University of Groningen

Targeting iron metabolism in drug discovery and deliveryCrielaard, Bart; Lammers, Twan; Rivella, Stefano

Published in:Nature Reviews Drug Discovery

DOI:10.1038/nrd.2016.248

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Citation for published version (APA):Crielaard, B. J., Lammers, T., & Rivella, S. (2017). Targeting iron metabolism in drug discovery anddelivery. Nature Reviews Drug Discovery, 16(6), 400-423. DOI: 10.1038/nrd.2016.248

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1

IronmetabolismasatherapeutictargetfordrugdevelopmentanddeliveryBartJ.Crielaard1,2,*,TwanLammers3,4,5,StefanoRivella6

1. DepartmentofPolymerChemistryandBioengineering,ZernikeInstitute

forAdvancedMaterials,FacultyofMathematicsandNaturalSciences,

UniversityofGroningen,Groningen,TheNetherlands

2. W.J.KolffInstituteforBiomedicalEngineeringandMaterialsScience,

UniversityMedicalCenterGroningen,Groningen,TheNetherlands

3. DepartmentofNanomedicineandTheranostics,Institutefor

ExperimentalMolecularImaging,UniversityClinicandHelmholtz

InstituteforBiomedicalEngineering,RWTHAachenUniversity,Aachen,

Germany

4. DepartmentofPharmaceutics,UtrechtInstituteforPharmaceutical

Sciences,UtrechtUniversity,Utrecht,TheNetherlands

5. DepartmentofTargetedTherapeutics,MIRAInstituteforBiomedical

TechnologyandTechnicalMedicine,UniversityofTwente,Enschede,The

Netherlands

6. Children’sHospitalofPhiladelphia,AbramsonResearchCenter,

Philadelphia,PA,UnitedStatesofAmerica

*Correspondence: BartJ.Crielaard,M.D.,Ph.D

ZernikeInstituteforAdvancedMaterials

UniversityofGroningen

Nijenborgh4,9747AGGroningen

TheNetherlands

E:b.j.crielaard@rug.nl

T:+31503637888

F:+31503634400

2

Abstract

Ironfulfilsacentralroleinmanyessentialbiochemicalprocessesinhuman

physiology,whichmakesproperprocessingofironcrucial.Althoughiron

metabolismissubjecttorelativelystrictphysiologicalcontrol,inrecentyears

numerousdisorders,suchascancerandneurodegenerativediseases,havebeen

linkedtoderegulatedironhomeostasis.Becauseofitsinvolvementinthe

pathogenesisofthesediseases,ironmetabolismconstitutesapromisingand

largelyunexploitedtherapeutictargetforthedevelopmentofnew

pharmacologicaltreatments.Severalironmetabolism-targetedtherapiesare

alreadyunderclinicalevaluationforhaematologicaldisorders,andtheseand

newlydevelopedtherapeuticagentswilllikelyhavesubstantialbenefitinthe

clinicalmanagementofironmetabolism-associateddiseases,forwhichfew

efficacioustreatmentsareoftenavailable.

3

Ironis,afteraluminium,thesecondmostabundantmetalonearth,andisan

essentialelementforallformsoflifeonearth.Numerousenzymesinvolvedin

DNAreplication,repairandtranslationrelyoniron,oftenintheformofiron-

sulphur(Fe-S)clusters,forproperfunctioninginanimals,plantsandfungi,as

wellasinorganismsfromthetwoprokaryoticdomainsoflife,Bacteriaand

Archea1.Thebiologicalactivityofironlies,toalargeextent,initsefficient

electrontransferringproperties,enablingittoacceptordonateelectronswhile

switchingbetweenitsferrousbivalent(Fe(II),Fe2+),ferrictrivalent(Fe(III),Fe3+)

anditsferryltetravalent(Fe(IV),Fe4+)states,therebyfunctioningasacatalysing

cofactorinvariousbiochemicalreactions2.Invertebrates,thesecondmainrole

ofironinvolvestheoxygen-bindingcharacteristicofporphyrin-complexediron,

betterknownashaem,whichiscrucialfortheoxygen-carryingcapacityof

haemoglobinandmyoglobin.

Takingintoaccountthesevitalfunctionsofironinhumanphysiology,itisclear

thatsystemicorcellulardisordersinironmetabolismmayhaveserious

consequences.Atthesystemiclevel,haemincorporatedinhaemoglobin(Hb)

andmyoglobinaccountsformorethanhalfoftheapproximately4gramsofiron

presentinthehumanbody,andbyfarthelargestshareofthetotalironturnover

isforhaemproduction3.Consequently,aninsufficientironsupply,unmet

demandforiron,orsubstantiallossofironwillleadtoashortageofHb,resulting

iniron-deficiencyanaemia4.Conversely,patientswithredbloodcelldisorders

suchasβ-thalassemiasufferfromanaemiathatisassociatedwithmalformedred

bloodcellsthathaveareducedlifespanduetodysfunctionalβ-globinexpression

andreducedHbproduction5.Inanattempttocompensatethechronicanaemia,

theseindividualsproducelargenumbersoferythroidprogenitors.Thishigh

erythroidactivityisaccompaniedbyagreatlyincreasedirondemand,which

promotesironabsorptionand,inturn,causesseriouscomorbidityresultingfrom

ironoverloading.

Atthecellularlevel,thepresenceofintracellularironhasastrongimpactonthe

cellularredoxstatus,contributingtooxidativestressinindividualcells.Reactive

oxygenspecies(ROS),suchassuperoxide(O2−)andhydrogenperoxide(H2O2),

whichareformedbyasingleanddoubleunivalentreductionofmolecular

4

oxygen(O2),respectively,areknowntocatalysespecificcellularredoxreactions

andarethereforeinvolvedinanumberofsignallingpathways.However,further

reductionofrelativelyharmlessH2O2resultsintheformationofhydroxyl

radicals(OH�)thatarehighlyreactive,causingnonspecificoxidationand

damagetonucleicacids,lipidsandproteins6.Iron,aswellasothermetals,

catalysestheformationofOH�fromotherROSbyFentonchemistry7,which

involvestheoxidationofFe(II)(toFe(III))andelectrontransfertoH2O2.The

presenceofsuperoxidefurtherassiststhisprocessbypromotingthereduction

ofFe(III)toformFe(II)(andO2)tocompletethecatalyticelectrontransport

cycleofironknownastheHaber−Weissreaction8.

Asaconsequenceofitswell-establishedrolesiniron-deficiencyanaemiaand

iron-loadinganaemia,ironmetabolismhashistoricallyremainedwithinthe

scopeofhaematologicalpathologies.However,overthepastdecade,arangeof

ageing-related,non-haematologicaldisordershasbeenassociatedwith

deregulatedironhomeostasisaswell.InthisReview,wediscussiron

metabolismasatargetforthedevelopmentofnewtherapeuticsordrugdelivery

strategiesinthesediseases.Weprovideasystematicoverviewoftheiron

regulatorypathwaysanditskeyplayers,aswellasthemajorpathophysiologies

associatedwithdysfunctionalironhomeostasis,andthenreviewsomethemost

promisingironmetabolism-targetedtherapeuticsthusdeveloped,whichcould

providenewtherapeuticoptionsfortheseoftendifficulttotreatdisorders.

5

Physiologyofironmetabolism

Systemicironregulation–thehepcidin−ferroportinaxis

Hepcidinisapeptidecomprising25aminoacidsthatisencodedbytheHAMP

geneandnamedforitshighexpressionintheliver9.Hepcidinwasoriginally

thoughttobeapeptidewithmoderateantimicrobialactivity9,10,butitwassoon

recognizedtobethemasterregulatorofsystemicironmetabolism11.Hepcidin

regulatesthesystemicfluxofironbymodulatingthelevelsofferroportinonthe

cellsurface,theonlyknowncellularexporterofunboundironinvertebrates12.

Bydirectlybindingtotheextracellulardomainofferroportin,hepcidininduces

endocytosisanddegradationofthetransmembraneprotein,therebypreventing

ironegressfromthecell13.Highlevelsofferroportinarefoundinenterocytesin

theduodenum(totransportabsorbediron),inhepatocytes(totransportstored

iron),andinmacrophages(totransportrecyclediron),whichtogethercontrol

systemicironlevels14–16.Byreducingsurfaceferroportin,theexpressionof

hepcidinlimitstheabsorption,remobilizationandrecyclingofiron,thereby

reducingironplasmalevels(Figure1).

Withhepcidinasthecentralorchestrator,allmajorregulatorypathwaysof

systemicironhomeostasisconvergeonHAMPtranscription,mostlyviaSMAD

signalling(Figure2).Asaresult,alterationorgeneticmutationofproteinsthat

actwithinthesepathwayshavebeenassociatedwithavarietyofdisorders,

includingprimaryandsecondaryformsofironoverload,andchronicanaemia.

Consequently,theseproteins,togetherwithferroportin,aresomeofthemost

interestingtherapeutictargetsthatcouldbeexploitedforavarietyofdisorders

(Table1).

Cellularironregulation

Cellularirontraffickingcanbeseparatedintothreeprocesses:intake,utilization

andefflux(Figure3,Table2).Therearefourgeneralpathwaysbywhich

individualcellsinternalizeiron.Themostimportantpathwayiscentredaround

transferrin(Tf),anabundantplasmaproteinthatactsanaturalchelatingagent

withtwohigh-affinitybindingsitesforferriciron17.Tffulfilsseveralcrucial

physiologicalrolesthatincludethesolubilisationofrelativelyinsolubleFe(III),

thepreventionofiron-mediatedredoxtoxicity,andthesystemictraffickingof

6

iron.Althoughundernormalcircumstanceslessthan0.1%oftotalbodyironis

Tf-bound,thehighturnoverofTfensuresthateachday20-30mgofironis

deliveredtothebonemarrowforerythropoiesis.Cellularinternalizationof

transferrin-boundirontypicallyoccursafterdockingwiththemembrane-bound

transferrinreceptor1(TfR1),whichhasnanomolaraffinityforiron-boundTf,

butnotforapotransferrin(apoTf)(i.e.iron-freeTf)atphysiologicalpH18.TheTf-

TfR1complexisinternalizedviaclathrin-mediatedendocytosis,andFe(III)is

liberatedfromTfasaresultofadropinpHto5.5.Subsequently,TfR1andapoTf,

whichremainassociatedatthislowpH,arerecycledbacktothecellsurface,

wherethepHisphysiologicalandapoTfisthusreleased19.InadditiontotheTfR-

mediateduptakeoftransferrin,cellsmayalsoutilizeanalternative,low-affinity

routefortransferrininternalization20.

Thesecondroutebywhichcellscanacquireironisthroughtheuptakeofnon-

transferrinboundiron(NTBI),whichcanberegardedas‘freeiron’.Currently,

threecellularmembranetransportershavebeenshowntobeinvolvedinthe

uptakeofNTBI:divalentmetal-iontransporter1(DMT1)21,Zrt-andIrt-like

protein14(ZIP14)22,and,mostrecently,ZIP823.

In1997,DMT1wasidentifiedasakeymammalianmetal-iontransporterthatis

responsiblefortheintestinalironabsorptionofvariouscationicbivalentmetals,

suchasFe(II),butalsoCd(II),Co(II),Cu(II),Pb(II),Mn(II),Ni(II)andZn(II)21.

