AssignedReading:Chapter55,ChemicalToolsforInhibitingGlycosylationEssentialsinGlycobiology,3rdeditionChapter57,GlycansinBiotechnologyandthePharmaceuticalIndustryEssentialsinGlycobiology,3rdedition

Chapter55,ChemicalToolsforInhibitingGlycosylationEssentialsinGlycobiology,3rdedition

Chapter55

ChemicalToolsforInhibitingGlycosylation

Authors:JeffreyD.Esko,CarolynBertozzi,andRonaldSchnaar

Theuseofchemicaltoolstoinhibitglycosylationprovidesapowerfulapproachforstudyingglycanfunctionsandservesasastartingpointfordrugdiscovery.Thischapterdiscussesvarioustypesofinhibitors,includingnaturalproducts,substrate-basedtight-bindinginhibitors,glycosideprimers,inhibitorsfoundthroughscreeningchemicallibraries,andexamplesofrationallydesignedinhibitorsbasedonthree-dimensionalstructuresofenzymes.

ADVANTAGESOFINHIBITORS

Chapters44,45,and49describevariousnaturalandinducedmutantswithdefectsinglycosylation.Thesemutantshavehelpedtodefinegenesthatencodevarioustransferasesandglycosidases,andinsomecasesalternatebiosyntheticpathwayshavebeenuncovered.Mutantsalsoprovideinsightsintothefunctionofglycosylationincellsandtissuesandmodelsforhumaninbornerrorsinmetabolismanddisease.However,onelimitationofstudyingmutantsisthattheanalysesareusuallyrestrictedtothecellororganismfromwhichthemutantstrainwasisolated.Additionally,manymutationsarelethalinanimals,whichmakesthestudyofthegeneinadultanimalsmoredifficult.Inhibitorsofglycosyltransferasesandglycosidasesprovideanotherapproachforstudyingglycosylationincells,tissues,andwholeorganismsthatavoidssomeoftheproblemsassociatedwithstudyingmutants.Manyofthesecompoundsaresmallmoleculesthataretakenupreadilybyavarietyofcelltypesandsomecanbeabsorbedthroughthegut,providinganopportunityfordesigningdrugstotreathumandiseasesanddisorderscorrelatedwithalteredglycosylation(Chapter57).Becausethefieldisquitelarge,onlyaselectionofinhibitorsthatactonspecificenzymesormetabolicpathwaysandthatillustratecertainbasicconceptsarediscussedhere(Table55.1).Agentsthatblockprotein/carbohydrateinteractionsaresurveyedinChapter29,andtheaminoglycosideantibioticsarediscussedbrieflyinChapter57.

METABOLICINHIBITORS

Anumberofinhibitorshavebeendescribedthatblockglycosylationbyinterferingwiththemetabolismofcommonprecursorsorintracellulartransportactivities.Someofthesecompoundsactindirectlybyimpedingthetransitofproteinsbetweentheendoplasmicreticulum(ER),Golgi,andtrans-Golginetwork.Forexample,thefungalmetabolitebrefeldinAcausesretrogradetransportofGolgicomponentslocatedproximaltothetrans-GolginetworkbacktotheER.Thus,treatingcellswithbrefeldinAseparatesenzymeslocatedinthetrans-GolginetworkfromthosefoundintheERandGolgianduncouplestheassemblyofthecorestructuresofsomeglycansfromlaterreactions,suchassialylationorsulfation.Thedrugcanbeusedtoexamineiftwopathwaysresideinthesamecompartmentorsharethesameenzymes.Becausethelocalizationandarrayoftheenzymesvaryconsiderablyindifferentcelltypes,extrapolatingtheeffectsofbrefeldinAfromonesystemtoanotherisoftendifficult.Someinhibitorsactatkeystepsinintermediarymetabolismwhereprecursorsinvolvedinglycosylationareformed.Forexample,aglutamineanalog,6-diazo-5-oxo-L-norleucine(DON),blocksglutamine:fructose-6-phosphateamidotransferase,theenzymeofthehexosaminebiosyntheticpathwaythatformsglucosaminefromfructoseandglutamine(Chapter5).DepressingglucosamineproductioninthiswayhasapleiotropiceffectonglycanassemblybecauseallofthemajorfamiliescontainN-acetylglucosamineorN-acetylgalactosamine.DONalsoaffectsotherglutamineutilizingenzymesandtherefore,careshouldbetakentolimitnon-specificsideeffects.Chlorateisanothertypeofgeneralinhibitorthatblockssulfation.Thechlorateanion(ClO42−)isananalogofsulfate(SO42−)anditformsanabortivecomplexwiththesulfurylaseinvolvedintheformationofphosphoadenosine-5’-phosphosulfate(PAPS),theactivesulfatedonorforallknownsulfationreactions.Thus,treatingcellswithchlorate(usually10–30mM)inhibitssulfationbymorethan90%,buttheeffectisnotspecificforanyparticularclassofglycanorsulfationreaction(e.g.,tyrosinesulfationisalsoaffected).Anumberofsugaranalogshavebeenmadewiththehopethattheymightexhibitselectiveinhibitionofglycosylation.2-Deoxyglucoseandfluorinatedanalogsofsugars(3-deoxy-3-fluoroglucosamine,4-deoxy-4-fluoroglucosamine,6-deoxy-6-fluoro-N-acetylglucosamine,2-deoxy-2-fluoroglucose,2-deoxy-2-fluoromannose,2-deoxy-2-fluorofucose,and3-fluorosialicacid)inhibitglycoproteinbiosynthesis,butthemechanismunderlyingtheinhibitoryeffectisunclearinmanycases.Earlystudiesof2-deoxyglucoseshowedthattheanalogwasconvertedtoUDP-2-deoxyglucoseaswellastoGDP-2-deoxyglucoseanddolichol-P-2-deoxyglucose.Inhibitionofglycoproteinformationapparentlyoccursasaresultofaccumulationofvariousdolichololigosaccharidescontaining2-deoxyglucose,whichcannotbeelongatedortransferredtoglycoproteinsnormally.Similarly,4-deoxy-N-acetylglucosamineisconvertedtoUDP-4-deoxy-N-acetylglucosamine,resultingininhibitionofheparansulfateformationwithoutincorporationoftheanalog.4-

deoxy-xylosealsoinhibitsglycosaminoglycanassemblypresumablybycompetingwithnaturallyoccurringxylosylatedsubstrates.Caremustbetakenininterpretingtheresultsofexperimentsemployingthesecompoundsbecausetheymayhavepleiotropiceffectsonglycanassemblyduetooverlapofnucleotideprecursors.

TUNICAMYCIN:INHIBITIONOFDOLICHOL-PP-GlcNAcASSEMBLY

Anumberofnaturalproductshavebeenfoundtoalterglycosylation.Tunicamycinbelongstoaclassofnucleosideantibioticscomposedofuridine,an11-carbondisaccharidecalledaminodeoxydialdose(tunicamine),andafattyacidofvariablelength(13–17carbons),branching,andunsaturation(Figure55.1).TunicamycinwasfirstidentifiedinStreptomyceslysosuperificus,andrelatedcompoundswerefoundlaterinothermicroorganisms.Itderivesitsnamefromitsantiviralactivity,whichoccursbyinhibitingviralcoat(or“tunica”)formation.TunicamycininhibitsN-glycosylationineukaryotesbyblockingthetransferofN-acetylglucosamine-1-phosphate(GlcNAc-1-P)fromUDP-GlcNActodolichol-P(catalyzedbyGlcNAcphosphotransferase;GPT),therebydecreasingtheformationofdolichol-PP-GlcNAc(Chapter9).OtherGlcNActransferasereactionsarenotinhibited(e.g.,GlcNAcTI–V),butthetransferofGlcNAc-1-Ptoundecaprenyl-Pandtheformationofundecaprenyl-PP-MurNAcpentapeptide(whichisinvolvedinbacterialpeptidoglycanbiosynthesis)aresensitivetotunicamycin(Chapter21).Tunicamycinactsasatight-bindingcompetitiveinhibitor,presumablybecauseitresemblesthedonornucleotidesugar.TheKivaluefortunicamycinisabout5×10−8M,whereastheKmvalueforUDP-GlcNAcisapproximately3×10−6M.Theactualamountoftunicamycinneededtoinhibitglycosylationvariesindifferentcells(0.1–10μg/ml),possiblybecauseofvariableuptakeandcultureconditionsordifferencesinthelevelofexpressionofthephosphotransferase.GiventhekeyroleofN-glycosylationinproteinfoldingandqualitycontrolintheER(Chapter39),itisnotsuprisingthattunicamyciniscytotoxictocells,andthatresistantmutantsoverproduceGPT.Similarly,transfectionofcellswiththeclonedGPTconfersresistance,suggestingthatthevariabledoseofinhibitorrequiredindifferentcellsmayreflectvariationinenzymelevels.TunicamycinhasbeenusedextensivelyforstudyingtheroleofN-glycansinglycoproteinmaturation,secretion,andfunction,sinceitsfirstdiscoveryin1973.Thedruginducesapoptosispreferentiallyincancercells,presumablybecauseofalterationsinglycosylationofvariouscell-surfacereceptorsandsignalingmoleculesandbyinducingERstress(Chapter39).Thus,inhibitionofN-glycanformationcouldbeusefulfortreatingcancerpatients.Otherpotentialapplicationsincludesubstratereductiontherapyfortreatmentoflysosomalstoragedisorders(Chapter44),congenitaldisordersofglycosylation(Chapter45),ornaturallyoccurringmutationsthatcreateN-glycosylationsitesincell-surfacereceptors(gain-of-glycosylationmutants;Chapter45).

Amphomycin,alipopeptide,inhibitsdolichol-P-mannosesynthesisbyapparentlyformingcomplexeswiththecarrierlipiddolichol-P.Otherlipophiliccompoundsthatbindlipidintermediatesinbacterialcellwallsynthesisalsohavealsobeenstudied(Chapter21).

