x105ꢀphotonsꢀperꢀsecond,ꢀp/s).ꢀAsꢀexpected,ꢀmiceꢀinoc-

16 ulatedꢀ withꢀ virusꢀ thatꢀ wereꢀ preincubatedꢀ withꢀ carragee-

17 nanꢀ orꢀ heparinꢀ wereꢀ notꢀ infectedꢀ (Fig.ꢀ 5;ꢀ carrageenan:ꢀ

18 1.5x104ꢀ±ꢀ3.0x103ꢀp/s,ꢀheparin:ꢀ2.1x104ꢀ±ꢀ6.4x103ꢀp/s;ꢀpho-

19 tonꢀemissionꢀcomparableꢀtoꢀuninfectedꢀcontrols:ꢀ1.8x10⁴ꢀ±ꢀ

20 3.3x10³).ꢀ Theꢀ glycopolymerꢀ PG2,ꢀ usedꢀ asꢀ prototypicalꢀ

21 glycopolymer,ꢀ alsoꢀ completelyꢀ blockedꢀ infectionꢀ inꢀ allꢀ

22 miceꢀ(1.8x104ꢀ±ꢀ4.5x103ꢀp/s),ꢀsupportingꢀtheꢀinꢀvitroꢀresultsꢀ

23 andꢀfurtherꢀdemonstratingꢀtheꢀefficacyꢀofꢀthisꢀcompoundꢀ

24 asꢀanꢀinhibitorꢀofꢀHPV16ꢀinfection.ꢀHowever,ꢀO1ꢀandꢀO4ꢀ

25 showedꢀ littleꢀ orꢀ noꢀ antiviralꢀ effect,ꢀ respectively,ꢀ inꢀ lineꢀ

26 withꢀtheirꢀreducedꢀantiviralꢀactivityꢀinꢀvitroꢀ(SB1:ꢀ2.1x105ꢀ±ꢀ

27 3.8x105ꢀp/s,ꢀSB4:ꢀ1.5x105ꢀ±ꢀ9.8x104ꢀp/s).ꢀInꢀconclusion,ꢀtheꢀ

28 glycopolymerꢀ testedꢀ blockedꢀ HPV16ꢀ infectionꢀ efficientlyꢀ

29 inꢀvitroꢀandꢀinꢀvivo,ꢀwhichꢀisꢀremarkable.ꢀToꢀtheꢀbestꢀofꢀ

30 ourꢀ knowledge,ꢀ thisꢀ isꢀ theꢀ firstꢀ studyꢀ demonstratingꢀ inꢀ

31 vivoꢀefficacyꢀofꢀsulfatedꢀglycopolymersꢀasꢀantivirals.ꢀ

367 Twoꢀdistinctꢀmechanismsꢀmediateꢀinhibitionꢀofꢀinfectionꢀ

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368 byꢀglycooligomersꢀandꢀglycopolymersꢀ

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ꢀ

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70 Sinceꢀ theꢀ differentꢀ glycomimeticsꢀ displayedꢀ unexpectedꢀ

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71 effectsꢀ inꢀ inhibitingꢀ viralꢀ infection,ꢀ weꢀ aimedꢀ toꢀ deter-

72 mineꢀ howꢀ theyꢀ interferedꢀ withꢀ infection.ꢀ Sinceꢀ weꢀ as-

73 sumedꢀ thatꢀ initialꢀ bindingꢀ wasꢀ blocked,ꢀ weꢀ usedꢀ fluoro-

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74 phore-labelledꢀHPV16ꢀparticlesꢀthatꢀprovidedꢀaꢀvisualꢀandꢀ

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75 quantitativeꢀassessmentꢀofꢀbindingꢀtoꢀcellsꢀwhileꢀretainingꢀ

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76 theirꢀinfectivity. ꢀGlycopolymers,ꢀglycooligomersꢀorꢀnatu-

77 ralꢀ polysaccharidesꢀ wereꢀ incubatedꢀ withꢀ fluorophore-

78 labelledꢀHPV16,ꢀandꢀsubsequentlyꢀaddedꢀtoꢀcellsꢀtoꢀallowꢀ

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379 forꢀ bindingꢀ toꢀ cellꢀ surfaceꢀ HS.ꢀ Microscopicꢀ analysisꢀ

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380 showedꢀthatꢀinꢀuntreatedꢀcells,ꢀboundꢀvirusꢀparticlesꢀareꢀ

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381 visibleꢀasꢀbrightꢀdotsꢀ(Fig.ꢀ4),ꢀwhereasꢀtheꢀnaturalꢀglycans,ꢀ

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382 heparinꢀ andꢀ carrageenan,ꢀ blockedꢀ attachmentꢀ ofꢀ HPV16ꢀ

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383 toꢀ cellsꢀ asꢀ expectedꢀ (Fig.ꢀ 4, ).ꢀ ꢀ Similarly,ꢀ PG1ꢀ andꢀ PMꢀ

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384 dramaticallyꢀreducedꢀvirusꢀbindingꢀtoꢀcellsꢀ(Fig.ꢀ4).ꢀHow-

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385 ever,ꢀtheꢀshortꢀglycooligomersꢀunexpectedlyꢀdidꢀnotꢀaffectꢀ

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ꢀ

386 bindingꢀ toꢀ aꢀ significantꢀ extent,ꢀ evenꢀ ifꢀ infectionꢀ wasꢀ re-

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Figureꢀ 4.ꢀ Differentꢀ mechanismsꢀ ofꢀ inhibitingꢀ HPV16ꢀ infectionꢀ byꢀ sulfatedꢀ glycopolymersꢀ andꢀ glycooligomers.ꢀ Fluoro-

phore-labeledꢀHPV16ꢀ(white)ꢀwasꢀincubatedꢀwithꢀ1mg/mlꢀofꢀtheꢀindicatedꢀcompoundsꢀforꢀ1h.ꢀPreincubatedꢀPsVꢀwereꢀal-

lowedꢀtoꢀbindꢀtoꢀHeLaꢀcellsꢀandꢀfixedꢀafterꢀ2h.ꢀRepresentativeꢀmaximumꢀintensityꢀprojectionsꢀofꢀstackꢀimagesꢀforꢀeachꢀ

conditionꢀareꢀshown.ꢀCellꢀoutlinesꢀ(yellow)ꢀwereꢀdrawnꢀinꢀFijiꢀ(ImageJ)ꢀbasedꢀonꢀphalloidinꢀstaining.ꢀScaleꢀbarsꢀcorre-