Aroundthesametime,theinheritablemicrocytic,hypochromicanaemiafoundin

mk/mkmiceandBelgrade(b)rats,whichwasalreadyknowntobeassociated

withanimpairedintestinaluptakeanderythroidutilizationofiron,wasshown

tooriginatefromhomozygousmutationsinthegeneencodingDMT1,Slc11a224,25.ThecriticalroleofDMT1intheabsorptionofdietarynon-haemironhas

recentlybeenconfirmedusingintestinalDMT1knockoutmice26,andinhumans,

homozygousorcompoundheterozygousmutationsintheSLA11A2genehave

beenassociatedwithmicrocyticanaemia27.However,incontrasttotherodent

models,patientswithDMT1mutationsareoverloadedwithiron,ratherthan

deficient.Thiseffectislikelycausedbyaresidualcapacityforironabsorption,

fulfilledeitherbytheDMT1mutantorbyanalternative(haem)absorption

pathwaythatisfurtherstimulatedbyaregulatoryfeedbackmechanismdriven

bythemicrocyticanaemia.Themicrocytichypochromicanaemiainthese

7

patients,andinthemk/mkmiceandBelgrade(b)rats,isthereforeprimarily

relatedtotheothermainroleofDMT1:thetransportofFe(II)throughthe

endosomalmembrane.Asdemonstratedinmk/mkmice,DMT1co-localizeswith

TfR1intheendosometohandlethecellularimportofironthatisliberatedfrom

theTf-TfR1complex.DMT1dysfunctionparticularlyaffectsthedevelopingred

bloodcells,astheyrequireasubstantialsupplyofironandareheavily

dependentonTf-TfR1-mediatedimport28.

Morerecently,ZIP14hasbeenshowntofulfilanimportantroleinthetissue

accumulationofironbyNTBI.Ingeneticmodelsofironoverloadinginmice,

ZIP14deficiencyreducedironcontentintheliverandpancreasandprovided

somelevelofprotectionagainsthereditaryhemochromatosis29.Eachoftheiron

transportersonlytransportsolubleFe(II),andthereforerequireenzymeswith

ferroreductiveactivitytoreduceFe(III)priortointernalization.Familymembers

ofcytochromeb561,thebestknownofwhichisDcytb,whichisexpressedinthe

duodenum30,aswellasSTEAP,whichisfoundinerythroidprogenitorcells31,

havedemonstratedsuchferroreductiveactivity32.Recently,prionproteins

presentonthesurfaceofneuronalcellshavebeenshowntopossessiron-

reducingcapacityaswell,therebyassistingcellularuptakeofNTBIinthe

brain33,34.

Thethirdmechanismforironinternalizationistheuptakeofporphyrin-bound

ironintheformofHbandhaem.ThescavengerreceptorCD163isalmost

exclusivelyexpressedbymacrophagesandisspecializedtotakeinHbin

complexwiththeglycoproteinhaptoglobin35,36.Similarly,CD91orchestratesthe

uptakeofhaemthatisincomplexwiththeglycoproteinhemopexin37,and

althoughCD91isconsideredapromiscuousreceptorrecognisingotherligands,

itishighlyexpressedbyCD163-positivemacrophagesandmaythereforebean

essentialroutefortheclearanceofironthatwasincorporatedintoHbandother

haem-containingproteins.HRG138andFLVCR239arealsohaem-importing

proteins,andalthoughtheirroleincellularirontraffickingneedstobefurther

elucidated,ithasbeenshownthatHRG1inparticularisinvolvedinthetransport

ofhaemacrossthelysosomalmembraneuponinternalizationanddegradationof

erythrocytesandhaemoglobinbymacrophages40.

8

Thefourthgeneralpathwayforironinternalizationconsistsoftheuptakeofthe

iron-storageproteinferritinbyreceptorsthatincludeScara541,TfR142,anda

currentlyuncharacterizedhumanferritinreceptorthatcouldberelatedtoTIM-

2,awell-characterisedferritinreceptorinmice43,44.

Onceinsidethecell,ironcanbeutilizedandstored.Mitoferrin1and2are

specializedtransportersthatmoveironintothemitochondria,whereitassists

withcellularrespirationandisusedforthesynthesisofFe-Sclustersand

haem45.Intracellularironcanbe‘stored’intwoforms:stablyincorporatedinto

ferritin,aniron-sequesteringproteinthatcancontainupto4,500ironatoms46,

orasactiveunboundspecies,knownasthelabileironpool(LIP).Becausean

excessofLIPmaycauseoxidativestress-relatedtoxicity,ironisgenerallystored

ascytosolicferritin,withtheliverfunctioningastheprimaryorganforsystemic

ironstorage.TheLIPislargelyresponsiblefortheregulationofcellulariron

homeostasisthroughtheironregulatoryprotein(IRP)/iron-responsiveelement

system,whichpost-transcriptionallymodulatesthetranslationmRNAsof

severalproteinsthatareinvolvedincellularirontrafficking,includingDMT1,

TfR1,ferritinandFpn47,48.

Theexportofironfromcellsisalmostexclusivelymanagedbyferroportin(Table

1).Asaresult,hepcidinregulatessystemicironuptakeandmobilizationby

inducingthedegradationofferroportinasdiscussedabove.Themulti-copper

oxidasesceruloplasmin(inserumandastrocytes),hephaestin(intheintestine,

placenta,heart,brainandpancreaticβ-cells),andzyklopen(inplacenta),assist

theexportofironbyferroportinbyoxidizingferrousirontoferriciron,thereby

allowingittobeloadedontotransferrin49.Thereare,however,otherpossible

mechanismsforcellularexportofcomplexediron.Theseincludethesecretionof

iron-containingferritinbyhepatocytes50,macrophagesandkidneyproximal

tubulecells51,whichislikelymediatedbyautophagyofcytoplasmicferritinand

subsequentreleaseviathesecretory-lysosomalpathway51,52.Anotherpathwayis

thesecretionofhaembyFelineleukaemiavirus,subgroupC,receptor1b

(FLVCR1b),whichontheonehandmayfunctionasan‘emergencyvalve’to

preventhaemoverload,especiallyinerythroidcells,andontheotherhandasa

9

potentialrouteformacrophagestodirectlysupplyhaemtodeveloping

erythroblasts53.

10

Ironandthepathophysiologyofdisease

Ironoverload,irondeficiencyandanaemiaofinflammationandchronicdisease

Ironoverloadisamajorclinicalchallengeinmanagementofhereditary

hemochromatosisandhaemoglobinopathy-relatedanaemia,alsoknownasiron-

loadinganaemia.Primaryironoverload,suchasoccursinpatientswith

hereditaryhemochromatosis,isdirectlycausedbydisorderedironmetabolism

thatisassociatedwithgeneticmutationsinregulatoryproteins.Bycontrast,

secondaryironoverloadinpatientswithiron-loadinganaemiaisnotintrinsically

relatedtoironmetabolism,butratherresultsfromtheanaemiathatstimulates

erythropoiesisandironabsorption.Moreover,theironoverloadinseverely

anaemicpatientsisfurtheraggravatedbyregularbloodtransfusionstothataim

toimprovehaemoglobinlevels.Tissuescommonlyaffectedbyironoverload

includetheliver,spleen,heart,pancreas,andpituitarygland,andexcessiron

ofteneventuallyleadstocomplicationssuchasliverfailure,myocardialdisease,

diabetesmellitusandothermetabolicdisorders54.

Irondeficiencyoccurswhenthesystemicdemandforironsurpassesitssupply.

Aserythropoiesisis,intermsofironrequirements,themostimportant

physiologicalprocess,irondeficiencywilltypicallypresentasirondeficiency

anaemia.Increasingorsupplementingthedietaryironintakecorrectsiron

deficiencyinmostcases,althoughinarareinheritedformofanaemiaknownas

iron-refractoryirondeficiencyanaemia(IRIDA),mutationsinTMPRSS6leadto

inappropriatelyhighhepcidinexpressionandrestrictedintestinaliron

absorption,sointravenousironadministrationisoftenrequired55.Ahigh

hepcidinlevelisalsooneofthehallmarksinanaemiaofinflammationand

chronicdisease(AI/ACD).Underconditionsofchronicinflammation,suchas

inflammatorydiseasesandcancer,increasedexpressionofIL-6andstructurally

relatedcytokinesleadtoactivationoftheIL-6receptorpathway,resultingin

STAT3-mediatedhepcidinexpressionand,consequently,limitedironabsorption

andavailability56.Additionally,activinB,amemberoftheTGF-βsuperfamily

thatisexpressedintheliverunderinflammatoryconditions,wasshownto

induceHAMPtranscriptionviatheSMAD1/5/8signallingpathway57.Although

theexpressionofhepcidincanbeconsideredausefuldefencemechanism

againstinfections,asitlimitstheavailabilityofirontoinvadingpathogens,the

11

presenceofAI/ACDinpatientswithchronicdiseasesisacommonand

substantialclinicalproblemthatcannegativelyaffecttherecoveryandqualityof

lifeinthesepatients58.

Ironandcellularredoxstatus

ThemitochondrialgenerationofATPishighlydependentontheformationand

presenceofROS,andmitochondriaareamajorsourceofcellularROS.

Mitochondrialironhomeostasisisthereforeofcrucialimportanceinthecontrol

ofcellularredoxandpotentialoxidativedamage.Forexample,inFriedreich’s

ataxia,ageneticdisorderaffectingtheexpressionofthemitochondrialiron-

chaperonefrataxin,cardiomyopathyisamajorcomplicationthathasbeen

associatedtomitochondrialironoverload.Additionally,complicationssecondary

tohereditaryhemochromatosis,includingliverandcardiactoxicity,havebeen

relatedtoROS-mediateddamageandmitochondrialpathology59.

Ferroptosisisarecentlydescribedformofcontrollednon-apoptoticcelldeath

thatheavilydependsonironandROS60.Ferroptosishasbeenassociatedwitha

diverserangeofpathologiesincludingneurologicaldisorders61,62,ischaemia-

reperfusioninjury63,64,andcancer65,66.Althoughtheferroptoticpathwayandits

functionstillhaveseveralunknownfeatures,peroxidationoflipidsbycytosolic

ferrousironplaysacentralrole.Ferroptosiscanbeinducedbyblockingthe

systemXc−cystine/glutamateantiporter,whichlimitsglutathioneproduction

andthusreducesoxidativeprotectionbyglutathioneperoxidase467,while

chelationofironprotectscellsfromferroptosis60.Theinterplaybetween

mitochondrialiron,ROSandferroptosishasrecentlybeenreviewedindepth68–

70,andwillundoubtedlyuncoverkeytherapeutictargetsthatcanbeexploitedto

designnoveltherapiesfordiseasesassociatedwithabnormalferroptoticactivity.