PLANTALKALOIDS:NATURALINHIBITORSOFGLYCOSIDASES

PlantalkaloidsblockN-linkedglycosylationbyinhibitingtheprocessingglycosidases(α-glucosidasesandα-mannosidases)involvedintrimmingnascentchains(Table55.2).Unliketunicamycin,whichblocksglycosylationofglycoproteinsentirely,thealkaloidsinhibitthetrimmingreactionsthatoccuraftertheGlc3Man9GlcNAc2oligosaccharideisattachedtoaglycoprotein(Chapter9),resultingintheappearanceofglycoproteinsonthecellsurfacelackingthecharacteristicterminifoundonmatureN-glycans(Chapter14).α-GlucosidaseinhibitorsinvolvedintheinitialprocessingofN-glycansandinqualitycontrolofproteinfolding(Chapters32and39)includecastanospermine(fromtheseedoftheAustralianchestnuttree,Castanosperumaustrale),whichinhibitsα-glucosidasesIandII,australine(alsofromC.australe),whichpreferentiallyinhibitsα-glucosidaseI,anddeoxynojirimycin(fromStreptomycesspecies),whichpreferentiallyinhibitsα-glucosidaseII(Table55.2).Castanospermineandaustralinecauseaccumulationoffullyglucosylatedchains,whereasdeoxynojirimycinresultsinchainscontainingonetotwoglucoseresidues.Unexpectedly,treatingcellswiththeseinhibitorsrevealedthatsometrimmingofthemannoseresiduescouldoccurindependentlyofremovaloftheglucoseresidues(Chapter9).SwainsoninewasfirstdiscoveredinplantsfromthewesternUnitedStates(Astragalusspecies,alsoknownaslocoweed)andAustralia(Swainsonacanescens),andwaslaterfoundinthefungusRhizoctonialeguminocolathatinfectsredclover.Consumptionoftheseplantsinanimalscausesasevereabnormalitycalledlocoismandaccumulationofglycoproteinsinthelymphnodes.Swainsonineinhibitsα-mannosidaseII,causingtheaccumulationofhigh-mannoseoligosaccharides(Man4GlcNAc2andMan5GlcNAc2)andhybrid-typechainsattheexpenseofcomplexoligosaccharides.Inaddition,swainsoninealsoinhibitsthelysosomalα-mannosidase.MannostatinAworksinasimilarway,butdifferssignificantlyinstructurefromswainsonine(Table55.2).Othermannosidaseinhibitorsincludedeoxymannojirimycinandkifunensin,whichselectivelyinhibitα-mannosidaseI.TheseagentscausetheaccumulationofMan7–9GlcNAc2oligosaccharidesonglycoproteins.Alloftheabovelistedinhibitorshaveincommonpolyhydroxylatedringsystemsthatmimictheorientationofhydroxylgroupsinthenaturalsubstrates,butastrictcorrelationbetweenstereochemistryandenzymetarget(α-glucosidasevs.α-mannosidase)doesnotexist.Thecompoundscontainnitrogen,usuallyinplaceof

theringoxygen.OneideaisthatthenitrogenintheprotonatedstatemaymimicthepositivechargeontheringoxygenthatarisesfromdelocalizationofchargefromthetentativecarbocationatC-1generatedduringthehydrolysisreaction.Crystalstructuresfortheα-mannosidaseareavailablewithboundinhibitors.Alkylatedandacylatedanalogsofthealkaloidshaveinterestingandusefulproperties.N-Butylationofdeoxynojirimycinactuallyconvertstheglucosidaseinhibitorintoaninhibitorofglycolipidbiosynthesis.Alkylationoftheaminogrouporacylationofthehydroxylgroupscanimprovethepotencyofthecompound,presumablybyfacilitatinguptakeacrosstheplasmaandGolgimembranes.Someofthesecompoundshaveshownpositiveeffectsfortreatingdiabetes,lysosomalstoragediseases,cancer,andHIVinfection,butalsoinducesmalesterility(seeChapter57).

INHIBITIONOFO-GalNAcINITIATIONOFMUCIN-TYPEGLYCANS

FewinhibitorsareavailablethatblockO-linkedglycanscomparedtoN-linkedglycanbiosynthesis.Mucin-typeO-linkedglycanbiosynthesisisinitiatedbypolypeptidylN-acetylgalactosaminyltransferases(ppGalNAcTs),alargefamilyofenzymesthatuseUDP-GalNAcasacommondonorandvariousglycoproteinacceptors(Chapter10).ScreeningasyntheticlibraryofuridineanalogsagainstmembersoftheppGalNAcTfamilyyieldedtwocompoundsthatdisruptO-GalNAcaddition(Figure55.2).ThesecompoundshaveKivaluesofapproximately8μMwithrespecttoUDP-GalNAc.Liketunicamycin,theseinhibitorssuppressglycosylationwithoutselectivityfordifferentglycoproteintargets.ThesefirstgenerationenzymeinhibitorsworkonO-linkedglycans,andraisethepossibilitythatinhibitorsofspecificppGalNAcTisoformsaswellasothertypesofO-linkedglycans,suchasO-xylose(Chapter17),O-glucose(Chapter18),andO-GlcNAc(Chapter19)maybedevelopedeventually.

INHIBITIONOFO-GlcNAcMODIFICATION

TheimportanceofO-GlcNAcadditiontomanycytoplasmicandnuclearproteins(Chapter19)hasstimulatedgreatinterestindevelopingagentstoinhibititsadditionbyO-GlcNActransferase(OGT)oritsremovalbyO-GlcNAc-specificβ-hexosaminidase(O-GlcNAcase).AlloxanandstreptozotocinaffectO-GlcNAcaddition,butthesecompoundslackspecificity.ThefirstpotentiallyusefulOGTinhibitorswereobtainedbyscreeningchemicallibrariesforcompoundsthatdisplacedafluorescentderivativeofthedonorsugar,UDP-GlcNAc.TheactivecompoundsdonotblockotherN-acetylglucosamineadditionreactions,forexampleoneinvolvedinformationofthepolysaccharidebackboneofbacterialpeptidoglycan(Chapter21).Inaddition,5-S-GlcNAcisanotherinhibitorofOGT(Figure55.3).Itsperacetylatedform(Ac-5SGlcNAc),aswellasasimilarchemicalinhibitor,peracetyl

4-thio-GlcNAc(Ac-4SGlcNAc),crossescellmembranesandundergoesdeacetylationbynon-specificesterasestogenerateactiveinhibitors.SeveralO-GlcNAcaseinhibitorsarebasedonN-acetylglucosamine.Thefirstcompoundinthisclass,PUGNAc(O-[2-acetamido-2-deoxy-D-glucopyranosylidene]amino-N-phenylcarbamate;Figure55.3)inhibitsO-GlcNAcaseatnanomolarconcentrations,butalsoinhibitslysosomalβ-hexosaminidases(HexAandHexB;Chapter44).Thiamet-GandtherelatedN-acetylglucosamine-thiazoline(NAG-thiazoline)aremorespecificandinhibitatlowerconcentrationsthanPUGNAc.Arationallydesignedglucoimidazole,GlcNAcstatin,inhibitsO-GlcNAcasewithaKiof4.6pMandexhibits105-foldselectivityoverHexAandHexB.Thesecompoundsinhibittheenzymeincellsandtissues,providingnewtoolstostudythefunctionofO-GlcNAc,andarepotentialcandidatesfordrugtherapy.

SUBSTRATEANALOGS:DIRECTEDSYNTHESISOFINHIBITORS

Anumberofinhibitorsofspecifictransferaseshavebeendevelopedbasedontheconceptthatsubstrateanalogsmightactastight-bindinginhibitors.Thegeneralstrategyistomodifythehydroxylgroupthatactsasthenucleophileduringformationoftheglycosidicbondorgroupsinitsimmediatevicinity(Table55.3).Manydesignercompoundslackinhibitoryactivity,sincemodificationofthetargetedhydroxylgrouppreventsbindingoftheanalogtotheenzymebyinterferingwithhydrogenbondingnetworksthatpositionthesubstrate.Inothercases,theanalogsexhibitKivaluesintheapproximaterangeoftheKmvaluesfortheunmodifiedsubstrate.Asonemightexpect,theanalogsusuallyactcompetitivelywithrespecttotheunmodifiedsubstrate,butinafewcasestheinhibitionpatternismorecomplex,suggestingpossiblebindingoutsidetheactivesite.Nucleotidesugaranalogsprovideopportunitiesforblockingclassesofenzymesthatutilizeacommondonor(e.g.,allfucosyltransferasesutilizeGDP-fucose).Alargenumberofnucleotidesugarderivativeshavebeenmade(e.g.,N-andO-substitutedanalogsofUDP-GalNAc)andseveralinhibittheenzymesinvitro,buthaveprovenlessusefulinlivingcellsduetopooruptake.“Bisubstrate”analogsconsistofthenucleosidesugardonororPAPScovalentlylinkedtotheacceptorsubstratebywayofaneutralbridginggroup.Thisarrangementmaygenerateinhibitorswhosebindingcharacteristicsreflecttheproductoftheaffinityconstantsfordonorandacceptor(approximatedbytheproductoftheindividualKmvalues).BisubstratesthathavebeenmadehaveKivaluesintherangeoftheKmvaluesforthenucleotidedonors,suggestingthatthecorrectgeometryforthebridginggroupmayhavenotbeenattainedorthattheanalogbindsinwaysthatdifferfromthenaturalsubstrates.Thesearchforactivecompoundsoftenbenefitsfromserendipidyandthesynthesisofdi-,tri-,andtetrasaccharideswiththedesiredmodificationsisratherlabor

intensive.Nevertheless,theapproachhasyieldedinsightsintothebindingandreactivityoftheenzymes,andsubstrateanalogswithselectivityforparticularenzymeshavebeendevelopedinthisway.Becausemanyofthetransferaseshavenowbeenpurifiedandcloned,wecanlookforwardtomoredetailedkineticandcrystallographicstudies,whichwillprovidecluesforderivingmechanism-basedinhibitorsinthefuture(Chapter6).