spondꢀtoꢀ20µm.ꢀꢀ

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ꢀ

Figureꢀ5.ꢀPG2ꢀeffectivelyꢀinhibitsꢀHPV16-luciferaseꢀinfectionꢀinꢀvivo.ꢀ(A)ꢀBalbCꢀmiceꢀ(5ꢀperꢀgroup),ꢀintravaginallyꢀinocu-

latedꢀwithꢀHPV16-luciferaseꢀpreincubatedꢀwithꢀtheꢀrespectiveꢀcompounds,ꢀwereꢀmeasuredꢀwithꢀanꢀIVISꢀSpectrumꢀtoꢀas-

sessꢀinfectionꢀtwoꢀdaysꢀafterꢀvirusꢀinoculation.ꢀ(B)ꢀTheꢀsignalꢀwasꢀquantifiedꢀasꢀphotonsꢀperꢀsecondꢀ(p/s).ꢀTheꢀdataꢀwasꢀ

analysedꢀwithꢀGraphpadꢀandꢀinfectionꢀlevelꢀofꢀeachꢀmouseꢀisꢀshownꢀasꢀaꢀsingleꢀdataꢀpoint.ꢀTheꢀlineꢀrepresentsꢀmedianꢀofꢀ

theꢀvalues.ꢀ**:ꢀPꢀ<ꢀ0.01ꢀrelativeꢀtoꢀuninhibitedꢀcontrol.ꢀ

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47

ꢀ

458 Toꢀtestꢀthisꢀnotion,ꢀseveralꢀvirusesꢀwereꢀsubjectedꢀtoꢀin-

4

59 fectivityꢀassaysꢀsimilarꢀtoꢀthoseꢀdescribedꢀforꢀHPV16.ꢀꢀWeꢀ

60 selectedꢀ virusesꢀ withꢀ differentꢀ requirementsꢀ forꢀ sulfatedꢀ

61 GAGsꢀforꢀsuccessfulꢀviralꢀentry:ꢀfullyꢀdependentꢀ(Herpesꢀ

62 SimplexꢀVirus-1ꢀandꢀMerkelꢀCellꢀPolyomavirus),ꢀpotential-

63 lyꢀ dependentꢀ (Simianꢀ Virusꢀ 40ꢀ (SV40))ꢀ orꢀ independentꢀ

64 (Influenzaꢀ Aꢀ Virus).ꢀ HSV-1ꢀ isꢀ anꢀ envelopedꢀ virusꢀ thatꢀ

65 causesꢀ coldꢀ soresꢀ andꢀ inꢀ severeꢀ casesꢀ viralꢀ encephalitisꢀ

66 withꢀ potentialꢀ fatalꢀ outcome.ꢀ HSV-1ꢀ requiresꢀ HSPGsꢀ forꢀ

448 Glycopolymersꢀ areꢀ potentialꢀ broadbandꢀ antiviralꢀ com-

449 poundsꢀ

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51 Soꢀfar,ꢀourꢀworkꢀhasꢀdemonstratedꢀthatꢀsulfatedꢀglycopol-

52 ymersꢀareꢀgoodꢀcandidatesꢀasꢀantiviralꢀcompoundsꢀagainstꢀ

53 HPV16.ꢀSinceꢀtheꢀmechanismsꢀbyꢀwhichꢀtheseꢀglycopoly-

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54 mersꢀ andꢀ glycooligomersꢀ blockꢀ HPV16ꢀ areꢀ likelyꢀ toꢀ beꢀ

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55 applicableꢀ toꢀ furtherꢀ HSPG-bindingꢀ viruses,ꢀ theyꢀ poten-

56 tiallyꢀconstituteꢀbroad-spectrumꢀantivirals.ꢀ

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67 attachmentꢀtoꢀcells. ꢀMCPyVꢀhasꢀbeenꢀassociatedꢀwithꢀ

68 theꢀdevelopmentꢀofꢀMerkelꢀcellꢀcarcinoma,ꢀaꢀsevereꢀandꢀ

56

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469 aggressiveꢀ carcinomaꢀ ofꢀ theꢀ skin. ꢀ Thisꢀ non-envelopedꢀ

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70 polyomavirusꢀrequiresꢀsequentialꢀengagementꢀofꢀsulfatedꢀ

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71 GAGsꢀ(suchꢀasꢀHS)ꢀandꢀsialylatedꢀglycans. ꢀTheꢀanimalꢀ492 contrastꢀ toꢀ HPV16,ꢀ theꢀ unsulfatedꢀ PG-OHꢀ decreasedꢀ

72 polyomavirusꢀ SV40ꢀ requiresꢀ bindingꢀ toꢀ sialic-containingꢀ493 infectivityꢀofꢀHSV-1ꢀandꢀSV40ꢀtoꢀaꢀminorꢀextent,ꢀsuggest-

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473 gangliosideꢀ GM1ꢀ butꢀ mayꢀ alsoꢀ engageꢀ HSPGs.

ꢀ Asꢀ aꢀ494 ingꢀ thatꢀ theꢀ polymericꢀ backboneꢀ orꢀ non-sulfatedꢀ sugarsꢀ

474 potentialꢀ negativeꢀ control,ꢀ IAV,ꢀ aꢀ causativeꢀ agentꢀ ofꢀ se- 495 mayꢀassistꢀinꢀengagingꢀcertainꢀviruses.ꢀ

475 vereꢀ respiratoryꢀ infections,ꢀ wasꢀ used.ꢀ IAVꢀ strictlyꢀ bindsꢀ

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476 sialylatedꢀreceptorsꢀonꢀtheꢀhostꢀcellsꢀtoꢀinitiateꢀviralꢀen-

477 try. ꢀ MCPyVꢀ isꢀ aꢀ BSL2ꢀ organism,ꢀ butꢀ sinceꢀ itꢀ wasꢀ pre-

478 paredꢀ asꢀ aꢀ PsV,ꢀ workꢀ couldꢀ beꢀ conductedꢀ underꢀ BSL1ꢀ

479 conditions.ꢀ Allꢀ otherꢀ virusesꢀ areꢀ BSL2ꢀ organisms,ꢀ andꢀ

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497 Asꢀ mentionedꢀ before,ꢀ IAVꢀ strictlyꢀ dependsꢀ onꢀ sialylatedꢀ