Ironandimmunity

Therelationshipbetweenironandtheimmunesystemiswellestablished.As

partoftheinnateimmunesystem,macrophagesarehighlyefficientphagocytes

thatfulfilacentralroleinthefirst-linedefenceagainstmicroorganisms.In

parallel,theyareresponsibleforironrecyclingfromredbloodcells,andare

therebysolelyaccountablefor>95%oftheironsupplyneededforsteady-state

12

erythropoiesis.Inaddition,theyhaveaniron-independentgrowth-supporting

functioninerythroiddevelopment71,72.Althoughthesemacrophageactivities

appeartobequitedissimilar,aconnectionbetweenironstatusandmacrophage

phenotypehasbeenfirmlydemonstrated.Firstofall,multiplereportshave

shownthathighintracellularironlevelsstimulatethesecretionofinflammatory

cytokinessuchasTNF-αbymacrophages73,74;theclinicalsignificanceofthishas

beenillustratedbyseveralpreclinicalstudies.Forexample,intravenous

administrationofferumoxytolironoxidenanoparticles(alsoknownas

Feraheme;AMAGPharmaceuticals),anFDA-approvedforironreplacement

therapyinchronickidneydisease(seealsoBox1),significantlyreducedtumour

growthandsuppressedlivermetastasisintumour-bearingmice,andwas

associatedwithaproinflammatoryphenotypeintumour-associated

macrophages75.Similarly,iron-overloadedmacrophagesinchronicvenous

ulcershaveastrongproinflammatoryphenotype,producingofTNF-αandROS,

therebydelayingwoundhealingandsupportingfurthertissuedamage76.Finally,

unboundhaemandironcaninduceaswitchinthephenotypeofmacrophages

fromgrowth-supportingtoinflammatory,whichcanbepreventedbyiron

chelationorscavengingofhaembyhemopexin77.Becausevariouscomplications

inpatientswithiron-loadinganaemiashaveachronicinflammatorycomponent,

suchasliverfibrosis,legulcers,pulmonaryhypertensionandmusculoskeletal

inflammation,itistemptingtohypothesizethatiron-mediatedinflammatory

activationofmacrophageshasaroleinthepathogenesisofthesecomplications5.

Ironhomeostasisinfuencesadaptiveimmunityaswell.Forexample,aformof

immunodeficiencycharacterizedbyimpaireddevelopmentandfunctionof

lymphocyteshasbeenattributedtomutationsinthegeneencodingTfR1,TFRC,

whichaffectsinternalizationofthesurfacereceptor78.Althoughirondeficiencyis

theunderlyingcauseofthisdisorder,illustratedbythereversalofdefective

lymphocyteproliferationbyFe(III)supplementation,interestingly,patientsare

onlymildlyanaemic.

Anotherconnectionbetweenironandimmunityisrelatedtothedefenceagainst

invadingpathogens.Microorganisms,asforallotherformsoflife,arehighly

dependentonthepresenceofirontosupportgrowthandproliferation79.Asa

consequence,tolimitinfectivityofinvadingmicroorganisms,hostorganisms

13

havedevelopedstrategiestorestrictironsupplytopathogens.NTBIis

consideredanimportantsourceofironformicrobes,whichsomeofthem

sequesterbyreleasingiron-bindingproteinsknownassiderophores.Captureof

thesesiderophores,aswellasNTBI,bymacrophage-producedlipocalin-2is

thereforeaneffectivedefensiveresponsetoinfection80,81.Anotherhost

mechanismtolimitNTBIavailabilityunderinflammatoryconditionsistoblock

cellularironegressbyreducingferroportinexpression82.Thisinflammation-

inducedreductionofferroportinismainlydrivenbyupregulationofhepcidin

transcriptionviaIL-6R-activatedJAK/STAT3-signalling(Figure2,Table1),and

viaTGF-β1−TGF-β1R-mediatedactivationofSMADsignalling,whichisdistinct

fromBMP6-inducedSMADsignalling83.Alternatively,ferroportinreductionis

alsocontrolledbyhepcidin-independentmechanisms,suchassignallingviaToll-

likereceptors2and6,allowingforarapidsystemicresponseunder

inflammatoryconditions84.

Ironandcancer

Changesincellularredoxstatushaveaconsiderableroleinmalignant

transformationandmetastasisofcancercells85,andconsequently,many

researchershaveproposedandinvestigatedthepotentialrelationshipbetween

malignanciesandiron86,87.Althoughseveralstudieshaveindeedshowna

correlationbetweentheincidenceandmortalityofcancerandsystemiciron

levels,asmeasuredbytransferrinsaturationorserumferritin,thiscorrelationis

generallynotstrong,hasshownconflictingoutcomes,anditisoftenconfounded

bythepresenceofotherpathology88,89.Thereis,however,clearevidencethat

deregulatedironhomeostasisonalocal—thatis,microenvironmentaland/or

cellular—levelisassociatedwithtumourprogression,asillustratedbychanges

inexpressionofiron-relatedgenesincancercells(Figure4),thelevelsofwhich

areinverselycorrelatedwithpatientsurvival90–92.Inthecontextoftumour

progression,onecanenvisionthatproliferatingcancercells,whichrequire

substantialamountsofirontosupporttheirgrowth,willattempttoincrease

theirironintake,forexamplebyincreasingtheexpressionofTfR193–95and

STEAP396,97;tomodulateironstoragecapacitybyproducingferritin94,96;andto

limittheirironexportbydownregulatingferroportin91,98–100.However,the

14

additionalintracellularoxidativestressthatisaccompaniedbytheincreased

presenceoflabileironmayalsomakethemmoresensitivetoferroptosis65,67and

hindertumourmetastasis85,illustratingthecomplexrelationshipbetweeniron,

ROSandcancercellsurvival.Furthermore,althoughaconnectionbetweeniron

andcancergrowthhasbeendemonstratedinseveralformsofcancers,with

breastcancer90,91,99–101,prostatecancer98,102,103andglioblastomaasestablished

examples94,104,itseemsreasonablethatmany,butnotall,cancertypeswill

demonstrateasimilarirondependency.

Ironandneurodegenerativediseases

Ironhomeostasisinthecentralnervoussystem(CNS)differsfromthatinother

tissues105.Endothelialcellsoftheblood−brainbarriermodulateironinfluxinto

theCNSbyimportingironfromthesystemiccirculationviaTfR1andDMT1,and

subsequentlyreleasingironviaferroportinatthebasalsideintothebrain

interstitium,formingacontrolmechanismreminiscentoftheabsorptionofiron

intheduodenum106,107.Thepresenceofanadditionallayerofhomeostatic

regulationsuggeststhatthebrainmaybesparedfromexcessiveiron

accumulationiniron-overloadedpatients,whichissupportedbythefactthat

largeirondepositsinthebrainarenottypicallyfoundinpatientswith

hemochromatosis108.Nevertheless,geneticmutationsassociatedwithan

inheritedformofhemochromatosisareoverrepresentedinpatientswith

Parkinson’sdisease(PD),parkinsonismandamyotrophiclateralsclerosis

(ALS)109,110,indicatingthatcellular,ratherthansystemic,disturbancesinbrain

ironhomeostasismayhavearoleinthepathogenesisinthesediseases.

Althoughprogressiveneurologicaldisordershavebeenlinkedtodisturbancesin

othermetalssuchascopper(ahallmarkofWilson’sDisease,butalsofoundin

Alzheimer’sDisease(AD)111)andzinc(e.g.inPD112),manystudieshave

implicatedcellularironoverloadandiron-inducedoxidativestressinthe

developmentofPD,ALS,AD,Huntington’sdisease,multiplesclerosis(MS)and

severalotherneurodegenerativediseases(Figure4)113,114.Asaninteresting

example,prionprotein(PrPC)hasbeenshowntopromotethereductionof

Fe(III)toFe(II),enablingitsuptakeintocellsbyZIP14andDMT133,34.However,

theconversionofPrPCtoitsisoformPrPSc,whichisimplicatedinneurological

15

priondiseasessuchasCreutzfeldt−Jakobdisease,ispromotedbyunbound

iron115,andPrPSccanbindtoferritintoformaninsolublecomplex,thereby

stimulatingthecellularintakeofironandpromotingtheformationofadditional

PrPSc116.

Astrocytes,glialcellsintheCNSthathaveafunctioninthecontrolofnutrient

supplyandbrainremodelling,arethoughttofulfil,inconcertwithendothelial

cellsoftheblood−brainbarrier,acriticalroleinregulatingironhomeostasisin

thebrain106,117.Astrocytesproducehepcidininresponsetoinflammatory

signallingandintracellulariron118,suggestingacontrolmechanismvia

ferroportin,similartotheregulationofsystemicironlevelsbyhepatocytes.This

regulatoryfeedbackloopallowsforlocalcontroloverirontraffickingwithinthe

CNS,althoughtheabundanceofhepcidininthebraininterstitiumsuggeststhat

atleastsomefractionofitisofsystemic(likelyhepatic)origin119.Lowlevelsof

ferroportinhavebeenfoundinbrainsofpatientswithAD120,aswellasinrat

modelsofPD121andALS122.InamousemodelofMS,reducedexpressionof

ferroportinandintracellularironoverloadwerespecificallyfoundin

macrophage-likemicroglia,butnotinastrocytes123.Moreover,iron-overloaded

microgliacanbefoundinactiveMSlesionsandhaveapro-inflammatory

phenotypethatinhibitsCNSrepair,thuscontributingtodisease

progression124,125.Neurologicaldysfunctionsarealsofoundinpatientswith

aceruloplasminaemia:theseindividualslackfunctionalceruloplasmin,aprotein

thatfacilitatesironremovalfromtissuesbyoxidisingexportedFe(II)toFe(III),

therebypromotingitsassociationwithtransferrin.Thisgeneticdiseaseis

characterizedbyalossofneurons,particularlyinthebasalganglia,andby

astrocyteswithanattenuatedironeffluxcapacityandiron-overload126.Finally,

inaclinicalprospectivestudy,higherferritinlevelsinthecerebrospinalfluid

werefoundtobecorrelatedwithalowercognitivefunctionandbrainstructural

changesinpatientswithAD,butthiscorrelationseemedtoalsobepresentin

individualsthatwereconsideredcognitivelynormal127.Interestingly,genetic

mutationsthataffecttheferritinlightchain,whichprobablymakestheiron-

storageprotein‘leaky’,leadtoaheterogeneousneurologicalclinical

presentation,knownasneuroferritinopathy114,128.Thisraisesthequestionof

whetherdysfunctionalferritinormalfunctionsinitscellularprocessingmay

16

havesomeinvolvementinthepathogenesisofotherneurodegenerative

diseases129.

Ironandatherosclerosis

Inatheroscleroticplaques,ironispresentinmuchhigherconcentrationsthatit

isinhealthytissues(Figure4)130.Thefunctionoftheseirondepositsin

atherosclerosis,andwhetherironaccumulationhasacausativeroleorisamere

consequenceofthepathogenicprocess,forexampleduetohaemolysis,isstill

underdebate.Itseemsreasonablethatironcouldbedirectlyinvolvedby

catalysingtheformationofoxidizedlow-densitylipoprotein(LDL)inthearterial

wall131,whichisconsideredoneofthehallmarksofatherosclerosis.However,in

iron-overloadedpatientswithtype1hemochromatosistheincidenceof

atherosclerosisisactuallylower,ratherthanhigher,thanitisintherestofthe

population132.Interestingly,theironstatusofmacrophages,whichfulfilakey

roleinthedevelopmentandprogressionofatheroscleroticplaques133,134,could

explainthisparadox,asmacrophagesofpatientswiththisformof

hemochromatosiscontainlessironthandothoseofhealthyindividuals.

Conversely,patientswithβ-thalassemia,adisorderthatisaccompaniedbyiron-

loadingespeciallyofmacrophages,indeeddohaveahigherriskofdeveloping

atherosclerosis,confirmingtheso-called‘ironhypothesis’in

atherosclerosis135,136.Arelationshipbetweenmacrophages,ironand

atherosclerosiscouldpartiallybeowingtoiron’sdirectroleintheoxidationof

LDLbymacrophages,aswellasaphenotypicchangeinmacrophagesduetoiron

sequestrationasdescribedabove.Nevertheless,variousexperimentalstudies

thathaveinvestigatedintracellularironaccumulationinmacrophagesasa

drivingmechanismforatherosclerosishaveproducedconflictingresults,

suggestingthatirontakespartinacomplexnetworkinvolvingseveral

underlyingmechanisms137–141.