GLYCOSIDEPRIMERS:MIMICKINGWHATALREADYWORKS

TheutilityofanyglycosyltransferaseinhibitorultimatelydependsonitsabilitytocrosstheplasmamembraneandentertheGolgiwheretheglycosyltransferasesreside.Unfortunately,manyofthecompoundsdescribedabovelackactivityinlivecells,presumablybecausetheirpolarityandchargepreventstheiruptake.Morethan40yearsago,OkayamaandcolleaguesfoundthatD-xyloseinβ-linkagetoahydrophobicaglycone(thenoncarbohydrateportionofaglycoside;e.g.,p-nitrophenol)wastakenupratherefficientlyandinhibitedtheassemblyofglycosaminoglycansonproteoglycans.Xylosidesmimicthenaturalsubstrate,xylosylatedserineresiduesinproteoglycancoreproteins,andthusactasasubstrate.“Priming”ofchainsoccursontheaddedxyloside,whichdivertstheassemblyprocessfromtheendogenouscoreproteinsandcausesinhibitionofproteoglycanformation.Ingeneral,cellsincubatedwithxylosidessecretelargeamountsofindividualglycosaminoglycanchainsandaccumulateproteoglycanscontainingtruncatedchains.Theenormoussuccessofβ-D-xylosidesinalteringproteoglycanbiosynthesissuggestedthatotherglycosidesmightactas“primers”aswell(Table55.4).Subsequentstudieshaveshownthatβ-N-acetylgalactosaminidesprimeoligosaccharidesfoundonmucinsandinhibitO-glycosylationofglycoproteins.Otheractiveglycosidesincludeβ-glucosides,β-galactosides,β-N-acetylglucosaminides,andevendisaccharidesandtrisaccharides.Thesecompoundsrequireconjugationtoappropriateaglyconesandacetylationtoneutralizethepolarhydroxylgroupsonthesugars.CellscontainseveralcarboxyesterasesthatremovetheacetylgroupsandrenderthecompoundsavailabletothetransferasesintheGolgi.Primingbyglycosidesoccursinaconcentration-dependentmanner,buttheefficiencyvarieswidelyamongdifferentcompoundsandcelltypes.Thesevariationsmayrelatetotherelativeabundanceofendogenoussubstrates,enzymeconcentrationandcomposition,thesolubilityofdifferentglycosides,theirsusceptibilitytohydrolysis,theiruptakeacrosstheplasmamembraneandintotheGolgi,andtheirrelativeaffinityfortheglycosyltransferases.Thetypeofchainmadeonagivenprimeralsodependsonconcentrationandaglyconestructure,whichmayreflectselectivepartitioningofprimersintodifferentintracellularcompartmentsorintodifferentbranchesofbiosyntheticpathways.Likepriming,inhibitionofglycoprotein,glycolipid,orproteoglycanformationoccursinadose-dependent

fashion,buttheblockadeisrarelycomplete,probablybecauseoftheinabilityofglycosidestomimictheentireendogenoussubstrates.Primersrepresentstartingpointsformakinganalogswithtight-bindinginhibitorpropertiesdescribedabove.ManyofthecompoundsdescribedinTable55.3couldbeconvertedtopermeableacylatedglycosidesandtestedinlivecellsforinhibitoryactivity.Activecompoundscouldpotentiallybecomecarbohydrate-baseddrugstotreatglycosylation-dependentdiseases.Oligosaccharideprimingmayhavebeneficialeffectsaswell.Xylosides,forexample,canbeabsorbedthroughthegut,andwhenconsumedatsufficientconcentration,theyexhibitantithromboticactivity.Manyglycosidesoccurnaturally,sincevariousorganisms(especiallyplants)producehydrophobiccompoundsaspartofchemicaldefenseandconjugatethemtosugarsinordertorenderthemsoluble.Thus,thehumandietmaycontainvarioustypesofglycosideswithinteresting(andunknown)biologicalactivities.Caremustbetakenininterpretingtheresultsofexperimentsutilizingglycosideprimers.Forexample, β-D-xylosidesalsoprimeglycansrelatedinstructuretoglycosphingolipidsandHNK-1.Insomecasesprimingperseisnotthemechanismresponsibleforinhibitionofglycosylation,butratherinhibitionoccursduetocompetitivebindingoftheprimertoatargetenzyme.Finally,primerscoulddepletecellsofnucleotidesugarsandhavemultipleeffectsonglycosylation.Forexample,4-methyl-umbelliferoneisoftenusedtoblockhyaluronanbiosynthesis.Theprecisemechanismofactionisunknown,butisthoughttoinvolvedepletionofUDP-GlcAduetoglucuronidationofthecompound.ReductionofUDP-GlcAinturncouldaffectformationofsulfatedglycosaminoglycansandotherglucuronicacidcontainingglycansandalterthepoolsofothernucleotidesugars,suchasUDP-GlcNAc.

INHIBITORSOFGLYCOLIPIDSANDGPIANCHORS

Reagentsthatcanaltertheassemblyofglycolipidsincellshavebeendescribed.Xylosideshaveamildeffectonglycolipidformation,possiblybecauseofthesimilaritybetweenxyloseandglucoseandtheassemblyofaGM3-likecompound(Neu5Acα2–3Galβ1–4Xylβ-O-R)ontheprimer.Becausecellswilltakeupintermediatesinglycolipidbiosynthesis,theybehavelikesyntheticglycosideprimers.Forexample,glucosylceramidewillgiverisetocomplexglycolipidswhenfedtocells.Onthebasisofthisobservation,ananalogcontainingareactiveexocyclicepoxidegroupwasprepared.Thecompoundinhibitsglycolipidformation(IC50~8μM),presumablybyreactionoftheepoxidewithanucleophileintheactivesiteoflactosylceramidesynthase.D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol(D-PDMP),ananalogofglucosylceramide,wassynthesizedoriginallytoinhibitglucosylceramidesynthesisinpatientswithGaucherdisease.However,thiscompounddirectlyinhibitstheactivityofpurifiedlactosylceramidesynthase.

Asmentionedabove,theα-glucosidaseinhibitor,N-butyldeoxynojirimycin,hasbeenusedtoinhibitglycosphingolipidformationbecauseitblocksglucosylceramidesynthesis.ThiscompoundisnowinclinicalusefortreatingtypeIGaucher’sdisease,alysosomalstoragedisorderinwhichglucocerebrosidaseismissing(Chapter44).Itsbeneficialactivityoccursthrough“substratedeprivation”byblockingsynthesisofglycosphingolipids,thereby“depriving”thelysosomeofsubstrate.Sphingolipidanalogs(Figure55.4)aremorepotentinhibitors.Lengtheningthehydrocarbonchainfrom10to16carbonsfurtherenhancesefficacy.Thesecompoundscanalmostcompletelydepleteglycolipidsincells,andrequireonlymicromolarconcentrationsforactivity.InhibitorsofGPIanchorformationhavealsobeendescribed.Mannoseanalogs(2-deoxy-2-fluoro-D-glucoseand2-deoxy-D-glucose)inhibittheformationofdolichol-P-mannoseinvivoandthusinhibitGPIbiosynthesis.However,theylackspecificitybecausetheseagentsalsocanaffectotherpathwaysdependentondolichol-P-mannose.MannosamineinhibitsGPIanchorformationbothinTrypanosomabruceiandinmammaliancellsbytheformationofManNH2-Man-GlcN-PI.Apparently,mannosamineinitsactivatedform(GDP-ManNH2)isusedasasubstrateinthesecondmannosyltransferasereaction,buttheManNH2-Man-GlcN-PIintermediatewillnotactasasubstrateforthenextα2-mannosyl-transferase(Chapter12).GlcNR-phosphatidylinositolswithdifferentsubstituents(R)actassubstrateanalogsandsomeactassuicideinhibitorsinvitro.AnotherclassofinhibitorsisbasedonfattyacidanalogsthatonlytrypanosomesincorporateintoGPIanchors.Trypanosomes,unliketheirmammalianhosts,incorporatemyristicacidintoGPIanchorsbyexchangingmyristicacidforotherfattyacidsinthephosphatidylinositolmoiety.Bymakingaseriesofanalogs,aninhibitorwasfoundthatishighlytoxictotrypanosomesincultureandnontoxictomammaliancells(10-[propoxy]decanoicacid).Suchreagentsaredrugcandidatesfortreatingtrypanosomiasis,whichisendemicinsub-SaharanregionsofAfrica.

RATIONALDESIGNUSINGCRYSTALSTRUCTURES

Neuraminidase(Sialidase)Inhibitors

Studiesofinfluenzaneuraminidaseexemplifythepowerofrationallydesigneddrugs.Thecrystalstructureforinfluenzaneuraminidasewasobtainedin1983,andsincethenmanyotherenzymeshavebeencharacterizedfromothersources.Evenbeforethecrystalstructurehadbeenobtained,aneuraminidaseinhibitorwasdeducedbyassumingthatthehydrolysisreactionprobablyinvolvedatransitionstatewithacarbocationintermediateatC-2.ThiswouldresultinC-2andC-3adoptingatrigonalplanar(sp2)configuration,andthereforecompoundsthatmimickedthisgeometrywerehopedtohaveinhibitoryactivity.Indeed,Neu5Ac-2-

ene(DANA;Figure55.5)hasamicromolarKivalue.Interestingly,thiscompoundworksonmostsialidases,butnotonthetrypanosometrans-sialidaseandonlyweaklyonbacterialsialidases.Visualinspectionofinfluenzaneuraminidasewiththeinhibitorboundshowedthattwoglutamateresidueslinedapocketnearcarbon4ofthesialicacidanalog(Figure55.6).Thepocketwasfairlyopen,suggestingthatabulkiersubstituentatthispositionmightbetolerated,atleaststerically.Asubstrateanalogwasproducedcontainingapositivelychargedguanidiniumgroupinsteadofthehydroxylatcarbon4(4-guanidino-DANA;Figure55.5),creatingananalogwithremarkableinfluenzaneuraminidaseinhibitorpotency(Kivalueof10−11M).Thehigheraffinitywaspresumablyduetoanadditionalsaltbridgeformedbetweenthechargedguanidiniumgroupandthecarboxylatesliningthepocket.Theanalogisnearlyamilliontimeslesspotentonhumansialidases,leadingtoitsuseastheanti-influenzadrugRelenza®(Chapter57).Itdoesnotworkonbacterialsialidases,however,becausetheequivalentpocketisfilledwithanargininegroup.Subsequentstudiessubstitutedacyclohexenetomimictheplanarringoftheproposedintermediateinhydrolysis.Goodinhibitorswerefoundandanorallyactiveanalogiswidelyused,theoralanti-influenzadrugTamiflu®(Figure55.5,Chapter57).Asthecrystalstructuresforothersialidasesaresolved,thedesignofspecies-specificanalogsmaybepossible.Thisrationalapproachtoinhibitordesignholdsgreatpotential,notonlyforneuraminidaseinhibitors,butalsoforthedesignofcompoundsthatmightblocktheactivityofvariousglycosyltransferases.