61

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98 glycansꢀforꢀbindingꢀandꢀinfection. ꢀSialylatedꢀglycansꢀareꢀ

99 structurallyꢀ veryꢀ differentꢀ fromꢀ HS:ꢀ HSꢀ areꢀ long,ꢀ un-

00 branchedꢀandꢀhighlyꢀsulfatedꢀchains,ꢀwhereasꢀsialicꢀacidꢀisꢀ

01 generallyꢀ foundꢀ asꢀ aꢀ terminalꢀ carbohydrateꢀ residueꢀ onꢀ

02 short,ꢀ branchedꢀ and/orꢀ lowꢀ orꢀ non-sulfatedꢀ glycans.ꢀ

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80 workꢀwasꢀconductedꢀaccordingly.ꢀꢀ

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82 Asꢀ indicatedꢀ byꢀ reductionꢀ ofꢀ infectivity,ꢀ glycopolymersꢀ503 Hence,ꢀ weꢀ reasonedꢀ thatꢀ IAVꢀ shouldꢀ beꢀ lessꢀ affectedꢀ byꢀ

83 successfullyꢀblockedꢀallꢀHS-dependentꢀviruses.ꢀHSV-1ꢀwasꢀ504 theꢀ glycopolymers.ꢀ Surprisingly,ꢀ glycopolymers,ꢀ despiteꢀ

84 blockedꢀwithꢀsimilarꢀefficiencyꢀasꢀHPV16ꢀ(Fig.ꢀ6A),ꢀwhere- 505 resemblingꢀ HS,ꢀ inhibitedꢀ IAVꢀ infection.ꢀ Ofꢀ note,ꢀ whileꢀ

85 asꢀMCPyVꢀrequiredꢀ100-foldꢀhigherꢀdosesꢀofꢀPMꢀandꢀPG1ꢀ506 infectivityꢀ wasꢀ reducedꢀ byꢀ 50%ꢀ atꢀ aꢀ comparativelyꢀ highꢀ

86 (Fig.ꢀ6B).ꢀInterestingly,ꢀSV40ꢀwasꢀalsoꢀsusceptibleꢀtoꢀgly- 507 concentrationꢀ ofꢀ 0.1mg/ml,ꢀ itꢀ remainedꢀ unaffectedꢀ byꢀ

87 copolymer-mediatedꢀinhibitionꢀ(Fig.ꢀ6C).ꢀWhileꢀinfectionꢀ508 heparinꢀ (Fig.ꢀ 6D).ꢀ Thisꢀ mayꢀ suggestꢀ thatꢀ theꢀ displayꢀ ofꢀ

488 wasꢀreducedꢀtoꢀ50%ꢀatꢀaꢀlowꢀglycopolymerꢀconcentrationꢀ509 saccharidesꢀasꢀsideꢀchainsꢀofꢀaꢀpolymericꢀbackboneꢀratherꢀ

489 ofꢀ 0.0001mg/ml,ꢀ aꢀ basalꢀ levelꢀ ofꢀ infectionꢀ ofꢀ aboutꢀ 10%ꢀ510 thanꢀconstitutingꢀtheꢀbackboneꢀitselfꢀisꢀadvantageousꢀforꢀ

490 wasꢀresistantꢀtoꢀinhibition.ꢀThisꢀmayꢀreflectꢀdirectꢀbindingꢀ511 aꢀbroadꢀefficacyꢀtowardsꢀglycanꢀbindingꢀviruses.ꢀꢀ

491 toꢀ cellsꢀ byꢀ lowꢀ affinityꢀ interactionsꢀ withꢀ sialicꢀ acids.ꢀ Inꢀ

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Figureꢀ6.ꢀBroad-spectrumꢀantiviralꢀeffectsꢀofꢀsulfatedꢀglycopolymers.ꢀHSV-1ꢀ(A),ꢀMCPyVꢀ(B),ꢀSV40ꢀ(C)ꢀorꢀIAVꢀ(D)ꢀwereꢀ

incubatedꢀwithꢀglycopolymersꢀPM,ꢀPG1ꢀandꢀPG-OHꢀ(asꢀshownꢀinꢀTab.ꢀ1)ꢀatꢀtheꢀindicatedꢀconcentrations.ꢀForꢀmoreꢀin-

formationꢀonꢀtheꢀinfectionꢀassaysꢀofꢀeachꢀvirus,ꢀseeꢀMaterialꢀandꢀMethodsꢀ(SI).ꢀResultsꢀareꢀshownꢀinꢀrelativeꢀinfectionꢀ(%)ꢀ

toꢀanꢀuntreatedꢀcondition.ꢀAllꢀinfectionꢀvaluesꢀareꢀtheꢀmeanꢀ±ꢀSDꢀofꢀatꢀleastꢀ3ꢀindependentꢀexperiments.ꢀ

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18 Asꢀ glycooligomersꢀ displayedꢀ someꢀ antiviralꢀ activityꢀ540 Inꢀcontrast,ꢀMCPyVꢀinfectionꢀwasꢀunaffectedꢀinꢀtheꢀpres-

19 againstꢀ HPV16,ꢀ weꢀ alsoꢀ testedꢀ theirꢀ potentialꢀ broad- 541 enceꢀ ofꢀ glycooligomersꢀ suggestingꢀ thatꢀ theꢀ oligomersꢀ

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520 spectrumꢀeffect.ꢀHSV-1ꢀwasꢀblockedꢀbyꢀallꢀglycooligomersꢀ542 displayedꢀaꢀlowꢀaffinityꢀtoꢀtheꢀvirionꢀ(Fig.ꢀ7B).ꢀ

521 atꢀ theꢀ highestꢀ concentrationꢀ tested.ꢀ Atꢀ 0.1ꢀ mg/mlꢀ theꢀ

522 intermediate-sizedꢀstructureꢀ(O2)ꢀwasꢀtheꢀmostꢀefficientꢀ

523 asꢀcomparedꢀtoꢀtheꢀunsulfatedꢀcontrolꢀ(Fig.ꢀ7A)ꢀindicatingꢀ

524 thatꢀtheꢀdpꢀdoesꢀnotꢀinfluenceꢀtheꢀinhibitoryꢀpotentialꢀinꢀ

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44 Inꢀsummary,ꢀsulfatedꢀglycopolymersꢀdisplayedꢀaꢀgoodꢀandꢀ