Ironandotherageing-associateddiseases

Besidescancer,neurodegenerativedisordersandatherosclerosis,variousother

pathologieshavebeenlinkedtoironhomeostasis.Indiseasessuchastype2

diabetesmellitus142,osteoporosis143,osteoarthritis144,maculardegeneration145,

17

andliverfibrosis146,highlevelsofironhavebeenassociatedwithdisease

severity,suggestingapossibleroleinpathogenesisordiseaseprogression.

Importantly,manyofthesedisordersareconsideredchronicageing-associated

diseasesofmultifactorialaetiology,andaresubstantialcausesofmortalityand

morbidityintheageingWesternsociety.Thisstressestheneedforathorough

explorationoftheroleofirondyshomeostasisinthesediseases,aswellthe

utilityoftargetingironmetabolismasatherapeuticapproachorasastrategyto

deliverpharmaceuticalagents.

18

Ironmetabolismasatherapeutictarget

Therapeuticstargetingsystemicironavailability

Theuseofiron-chelatingagentsisastraightforwardapproachforlimitingand

redistributingsystemicironavailability,andisthereforefrequentlyusedinthe

clinicalmanagementofprimaryorsecondaryironoverload.Thecurrently

availablechelatorsaredeferoxamine(Desferal,Novartis),deferiprone

(Ferriprox,ApoPharma),anddeferasirox(Exjade,Novartis).Morethan50years

ago,desferoxamine(DFO)wasthefirstchelatorthatshowedclinicalpromise147.

Ironchelationtherapywasthenrapidlydemonstratedtobeaneffectivestrategy

formobilizingandremovingironviafaecalandurinaryexcretion,illustratedby

amarkeddecreaseinmortalityandiron-relatedcomplicationsinpatientswith

thalassemiamajor148.Despiteitssuccess,DFOhaspoorbioavailabilityand

requiresparenteraladministrationthatisoftenpainful,andwhichshouldbe

executedslowly(upto10hinfusion)andregularly(5-7timesaweek),making

patientcomplianceamajorlimitation.Inthefollowingdecades,theoraliron

chelatorsdeferiprone(DFP)149anddeferasirox(DFX)150weredevelopedand

approvedforthetreatmentofironoverload,improvingironchelationtherapy

patientsatisfactionascomparedtotreatmentwithDFO151.BothDFOandDFX

arecurrentlyrecommendedasfirst-linechelationtherapyinironoverload,but

thetoxicityprofileofthesecompounds,whichincludehypersensitivityreactions,

liverdysfunction,renaldysfunctionandneuronalhearingloss152,warrant

furtherdevelopmentsinironchelationtherapies.Forexample,inpreclinical

modelsofiron-loadinganaemia,theadministrationofunmodifiedTf,whichcan

beconsideredthephysiologicalironchelator,haseffectivelyredistributediron

todevelopingerythroidcells153.

Inadditiontoironoverloaddisorders,ironchelationhasalsobeenextensively

investigatedasanadjuvanttherapyinotherdisorders.Inthe1990s,clinical

trialsofironchelatorsinpatientswithneuroblastomashowedantitumor

efficacy154,155,whichencouragedfurtherexplorationofironchelationtherapyin

cancer.Severalnovelchelatingagentshavebeenexplored,ofwhichoneis

currentlyunderclinicaldevelopment(DpC,OncochelTherapeutics)156.In

parallel,ironchelationhasalsoshownclinicalpotentialinneurodegenerative

disorders,includingmultiplesclerosis157,Alzheimer’s158andParkinson’s

19

disease159,therebyconfirmingtheinvolvementofironinthepathogenesisof

thesedisorders.

Sincethediscoveryofhepcidininthebeginningofthismillennium,asubstantial

numberofagentsstimulatingorblockingitsexpressionoractivityhavebeen

investigated(Table3).Severalshorthepcidinderivativeshavebeendesignedas

hepcidin-mimickingagentswithanimprovedpharmacokineticprofiles,andare

currentlyunderdevelopmentbyMerganserBiotech(M012,phase1)160,LaJolla

PharmaceuticalCompany(LJPC-401,phase1,seeFurtherinformation),and

ProtagonistTherapeutics(PTG-300,preclinical,seeFurtherinformation).

Interestingly,preclinicalstudieshavenotonlydemonstratedbeneficialactivity

ofhepcidinderivativesinseveralmodelsforredbloodcellandironoverload

disorders,suchasβ-thalassemia,polycythaemiavera,and

hemochromatosis161,162,butalsoasanantimicrobialagent,asitlimitsthe

availabilityofirontocertaintypesofmicroorganisms82.Conversely,therapeutics

thatbindtoandinactivatehepcidinhavebeendevelopedandstudiedforthe

treatmentofanaemiaofinflammationandchronicdisease.Suchhepcidin-

bindingagentsincludehumanizedmonoclonalantibodies(LY2787106,

discontinuedafterphase1in2015,EliLilly163;and12B9m,AmgenInc164),an

anticalin(PRS-080,PierisPharmaceutical)165andaL-RNAspiegelmer

(Lexaptepidpegol(NOX-H94),NOXXONPharma)166,167.

Asimilarferroportin-reducingeffectcanbeachievedwithagentsthattarget

ferroportindirectly,blockingthebindingofhepcidinandpreventing

internalizationanddegradationoftheironexporterprotein.Thestructureof

ferroportinhasnotyetbeenresolved,butthesolvedcrystalstructureofa

bacterialhomologueandmodellingofhumanferroportinstronglysuggestthat

thetransportercomprises12transmembranedomainswithacentralpore168,169,

sharingstructuralfeatureswiththemajorfacilitatorsuperfamily(MFS)of

transporters.Thecys326residuethatisessentialforthebindingofhepcidinis

likelylocatedinsidethecentralpore,whichcouldindicatethatbindingof

hepcidincouldstructurallyblockferroportinfunctionbeforeinternalizationhas

takenplace169,170.Fursultiamine,asmallmoleculealsoknownasthiamine

tetrahydrofurfuryldisulfidethattargetsthecys326residueofferroportin,

indeedreducedhepcidin-mediatedinternalizationofferroportinandstimulated

20

cellularexportofiron,howeveritsfunctioninvivoislimitedduetorapid

conversionoffursultiamineintothiamine171.Anantibodyagainstferroportin

(LY2928057,EliLilly),whichhasbeenexploredclinically,hashadmoresuccess

invivo172.However,thelargestchallengeofanystrategydirectlytargeting

hepcidinoritsreceptor,ferroportin,isanunderlyinghomeostaticfeedback

mechanismthatmaycauseareboundinhepcidinexpression,therebylikely

abolishingalargepartofanylong-termtherapeuticeffect164.Thisfeedbackloop

mayexplainwhythedevelopmentofLY2928057seemstohavebeen

discontinueduponcompletionofaphase2trialin2015(NCT01991483).

Duetothehomeostaticcontroloverhepcidinexpression,approachesthat

interferewiththeunderlyingregulatorypathwaysmaythereforebemoreuseful,

althoughnonehasthusprogressedpastphase2ofpharmaceuticaldevelopment.

Severalstrategieshaveinhibitedhepcidinexpressionbymodulatingthe

BMP6/SMADpathway.CapturingBMP6topreventitsbindingtotheBMP6

receptor(BMP6R)hasbeeninvestigatedusinganhumanizedantibody

(LY3113593,EliLily,lostthroughattritionin2016(seeFurtherinformation,

NCT02604160),afusionproteinofhemojuvelin(HJV)andtheFcdomainofIgG

(FMX-8,phase2trialshavebeenterminatedin2016dueaninabilitytorecruit

patientsmeetingeligibilitycriteria,FerruMaxPharmaceuticals173),ornon-

anticoagulantheparin-derivativesthatbindBMP6inpreclinicalstudies174.A

secondstrategyaimstoreduceBMP6-inducedhepcidintranscription

downstreamoftheBMP6−BMP6Rinteraction,byblockingSMAD

phosphorylationusingdorsomorphin175oritsderivatives176,orbysiRNA-

induceddegradationofmRNAencodingforTfR2(ALN-HPN,discontinuedfor

unknownreasons,AlnylamPharmaceuticalsInc)177.

Ahepcidinagonisticstrategycomprisesinhibitionofmatriptase2(MT-2)-

inducedcleavageofHJV.HJVisacoreceptorintheBMP6Rcomplexthatis

requiredforironsensingandhepcidininduction178,andMT-2cleavesHJV,

therebysuppressingBMP/SMAD-inducedhepcidinexpression179,180.MT-2isa

typeIItransmembraneserineproteasethatisstructurallysimilartomatriptase,

anendothelialenzymeassociatedwithtumourdevelopment.Althoughacrystal

structureofMT-2isnotyetavailable,modellingoftheactivedomainhas

identifiedanumberofresiduesthatcanbetargeted,whichallowsthe

21

developmentofsmall-molecularinhibitorsagainstMT-2181,182.Serum-stabilized

antisenseDNA(IONIS-TMPRSS6-LRx,IonisPharmaceuticals183,184)orliposomal

siRNA(ALN-TMP,AlnylamPharmaceuticalsInc185),blockMT-2mRNA

translation,andhaveshownpreclinicalefficacyinanimalmodelsofβ-

thalassemiaandironoverload.Inaddition,severalphysiologicalprotease

inhibitorshavedemonstratedspecificMT-2-inhibitingactivity186,andsomeof

themhaveaKunitzdomainthatcouldbeanimportantleadforfurther

developmentofhepcidin-inducingpeptides187.

TheIL6-JAK1/2-STAT3pathwayisasecondregulatorypathwaythatinduces

hepcidinexpression.Theisoflavonegenisteinpromotesphosphorylationof

STAT3,inadditiontoSMAD4,andtherebyinduceshepcidinexpressionin

zebrafish,butitsactivityinapreclinicalorclinicalsettingneedstobe

evaluated188.Conversely,inhibitionofJAKbykinaseinhibitorseffectively

reduceshepcidinexpressioninvitro(1,2,3,4,5,6-hexabromocyclohexane189)and

invivo(AG490190),andsimilareffectshavebeenobtainedbydisruptingSTAT3

dimerizationusingaSTAT3-bindingpeptide191.

Therapeuticstargetingcellularirontrafficking

Formanydiseasesdisorderedironmetabolismtypicallyoccursonlyonacellular

level,whereassystemicironhomeostasisseemsunaffected.Therapiesthat

targetcellularironuptake,storageorefflux,maythereforebeparticularly

promising.Severalcompoundstargetcellularuptakeofiron,andDMT1in

particularhasbeenafrequenttargetinpreclinicalstudies.Structuralmodelling

ofhumanDMT1192,193,aswellasthecrystalstructureofScaDMT,aprokaryotic

DMT1homologuefoundinStaphylococcuscapitis194,suggestthatDMT1isa12

transmembraneproteinof2-foldsymmetryandcontainsahydrophobiccore

thatcoordinatesthecoupledtransferofabivalentmetalionandaprotoninto

thecytoplasm.