SulfotransferaseInhibitors

AlargefamilyofGolgisulfotransferasesinstallssulfateestersonavarietyofglycansusingPAPSastheactivesulfatedonor.High-resolutioncrystalstructuresarenowavailableforbothglycan-modifyingsulfotransferasesaswellassolubledrugdetoxifyingsulfotransferases.TheseenzymeshaveincommonaconservedfoldthatbindsPAPS(seeChapter6).Aseriesofsmallaromaticcompounds(suchas2,6-dichloro-4-nitrophenolandpentachlorophenol)andseveraldisaccharideanalogsofGlcNAc-6-sulfotransferasesubstrateshaveinhibitoryactivity.Screeningtargetedlibrariesofpurinederivativesyieldedcompoundswithhighselectivitytowardsindividualsulfotransferases,suggestingthatsubtledifferencesinthePAPS-bindingsitescanbeexploited.Theabilitytoscreenlargelibrariesagainstsulfotransferaseshasbeenfacilitatedbythedevelopmentofhigh-throughputassays.Mostlow-throughputassaysmeasurethetransferof[35S]from[35S]PAPSormeasuretheradiolabeltransfertoacarbohydratesubstratebearingahydrophobictailthatcaneasilybeisolatedusingareverse-phasecartridge.Somesulfotransferaseswillcatalyzethereversereactioninthepresenceofhighconcentrationsofasulfateddonor.Forexample,β-

TABLE55.1ClassesofinhibitorsClassofinhibitor/modulator TargetMetabolicinhibitors Stepsinvolvedinformationofcommonintermediates

suchasPAPSornucleotidesugarsTunicamycin N-linkedglycosylationthroughinhibitionofdolichol-PP-

GlcNAcformation;peptidoglycanbiosynthesisthroughinhibitionofundecaprenyl-PP-GlcNAcassembly

Plantalkaloids N-linkedglycosylationthroughinhibitionofprocessingglycosidases

Substrateanalogs SpecificglycosyltransferasesorglycosidasesGlycosideprimers Glycosylationpathwaysbydivertingtheassemblyof

glycansfromendogenousacceptorstoexogenousprimers

Table55.2ExamplesofalkaloidsthatinhibitglycosidasesinvolvedinN-linkedglycanbiosynthesisAlkaloid Source TargetAustraline

α-glucosidaseI

Castanospermine

α-glucosidaseIandII

Deoxynojirimycin

α-glucosidaseII(andI)

Deoxymannojirimycin

α-mannosidaseI

arylsulfotransferaseIV(β-AST-IV)willcatalyzethereversetransferofthesulfurylgroupfromp-nitrophenolsulfatetoPAP,generatingPAPSandp–nitrophenolateion.Whencoupledtoanothersulfotransferaseofinterest,β-AST-IVregeneratesPAPSandstoichiometricamountsoftheion,whichcanbemonitoredbyUVabsorbance.Theenzymealsowilltransfersulfatetowater.Alibraryof35,000compoundswithpurineandpyrimidinescaffoldswasscreenedusingβ-AST-IVandafluorescenceassaythatmeasureddesulfationof4-methylumbelliferonesulfate.Multiplehitswereobtainedwithmoderateinhibition,andsubsequentstructureelaborationofthelibraryresultedinthegenerationofaverytightbindingsmallmoleculeinhibitorwithaKmvaluefiveordersofmagnitudelowerthenthenaturalsubstrate.Intheory,thisapproachcanbeexploitedforotherenzymesforwhichhigh-throughputassayscanbedeveloped.

ACKNOWLEDGEMENTS

TheauthorsappreciatehelpfulcommentsandsuggestionsfromJarrodBarnesandMarildaLisboa.

FURTHERREADING

KallemeijnWW,WitteMD,WennekesT,AertsJM.2014.Mechanism-basedinhibitorsofglycosidases:designandapplications.AdvCarbohydrChemBiochem71:297-338.

BrownJR,CrawfordBE,EskoJD.2007.Glycanantagonistsandinhibitors:afountfordrugdiscovery.CritRevBiochemMolBiol42:481-515.

ChapmanE,BestMD,HansonSR,WongCH.2004.Sulfotransferases:structure,mechanism,biologicalactivity,inhibition,andsyntheticutility.AngewChemIntEdEngl43:3526-3548.

ChaudharyPM,TupeSG,DeshpandeMV.2013.Chitinsynthaseinhibitorsasantifungalagents.MiniRevMedChem13:222-236.

DorfmuellerHC,BorodkinVS,SchimplM,ShepherdSM,ShpiroNA,vanAaltenDM.2006.GlcNAcstatin:apicomolar,selectiveO-GlcNAcaseinhibitorthatmodulatesintracellularO-glcNAcylationlevels.JAmChemSoc128:16484-16485.

GalleyNF,O'ReillyAM,RoperDI.2014.Prospectsfornovelinhibitorsofpeptidoglycantransglycosylases.BioorgChem55:16-26.

GouinSG.2014.Multivalentinhibitorsforcarbohydrate-processingenzymes:beyondthe"lock-and-key"concept.Chemistry20:11616-11628.

KimEJ,BondMR,LoveDC,HanoverJA.2014.Chemicaltoolstoexplorenutrient-drivenO-GlcNAccycling.CritRevBiochemMolBiol49:327-342.

ShaymanJA,LarsenSD.2014.Thedevelopmentanduseofsmallmoleculeinhibitorsofglycosphingolipidmetabolismforlysosomalstoragediseases.JLipidRes55:1215-1225.

TuZ,LinYN,LinCH.2013.Developmentoffucosyltransferaseandfucosidaseinhibitors.ChemSocRev42:4459-4475.

FigureLegends

FIGURE55.1.Structureoftunicamycin,whichconsistsofuridineconjugatedtothedisaccharide,tunicamine.

FIGURE55.2.Broad-spectruminhibitorsoftheppGalNAcTsidentifiedfromscreeningauridine-basedlibrary.

FIGURE55.3.InhibitorsofO-GlcNAc-specificβ-hexosaminidase(OGA)andO-GlcNActransferase(OGT).

FIGURE55.4.Inhibitorsofglycosphingolipidformation.Theseanalogsarepotentinhibitorsoftheglucosyltransferasethatinitiatesglycosphingolipidformation.

FIGURE55.5.Structureofinfluenzaneuraminidaseinhibitors.ChemicalstructureofNeu5Ac,NANA;2-deoxy-2,3-dehydro-N-acetylneuraminicacid,DANA;4-amino-DANA;4-guanidino-DANA(Relenza®,zanamivir);(3R,4R,5S)-4-acetamido-5-amino-3-(1-ethylpropoxyl)-1-cyclohexane-1-carboxylicacidethylester(GS4104,Tamiflu®,oseltamivir).DANAisthoughttoresemblethetransitionstateinhydrolysis,andadditionoftheguanidiniumgroupinRelenzaprovideshigheraffinitybindingtotheactivesite.TheethylesterinTamifluenhancesoralavailability,thenisquicklyremovedinthebodybynon-specificesterases.

FIGURE55.6.Crystallographicstructuresofinfluenzavirusneuraminidase(N9subtype)withtwodifferentrationallydesignedinhibitorsboundintheactivesite.Theinhibitorsareshownascoloredballandstickmodels,whereyellowiscarbon,blueisnitrogen,andredisoxygen.Thecatalyticsiteoftheenzymeisshownwiththeclosercarbonatomsindarkgreenandthosefurtherawayinlightgreen.(a)Relenza(4-guanidino-Neu5Ac2en).(b)De-esterifiedTamiflu.(Redrawn,withpermission,fromGarmanE.andLaverG.2004.Curr.DrugTargets5:119–136.)

Alkaloid Source TargetKifunensin

α-mannosidaseI

Swainsonine

α-mannosidaseII

MannostatinA

α-mannosidaseII

TABLE55.3Syntheticsubstrate-basedinhibitorsofglycosyltransferases

Enzyme Substrate InhibitorSubstrateKm(μM)

InhibitorKi(μM)

α2FucT β3GlcNAcβ-O-R 2-deoxyGalβ3GlcNAcβ-O-R

200 800

β4GalT GlcNAcβ3Galβ-O-R 6-thioGlcNAcβ3Galβ-O-Me

1000 1000

α3GalT Galβ4GlcNAcβ-O-R 3-aminoGalβ4GlcNAcβ-O-R

190 104a

β6GlcNAcT Galβ3GalNAcα-O-R Galβ3(6-deoxy)GalNAcα-O-R

80 560

β6GlcNAcT-V

GlcNAcβ2Manα6Glcβ-O-R

GlcNAcβ2(6-deoxy)Manα6Glcβ-O-R

23 30

β6GlcNAcT-V

GlcNAcβ2Manα6Glcβ-O-R

GlcNAcβ2(4-O-methyl)Manα6Glcβ-O-R

23 14

β6GlcNAcT-V

GlcNAcβ2Manα6Glcβ-O-R

GlcNAcβ2(6-deoxy,4-O-Me)Manα6Glcβ-O-R

23 3

α6SialylT Galβ4GlcNAcβ-O-R 6-deoxyGalβ2GlcNAcβ-O-R

900 760a

α3GalNAcT-A

Fucα2Galβ-O-R Fucα2(3-deoxy)Galβ-O-R

2 68

α3GalNAcT-A

Fucα2Galβ-O-R Fucα2(3-amino)Galβ-O-R

2 0.2a

Theaglycone(R)variesinthedifferentcompounds.aInhibitionmixedornoncompetitive.TABLE55.4ExamplesofglycosideprimersGlycoside PathwayaffectedXylβ-O-R Glycosaminoglycans,glycolipidsGalβ-O-R GlycosaminoglycansGalNAcα-O-R O-linkedchainsfoundonglycoproteinsand

mucinsGlcNAcβ-O-R PolylactosaminoglycansPeracetylatedGalβ4GlcNAcβ-O-R

SialylLewisx

PeracetylatedGlcNAcβ3Galβ-O-R

SialylLewisx

Chapter57,GlycansinBiotechnologyandthePharmaceuticalIndustryEssentialsinGlycobiology,3rdedition