45 broadꢀanti-viralꢀactivity,ꢀwhereasꢀsulfatedꢀglycooligomersꢀ

46 showedꢀinterestingꢀpotential.ꢀMoreꢀimportantly,ꢀtheꢀgly-

47 cooligomersꢀexhibitedꢀunexpectedꢀbiologicalꢀeffectsꢀsuchꢀ

48 asꢀpost-bindingꢀinhibitionꢀandꢀnon-linearꢀsizeꢀeffects.ꢀTheꢀ

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525 aꢀstraightforwardꢀmanner.ꢀ

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27 Similarꢀ effectsꢀ wereꢀ observedꢀ forꢀ SV40.ꢀ Infectionꢀ wasꢀ549 latterꢀwillꢀneedꢀfurtherꢀin-depthꢀcharacterizationꢀtoꢀallowꢀ

28 blockedꢀbyꢀtheꢀshortꢀglycooligomerꢀO1ꢀonlyꢀatꢀtheꢀhighestꢀ550 forꢀaꢀmoreꢀrationalꢀdesignꢀofꢀnextꢀgenerationꢀglycooligo-

29 concentrationꢀ (Fig.ꢀ 7C).ꢀ O2ꢀ wasꢀ theꢀ mostꢀ efficientꢀ com- 551 mericꢀantivirals.ꢀ

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30 poundꢀandꢀreducedꢀinfectionꢀbyꢀ50%ꢀatꢀ0.1mg/ml,ꢀwhere-

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ꢀ

31 asꢀlongerꢀglycooligomersꢀO3ꢀandꢀO4ꢀdidꢀnotꢀaffectꢀinfec-

32 tion.ꢀSimilarꢀtoꢀtheꢀglycopolymers,ꢀaꢀbasalꢀlevelꢀofꢀinfec-

33 tionꢀwasꢀresistantꢀtoꢀinhibition.ꢀꢀ

554 DISCUSSIONꢀ

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55 Theꢀstrategyꢀtoꢀinterfereꢀwithꢀviralꢀattachmentꢀusingꢀsul-

56 fatedꢀpolymericꢀglycansꢀisꢀnotꢀwithoutꢀprecedent.ꢀNaturalꢀ

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35 AsꢀIAVꢀwasꢀblockedꢀbyꢀglycopolymersꢀatꢀhighꢀconcentra-

36 tions,ꢀitꢀwasꢀnotꢀsurprisingꢀthatꢀglycooligomersꢀalsoꢀexert-

37 edꢀantiviralꢀfunctions.ꢀInterestingly,ꢀforꢀIAVꢀtheꢀincreaseꢀ

38 inꢀglycooligomerꢀchainꢀlengthꢀcorrelatedꢀwithꢀbetterꢀneu-

39 tralizationꢀofꢀtheꢀvirusꢀ(Fig.ꢀ7D).ꢀꢀ

557 polysaccharidesꢀ haveꢀ longꢀ beenꢀ usedꢀ toꢀ interfereꢀ withꢀ

558 viralꢀattachment,ꢀmostlyꢀtoꢀcharacterizeꢀtheꢀglycanꢀbind-

559 ingꢀpropertiesꢀofꢀaꢀparticularꢀvirusꢀbutꢀalsoꢀinꢀlightꢀofꢀtheirꢀ

560 antiviralꢀpotential.ꢀꢀ

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Figureꢀ7.ꢀSulfatedꢀglycooligomerꢀinhibitionꢀprofileꢀofꢀvariousꢀviralꢀinfections.ꢀHSV-1ꢀ(A),ꢀMCPyVꢀ(B),ꢀSV40ꢀ(C)ꢀorꢀIAVꢀ(D)ꢀ

wereꢀincubatedꢀwithꢀglycooligomersꢀO1-O4ꢀandꢀO2-OHꢀ(asꢀshownꢀinꢀTab.ꢀ2)ꢀatꢀtheꢀindicatedꢀconcentrations.ꢀForꢀmoreꢀ

informationꢀonꢀtheꢀinfectionꢀassaysꢀofꢀeachꢀvirus,ꢀseeꢀMaterialꢀandꢀMethodsꢀ(SI).ꢀResultsꢀareꢀshownꢀinꢀrelativeꢀinfectionꢀ

(%)ꢀtoꢀanꢀuntreatedꢀcondition.ꢀAllꢀinfectionꢀvaluesꢀareꢀtheꢀmeanꢀ±ꢀSDꢀofꢀatꢀleastꢀ3ꢀindependentꢀexperiments.ꢀ

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69 Inꢀ thisꢀ manuscript,ꢀ weꢀ demonstrateꢀ forꢀ theꢀ firstꢀ time,ꢀ628 mentꢀofꢀsitesꢀindependentꢀofꢀHSꢀinteraction,ꢀwhichꢀseemsꢀ

70 proof-of-principleꢀ thatꢀ syntheticꢀ sulfatedꢀ glycopolymersꢀ629 lessꢀlikely.ꢀꢀ

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571 andꢀglycooligomersꢀcanꢀserveꢀasꢀpowerfulꢀalternativesꢀtoꢀ

572 naturallyꢀ occurringꢀ polysaccharides,ꢀ andꢀ thatꢀ theyꢀ pro-

573 videꢀaꢀsuitableꢀplatformꢀtoꢀdevelopꢀbroadꢀantiviralsꢀwithꢀ

574 theꢀcapacityꢀtoꢀfine-tuneꢀtheꢀinhibitoryꢀmechanism.ꢀOurꢀ

575 workꢀ revealedꢀ thatꢀ sulfatedꢀ glycopolymersꢀ efficientlyꢀ

576 blockedꢀHPV16ꢀandꢀotherꢀvirusesꢀwithꢀaꢀsimilarꢀefficacyꢀasꢀ

577 carrageenanꢀ(Figꢀ1,ꢀ2,ꢀ5,ꢀ6).ꢀMoreover,ꢀglycopolymersꢀandꢀ

578 carrageenanꢀinhibitedꢀviralꢀinfectivityꢀmoreꢀstronglyꢀthanꢀ

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31 Anotherꢀ intriguingꢀ observationꢀ wasꢀ thatꢀ withꢀ increasingꢀ