SeveralsmallmoleculeinhibitorsofDMT1-mediatedirontransporthavebeen

investigated.Ferristatinrestrictscellularintakeoffreeironinvitro,butits

efficacyinvivohasnotyetbeenevaluated195.Althoughtheinhibitoryeffectof

ferristatinwasatfirstattributedtodegradationofTfR1196,subsequentwork

demonstratedthatferristatindirectlyinhibitedactivityofDMT1195.Similarly,

22

ferristatinIIisastructurallyunrelatedcompoundthathasshownpotentTfR1-

inhibitingproperties,mediatedviaDMT1internalization,invitro,aswellasin

vivo197,198.Interestingly,ferristatinIIalsoinduceshepcidinexpressionvia

JAK/STAT3signallinginvivo199.However,themetabolisationofferristatinII,

alsoknownasdirectblack38orchlorazolblack,includestheformationof

benzidine,whichisclassifiedbytheWHOasGroup1carcinogen(carcinogenicto

humans)200,therebylimitingitstranslationfortherapeuticpurposes.The

selenium-containingdrugebselen,whichisknownforitsantioxidantactivity

thatresemblesglutathioneperoxidase,stronglyinhibitsDMT1-mediateduptake

ofiron(IC50≈0.2uM)201,socouldhavetherapeuticpotentialintreatingiron-

relatedoxidativestressinarangeofdisorders,ashasbeenshownpreclinically

foriron-inducedcardiomyopathy202.ComparablypotentDMT1inhibitionwas

achievedwithpyrrolidinedithiobarbamate(PDTC,IC50≈1.5μM)andΔ9-

tetrahydrocannabinol(THC),whichalsohaveantioxidantactivity201.Recent

screeningstudieshaveidentifiedaseriesofpyrazolederivativesand

benzylisothioureasaspotentinhibitorsofDMT1bothinvitro(IC50<1μM)and

invivo(30-85%inhibition,50mg/kg,takenorally)203–205.Aparticularly

interesting,butnotyetexploredstrategytolimitironoverloadmaybetheoral

administrationofanon-bioavailableDMT1inhibitor,i.e.acompoundthatisnot

systemicallyabsorbedandthereforelikelyhasnosystemicoff-targeteffects,but

wouldinhibitabsorptionofironfromtheintestine.Althoughthecapacityofthe

intestinetoabsorbhaem-boundiron206andnanosizedparticulateiron(suchas

theferritincore)207,whichbypasstheDMT1absorptionpathwayviaunknown

mechanisms,clearlyformsapotentiallimitationofthisapproach,combining

intestinalDMT1inhibitionwithdietaryrestrictionscouldbearelativelysafe

therapeuticoptioninthetreatmentofprimaryorsecondaryironoverload.

ThecentralroleofZIP14intransportofNTBIintotissuesthataretypically

affectedbyironoverload,especiallytheliverandpancreas29,makesthe

inhibitionofZIP14anappealingstrategyfortheclinicalmanagementofiron

overload.SimilartoothermembersoftheZIPtransporterfamily,ZIP14is

predictedtocompriseeighttransmembranedomains,whichareallinvolvedin

thetransportofbivalentmetals208.WhetherZIP14isasufficientlydruggable

targetstillneedstobeevaluated.

23

Thehypoxia-induciblefactor(HIF)transcriptionfactors,whicharecomposedof

aheterodimerizedα-andβ-subunit,maybeanotherveryattractivetargetfor

interferingwithironmetabolism209.HIFsmodulate,bybindingtoahypoxia

responseelement(HRE)inthepromoterofatargetgene,theexpressionofa

widerangeofproteinsthatareinvolvedincellularoxygenstatus,glucose

metabolism,angiogenesisanderythopoiesis.Inparallel,thepromotersofseveral

genesencodingkeyirontraffickingproteins,namelyDMT1210,211,DCytB211,

TfR1212,andFPN213,especiallythosewithrolesinintestinalcells,alsocontain

suchHREsandhavebeenshowntobesusceptibletoHIFregulation.Asa

consequence,inhibitorsofHIFs,forexamplethosethatbindwithintheC-

terminalPASdomainsofHIF-2αtopreventHIF-2heterodimerization214,may

formanappealingstrategyforlimitingironabsorptionandironoverload215.The

oxygen-sensitiveprolyl-4-hydroxylases(PHDs)arephysiologicalintracellular

inhibitorsthatregulatethepost-translationalexpressionofHIFsbypromoting

thehydroxylationoftwoprolineresiduesinitsα-subunit,labellingitfor

recognitionbytheVHL-E3-ubiquitinligasecomplexandsubsequent

degradation216,217.PHD2,whichseemstobemostdominantofthethreePHDs

withrespecttoHIFregulation,isahomotrimericproteinthatcontainsa

coordinatedFe(II)andahydrophobicpocketatitsactivesite,andrequires2-

oxoglutarate(2-OG)asaco-substrate218.Byusing2-OGanaloguesascompetitive

substratestopharmacologicallyinhibitPHDs,itispossibletostabilizeHIF

expression,whichisaverypromisingstrategyforpromotingerythropoiesisin

patientswithanaemia219.AlthoughthedirecteffectofHIFstabilizationonthe

expressionoferythropoietinisakeycharacteristicofthisstrategy,PHD

inhibitionalsoimprovesduodenalironadsorptionandutilization,asillustrated

byanimprovedmeancorpuscularvolume(MCV)andmeancorpuscular

haemoglobin(MCH)oferythrocytesinaratmodelforanaemiaofinflammation

andchronicdisease220.Moreover,increasedHIF-1expressionbyinhibitionof

PHD2improvedneuronalsurvivalinmodelsofPD,likelyduetoanincreased

expressionofthelysosomalprotonpumpATP13A2,whichreducedthecytosolic

ironpoolbyimprovinglysosomalironstorageconditions221.

Asubstantialnumberofpharmaceuticalcompanies,aswellasonetobacco

company,areactivelypursuingthedevelopmentofsuchinhibitorsforthe

24

treatmentofanaemiainchronickidneydiseaseandend-stagerenaldisease.

Promisingcandidatesthathavealreadyenteredphase3clinicaltrialsinclude

FG-4592(Roxadustat,Fibrogen/AstellasPharma/AstraZeneca222),GSK1278863

(Daprodustat,GlaxoSmithKline223),andAKB-6548(Vadadustat,Akebia

Therapeutics224),andseveralothersarecurrentlybeingevaluatedinaphase2

(BAY85-3934,Molidustat,BayerPharmaAG225;JTZ-951,JapanTobacco226),

phase1(DS-1093,DaiichiSankyo(discontinuedin2016));ZYAN1,Zydus

Cadila227),orpreclinical(JNJ-42905343,Johnson&Johnson220)setting.

Drugtargetingstrategiesexploitingironmetabolism

Ironandiron-basedmaterialshavebeenwidelyresearchedfordrugtargeting

anddiagnosticapplications,andironoxidenanoparticleshaveespecially

receivedattentionduetotheirsuperparamagneticpropertiesthatcanbeused

fortherapeuticanddiagnosticpurposes(Box1).Themetabolismofironcanbe

utilizedforthedeliveryoftherapeuticstospecificsitesinthebodybytargeting

iron-relatedproteinsthatareupregulatedincertaintissuesandpathological

processes(Figure5).TargetingTfR1usingdrugsanddrugdeliverysystems

functionalizedwithtransferrin,antibodies,antibody-derivatives,orTfR-targeted

peptides,isacommonlyemployedandarguablythebest-knownstrategy,as

illustratedbyitsusageasaprototypicalsystemformechanisticstudiesofactive

targetingtocancercells228.AnimpressiveamountofdifferentTfR-targeted

deliverysystemshavebeendesignedandevaluatedforcancertherapyby

exploitingtheincreasedexpressionofTfRinsometumours,andthisstrategyhas

shownpreclinicalbenefitwhenusingalow-doseregimen229.AtleastthreeTfR-

targetedsystemsarecurrentlyunderclinicaldevelopment(Table3)(MBP-426,

Mebiopharm;SGT-53,SGT-94,SynerGeneTherapeutics)230,231.

Anelegantstrategythathasbeenevaluatedinpreclinicalcancermodelsisthe

applicationoftransferrin-artemisininconjugatesascytotoxicagents232.

Artemisinin,discoveredanddevelopedbyTuYouyou,forwhichshewas

awardedthe2015NobelPrizeinphysiologyandmedicine,isadrugwith

antimalarialactivitythatisisolatedfromtheherbArtemisiaannuaandcontains

anintramolecularperoxidebridge.Fe(II)isinvolvedintheactivationof

artemisininbycleavageofthisendoperoxidebridge,resultingintheformationof

25

reactivecarbon-centredradicalsandsubsequentcytotoxicity233.Byusing

transferrinasatargetingmoiety,bothironandtheinactiveartemisinincanbe

deliveredtoTfR-overexpressingcancercells;uponinternalizationofthe

complexthereleasedFe(III)isoxidizedtoFe(II),whichinturnreducesand

activatesartemisininintoitscytotoxicform232.However,theeffectofsuch

artemisinin-constructsonothertissueswithhighTfR1expression,notablythe

bonemarrow,hasnotyetbeenreported.AsecondnotableapplicationofTfR-

targetingtherapeuticsisthetransportofdrugsanddrugdeliverysystemsacross

theblood−brainbarrier.Theendothelialcellsoftheblood−brainbarrier

transcytoseTfR-transferrincomplexestothebrainparenchymatofulfiltheiron

requirementsoftheCNS.Exploitingthismechanism,transferrinandantibodies

thattargetTfRcanbeusedasaTrojanhorsetodeliverdrugcarriersand

macromoleculestothebrain,butfutureevaluationshoulddemonstrateits

practicalpotentialinaclinicalsetting234,235.

Ferritinisacommonlyusedbiomoleculefordrugtargeting,asitcanefficiently

encapsulatelargeamountsofdrugsbecauseofits‘nanocage’structure236.

Ferritincanfunctionasaligandaswelltotargetcellsthathighlyexpress

ferritin-internalizingreceptors,suchasScara5andTfR1237.Moreover,ferritin

canbedisassembledandreassembledbychangingthepHtolowor

physiological,respectively,whichfacilitatesdrugloadingandendowsthe

proteinwithpH-triggereddrugreleaseproperties,enablingdischarge

specificallyuponuptakeandlysosomalprocessingbythetargetcell238.

Arelativelyspecificapproachtotargetasubsetofmacrophagesistheuseof

haemoglobinclearancemechanisms.Thehaemoglobin-scavengerreceptor,

CD163,isexclusivelyexpressedbymacrophagesandmonocytesthatrecycle

iron35,anditcanalsobefoundinsolidtumours239andinflamedtissues240,for

example,asthesetissueshaveincreasedironrequirements.Consequently,a

liposomaltargetingstrategyexploitingtissue-specificexpressionofCD163using

ananti-CD163antibodyhasdemonstratedeffectivemacrophagetargetinginvivo

ascomparedtountargetednanocarriers241.

Thevalueofhaemasatargetingligand,whichusesdifferencesincellularaffinity

forhaemtospecificallytargetsubsetsofcells,hasnotyetbeenfullyexplored.As

invitrostudieshaveshownthatPLGAnanoparticlesfunctionalizedwith

26

haematin,theoxidizedFe(III)formofhaem,localizeintumourcells242,and

haem-targetingimprovestheuptakeofoligonucleotidesinhepatocytes243,

furtherevaluationofhaem-targetingiscertainlywarranted.

Meritsandchallengesoftargetingironmetabolismastherapeuticstrategy

Themajorityoftherapeuticsthattargetironmetabolismhasbeendevelopedin

thecontextofhaematologicaldiseasesandironhomeostasisdisorders,suchas

irondeficiencyandoverload.However,consideringtherapidlygrowingevidence

implicatingironinavarietyofother(oftenageing-related)disorders,suchas

neurodegenerativediseasesandatherosclerosis,furtherexplorationofthese

therapeuticsagentsandtheirtargetsinotherpathologiesiscertainlywarranted.