Chapter57

GlycansinBiotechnologyandthePharmaceuticalIndustry

Authors:PeterH.Seeberger,RichardD.CummingsSeveralclassesofsuccessfulcommercialproductsarebasedonisolatedorsyntheticglycans.Thischaptersummarizestheuseofglycansasvaccinesandtherapeutics.Applicationsofglycanmimicsasdrugsisalsodiscussed.GLYCANSASCOMPONENTSOFSMALL-MOLECULEDRUGSManywell-knownsmall-moleculedrugs,suchasantibioticsandanticancertherapeuticagents,arenaturalproductsthatcontainglycansaspartoftheircorestructureand/orasasugarsidechain(i.e.,aglycoside).SomeexamplesofnaturalproductsthatbearglycansidechainsareshowninFigure57.1.Thewell-establishedareaofnaturalproductchemistrywillnotbereviewedindetailhere.Modifiedglycansgaverisetosyntheticdrugssuchassmall-moleculeinhibitorsofinfluenzavirusneuraminidase(seealsoChapter55).Recentadvancesinthefunctionalunderstandingofcarbohydrate–proteininteractionshaveenabledthedevelopmentofglycomimetics,anewclassofsmall-moleculedrugsthatarebrieflydescribed.Small-MoleculeInhibitorsofInfluenzaVirusNeuraminidaseInfluenzavirushastwomajorsurfaceproteins,hemagglutininandneuraminidase(seeChapter34).Thehemagglutinininitiatesinfectionbybindingtocell-surfacesialicacids.Theneuraminidaseassistsvirusreleasebycleavingsialicacidstopreventunwantedretentionofnewlysynthesizedvirusonthecellsurface.Neuraminidasemayalsofunctionduringtheinvasionphasebyremovingsialicacidsonsolublemucinsthatwouldotherwiseinhibitcell-surfacebinding.Sinceneuraminidaseisessentialtothevirallifecycle,basedonthecrystalstructureoftheenzymearationaldrugdesignprogramyieldedZanamivir(Relenza™).TheadditionofabulkyguanidinosidechainatC-4ofapreviouslyknownneuraminidaseinhibitor,2-deoxy-2,3-dehydro-N-acetyl-neuraminicacid,markedlyincreasedtheaffinityforinfluenzaneuraminidase,withoutaffectinghost-cellneuraminidases(Figure57.2;seealsoChapter55).Relenzablockstheinfluenzaviruslifecyclebypreventinginfectionandbyinterruptingthespreadofthevirusduringtheearlyphaseofaninfection.Duetopoororalavailability,Relenzahastobeinhaledtoworkatthemucosalsitesofinfectionintheupperairway.TheorallyavailabledrugOseltamivir(Tamiflu™)achievesthesameeffectsandhastakenovermostofthemarketduetotheeaseofuse.Thefearofavianinfluenzavirus(“birdflu”)spreadingintohumanpopulationshaspromptedstockpilingofTamiflu.Fortunately,widespreaduseofthedrughasnotbecomenecessarytodate.ThedevelopmentofTamifluisatextbookexampleforrationaldrugdesignresultinginapowerfuldrugagainstadevastatingdisease.

THERAPEUTICGLYCOPROTEINSMostbiotherapeuticproductsareglycoproteinsandincludeerythropoietinaswellasvariousothercytokines,antibodies,glycosyltransferases,andglycosidases.ThisclassofmoleculessellsinthetensofbillionsUSDperyearworldwide.Therapeuticglycoproteinsaretypicallyproducedasrecombinantlyincellculturesystemsor,lesscommonly,inthemilkoftransgenicanimals.Controlofglycosylationisofmajorimportanceduringthedevelopmentofthesedrugs,becausetheirglycanchainshavemarkedeffectsonstability,activity,antigenicity,andpharmacodynamicsinintactorganisms.Inmostcases,glycosylationmustbeoptimizedtoensureprolongedcirculatoryhalf-lifeintheblood.Manipulationofglycanstopromotetargetingtospecifictissuesandcelltypeshasalsobeenausefulelementofdrugdesign.OptimizingGlycansofTherapeuticGlycoproteinsforProlongedSerumHalf-lifeErythropoietin(EPO)isthemostsuccessfulbiotechnologyproducttodate.Itisacirculatingcytokinethatbindstotheerythropoietinreceptor,inducingproliferationanddifferentiationoferythroidprogenitorsinthebonemarrow.EPOwasdevelopedtotreatanemiascausedbybonemarrowsuppressionafterchemotherapyorlackoferythropoietin(e.g.,renalfailure).NaturalandrecombinantformsoferythropoietincarrythreesialylatedcomplexN-glycansandonesialylatedO-glycan.Althoughinvitrotheactivityofdeglycosylatederythropoietiniscomparabletothatofthefullyglycosylatedmolecule,itsactivityinvivoisreducedbyabout90%,becausepoorlyglycosylatederythropoietinisrapidlyclearedbyfiltrationinthekidney.Undersialylatederythropoietinisalsorapidlyclearedbygalactosereceptorsinhepatocytesandmacrophages(seeChapter31).Fullysialylatedchainsandincreasedtetra-antennarybranchingreducestheseproblemsandincreasesEPOactivityinvivonearlytenfold.AdditionofanN-glycosylationsitealsoincreaseshalf-lifeandactivityinvivo.Covalentlylinkingpolyethyleneglycoltotheproteinalsoreducesclearancebythekidney.Erythropoietinisunusualbecauseitissmallenoughtobeclearedbythekidneyifitisunderglycosylated.Formostglycoproteintherapeutics,amoreimportantconsiderationisminimizingclearancebygalactose-bindinghepaticreceptorsbyensuringfullsialylationofglycans.Becauseglycansgreatlyinfluencetheefficacyofthesedrugs,controloverglycosylationduringproductionisvryimportantinlightofregulatoryrequirementsforbatch-to-batchproductconsistency.ChangesinculturepH,theavailabilityofprecursorsandnutrients,andthepresenceorabsenceofvariousgrowthfactorsandhormonescaneachaffecttheextentofglycosylation,thedegreeofbranching,andthecompletenessofsialylation.Sialidasesandotherglycosidasesthatareeithersecretedorreleasedbydeadcellscanalsocausedegradationofthepreviouslyintactproductintheculturemedium.Theseissueswerehotlydebatedinrecentyearswiththeadventof“biosimilars”orgenericversionsofglycoproteins.Theneedtoproofcompositionhasfueledeffortsdevotedtoglycananalysisandsequencing.

ImpactofGlycosylationonLicensingandPatentabilityofBiotherapeuticAgentsTherapeuticGlycoproteinsforProlongedSerumHalf-lifePatentingofnewtherapeuticsistypicallybasedonthecompositionofmatterintheclaimedmolecule.Smallmoleculesofdefinedstructureandnonglycosylatedproteinsareeasilycapturedinthismanner.However,glycoproteins,especiallythosewithmultipleglycosylationsites,renderitvirtuallyimpossibletoobtainpreparationsthatcontainonlyasingleglycoform.Thus,mostbiotherapeuticglycoproteinsconsistofamixtureofglycoforms.Licensingbodiesallowforacertainrangeofvariationinglycoformsandthecomplexityofthemixture.However,themanufacturerandtheagencymustagreeontheextentsuchvariationisacceptableforagivendrugformulation.Biopharmaceuticalcompaniesthereforespendconsiderableeffortinassuringthattheirproductsfallwithinthesedefinedranges,oncetheseareapprovedbylicensingbodies.Theinherentdifficultyinreproducingcomplexglycoformmixturesalsocomplicateseffortstomakegenericformsofrecombinantglycoproteindrugs.Giventhecomplexitiesofproducingglycotherapeuticagentsinmammaliancells,eventhesmallestchangesingrowthconditionscanhavesignificanteffectsontherangeofglycoformsfoundinanygivenproductbatch.Thelicensingagenciesuseconsistencyinglycoformcompositionasanindirectmeasureofthequalityofprocesscontrolinproduction.Differencesinglycosylationcanhaveimplicationsforthepatentabilityofagentswherethepolypeptideremainsconstant.Markeddifferencesinglycosylationhavebeenusedtodefineagentsasbeinguniquelydifferent.However,itisusuallynecessarytoshowthatthedifferencesinglycosylationbeingclaimedalsohaveasignificanteffectinchangingthefunctionalityofthedruginquestion.Theassociatedpharmaceuticallicensingandlegalissuesarerapidlyevolvingtokeeppacewithscientificadvancesinthisarea.

GLYCOSYLATIONENGINEERING

TTherearelimitsastohowmuchofabiotherapeuticglycoproteinananimalcelllinecanproduce.Productionbecomesanissueincaseswhereverylargeamountsofaparticularglycoproteinisneeded.Glycoproteinproductioninplantsoryeastisattractivebutmakesitnecessarytoeliminaterisksarisingfromthenonhumanglycansofplantandfungalcellsthatcouldcauseexcessivelyrapidclearanceand/orantigenicreactions.Manyplantandyeastglycansareimmunogenicandelicitglycan-specificIgEandIgGantibodiesinhumanswhendeliveredparenterally.Avarietyofmammaliangeneshavebeenaddedbackintoyeastand/orgenesthatareproducingnonhumanglycosylationhavebeneliminated.ExtensivelyengineeredyeaststrainsarecapableofproducingbiantennaryN-linkedglycanswiththehumansialicacidN-acetylneuraminicacid(Neu5Ac)buttheproductivityofsuchyeaststrainsareoftenlow.Effortstoengineeryeasttomakehuman-likeO-glycansareunderway,butglycosaminoglycanshavenotyetbeenaddressed.

Plantsandalgaehavealsobeenusedtoengineerrecombinantglycoproteins,but,asinyeast,theglycansproducedbyplantsdifferfromthosefoundinvertebrates.Theantigenicdifferencesthatariseinrecombinantglycoproteinsproducedinplantsbecomelessproblematicifusedfortopicalororaladministration,sincehumansarenormallyexposedtoplantglycansinthediet.Thecostofproductionismuchlowerthaninanimalcellculturesystemsandanimalseraarenotneeded.Asinyeast,“humanizingplants”withrespecttoglycosylationmayallowtheproductionofnonimmunogenicglycoproteins.ChemicalmethodsforsynthesizingentireglycoproteinsfromscratchhavebeendevelopedandsingleglycoformsofEPOhavebeenpreparedbytotalsynthesis.Giventhecomplexityofglycoproteinsynthesis,scaleupoftheseprocessesischallengingandanareaofintenseresearchactivity.(seeChapter49).