32 lengthꢀofꢀtheꢀglycooligomers,ꢀvirionsꢀclusteredꢀonꢀtheꢀcellꢀ

33 surfaceꢀ (Fig.ꢀ 4).ꢀ Possibly,ꢀ longꢀ glycomimeticsꢀ bindꢀ toꢀ

34 severalꢀvirusꢀparticlesꢀthusꢀlinkingꢀthem,ꢀwhereasꢀshorterꢀ

35 oligomersꢀmayꢀbeꢀunableꢀtoꢀbridgeꢀvirions.ꢀAlternatively,ꢀ

36 theꢀlongerꢀoligomersꢀmayꢀadditionallyꢀengageꢀotherꢀcellu-

37 larꢀproteins,ꢀwhichꢀwouldꢀleadꢀtoꢀclusteringꢀonꢀcells.ꢀꢀWeꢀ

38 alsoꢀ observedꢀ someꢀ viralꢀ clusteringꢀ withꢀ glycopolymersꢀ

39 whichꢀabsorbedꢀtoꢀtheꢀcoverslipsꢀduringꢀmicroscopyꢀ(Fig.ꢀ

40 4,ꢀ PG).ꢀ Thisꢀ indicatesꢀ thatꢀ clusteringꢀ alsoꢀ occurredꢀ withꢀ

41 glycopolymers,ꢀ butꢀ wasꢀ notꢀ easilyꢀ identifiableꢀ asꢀ virionsꢀ

42 wereꢀunableꢀtoꢀbindꢀcells.ꢀ

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79 heparin,ꢀ whichꢀ servesꢀ asꢀ aꢀ mimicꢀ forꢀ theꢀ naturalꢀ ligandꢀ

80 (Fig.ꢀ1,ꢀ2).ꢀThisꢀprovidesꢀanꢀexcellentꢀfoundationꢀforꢀfutureꢀ

81 optimizationꢀofꢀsyntheticꢀmimeticsꢀforꢀantiviralꢀtherapies.ꢀ

82 Inꢀ contrastꢀ toꢀ naturalꢀ polysaccharides,ꢀ syntheticꢀ glyco-

83 mimeticsꢀareꢀdefined,ꢀhomogeneous,ꢀreplicableꢀandꢀtuna-

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84 bleꢀ compoundsꢀ thatꢀ canꢀ beꢀ designedꢀ toꢀ varyꢀ inꢀ length,ꢀ

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85 sizeꢀandꢀpresentation.ꢀInꢀaddition,ꢀdifferentꢀsugars,ꢀlink-

62–64

86 ersꢀandꢀspacingsꢀcanꢀalsoꢀbeꢀexplored.

ꢀMoreover,ꢀgly- 644 Irrespectiveꢀofꢀtheꢀdifferentꢀmodesꢀofꢀinhibitionꢀbetweenꢀ

87 comimeticsꢀprovideꢀtheꢀadvantageꢀthatꢀtheyꢀhave,ꢀtoꢀtheꢀ645 glycopolymersꢀ andꢀ glycooligomers,ꢀ ourꢀ resultsꢀ demon-

88 bestꢀ ofꢀ ourꢀ knowledge,ꢀ noꢀ furtherꢀ bioactiveꢀ propertiesꢀ646 strateꢀ thatꢀ additionalꢀ workꢀ needsꢀ toꢀ beꢀ conductedꢀ toꢀ

89 avoidingꢀe.g.ꢀtheꢀanti-coagulatingꢀpropertiesꢀofꢀheparinꢀinꢀ647 determineꢀ theꢀ rulesꢀ ofꢀ engagementꢀ inꢀ orderꢀ toꢀ deviseꢀ

65

90 vivo ,ꢀwhichꢀmayꢀcauseꢀinherentꢀrisksꢀforꢀcertainꢀapplica- 648 rationalꢀ guidelinesꢀ forꢀ theꢀ futureꢀ designꢀ ofꢀ sulfatedꢀ gly-

91 tions.ꢀ Whileꢀ theꢀ incorporationꢀ ofꢀ differentꢀ sugarsꢀ intoꢀ649 comimeticꢀantivirals.ꢀAꢀvarietyꢀofꢀvirusesꢀengageꢀcellularꢀ

92 glycopolymersꢀhadꢀonlyꢀslightꢀeffectsꢀonꢀtheirꢀefficacyꢀinꢀ650 glycansꢀforꢀinfectivity.ꢀHere,ꢀsulfatedꢀglycomimeticꢀcom-

93 thisꢀstudy,ꢀitꢀhasꢀbeenꢀpreviouslyꢀnotedꢀthatꢀoptimizationꢀ651 poundsꢀinterferedꢀwithꢀinfectionꢀofꢀdiverseꢀviruses,ꢀenvel-

94 ofꢀaspectsꢀsuchꢀasꢀlength,ꢀcompositionꢀandꢀdeliveryꢀmayꢀ652 opedꢀ vs.ꢀ non-enveloped,ꢀ HSPG-bindingꢀ vs.ꢀ non-HSPGꢀ

95 yieldꢀ increasedꢀ biologicalꢀ functionꢀ beyondꢀ naturalꢀ lig- 653 bindingꢀvirusesꢀ(Fig.ꢀ6,ꢀ7).ꢀInfectionꢀofꢀHSV-1ꢀandꢀMCPyV,ꢀ

18

96 ands. ꢀ Finally,ꢀ glycomimeticsꢀ offerꢀ veryꢀ highꢀ batchꢀ toꢀ654 whichꢀalsoꢀdependꢀonꢀheparanꢀsulfateꢀforꢀinfectionꢀofꢀhostꢀ

97 batchꢀreproducibilityꢀ(seeꢀSI)ꢀandꢀalsoꢀrobustꢀreproduci- 655 cells,ꢀwasꢀblocked.ꢀInterestingly,ꢀvirusesꢀsuchꢀasꢀSV40ꢀandꢀ