Althoughunbalancedirontraffickingshouldnotbeconsideredthesolecause,

thechronicandinflammatorycharacteroftheunderlyingpathologicprocesses

inironmetabolism-relateddiseasessuggestthataniron-inducedchangein

redoxstatewithintheaffectedtissuesmayformthemissinglinkinthe

pathogenesisofseveralmultifactorialdisorders86,105,244.Importantly,apartfrom

directlymodulatingthefunctionofcellsresponsibleforthecondition,suchas

neuronsandtumourcells,ironalsoimpactstheinflammatoryactivityoflocal

immunecells,especiallymacrophages134,245,246(SeealsoBox1).Consequently,

interferingwithironmetabolismmaybeapromisingtherapeuticstrategyin

thesedisordersforwhichtherearecurrentlyfeweffectivetreatmentsavailable.

Aclearadditionaladvantageofsuchanapproachisthatittypicallyaddresses

pathologicpathwaysthatarecomplementarytothosetargetedbyother

therapeuticapproaches,allowingforadditiveorpossiblyevensynergisticeffects

whenusedinacombinationstrategy.

However,thebroadinvolvementofironmetabolisminmany,ratherdiverse

physiologicalandpathologicalprocessespotentiallymakesitsuseasa

therapeutictargetadouble-edgedsword.Althoughiron-targetedtherapeutic

interventionsmayeffectivelychangetheironstatusindiseasedtissuestotreata

disorder,theyalsohaveaninherentriskofperturbingsystemiciron

homeostasisthatmayleadtosubstantialsideeffects,suchaserythropoietic

27

disorders.Usingatargetedstrategythatallowsthedeliveryoftherapeuticsto

specifictissuescouldlimitsuchoff-targetactivity.

Inaddition,mostironmetabolism-relateddisordersareassociatedwithcellular

ironaccumulation,which,duetothelateonsetofsymptoms,ismostlikelya

chronicprocessthattakesseveralyearstoestablish.Interferingwiththis

processonceiron-inducedcellulardamagehasalreadyoccurredmaynothavea

directtherapeuticeffect,andpreventionmaybemoreefficaciousinthiscontext.

Nevertheless,limitingfurtherdeteriorationandmodulatingtheinflammatory

statewithintheaffectedtissuecouldcertainlymakeironmetabolism-targeted

therapiesaneffectiveadjuvanttreatment124,247.

Conclusionandoutlook

Overthepastdecade,amultitudeofironmetabolism-targetingagentshavebeen

developedandclinicallyevaluatedindiseaseswithanobviousiron-related

background,suchasanaemia,ironoverloadandiron-loadinganaemia.However,

alterationsinironmetabolismhavedemonstratedinvolvementinawiderange

ofdisorders,suchascancer,neurodegenerationandatherosclerosis,butalso

type2diabetes,osteoporosis,osteoarthritis,retinaldisordersandliverfibrosis.

Thechronicinflammatoryandageing-relatedcharacterofmanyofthese

conditionssuggeststhataslowlychangingintracellularironlevel,causingashift

incellularredoxstatusthateventuallymayresultincelldamage,isan

underlyingmechanismthatcouldbeexploitedforthedevelopmentofnew

therapeuticapproaches.Furtherinvestigationisthereforerequiredtoexplore

currentandnewlydevelopedtherapiesthattargetironmetabolisminamuch

broadercontextthanhaematologicalpathologiesalone.Modulationof

macrophageactivitybytargetingtheirroleinironrecyclingmaybea

particularlyinterestingapproach.Similartoironmetabolism,theroleofan

alteredmacrophagephenotypehasbeenincreasinglyimplicatedinvarious

disorders248,andbothconceptsseemtobeinterconnected245.Targeted

approaches—forexamplethoseusingnanosizeddrugdeliverysystemsthat

preferentiallyaccumulateinphagocyticcells,tospecificallyinterferewithiron

traffickinginmacrophages—wouldtherebyenabletherapeuticmodulationof

macrophageactivityincancerandchronicinflammatorydisorders75,249,250.To

28

achievethis,thecellularmechanismsgoverningtherelationshipbetween

macrophagephenotypeanditsironloadneedtobefurtherelucidated.For

example,recentworkhasdemonstratedthatinternalizationofunboundhaem

inducesaninflammatoryphenotypeinmacrophages,whereasusinghaemin

complexwithhemopexin,whichisbescavengedbyCD91,suchaphenotypic

switchwasnotobserved77.Thisobservationindicatesthatthepathwayof

internalization,ratherthantheabsoluteloadofiron,hasadominantroleinthe

influenceofirononthephenotypeofmacrophages,therebyprovidingvaluable

cluesfortheidentificationofnewstrategiesformacrophage-modulating

therapies.

Overall,owingtothesubstantialadvancesinourunderstandingoftheroleof

ironinarangeofdiseases,ithasbecomeclearthattargetingironmetabolismis

acompellingnewtherapeuticstrategyforthesedisorders.Althoughfurther

researchisstillrequiredtosystematicallyexplorethevalueoftargetingthe

variousiron-relatedproteinsinindividualpathologies,theseeffortswillalmost

certainlyprovetobevaluableinthequestfornovelefficacioustherapiesfor

diseasesassociatedwithdisturbedironmetabolism.

29

Acknowledgments

TheauthorswishtoexpresstheirgratitudetoK.Römhildforherassistancein

theliteraturereview.Theauthors’workissupportedbytheEuropeanUnion’s

Horizon2020researchandinnovationprogrammeundertheMarie

Sklodowska−CuriegrantagreementNo707229(ADHERE)(B.J.C),theGerman

ResearchFoundation(DFG:La2937/1-2)(T.L),theEuropeanResearchCouncil

(ERC:StG-309495,PoC-680882)(T.L),theNIHgrantsR01DK095112and

R01DK090554(S.R.).

Competingfinancialinterests

S.R.hasrestrictedstocksinMerganserBiotech.S.R.isaconsultantforNovartis

Pharmaceuticals,BayerHealthcare,MerganserBiotechandKeryx

Pharmaceuticals.S.R.isamemberofscientificadvisoryboardofMerganser

BiotechandIonisPharmaceuticals.T.L.isamemberofscientificadvisoryboard

ofCristalTherapeuticsB.V..

Furtherinformation

Protagonist’spipeline.http://www.protagonist-inc.com/pipeline-main.php

LaJollaPharmaceuticalLJPC-401Phase1ResultsandDevelopmentUpdate.

http://lajollapharmaceutical.com/wp-content/uploads/2016/09/LJPC-401-

Phase-1-Results-and-Development-Update-9.7.16.pdf

EliLilly’sclinicalpipeline.

https://www.lilly.com/_assets/SiteCollectionDocuments/Pipeline/Clinical-

Development-Pipeline/index.html

30

Glossaryterms

Microcytic,hypochromicanaemia

LowsystemicHblevelsassociatedwithareducedHbconcentrationinsideeach

redbloodcell,makingthemsmall(microcytic)andpale(hypochromic).Thisis

mostcommonlycausedbyirondeficiency,butisalsofoundinpatientswithred

bloodcelldisorderssuchasthalassemia.

Hemochromatosis

Hemochromatosisisafamilyofhereditarydiseasesthatarecharacterisedby

geneticmutationsaffectinganironmetabolism-regulatingproteinandresulting

inexcessiveironabsorption.Sincethereisnophysiologicalmechanismforiron

excretion,thiswilleventuallyleadtoironoverload.

Anticalin

TechnologydevelopedbyPierisPharmaceuticalsthatmodifieslipocalins,which

arenaturalextracellularproteinsthatbindandtransportavarietyofligands,to

maketherapeuticsthattargetaspecificmolecule.

Spiegelmer

TechnologydevelopedbyNOXXONPharmathatusesL-RNAaptamersas

therapeuticagentsthatbindaspecificmolecule.L-RNA,thestereoisomericform

ofphysiologicalD-RNA,isnotsensitivetonucleaseactivityandthereforehas

improvedbiologicalstability.

Kunitzdomain

AKunitzdomain,orKunitz-typeproteaseinhibitordomain,istheactivedomain

ofvariousproteaseinhibitors.Theirrelativelyshortpeptidesequenceofaround

60aminoacidsmakesthemaninterestingleadforthedevelopmentnew

biopharmaceuticalproteaseinhibitors.

31

Box1–Ironoxidenanoparticles

Overthepastfewyears,anenormousvarietyofironoxidenanoparticleshave

beendevelopedfordiagnosticandtherapeuticapplications,althoughfewhave

reachedtheclinicandseveralhavebeenwithdrawnforvariousreasons251.Also

knownassuperparamagneticironoxidenanoparticles(SPIONs),thesesterically

stabilisedparticlestypicallycontainmagnetite(Fe3O4)andmaghemite(γFe2O3),

andtheresultingmagneticpropertiesmakethemparticularlyinterestingas

magneticresonanceimaging-contrastagents.Duetotheirnanosizeddimensions,

ironoxidenanoparticlesprimarilylocalizeinphagocyticcells,mostly

macrophages252,andintheliver,spleenandlymphatictissues,soironoxide

nanoparticlescanbeutilizedforimagingthesetissues.Forexample,Feraheme

(ferumoxytol,AMAGPharmaceuticals),whichisclinicallyapprovedforiron

replacementtherapyinchronickidneydisease,hasbeeninvestigatedasmarker

forthepresenceofmacrophagesinsolidtumours253,andhasrecentlybeen

showntomodulatetheactivityofthesemacrophagestowardsa

proinflammatory,anti-tumouralphenotype75.Thereareseveraladditional

promisingapplicationsofSPIONs,suchasusingtheirmagneticpropertiesand

localisationtumourstocreatelocalhyperthermiabyapplyinganalternating

magneticfield(NanoTherm,MagForceAG),orusingthemasdrugcarriersthat

releasetheirpayloadasaresultofmagneticallyinducedheating254.While

SPIONsaregenerallyconsiderednon-toxic,itisimportanttorealisethatthereis

nophysiologicalrouteofironexcretionfromthehumanbody,otherthana

passiveintestinallossthatistypicallylimitedto2mgironperday.Therefore,

intravenouslyadministeredironoxide,whichwouldbeintherangeof400mg

iron(Feraheme,NCT01336803),forexample,willaddtothetotalironpooland

may,inparallel,causeanproinflammatoryphenotypicswitchinthe

macrophagesclearingthesenanoparticles75.Furtherevaluationregardingthe

long-termtolerabilityofSPIONsasroutinelyusedimagingagentsmaybe

thereforewarranted.

32

Figure1–Systemicironmetabolism.Dietaryironisabsorbedbyduodenal

enterocytesandaddedtothesystemicironpooluponexportviaferroportin

(FPN,green).Thesystemicironpool,whichundernormalsteady-state

conditionsismainlyboundtotransferrin(orange),isprimarilyutilizedfor

erythropoiesis.Excesssystemicironisstoredasferritin(yellow)intheliver,and

toalesserextentinothertissues.Theironutilizedinsenescenterythrocytesis

recycledbymacrophagesandreleased,viaferroportin,backintothesystemic

ironpool.Ferroportinexpression,andconsequentlysystemicironavailability,is

inhibitedbyhepcidin(blue),apeptideprimarilyproducedbytheliver.Inturn,

transcriptionofthegeneencodinghepcidin,HAMP,isstimulatedbythepresence

ofironandinhibitedbyerythropoieticdemand.