GLYCANTHERAPEUTICAPPROACHESTOMETABOLICDISEASES

SalvageversusDeNovoSynthesisAllmonosaccharidesneededforcellularglycansynthesiscanbeobtainedfromglucosethroughmetabolicinterconversions(seeChapter4).Alternatively,monosaccharidescanbederivedfromthedietorsalvagedfromdegradedglycans.Therelativecontributionsofdifferentsourcescanvarywiththecelltype.Forinstance,eventhoughallmammaliancellsusesialicacid,onlysomecontainhighamountsofUDP-GlcNAcepimerase/N-acetylmannosaminekinase(GNE),whichisrequiredforthedenovosynthesisofCMP-sialicacid.Butsialicacidsalvagefromdegradedglycansisquiteefficient,decreasingthedemandonthedenovopathway.Similarly,galactose,fucose,mannose,N-acetylglucosamine,andN-acetylgalactosaminecancomefromthedietorbesalvagedforglycansynthesis,whereasglucuronicacid,iduronicacid,andxylosecannot.Allmonosaccharidesderivedfromthedietordegradedglycanscanbecatabolizedforenergy,andagain,cellsvaryintheirrelianceonthedifferentpathways.Thevariablecontributionsofthesepathwaysareimportantfortherapyofsomediseases.Forinstance,patientswithcongenitaldisorderofglycosylationtypeIb(CDG-Ib),whoaredeficientinphosphomannoseisomerase,benefitgreatlyfromoralmannosesupplementationtobypasstheinsufficientsupplyofglucose-derivedmannose-6-phosphate.AfewCDG-IIcpatientshavebeentreatedwithfucosetorestoresynthesisofsialylLewisxonleukocytes(seeChapter42).SomepatientswithCrohn’sdiseaseshowclinicalimprovementwithoralN-acetylglucosaminesupplementation,butthemechanismisunknown.MicedeficientinGNEactivityhavekidneyfailure,butprovidingN-acetylmannosamineinthedietpreventsthisoutcome.ClinicaltrialsusingN-acetylmannosaminetotreatGNE-deficientpatientswithhereditaryinclusionbodymyopathytypeII(HIBM-II)havebeenconductedbuthaveyieldedinconclusiveresults.SpecialDiets

Somemonosaccharidesanddisaccharidescanbetoxictohumanswholackspecificenzymes.Forexample,peoplewholackfructoaldolase(aldolaseB)accumulatefructose-1-phosphate,whichultimatelycausesATPdepletionanddisruptsglycogenmetabolism.Prolongedfructoseexposureinthesepeoplecanbefatal,andfructose-limiteddietsarecritical.Deficienciesintheabilitytometabolizegalactose(seeChapter4)aremostlyduetoaseverereductioningalactose-1-phosphateuridyltransferaseactivityandcausegalactosemia.Althoughthesepatientsareasymptomaticatbirth,ingestingmilkleadstovomitinganddiarrhea,cataracts,hepatomegaly,andevenneonataldeath.Low-galactoseorgalactose-freedietscanpreventtheselife-threateningsymptoms.However,eventhesedietsdonotpreventunexplainedlong-termcomplications,whichincludespeechandlearningdisabilitiesandovarianfailureinfemaleswithgalactosemia.Infantshydrolyzelactose(Galβ1-4Glc)quitewell,butthelevelofintestinallactasecanbemuchlowerorabsentinadultsbecauseofdown-regulationoflactasegeneexpression.Abouttwothirdsofthehumanpopulationhaslactasenonpersistence,makingmilkproductsadietaryannoyance.Unabsorbedlactoseprovidesanosmoticloadandismetabolizedbycolonicbacteria,causingdiarrhea,abdominalbloating,flatulence,andnausea.LactasepersistencehasevolvedincertainpastoralpopulationsfromnorthwesternEurope,India,andAfrica,allowingmilkconsumptioninadultlife.However,manyadultseitheravoidlactose-containingfoodsoruselactasetabletstoimprovelactosedigestion.SubstrateReductionTherapyThefailuretoturnoverglycansbylysosomaldegradationcausesseriousproblemsforpatientswithlysosomalstoragedisorders.Deficienciesinindividuallysosomalenzymesleadtopathologicalaccumulationoftheirsubstratesininclusionbodiesinsidethecells(seeChapter41).Oneapproachtotreatingthesedisordersistoinhibitinitialglycansynthesis,astrategytermedsubstratereductiontherapy(SRT).Reducedsynthesisoftheinitialcompounddecreasestheloadontheimpairedenzyme,andsomepatientsshowsignificantclinicalimprovement.AsmallmoleculedrugusedforSRTisN-butyldeoxynojirimycin(orN-butyl-DNJ)(Miglustat,Zavesca®),thatwasapprovedin2002fortreatmentofGaucher’sdisease(glucocerebrosidasedeficiency).LysosomalEnzymeReplacementTherapyAnotherapproachfortreatinglysosomalstoragedisordersisenzymereplacementtherapy.Unlikemosttherapeuticglycoproteinsthatinteractwithtargetreceptorsonthesurfaceofcells,lysosomalenzymesdevelopedforreplacementtherapymustbedeliveredintracellularlytolysosomes,theirsiteofaction.Duringthenormalbiosynthesisoflysosomalenzymes,theirN-glycansbecomemodifiedwithmannose-6-phosphate(Man-6-P)residues,whichtargetthemtolysosomesusingMan-6-Preceptors(seeChapter30).Thechallengeforenzymereplacementtherapyistogettheenzymestargetedproperlytolysosomes,wheretheycandegradeaccumulatedsubstrate.EnzymereplacementtherapyforGaucher’sdiseasetargetsthelysosomes

ofmacrophagesviathecell-surfacemannosereceptor(seeChapter31).ThefourrecombinantenzymeproductsImiglucerase(approvedin1995),Velaglucerase(approvedin2010),Taliglucerasealfa(Elelyso,approvedin2012),andEliglustat(Cerdelga,approvedin2014)aremarketed.ThesuccessofglucocerebrosidasetreatmentstimulatedthedevelopmentoflysosomalenzymesfortreatmentofotherlysosomalstoragediseasessuchasFabry’sdisease,mucopolysaccharidosestypeI,II,andVI,andPompe’sdisease.Thereplacementtherapiesclearlyhavebeneficialeffectsandprolonglifebutareextremelyexpensive.ChaperoneTherapyAthirdapproachfortreatinglysosomalstoragedisorderstakesadvantageofthefactthatsomegeneticdefectsleadtomisfoldingoftheencodedenzymeintheendoplasmicreticulum(ER).Low-molecular-weightcompetitiveinhibitorsofsomeoftheseenzymescanactas“chaperones,”thatstabilizethefoldedenzymeintheERandeffectivelyrescuethemutationandincreasethesteady-stateconcentrationofactiveenzymeinthelysosome.Thedoseoftheinhibitormustbecarefullyadjustedtoensurethattheinhibitoryeffectsonenzymefunctiondonotovershadowbeneficialeffectsonfolding.Onlyalowlevelofenzymerestorationisneededtosignificantlyreducetheaccumulationofundigestedglycansubstrates,indicatingthatlysosomalhydrolasesarenormallypresentinlargecatalyticexcess.

THERAPEUTICAPPLICATIONSOFGLYCOSAMINOGLYCANS

Theuseofpurifiedglycansastherapeuticshasreceivedlessattentionthanthedevelopmentofglycoprotein-basedtreatments.Difficultiesinestablishingstructure–activityrelationshipsduetothelargenumberofchiralcentersandfunctionalgroups,undesirablepharmacokineticsofavailableformulations,poororalabsorptionofthecompounds,andlow-affinityinteractionswithdrugtargetshavelimitedtheirdevelopment.Somesuccessfulglycandrugs,suchastheanticoagulantheparin,aregivenbyinjection,althougheffortsareunderwaytoconvertheparinintoanorallyabsorbableformbycomplexingitwithpositivelychargedmolecules.Itmaybepossibletodeliverotherhydrophilicand/ornegativelychargedglycandrugsinthiswaytoallowpenetrationoftheintestinalbarrier.Glycansarealsosometimesattachedtohydrophobicdrugstoimprovetheirsolubilityandaltertheirpharmacokinetics.TheanticoagulantheparinisasdiscussedinChapters16and43,oneofthemostwidelyprescribeddrugstoday.Heparinbindsandactivatesantithrombin,aproteaseinhibitorofthecoagulationcascade.AntithrombinactivationleadstorapidinhibitionofthrombinandfactorXa,shuttingdowntheproductionoffibrinclots.Billionsofdosesofheparin(severalmetrictons)isproducedbyautodigestionofpigintestines,followedbygradedfractionationoftheproducts.Unfractionatedheparinproducesavariableanticoagulantresponseasitalsobindstoseveralplasma,

platelet,andendothelialproteins.Low-molecular-weight(LMW)heparinsarederivedbychemicalorenzymaticcleavageofheparintoformsmallerfragments.ThepharmacologicalpropertiesandtherelativeefficacyofthevariousLMWheparinsaresuperiortothoseofunfractionatedheparinandfewersecondarycomplicationsarereported.LMWheparinshavereplacedunfractionatedheparinsastherapeuticofchoiceinvirtuallyalldevelopedcountries.Inpricesensitivemarketstheunfractionatedproductsarestillheavilyused.Thepreparationofrecombinantheparinbasedonheparinbiosynthesisenzymesisstillunderdevelopment.Arixtra,asyntheticheparinpentasaccharidethatbindsantithrombinexactlyasisolatedheparinisusedtopreventdeep-veinthrombosisandpulmonaryembolismandhasgainedmarketshareinrecentyears.Still,thehighercostofthesyntheticdrugArixtrahaspreventedanevenlargersuccesstherapeutic.Topreventexcessivebleeding,rapidneutralizationofheparinisdesirable.Administrationofthebasicproteinprotamine,whichbindstoheparin,neutralizesitsactivity,andresultsinclearanceofthecomplexbythekidneyandliver.Heparinisalsousedtotreatprotein-losingenteropathy(PLE),likelyworkingbycompetingforproinflammatoryheparin-bindingcytokinesthattriggerPLEinsusceptiblepatients(seeChapter43).Hyaluronan(seeChapter15)isanaturallyoccurringglycosaminoglycanthatisextensivelyusedinsurgicalapplications.Becauseofitsviscoelasticproperties,hyaluronanhaslubricatingandcushioningpropertiesthathavemadeitusefulforprotectingthecornealendotheliumduringocularsurgery.Hyaluronanhasantiadhesivepropertiesandisusefulinpostsurgicalwoundhealing.Themechanismofactionisnotwellunderstood,butitmayinvolvehyaluronan-bindingproteinsthatmediatecelladhesion(seeChapter15).Intra-articularinjectionsofhyaluronanareusedtotreatkneeandhiposteoarthritis.Modestimprovementinpatientstreatedwithhyaluronan,maybetheresultofamechanical(asaviscosupplement)and/orabiological(viasignalingpathways)effect.Hyaluronanisusedinverylargequantitiesasatissuefillerincosmeticmedicine.