98 bilityꢀofꢀantiviralꢀactivitiesꢀ(dataꢀnotꢀshown).ꢀꢀꢀ

656 IAVꢀthatꢀengageꢀsialicꢀacidsꢀasꢀmainꢀreceptorsꢀwereꢀalsoꢀ

6

57 blockedꢀbyꢀtheꢀglycopolymers,ꢀalthoughꢀtoꢀaꢀlesserꢀextent,ꢀ

58 especiallyꢀ IAV.ꢀ Thisꢀ isꢀ surprising,ꢀ asꢀ theꢀ interactionꢀ be-

59 tweenꢀsialicꢀacidsꢀandꢀthoseꢀvirusesꢀisꢀnotꢀdrivenꢀbyꢀtheꢀ

660 sulfationꢀ ofꢀ theꢀ sugars,ꢀ butꢀ ratherꢀ structuralꢀ constraintsꢀ

661 withinꢀtheꢀglycanꢀandꢀtheꢀvirus.ꢀNevertheless,ꢀSV40ꢀseemsꢀ

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00 Theꢀsulfatedꢀglycooligomersꢀusedꢀinꢀthisꢀstudyꢀdisplayedꢀaꢀ

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01 lowerꢀ efficacyꢀ toꢀ blockꢀ infectivityꢀ asꢀ comparedꢀ toꢀ theꢀ

02 glycopolymersꢀ(Fig.ꢀ3,ꢀ7).ꢀThisꢀisꢀnotꢀunexpected,ꢀsinceꢀtheꢀ

03 shorterꢀ oligomersꢀ displayꢀ lessꢀ sulfatedꢀ sugarsꢀ perꢀ mole-

04 cule,ꢀ whichꢀ wouldꢀ resultꢀ inꢀ lowerꢀ avidityꢀ towardsꢀ theꢀ

05 multipleꢀ bindingꢀ sitesꢀ onꢀ virusꢀ particles.ꢀ Nevertheless,ꢀ

06 theyꢀ blockedꢀ theꢀ infectivityꢀ ofꢀ severalꢀ virusesꢀ atꢀ higherꢀ

07 concentrationsꢀinꢀvitro.ꢀ

60

662 toꢀbeꢀsusceptibleꢀtoꢀblockingꢀbyꢀheparinꢀ(Fig.ꢀ6, ).ꢀInter-

663 estingly,ꢀthisꢀdoesꢀnotꢀholdꢀtrueꢀforꢀIAV.ꢀHeparinꢀdidꢀnotꢀ

664 showꢀinhibitionꢀofꢀIAVꢀinfection,ꢀwhileꢀtheꢀglycopolymersꢀ

665 blockedꢀinfectionꢀ(Fig.ꢀ6).ꢀThus,ꢀitꢀisꢀlikelyꢀthatꢀtheꢀflexi-

666 bilityꢀ ofꢀ theꢀ linkerꢀ betweenꢀ carbohydrateꢀ andꢀ polymericꢀ

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ꢀ

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67 backboneꢀallowsꢀforꢀanꢀeasierꢀfitꢀofꢀtheꢀsugarꢀresiduesꢀintoꢀ

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09 WhenꢀanalyzedꢀforꢀHPV16,ꢀglycopolymersꢀblockedꢀbind- 668 existingꢀ bindingꢀ sites.ꢀ Theꢀ specificꢀ mechanism(s)ꢀ byꢀ

10 ingꢀofꢀtheꢀvirusꢀtoꢀcellsꢀ(Fig.ꢀ4),ꢀindicatingꢀthatꢀglycopol- 669 whichꢀtheꢀglycopolymersꢀengageꢀtheseꢀvirusesꢀneedsꢀtoꢀbeꢀ

11 ymersꢀlikelyꢀengagedꢀheparanꢀsulfateꢀbindingꢀsitesꢀonꢀtheꢀ670 addressedꢀ inꢀ futureꢀ work,ꢀ butꢀ thisꢀ findingꢀ furtherꢀ sup-

12 virionꢀ asꢀ suggestedꢀ forꢀ carrageenanꢀ andꢀ otherꢀ naturalꢀ671 portsꢀanꢀadvantageousꢀroleꢀofꢀglycomimeticsꢀoverꢀnaturalꢀ

7

,21

13 sulfatedꢀ polysaccharides. ꢀ Interestingly,ꢀ glycooligomersꢀ672 polysaccharides.ꢀꢀ

14 blockedꢀ HPV16ꢀ infectivityꢀ atꢀ aꢀ post-bindingꢀ step,ꢀ asꢀ

673

ꢀ

15 shownꢀbyꢀtheꢀpresenceꢀofꢀcell-boundꢀvirusꢀatꢀconcentra-

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74 Importantly,ꢀ theꢀ antiviralꢀ effectꢀ ofꢀ glycooligomersꢀ doesꢀ