Duodenum

HAMP

Hepcidin

BoneMarrow

MacrophageErythrocytes

Liver

Systemicironpool

FPN

33

Figure2–Regulationofhepcidinexpressioninhepatocytes.Bone

morphogeneticprotein6(BMP6)isproducedbynon-parenchymalcellsinthe

liverinresponsetoironstoresandflux.BindingofBMP6totheBMPreceptor

complexonhepatocytes,inconjunctionwithhemojuvelin(HJV)resultsin

downstreamsignallingviaSMAD1/5/8andSMAD4,whichinturnstimulates

HAMPtranscriptionandexpressionofhepcidin.HJVisstabilizedbyneogenin,

andcleavedbymatriptase2(MT-2),allowingforfine-tuningofBMP6signalling.

SMADsignallingisadditionallyregulatedbytransferrinreceptor2(TfR2),upon

associationwithhumanhemochromatosisprotein(HFE)andHJV.HFEalso

interactswithTfR1,whereitcompeteswithholotransferrin.Consequently,when

ironsupplyishigh,HFEisdisplacedfromTfR1allowingittoassociatewithTfR2,

therebyincreasinghepcidinexpressionviaSMADsignalling.Underinflammatory

conditions,interleukin6(IL-6)andrelatedcytokinesbindIL-6receptor,

resultinginJAK1/2-STAT3activationandhepcidinexpressiontorestrictiron

availability.Finally,developingerythroidcellsconfertheirironrequirementsvia

secretionoferythroferrone(ERFE),whichinhibitshepcidinexpression,viaa

currentlyunknownreceptorandsignallingpathway,tostimulateironuptake

undercircumstancesofhigherythropoieticdemand.

BMP6

MT-2BMP6

R

HAMP

HFE

TFR2 TFR1

IL6

IL6R

ERFE

STAT3

Hepcidin

?Neo-genin

HJV

SMAD1/5/8SMAD4

JAK

34

Figure3–Cellularirontraffickingpathways.Ironintake.Non-transferrin

boundiron(NTBI)canbetransporteddirectly(solidarrow)intothecellby

DMT1,ZIP14andZIP8,afterreductionofFe(III)toFe(II)byferrireductases

STEAP,DcytBorSDR2.Transferrin-boundironistakenupviabindingtoTfR1

andendocytosis(dashedarrow)ofthereceptor,releaseandreductionofFe(III)

andtransportintothecytoplasmviaDMT1.Haemoglobinassociateswith

haptoglobintoallowsendocytosisbyCD163,uponwhichhaemoglobinis

degraded,andhaemtransportedintothecytosol.Systemichaemisscavengedby

complexationwithhaemopexinandendocytosisviaCD91.Haemcanbedirectly

transported,alsofromtheendosome,intothecytosolbyHRG1andFLVCR2,

whereitisprocessedbyhaemoxygenase1andironreleasedasFe(II).Scara5

internalizesferritinthatconsistsoflightchains,afterwhichironisliberatedand

transportedtothecytosol.Ferritincomposedofheavychainscanbe

endocytosedbyTfR1(notdepictedhere).

Ironutilization.Thelabileironpool(LIP)comprisescytosolicFe(II),whichcan

bestoredincytoplasmicferritinorutilizedforbiochemicalprocesses.Mostof

theseprocessestakeplaceinthemitochondria,forwhichironissuppliedvia

DMT1

FPN

TfR1ZIP14ZIP8

STEAPDcytBSDR2

Anchored ceruloplasmin (Cp)Hephaestin

ferro-reductase

ferroxidase

Cp

NTBI Transferrin Haemoglobin Ferritin

Ferritin

Mitochondrion

Haem

Fe(III)

Fe(II)

CD163

Haptoglobin

Haemopexin

CD91 HRG1 FLVCR2

FLVCR1a

FLVCR1b

Mitoferrin

Scara5

Haem oxygenase 1

LIP

IRP/IREcontrol

TfR1DMT1FerritinFPN1

HIF/HREcontrol

TfR1DMT1

PHD

O2

Iron

inta

keIro

n ut

iliza

tion

Iron

efflux

35

mitoferrin,whileFLVCR1ballowsexportofmitochondriallyproducedhaem.The

LIPcontrolstheexpressionofTfR1,DMT1,ferritinandferroportinviathe

posttranscriptionalIRP/IREregulatorysystem.Similarly,thetranscriptional

HIF/HREregulatorysystemcontrolsseveralintracellularprocesses,including

thetranscriptionofTfR1andDMT1.HIF2αtranslationisinhibitedbyIRP1,

while,conversely,IRP1transcriptionisinhibitedbyHIF.HIFexpressionis

regulatedbyPHD,whichstimulatesthedegradationofHIFsunderconditionsof

highoxygen.

Ironegress.Fe(II)isexportedbyferroportin,followedbyoxidationtoFe(III)by

theferroxidaseshephaestinorceruloplasmin,andsubsequentbindingto

transferrin.IntracellularhaemcanbedirectlyexportedviaFLVCR1a.

Notallpathwaysarepresentinallcells.

36

Figure4–Up-anddownregulationofseveralkeyplayersinnon-

haematologicalpathologies.Schematicoverviewillustratingthe

microenvironmentalandsystemicchangesinironandironmetabolism-related

proteinsinselectedpathologiesinpatients:multiplesclerosis(MS,sclerotic

plaques),Alzheimer’sdisease(AD,frontalcerebralcortex),Parkinson’sdisease

(PD,basalganglia),atherosclerosis(AS,scleroticplaques),andcancer(tumor

tissue).

A.UpregulationofDMT1hasbeenobservedinMS(preclinical)123,AD

(preclinical)255,PD256,andcancer(colorectal)257.B.TfR1upregulationhasbeen

foundinMS258,atherosclerosis259,andcancer(colorectal257,breast95,101,

glioblastoma94;normalTfR1levelshavebeenobservedinAD260,261,whileforPD

normallevels262,andlowlevelshavebeenreported263,264.C.Increasedlevelsof

themacrophage-associatedscavengerreceptorCD163havebeenfoundin

affectedtissuesofpatientswithMS265,AD266,PD266,AS267,andcancer(breast268,

prostate269,glioblastoma270.D.Tissueironstoredasferritinhasfoundtobe

increasedinMS271,AD272,PD264,AS259,273,andforcancerbothhighlevels

Systemic

Microenvironmental

utili

zatio

nTfR1 CD

163

TfSaturation

DMT1

FPN

Ferritin

Iron

inta

keeffl

ux

MS

Cancer

MS

PD

Cancer

AD

MS

PD

Cancer

Cancer

AD MS

Cancer

AD

PD

PD

AD=

Hepcidin

PD

Cancer

MS

AD

=

Iron

AS

AS

MS

PD

Cancer

AD

AS

MS

PD

Cancer

AD

AS

MS

Cancer

Cancer

ADAS AS

AS

AS

= AS

A B

E

C

Ferritin

MS

PD

Cancer

AD

AS

Cancer

D

F

G

H I

37

(glioblastoma)94andlowlevelshavebeenreported(breast)101.E.Similarly,

microenvironmentalirondepositshavebeenobservedinMS271,AD271,272,274,

PD256,264,274,AS273,andcancer(colorectal)257.F.UpregulationofFPNhasbeen

demonstratedintissuesofpatientswithPD264,AS273,andcancer(colorectal257,

breast101),whilelowlevelsofFPNhavebeenobservedinMS(preclinical)123,

AD120andalsoinbreast91andprostate98cancer.G.Hepcidinlevelsarenotinall

diseasesconsistentwith(aninversed)FPNexpression.Highhepcidin

concentrationshavebeenfoundinMS(preclinical,tissue)123,AS(serum)275,

breastcancer(tissue91,101andserum276)andprostatecancer(tissue)98,whereas

alowlevelhasbeenobservedinthetissuesofpatientswithAD120.H.Increased

systemicferritinconcentrationshavefoundinthecerebrospinalfluid(CSF)of

patientswithMS277,AD127,andglioblastoma278,aswellasintheserumof

patientswithAS279andbreastcancer276,althoughthelevelsofserumferritin

havealsobeenfoundtobenormalinpatientswithAS275andnon-skin

cancers280.I.Systemicironlevelsmeasuredbyironconcentrationand

transferrinsaturationhavebeenquiteinconsistent:higherthanaveragelevels

havebeenfoundinametaanalysisinPD281,normallevelshavebeenfoundin

MS282andAS275,andlowlevelshavebeenobservedinAD283,284.Withregardto

cancer,theassociationwithsystemicironlevelsisquiteunclear:womenwith

highlevelsofserumironwereathigherriskfordevelopingnon-skincancer,

whilemenwereatlowerrisk280.

38

Figure5–Proposedmechanismsfortargeteddrugdeliveryexploitingiron

metabolism-associatedcellulartargets.Thenaturalligandandthemeritsof

itsuseasadeliverystrategyarepresentedforeachtarget.TfR1,CD163and

Scara5allowendocytosisofdrugsassociatedwiththeirnaturalligands.

Subsequentlysosomalprocessingwouldthenliberatethetargeteddrug,which

thenentersthecytosolbypassivediffusionfromthelysosome.Thislikelyalso

occursforhaem-targetedconstructsuponinternalizationbyCD91,butpossibly

directtransportofthehaem-linkeddrugintothecytosolbyHRG1and/or

FLVCR2couldrepresentanalternativemechanism.DrugtargetingtoFPN,for

examplebyusinghepcidin,hasnotbeenexploreduntilnow,butformsan

interestingapproachfordirectingtherapeuticstocellthatoverexpressFPN,

suchassometypesoftumourcells.Tf,transferrin;Hb,haemoglobin;FPN,

ferroportin

CD163 FLVCR2

CD91TfR1

Scara5

FPN

Ligands: TfMerit: Blood-brain barrier & cancer

cell targeting

Ligands: HepcidinMerit: Iron recycling macrophage hepatocyte, enterocyte & cancer cell targeting?

Ligands: Hb Merit: Iron recycling macrophage targeting

Ligands: HaemMerit: Macrophage, hepatocyte & cancer cell targeting

Ligands: L-ferritinMerit: Drug carrier with pH-triggered release properties Cancer cell targeting

?

?

Drugs

39

Table1–Keyplayersinsystemicironmetabolism.Name Gene Characteristics Functioninsystemiciron

physiologyDysfunctionalphenotype Refs

Bonemorphogeneticprotein6(BMP6)

BMP6 • HomodimericmemberoftheTGF-βsuperfamily,expressedbynon-parenchymallivercells,cartilage,bone

• Majormediatorinnegativefeedbackloopbetweenironandhepcidin

• Expressedinliverinresponsetointracellularironstoresorfluxofiron

• InteractswiththeBMPreceptorcomplexonhepatocytestoinduceHAMPtranscriptionandexpressionofhepcidinthroughtheSMADsignallingpathway

• Inhumans,heterozygousmutationsinBMP6resultinrelativelymoderate,late-onsetironoverload

• Completedeficiencyinmiceleadstosevereironaccumulationinliver,pancreas,heartandkidney

173,285–

288

Erythroferrone(ERFE,alsoknownasmyonectinorCTRP15)

FAM132B • MemberoftheTNF-αsuperfamily,producedandsecretedbyerythroblasts

• Producedinresponsetoerythropoietin

• SuppressesHAMPtranscriptiontoincreaseironsupply

• Receptorandsignallingpathwayintheliverareunknown

• Noknownmutationsinhumans.• Inmice,ERFEdeficiencyisnotassociatedwithanabnormalphenotype,buttheerythropoieticresponsetoerythropoietinisdelayed

289,290

Ferroportin(FPN1,alsoknownasSLC11A3,MTP1orIREG1)

SLC40A1 • Transmembraneprotein,highlyexpressedinhepatocytes,enterocytes,macrophagesandplacentalcells

• ExportsFe(II)acrossthecellularmembrane

• Istheonlyknownironexporterinvertebrates

• Humanheterozygousmutationsleadtoautosomaldominanthemochromatosistype4,whichisassociatedwithliverfibrosis(Kupffercellironoverload),marginalanaemia.