“GLYCONUTRIENTS”

“Glyconutrient”isatermusedbythenutritionalsupplementindustrytodescribesomeoftheirproductswithwide-rangingclaimsconcerningpotentialbenefits.Inmostcases,theseclaimshavenotbeensubstantiatedthroughplacebo-controlled,double-blindtrialswithdefined,quantifiableoutcomes.Muchworkisneededinthisareatoobtaininsightintothepotentialroleofdietaryglycansonhumanhealthandtohelpconsumersmakewisedecisionsregardingtheiruse.Mixturesofplantpolysaccharidessuchaslarchbarkarabinogalactan,andglucomannanareoftentermed“glyconutrients”thatareclaimedtocontain“essentialmonosaccharides”neededfor“cellcommunication.”Becauseallmonosaccharidescanbemadefromglucose(exceptinpatientswithraregeneticdeficiencies;seeChapter42),noneoftheothermonosaccharidesareactuallyknowntobe“essential.”Moreover,thesepolysaccharidesarenotdegradedtoavailablemonosaccharidesinthestomachor

smallintestine.Instead,anaerobicbacteriainthecolonmetabolizethemandproduceshort-chainfattyacids.Nopeer-reviewedclinicalstudiessupporttheefficacyofsuch“glyconutrients”foranydiseaseorcondition.Nevertheless,thefollowingexamplesdemonstratehowdietaryglycansmighthavebeneficialeffects.GlucosamineandChondroitinSulfateGlucosamine(oftenmixedwithchondroitinsulfate)hasbeenpromotedtorelievesymptomsofosteoarthritis,whichinvolvestheage-dependenterosionofarticularcartilage.Cartilageprovidesacushionbetweenthebonestominimizemechanicaldamage,andanetlossofcartilageoccurswhenthedegradationrateexceedsthesyntheticrate.Anumberofclinicaltrialsreportthatglucosamineimprovesosteoarthritissymptoms,andsomeclaimtorestorepartiallythestructureoftheerodedcushion,inparticularintheknees.Superficially,thiswouldseemtomakesense,becauseprimaryglycansofcartilageincludehyaluronan(seeChapter15)andchondroitinsulfate,bothofwhichcontainhexosamineswithintheirstructure(seeChapter16).Conflictingreportssuggestthattheoutcomemaydependonstudydesignandthetypeandsourceofmaterial.Nevertheless,veterinariansreportpositiveresultsaftertreatinganimalswithglucosamineforovertwodecades.Double-blind,placebo-controlledstudiesinhumanshaveshownadecreasedrateofjointspacenarrowing.GlucosaminemightalsoalterUDP-GlcNAcandpotentiallyUDP-GalNAclevels,thusaffectingcellularresponsesinvolvingmajorclassesofglycans.Positiveeffectsofchondroitinsulfateonosteoarthritisarelesswell-documented.Itremainsunclearhowtheacidicchondroitinsulfatepolymercanbeabsorbedanddeliveredtoitsproposedsiteofaction.Furtherstudiesareneededtodeterminewhetherchondroitinsulfatesareabsorbedbythetargettissue,andiftheyactuallyleadtochangesincartilagemetabolism.XylitolandSorbitolinChewingGumManystudiessuggestthatchewinggumcontainingsugaralditolssuchasxylitolandsorbitol,canhelpcontrolthedevelopmentofdentalcaries.Motherswhochewxylitol-sweetenedgummayevenblocktransmissionofcaries-causingbacteriatotheirchildren.Thebenefitofthesereducedsugarsseemstobebasedonstimulationofsalivaryflow,butanantimicrobialeffectisalsopossible.XylitolalsoinhibitstheexpressionandsecretionofproinflammatorycytokinesfrommacrophagesandinhibitsthegrowthofPorphyromonasgingivalis,oneofthesuspectedcausesofperiodontaldisease.Childrenwhodrankxylitolsolutionsalsohadaloweroccurrenceofotitismedia.MilkOligosaccharidesHumanmilkcontainsabout70g/literoflactoseand5–10g/literoffreeoligosaccharides.Morethan130differentglycanspecieshavebeenidentifiedwithlactoseatthereducingend,includingpoly-N-acetyllactosamineunits.Someglycans

areα2-3-and/orα2-6-sialylatedand/orfucosylatedinα1-2,α1-3,and/orα1-4linkages.Incontrast,bovinemilk,thetypicalmainstayinhumaninfantformulas,containsmuchsmalleramountsoftheseglycans.Thesedifferencesmayaccountforsomeofthephysiologicaladvantagesseenforbreast-fedversusformula-fedinfants.Theglycansmayalsofavorgrowthofanonpathogenicbifidogenicmicrofloraand/orblockpathogenadhesionthatcausesinfectionsanddiarrhea.Surprisingly,asubstantialnumberofhumanmilkoligosaccharidesremainalmostundigestedintheinfant’sintestineandareexcretedintactintotheurine.Whethersupplementinginfantformulawithspecific,biologicallyactivefreeglycansenhancesinfanthealthisunknown.

GLYCANSASVACCINECOMPONENTS

MicrobialVaccinesPolysaccharidevaccinesconsistingsolelyofglycancomponentstypicallyelicitpoorimmunity,especiallyininfants.SinceglycansareT-cell-independentantigenstheydonoteffectivelystimulateT-helper-dependentactivationandclassswitchingofB-cell-mediatedimmunity.Conjugatevaccinesconsistingofglycanscoupledtocarrierproteinshaveproventobehighlyeffective.Threemajorconjugatevaccinesaremarketedtoday:Haemophilusinfluenzaetypeb(Hib)causesanacutelowerrespiratoryinfectionamongyoungchildren.ChildrenuptothisageconstituteahighriskgroupforHaemophilusinfluenzaetypeBinfections.Consequently,theHibPSVintroducedin1985waswithdrawnfromthemarketin1988andreplacedbyCPS-proteinconjugatevaccineformulations.AconjugatedformofanHib-derivedoligosaccharidecoupledtoaproteincarrierispartofroutinevaccinationschedulesandhasbeensosuccessfulthatinfectiousdiseasescausedbythisbacteriumarenearlyeradicatedinvaccinatedpopulations.Apotentsemi-syntheticHibglycoconjugatevaccinemarketedinCubacontainsglycanchainswithanaveragelengthof16monosaccharides.Pneumococcalconjugatevaccineshavebeendevelopedtocoveranincreasingnumberofserotypes,andcurrentformulationsare10-(Synflorix®,GSK)and13-valent(Prevnar13®,Pfizer).Prevnar13providesprotectionagainstserotypesthataccountformorethan70%ofcasesofinvasivepneumococcaldiseaseworldwideandisthebestsellingvaccineaxceedingrevenuesoffivebillionUS$in2015.ConjugatevaccinestoprotectfromNeisseriameningitidesarealsoverysuccessfulonthemarket.Duetothefastonsetandrapidprogressionofmeningococcalinfections,vaccinationisrequiredtoprotectagainstthisdisease.SeveralconjugatedCPSvaccinesarelicensedindifferentpartsoftheworld:ThetetravalentserogroupA,C,W,andY(Menactra,Menveo,andNimenrix)andafewmonovalentvaccinesbasedonserogroupCCPS(Meningitec,Menjugate,NeisVac-C).TwocombinationvaccinesforN.meningitidisandHaemophilusinfluenzaetypeb(Hib)areavailableagainstmeningococcalserogroupsC/Y(MenHibrix)(Haleetal.,2014)andagainstmeningococcalserogroupC(Menitorix).AmonovalentserogroupAvaccine(MenAfriVac)iswidelyusedinthesub-SaharanmeningitisbeltofAfrica.

Currently,severalnewvaccinesbasedonsyntheticoligosaccharideantigensarebeingdevelopedtoprotectchildrenandtheelderlyfromavarietyofbacterialinfections.Vaccinestoprotectfromhospitalacquiredinfectionsthatareincreasinglyantibioticresistantareinpreclinicalevaluation.CancerVaccinesSeveralcarbohydrate-basedcancervaccinesareatdifferentstagesofdevelopmenttotreatcancer.GangliosideimmunogenspresentoncertaintypesofcancercellssuchasgangliosidesGM2andGD2inmelanomasandgloboHinbreastcancerarebeingexplored.Theshorterglycansequencessuchassialyl-Tn(sialylα2-6GalNAcα-)foundoncancermucins(seeChapter44)hasseenlittleprogressintwentyyears.ThesyntheticGloboHhexasaccharide(seeFigure57.1)resemblingthebreastandprostatecancerantigenhasreachedPhase3clinicaltrialsandisexpectedtogainapprovalformarketinginlate2016followingtwodecadesofdevelopment.Thisfirstsuccessfulexampleisexpectedtoboosttoaverychallengingareaofvaccinedevelopment.