675 notꢀallowꢀpredictingꢀrulesꢀofꢀengagement,ꢀsinceꢀinhibitoryꢀ

676 effectsꢀ didꢀ notꢀ correlateꢀ forꢀ allꢀ ofꢀ theꢀ virusesꢀ weꢀ testedꢀ

677 (Fig.ꢀ 7).ꢀ Whileꢀ forꢀ SV40ꢀ theꢀ antiviralꢀ activityꢀ correlatedꢀ

678 withꢀ length,ꢀ theꢀ oppositeꢀ wasꢀ trueꢀ forꢀ HPV16,ꢀ whereasꢀ

679 MCPyVꢀ wasꢀ notꢀ blockedꢀ byꢀ anyꢀ ofꢀ theꢀ glycooligomers.ꢀ

680 Besidesꢀ aꢀ moreꢀ systematicꢀ analysisꢀ ofꢀ allꢀ functionalꢀ pa-

681 rametersꢀ ofꢀ glycooligomers,ꢀ itꢀ wouldꢀ beꢀ importantꢀ toꢀ

16 tionsꢀ inꢀ whichꢀ infectionꢀ wasꢀ blockedꢀ (Fig.ꢀ 3,ꢀ 4).ꢀ Hence,ꢀ

17 theꢀglycooligomersꢀdidꢀnotꢀeffectivelyꢀbindꢀtoꢀsitesꢀinꢀtheꢀ

18 virionꢀthatꢀmediatedꢀinitialꢀbindingꢀtoꢀcells.ꢀꢀHPV16ꢀinter-

19 actionꢀwithꢀheparanꢀsulfatesꢀinvolvesꢀseveralꢀdistinctꢀsitesꢀ

66

20 onꢀ theꢀ virion ,ꢀ whichꢀ areꢀ likelyꢀ engagedꢀ inꢀ aꢀ stepwiseꢀ

21 fashionꢀ facilitatingꢀ initialꢀ bindingꢀ andꢀ conformationalꢀ

44

22 changesꢀ inꢀ theꢀ virion .ꢀ Post-attachmentꢀ inhibitionꢀ ofꢀ

23 infectionꢀtherebyꢀpotentiallyꢀinvolvesꢀbindingꢀtoꢀsitesꢀthatꢀ

24 areꢀ typicallyꢀ engagedꢀ inꢀ aꢀ secondꢀ stepꢀ ofꢀ theꢀ HS-HPV16ꢀ

6

82 identify,ꢀhowꢀexactlyꢀtheꢀglycooligomersꢀbindꢀtoꢀdifferentꢀ

83 viruses.ꢀ

6

25 interaction,ꢀblockingꢀtheꢀconformationalꢀchangesꢀcrucialꢀ

26 forꢀsubsequentꢀengagementꢀofꢀtheꢀinternalizationꢀrecep-

684

ꢀ

2

9,32

27 tor. ꢀAlternatively,ꢀoneꢀwouldꢀhaveꢀtoꢀproposeꢀengage-

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85 Perhapsꢀ aꢀ ratherꢀ straightforwardꢀ approachꢀ toꢀ increaseꢀ742 accordingꢀ precursorsꢀ andꢀ intermediates.ꢀ Additionalꢀ infor-

86 broad-spectrumꢀantiviralꢀpotentialꢀwouldꢀbeꢀtoꢀsynthesizeꢀ743 mationꢀincludingꢀMaterialꢀandꢀMethodsꢀandꢀsupplementaryꢀ

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744 figuresꢀforꢀbiologicalꢀassays.ꢀThisꢀmaterialꢀisꢀavailableꢀfreeꢀofꢀ

745 chargeꢀviaꢀtheꢀInternetꢀatꢀhttp://pubs.acs.org.ꢀꢀ

87 multifunctionalꢀ compoundsꢀ containingꢀ differentꢀ biologi-

88 callyꢀ relevantꢀ glycans,ꢀ forꢀ exampleꢀ combiningꢀ GlcNAcꢀ

689 (monomerꢀ presentꢀ inꢀ HS)ꢀ andꢀ sialicꢀ acid.ꢀ Toꢀ increaseꢀ

7

46 AUTHORꢀINFORMATIONꢀ

690 avidity,ꢀitꢀmayꢀalsoꢀbeꢀreasonableꢀtoꢀpresentꢀtheꢀglycooli-

691 gomersꢀonꢀnanoparticles,ꢀasꢀhasꢀpreviouslyꢀbeenꢀdoneꢀforꢀ

7

47 CorrespondingꢀAuthorꢀ

6

7

692 heparin ꢀorꢀevenꢀonꢀmicelles.ꢀAꢀsimilarꢀapproach,ꢀusingꢀ

7

48 *ꢀlaura.hartmann@hhu.deꢀandꢀschelhaa@uni-muenster.de.ꢀꢀꢀ

693 nanogoldꢀparticles,ꢀimprovedꢀtheꢀefficacyꢀofꢀglycomimet-

68

694 ic-basedꢀvaccines ꢀ(andꢀisꢀalsoꢀavailableꢀforꢀtheꢀmultiva- 749 PresentꢀAddressesꢀ

ꢀ

6

95 lentꢀ presentationꢀ ofꢀ glycooligomersꢀ asꢀ usedꢀ inꢀ thisꢀ750 † SectionꢀofꢀVirology,ꢀDepartmentꢀofꢀClinicalꢀMicrobiology,ꢀ

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96 study ).ꢀ

751 UmeåꢀUniversity,ꢀUmeå,ꢀSwedenꢀ

52

7

ꢀ

697

ꢀ

6

7

98 Despiteꢀtheꢀfactꢀthatꢀimprovementsꢀwouldꢀbeꢀnecessaryꢀtoꢀ753 AuthorꢀContributionsꢀ

99 generateꢀ sulfatedꢀ glycomimeticsꢀ withꢀ moreꢀ efficientꢀ

7

54 Theꢀ manuscriptꢀ wasꢀ writtenꢀ throughꢀ contributionsꢀ ofꢀ allꢀ

00 broad-spectrumꢀ antiviralꢀ activity,ꢀ itꢀ isꢀ noteworthyꢀ thatꢀ

7

55 authors.ꢀAllꢀauthorsꢀhaveꢀgivenꢀapprovalꢀtoꢀtheꢀfinalꢀversionꢀ

01 theꢀ glycopolymersꢀ wereꢀ effectiveꢀ atꢀ preventingꢀ infectionꢀ756 ofꢀtheꢀmanuscript.ꢀ