BLOCKINGGLYCANRECOGNITIONINDISEASES

BlockingInfectionAsdiscussedinChapter34,manymicrobesandtoxinsbindtomammaliantissuesbyrecognizingspecificglycanligands.Thus,smallsolubleglycansorglycanmimeticscanbeusedtoblocktheinitialattachmentofmicrobesandtoxinstocellsurfaces(orblocktheirrelease),andthuspreventorsuppressinfection.Becausemanyoftheseorganismsnaturallygainaccessthroughtheairwaysorgut,theglycan-baseddrugscanbedelivereddirectlywithoutbeingdistributedsystemically.Milkoligosaccharidesarebelievedtobenaturalantagonistsofintestinalinfectionininfants(seeabove)andpolymersthatwillblockthebindingofvirusessuchasinfluenza.Althoughbackedbyastrongscientificrationaleandrobustinvitrostudies,such“antiadhesive”therapieshavenotyetfoundmuchpracticalapplication.InhibitionofSelectin-mediatedLeukocyteTraffickingWhenspecificglycan-proteininteractionsareresponsibleforselectivecell–cellinteractionsandaresultingpathology,thenadministrationofsmall-moleculeglycomimeticsofthenaturalligandisusefulmeansofintervention.Selectin-mediatedrecruitmentofneutrophilsandotherleukocytesintositesofinflammationorischemia/reperfusioninjuryinvolvesspecificselectin–glycaninteractionsinthevascularsystem(seeChapter31).TheuseofsialylLewisxtetrasaccharidederivativesfailedduetopoororalavailabilityandashortserumhalf-life.Glycomimeticsthatpreservetheessentialfunctionalityoftheparenttetrasaccharidebuteliminateunwantedpolarfunctionalgroupsandsynthetically

cumbersomeglycancomponentshavebeensuccessful.ThedesignofamonosaccharideglycomimeticstartingfromsialylLewisxisshowninFigure51.3.First,thesialicacidresiduewasreplacedwithachargedglycolicacidgroup,theN-acetylglucosamineresiduewasthenreplacedwithanethyleneglycollinker,andfinallythegalactoseresiduewasreplacedwithalinkermoiety.TheresultingglycomimetichadE-selectinbindingaffinitycomparabletosialylLewisx.SimplemonovalentsialylLewisxglycomimeticshaveproveneffectiveinvariousanimalinflammatorymodelsaswellasclinicaltrials.Bimosiamose(TBC-1269)anE-,P-andL-selectininhibitorisinphase2trialsastreatmentagainstasthmaandpsoriasis.GMI-1070istargetingthesamelectinsandisbeingexploredasatreatmentforsicklecellcrisisinphase2clinicaltrials.

TRANSFUSIONANDTRANSPLANTATIONREJECTIONBYANTIGLYCANANTIBODIES

AsdiscussedinChapter13,avarietyofglycans,includingtheclassicalAandBbloodgroupdeterminants,canactasbarrierstobloodtransfusionandtransplantationoforgans.Rejectionofmismatchedbloodororgansoccursbecausehostshaveahightiterofpreexistingantibodiesagainsttheglycanepitopes,presumablyasapriorreactiontorelatedstructuresfoundonbacteriaorothermicrobes.InthecaseoftheABObloodgroups,incompatibilityisroutinelymanagedbybloodandtissuetypingandfindinganappropriatedonorfortherecipient.BacterialenzymescanbeusedinvitrotoremovetheAandBbloodgroupdeterminantsfromAandBredcells,convertingtheminto“universaldonor”Oredcells.Arelatedproblemisfoundinxenotransplantation(i.e.,thetransplantationoforgansbetweenspecies)whichisactivelybeingpursuedasasolutionfortheshortageofhumanorgansforpatients.Theanimaldonorsofpreferencearepigs,becausemanyporcineorgansresemblethoseofhumansinsize,physiology,andstructure.However,unlikehumansandcertainotherprimates,pigsandmostothermammalsproducetheterminal“α-Gal”epitopeonglycoproteinsandglycolipids.Becausehumanshavenaturallyoccurringhigh-titerantibodiesinblooddirectedtowardthisepitope,thisresultsinhyperacuterejectionofporcineorgantransplants,viareactionoftheantibodieswithendothelialcellsofbloodvessels.Attemptstopreventthisreaction,includebloodfiltrationoverglycanaffinitycolumnstoremovexenoreactiveantibodiesandblockadeoftheinteractionbyinfusingsolublecompetingoligosaccharides.Transgenicpigslackingthereactiveepitopehavealsobeenproduced,ashaveanimalswithanexcessofcomplement-controllingproteinsontheircellsurfaces.Pigorgansalsohavehighlevelsofthenonhumansialicacid(Neu5Gc),againstwhichmosthumanshaveantibodies.Evenifthisproblemissolved,thereareotherglycanandproteinstructuraldifferencesbetweenhumansandpigsthatcauselaterstagesofgraftrejection,thusnecessitatingimmunosuppression.

ACKNOWLEDGMENTSTheauthorsappreciatehelpfulcommentsandsuggestionsfromWuDi,BenjaminSchulz,JonathanViolaandPaetonWantuch.

FURTHERREADINGKunzC,RudloffS,BaierW,KleinN,StrobelS.2000.Oligosaccharidesinhumanmilk:Structural,functional,andmetabolicaspects.AnnuRevNutr.20:699–722.PubMedPMID:10940350.GomordV,ChamberlainP,JefferisR,FayeL.2005.Biopharmaceuticalproductioninplants:Problems,solutionsandopportunities.TrendsBiotechnol.23:559–565.PubMedPMID:16168504.JoshiL,LopezLC.Bioprospectinginplantsforengineeredproteins.2005.CurrOpinPlantBiol.8:223–226.PubMedPMID:15753005.MhurchuCN,Dunshea-MooijC,BennettD,RodgersA.2005.Effectofchitosanonweightlossinoverweightandobeseindividuals:Asystematicreviewofrandomizedcontrolledtrials.ObesRev.6:35–42.PubMedPMID:15655037.PastoresGM,BarnettNL.2005.Currentandemergingtherapiesforthelysosomalstoragedisorders.ExpertOpin.Emerg.Drugs.10:891–902.PubMedPMID:16262569.BeckM.2007.Newtherapeuticoptionsforlysosomalstoragedisorders:Enzymereplacement,smallmoleculesandgenetherapy.HumGenet.121:1–22.PubMedPMID:17089160.BrownJR,CrawfordBE,EskoJD.Glycanantagonistsandinhibitors:Afountfordrugdiscovery.CritRevBiochemMolBiol.2007;42:481–515.PubMedPMID:18066955.ButtersTD.2007.Pharmacotherapeuticstrategiesusingsmallmoleculesforthetreatmentofglycolipidlysosomalstoragedisorders.ExpertOpinPharmacother.8:427–435.PubMedPMID:17309337.EklundEA,BodeL,FreezeHH.2007.Diseasesassociatedwithcarbohydrates/glycoconjugates.In:KamerlingJP,BooneGJ,LeeYC,editors.Comprehensiveglycoscience.Vol.4.Elsevier;NewYork:pp.339–372.HamiltonSR,GerngrossTU.2007.Glycosylationengineeringinyeast:Theadventoffullyhumanizedyeast.CurrOpinBiotechnol.18:387–392.PubMedPMID:17951046.SchultzBL,LaroyW,CallewaertN.2007.Clinicallaboratorytestinginhumanmedicinebasedonthedetectionofglycoconjugates.CurrMolMed.7:397–416.PubMedPMID:17584080.vonItzsteinM.2007.Thewaragainstinfluenza:Discoveryanddevelopmentofsialidaseinhibitors.NatRevDrugDiscov.6:967–974.PubMedPMID:18049471.SchnaarRL,FreezeHH.2008A“glyconutrientsham.Glycobiology.18:652–657.PubMedPMID:17855741.ErnstB,MagnaniJL.Fromcarbohydrateleadstoglycomimeticdrugs.NatRevDrugDiscov.2009;8:661-677.doi:10.1038/nrd2852.Anish,C,Schumann,B,Pereira,CL,Seeberger,PH.2014ChemicalBiologyApproachestoDesigningDefinedCarbohydrateVaccines;Chem.Bio.21:38-50.PubMedPMID:24439205

FIGURELEGENDS

FIGURE57.1.Examplesofnaturalproductsthatcontainglycancomponents.StreptomycinanderythromycinAareantibiotics,doxorubicinischemotherapeuticdrug,anddigoxinisusedtotreatcardiovasculardisease.

FIGURE57.2.ThesyntheticinfluenzaneuraminidaseinhibitorsRelenza™andTamiflu™.

FIGURE57.3.GlycomimeticE-selectininhibitorsbasedonsialylLewisx.

TABLE57.1Examplesofglycan-baseddrugs,theirtargetdiseases,andmodesofactionDrugTargetingsialicacids Zanamivir(Relenza®)

Biota/GlaxoSmithKline influenzatypeAandB inhibitsneuraminidase

Oseltamivir(GS4104,Tamiflu®)

Gilead/Roche chemoprophylaxis inhibitsneuraminidase

Targetingglycosaminoglycans Heparin multiplebrands anticoagulant;possible

valueincancermetastasisprevention

activatesantithrombin;inhibitsheparanaseandselectinsandblocksinteractionsbetweengrowthfactorsandheparansulfate

Hyaluronan(HA)

multiplebrands ocularsurgery;osteoarthritis;plasticsurgery

tissuespacefiller;anti-inflammatoryagent

Laronidase(Aldurazyme®)

Genzyme mucopolysaccharidosistypeI(MPSI);α-idu-ronidasedeficiency

enzymereplacementtherapy(ERT)

Galsulfase(Naglazyme®)

Biomarin mucopolysaccharidosistypeVI;arylsulfataseBdeficiency

ERT

Hyaluronidase(Cumulase®)

Halozyme invitrofertilization;indevelopmentasanadjuvantforcancerchemotherapy

degradesHAaroundoocytesimprovingfertilization;degradesHAintumorstodecreaseintratumorpressure

Tramiprosate(Alzhemed)

phaseIIItrials(Neurochem)

amyloiddiseases,Alzheimer’sdisease,andpossiblyotheramyloidoses

bindstoamyloidplaque,blocksitsformation

Eprodisate(Kiacta®)

phaseII/IIItrials(Neurochem)

amyloidAamyloidosis interfereswithglycosamino-glycan–amyloidinteractions

Targetingglycosphingolipids N-butyl-deoxynojirimycin(DNJ)(Miglustat,Zavesca®)

phaseIIItrials(Acetelion)

type1Gaucher’sdisease;Niemann–Pick’sdiseasetypeC;late-onsetTay–Sach’sdisease;type3Gaucher’sdisease

substratereductiontherapy;inhibitsglucosylceramidesynthase

Imiglucerase(Cerezyme®)

Genzyme type1Gaucher’sdisease ERT

β-Agalsidase(Fabrazyme®)

Genzyme Fabrydisease;α-galactosidaseAdeficiency

ERT

Others Acarbose(Glucobay®)

Bayer type2diabetes blocksintestinalα-glucosidasesinvolvedindigestionofdietaryglycans

Alglucosidasealfa(Myozyme®)

Genzyme Pompe’sdisease(glycogenstoragedisease);α-

ERT

glucosidaseAdeficiency Allosamidin IndustrialResearch insecticide chitinaseinhibitorModifiedfromBrownJ.R.,CrawfordB.E.,andEskoJ.D.2007.Crit.Rev.Biochem.Mol.Biol.24:481–515.Compoundstargetedatmicrobialglycans,suchastheaminoglycosideantibioticsorotherinhibitorsofcellwallassembly,havenotbeenincluded.