702 inꢀ aꢀ pre-clinicalꢀ mouseꢀ modelꢀ forꢀ vaginalꢀ infectionsꢀ ofꢀ

703 HPV16ꢀ(Fig.ꢀ5).ꢀMoreover,ꢀnoꢀcytotoxicityꢀofꢀsulfatedꢀgly-

7

57 FundingꢀSourcesꢀ

758 TheꢀauthorsꢀthankꢀtheꢀGermanꢀResearchꢀCouncilꢀ(DFG)ꢀandꢀ

04 copolymersꢀ andꢀ glycooligomersꢀ wasꢀ observedꢀ duringꢀ

7

59 theꢀFederalꢀMinistryꢀforꢀEducationꢀandꢀResearchꢀ(BMBF)ꢀforꢀ

60 financialꢀ support.ꢀ LHꢀ wasꢀ supportedꢀ throughꢀ theꢀ Researchꢀ

61 Unitꢀ FOR2327ꢀ (Virocarb,ꢀ grantꢀ HAꢀ 5950/5-1)ꢀ andꢀ largeꢀ

62 equipmentꢀgrantꢀ(INSTꢀ208/735-1).ꢀMSꢀwasꢀsupportedꢀwithinꢀ

63 theꢀInfectERAꢀinitiativeꢀbyꢀfundingꢀfromꢀtheꢀFederalꢀMinistryꢀ

64 forꢀEducationꢀandꢀResearchꢀ(BMBF,ꢀ031L0095A)ꢀandꢀthroughꢀ

65 theꢀResearchꢀUnitꢀFOR2327ꢀ(ViroCarb,ꢀgrantsꢀSCHEꢀ1552/3-1,ꢀ

66 3-2).ꢀꢀ

705 experimentationꢀ(Fig.ꢀ1B,ꢀ3B).ꢀThus,ꢀitꢀisꢀalreadyꢀpossibleꢀ

7

706 toꢀstartꢀassessingꢀessentialꢀpropertiesꢀforꢀclinicalꢀapplica-

707 tions.ꢀSomeꢀofꢀtheseꢀpropertiesꢀincludeꢀhowꢀlongꢀprotec-

708 tiveꢀeffectsꢀlastꢀafterꢀinitialꢀapplication,ꢀhowꢀtoꢀbestꢀdeliv-

7

709 erꢀtheꢀcompounds,ꢀwhetherꢀtheꢀglycomimeticꢀcompoundsꢀ

7

10 exhibitꢀ long-termꢀ adverseꢀ effectsꢀ inꢀ variousꢀ tis-

11 sues/organisms,ꢀandꢀtheꢀlongꢀtermꢀstabilityꢀofꢀtheseꢀcom-

12 pounds.ꢀ Itꢀ isꢀ conceivableꢀ thatꢀ theꢀ versatilityꢀ ofꢀ glycomi-

13 meticsꢀwillꢀallowꢀforꢀfine-tuningꢀallꢀtheseꢀparametersꢀandꢀ

14 deriveꢀ compoundsꢀ withꢀ superiorꢀ efficacyꢀ andꢀ versatilityꢀ

767 ACKNOWLEDGMENTꢀꢀ

7

68 WeꢀwouldꢀlikeꢀtoꢀthankꢀInesꢀFelsꢀ(InstituteꢀofꢀCellularꢀVirol-

769 ogy,ꢀ Universityꢀ ofꢀ Münster)ꢀ forꢀ technicalꢀ supportꢀ duringꢀ

770 virusꢀproduction.ꢀ

15 thanꢀnaturalꢀpolysaccharides.ꢀDespiteꢀtheirꢀimportanceꢀasꢀ

16 proof-of-conceptꢀ forꢀ theꢀ versatilityꢀ ofꢀ glycomimetics-

17 mediatedꢀ inhibition,ꢀ deliveryꢀ systemsꢀ forꢀ theꢀ polyoma-

18 virusesꢀMCPyVꢀandꢀSV40ꢀmayꢀbeꢀchallengingꢀtoꢀdevelop,ꢀ

7

71 REFERENCESꢀ

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7

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SYNOPSISꢀTOC.ꢀꢀ

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Figure 1. Natural polysaccharides and sulfated glycopolymers block infection of HPV16. (A) HPV16 was

preincubated with the indicated amounts of heparin,carrageenan, glycopolymers PM and PG1 and the

unsulfated control PG-OH for 1h at RT. HeLa cells were infected and infection was scored 48h p.i. by

microscopy (GFP signal). Results are shown in % relative to mock-incubated HPV16. All infection values are

the mean ± SD of at least 3 independent experiments. (B) Relative cell count of cells treated as in A). The

relative cell numbers after infection and treatment with glycopolymers or natural polysaccharides are shown

relative (in %) to the untreated mock (uninfected) condition of each experiment and indicate the absence of

toxicity by all treatments at 1mg/ml.

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Figure 2. Inhibitory efficiency of glycopolymers PM and PG1 and natural polysaccharides. HPV16 PsVs were

preincubated with the indicated compounds for 1h at increas-ing log concentrations. The inoculums were

added to HeLa cells for 2h and infection was scored 48h p.i. Inhibition curves and IC50 values for

carrageenan (A), heparin (B), PG1 (C) and PM (D) were calculated using GraphPad. Results are shown

relative (in %) to mock-infected samples. All infection values are mean ± SD of at least 3 independent

experiments.

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Figure 3. Antiviral activity by sulfated glycooligomers in HPV16 infection. (A) HPV16 infection assay was

performed as described in Fig. 1 with glycooligomers with 2, 6, 8 or 10 GlcNAc residues (O1-O4,

respectively) and un-sulfated glycooligomer O2-OH. Results are shown relative (in %) to the mock-infected

samples. All infection values are the mean ± SD of at least 3 independent experiments. (B) Relative cell

count of cells treated as in A). The rela-tive cell numbers after infection and treatment with glycooligomers

or natural polysaccharides are shown relative (in %) to the untreated mock (uninfected) condition of each

experiment and indicate the absence of toxicity by all treatments at 1mg/ml.

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Figure 4. Different mechanisms of inhibiting HPV16 infection by sulfated glycopolymers and glycooligomers.

Fluorophore-labeled HPV16 (white) was incubated with 1mg/ml of the indicated compounds for 1h.

Preincubated PsV were allowed to bind to HeLa cells and fixed after 2h. Representative maximum intensity

projections of stack images for each condition are shown. Cell outlines (yellow) were drawn in Fiji (ImageJ)

based on phalloidin staining. Scale bars correspond to 20µm.

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Figure 5. PG2 effectively inhibits HPV16-luciferase infection in vivo. (A) BalbC mice (5 per group),

intravaginally inoculated with HPV16-luciferase preincubated with the respective compounds, were

measured with an IVIS Spectrum to assess infection two days after virus inoculation. (B) The signal was

quantified as photons per second (p/s). The data was analysed with Graphpad and infection level of each

mouse is shown as a single data point. The line represents median of the values. **: P < 0.01 relative to

uninhibited control.

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Figure 6. Broad-spectrum antiviral effects of sulfated glycopolymers. HSV-1 (A), MCPyV (B), SV40 (C) or IAV

(D) were incubated with glycopolymers PM, PG1 and PG-OH (as shown in Tab. 1) at the indicated

concentrations. For more information on the infection assays of each virus, see Material and Methods (SI).

Results are shown in relative infection (%) to an untreated condition. All infection values are the mean ± SD

of at least 3 independent experiments.

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Figure 7. Sulfated glycooligomer inhibition profile of various viral infections. HSV-1 (A), MCPyV (B), SV40

C) or IAV (D) were incubated with glycooligomers O1-O4 and O2-OH (as shown in Tab. 2) at the indicated

(

concentrations. For more information on the infection assays of each virus, see Material and Methods (SI).

Results are shown in relative infection (%) to an untreated condition. All infection values are the mean ± SD

of at least 3 independent experiments.

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Prophylactic Antiviral Activity of Sulfated Glycomimetic Oligomers and Polymers

DOI: 10.1021/jacs.9b13484

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Article abstract of DOI:10.1021/jacs.9b13484

Full text of DOI:10.1021/jacs.9b13484

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