• Completelossoffunctionisembryoniclethalinmice.

12,14–

16,291–

293

40

Hemojuvelin(HJV,alsoknownasrepulsiveguidancemoleculeC;RGMc)

HFE2 • Heterodimericproteinexpressedonthecellsurfaceofhepatocytes

• CoreceptorintheBMPreceptorcomplex:alsointeractswithBMP6,possiblytoassistitsrecruitmenttothemembrane

• RequiredforSMADsignallingandHAMPtranscription

• Formsacomplexwithtransferrinreceptor2andHFEandfunctionsasironsensor

• Humanhomozygousorcompoundheterozygousmutationsleadtojuvenilehereditaryhemochromatosistype2A

• Severeironoverloadwithsimilarclinicalpresentationastype2B(HAMPmutations)

178,294–

296

Hepcidin(alsoknownasLEAP-1)

HAMP • 25aminoacidsecretedpeptide

• Producedprimarilyinliver,butalsobymacrophagesandseveralothercelltypes

• Masterregulatorofironmetabolism• Bindstoferroportin,inducingitsinternalizationanddegradation

• HumanhomozygousorcompoundheterozygousHAMPmutationsleadtojuvenilehereditaryhemochromatosistype2B,whichisassociatedwithsevereiron-overloadresultinginiron-relatedtoxicityearlyinlife:liverfibrosis,arthropathy,cardiomyopathy,hypogonadism,andreducedglucosetolerance

9–

11,13,29

7,298

Humanhemochromatosisprotein(HFE,alsoknownasHLA-H)

HFE • MonomericMHCclassI-typeprotein,expressedbyhepatocytes,intestinalepithelialcells,macrophages,placentalcells

• Competeswithtransferrinforbindingtotransferrinreceptortype1(TfR1)

• Associateswithtransferrinreceptortype2(TfR2),inconcertwithHJVtoinduceHAMPtranscriptionviaSMADsignalling

• HFEdisplacementfromTfR1anditssubsequentassociationwithTfR2andHJVlikelyfunctionasindependentextracellularsensors

• Homozygous(majorityC282Y)orcompoundheterozygousmutationsinHFEcauseadulthereditaryhemochromatosistype1:themostcommonformofhemochromatosis,associatedwithprogressiveironoverloadlaterinlife,leadingtoliverfibrosisandcardiomyopathy

296,299–

302

41

fortransferrin-boundiron,

Interleukin6receptor(IL-6R,alsoknownasCD126)

IL6R • TypeIcytokinereceptorexpressedonthesurfaceofhepatocytesandimmunecells

• SolubleIL-6R(sIL-6R)maybeinvolvedinIL-6(trans-)signallinginallcells

• BindingofIL-6orrelatedcytokinestoIL-6RpromotesHAMPtranscriptionthroughglycoprotein130(gp130)associationanddownstreamJAK1/2-STAT3activation.

• Hepcidinexpressionduringinflammationisconsideredoneofthehallmarksofanaemiaofinflammationandchronicdisease

• Noknownmutationsinhumans• MicedeficientforIL-6havenormalserumironlevels,butarelesssensitivetoinflammation-inducedanaemiaascomparedtowild-typemice

• TheironstatusinIL-6Rdeficientmicehasnotyetbeeninvestigated

56,246,3

03,304

Matriptase2(MT-2)

TMPRSS6 • TypeIItransmembraneserineprotease,expressedprimarilybyhepatocytes

• InhibitshepcidinexpressionbylimitingBMP/SMADsignalling,which,invitro,istheresultofMT-2-mediatedcleavageofHJV

• Geneexpression,stability,andenzymaticactivityofMT-2ispartofafeedbackloopthatrespondstoirondeficiency,hypoxiaanderythropoietin,butalsotochronicironloadingandBMP6

• HomozygousorcompoundheterozygousmutationsinTMPRSS6resultinhereditaryiron-refractoryirondeficiencyanaemia(IRIDA),whichismarkedbyinappropriatehighlevelsofhepcidin

55,179,1

80,305,3

06

Neogenin NEO1 • Transmembraneproteinwith4immunoglobulin-likeand6fibronectinIII-likeextracellulardomains

• StabilizesHJVonthemembranebybindingina2:2stoichiometryviainteractionsbetweentheRGDsequenceonHJVandthefibronectindomaininneogenin

• AssociateswiththeBMPreceptorcomplexviaBMPreceptortypeIa

• Noknownmutationsinhumans• Inmice,lossofinteractionofHJVwithneogeninimpairsBMP6-inducedhepcidinexpression

• Neogeninknockoutinmiceisperinatallethal

294,307,

308

Transferrin TFR2 • TypeIImembrane • Bindstransferrinsaturatedwith2 • Homozygousorcompound 296,300,

42

receptor2(TfR2,alsoknownasHFE3)

proteinthatisrelativelysimilar(66%)toTfR1,andisprimarilyexpressedintheliveranderythroidcells

Fe(III)ions(holotransferrin)andassociateswithHFEandHJVtoformanextracellulartransferrin-boundironsensorandregulatehepcidinexpressionviatheSMADsignallingpathway

• TfR2canbecleavedandreleasedfromthesurfaceunderirondeficientconditions,butthefunctionofthissoubleTfRremainsunclear

heterozygousmutationsintheTFR2geneunderlieadulthereditaryhemochromatosistype3:theironoverloadisusuallymoresevereandpresentsitselfearlierinlifethaninHFE-associatedhemochromatosistype1,butlessseverethanHJV-associatedhemochromatosistype2B

• Similarobservationshavebeenmadeinknockoutmice.

301,309–

311

43

Table2–Keyplayersincellularironhomeostasis.

Name Gene Characteristics Functionincellularironphysiology

Dysfunctionalphenotype Refs

Ironintake

ClassAscavengerreceptor5(Scara5,alsoknownasTESR,NET33orSR-A5)

SCARA5 • Membrane-boundmemberoftheclassAscavengerreceptorfamily

• Primarilyexpressedbyfibroblastsintherespiratorytract,theurinarytract,skin,retina,testesandovaries

• BindsandinternalizesL-ferritin• Tissuedistributionsuggestsaroleinanti-bacterialdefence,similartootherclassAscavengerreceptors

• Scara5-deficientmicedevelopnormally,butlaterinlifeantinuclearautoantibodiesareformedandlymphocyteinfiltrationsoccurinvariousconnectivetissues,reminiscentofhumanconnectivetissueautoimmunediseases

41,312,313

Lowdensitylipoproteinreceptor-relatedprotein(LRP,alsoknownasCD91

LRP1 • MemberoftheLDLreceptorfamilythatbindsawidearrayofligands

• Expressedbymanydifferentcells,includingmacrophages,hepatocytes,fibroblastsandneurons

• Scavengesandendocytoseshaemincomplexwithhaemopexin

• Inducesexpressionofhaemoxygenase-1

• LRP1deficiencyisembryoniclethalinmice

• Inmice,macrophage-specificdeletionpromotedatherosclerosis,buttherelationshipwithironwasnotevaluated.

37,314,315

CD163(alsoknownasM130,p155,haemoglobinscavengerreceptororSR-I1)

CD163 • Familymemberofthescavengercysteine-richdomain-containingproteins

• Receptorexpressedbymonocyte-likecells,includingmacrophages

• Canbeshedfrom

• Endocytoseshaemoglobinboundtohaptoglobinasdimericormultimericcomplexes

• InducesIL-10releaseandhaemoxygenase-1synthesis

• Inmice,CD163deficiencydoesnotleadtophenotypicchanges

35,36,316

44

membraneassolublesCD163

Cytochromeb561,familyincludingduodenalcytochromeb561(Dcytb)andferricchelatereductase1(alsoknownasstromalcell-derivedreceptor2;SDR2)

CYBRD1FRRS1

• Familyoftransmembraneproteins,containingtwohaemcofactors

• Isoformsarehighlyexpressedinduodenum(Dcytb),liver,andkidney(SDR2),aswellasinothertissuesincludingthebrain

• EnzymaticallyreducesFe(III)toFe(II)usingascorbate(vitaminC)asanelectrondonor

• Enablesirontransportacrossthecell/lysosomalmembranebyDMT,ZIP14orZIP8

• Inmice,Cybrd1knockoutdoesnotchangesteady-stateserumironlevelsorironstores,butintestinalironabsorptionislimitedduringerythropoieticstress

30,317–319

Divalentmetal-iontransporter1(DMT1,alsoknownasDCT1orNramp2)

SLC11A2 • 12-transmembraneprotein,ubiquitouslyexpressed,withhighlevelsinduodenum,erythroidcells,brainandkidney

• Transportsunboundextracellularbivalentmetals,includingFe,intothecell

• Inhumans,mutationsinDMT1havebeenfoundinpatientswithmicrocyticanaemiaandironoverload

• CompleteDMT1disruptionislethalshortlyafterbirthinmice

21,27,194,32

0

Felineleukaemiavirus,subgroupC,receptor2(FLVCR2,alsoknownasSLC49A2)

FLVCR2 • Transmembraneproteinhighlytranscribedinvarioustissues,includingthebrain,placenta,lung,liver,kidney,spleenandpancreas

• Importshaemintothecell• Exactfunctioninoverallironhomeostasisnotyetclear

• HomozygousorcompoundheterozygousmutationsinFLVCR2underlieFowlersyndrome,asyndromethatoftenresultsinprenatallethalitymarkedbyCNSmalformations,includinghydrocephalyandhydranencephaly.

39,321

45

Haemresponsivegene-1(HRG-1)

SLC48A1 • Membranetransporterwidelyexpressedinbrain,kidney,heartandmuscle

• Lowexpressioninliver,lung,placentaandintestine

• Transportshaemacrosstheendosomalmembrane,primarilyinmacrophagestorecyclehaemaftererythrophagocytosis.

• Noknownorgeneratedmutationsinmammals

• Inzebrafish,HRG-1knockoutleadstoseveredevelopmentaldefects,suchashydrocephalusanderythropoieticimpairment

38,40,322

Six-transmembraneepithelialantigenofprostate(STEAPfamilyproteins)

STEAP1-4 • Transmembraneproteinswith4homologousmembers

• Ubiquitousexpression;elevatedlevelsofSTEAP1inparticularcanbefoundinavarietyofsolidtumours,includingprostate

• STEAP2-4canreduceFe(III)toFe(II)

• STEAP3hasbeenimplicatedasthemainferrireductaseintheTfcycle,enablingirontransportoutoftheendosome;STEAP2andSTEAP4haveshownasimilarcapacity

• STEAP2-4arealsoinvolvedincopperreduction

• Inhumans,loworabsentexpressionoffunctionalSTEAP3leadstohypochromicanaemia,duetoaninsufficientironsupplyforHbsynthesis.

• STEAP3-deficientmiceretainsomeferrireductiveactivity,possiblyviaSTEAP2andSTEAP4

31,32,323–

325

Transferrin(Tf) TF • Abundantmonomericserumglycoprotein,primarilyproducedintheliver

• Tfcomprisestwolobes,eachcapableofbindingaFe(III)ionwithhighaffinity(Kd≈10-20MatpH7.4).

• Homologousfamilymembersinmammalsincludelactoferrin,melanotransferrinandovotransferrin

• ChelatesandtransportsinsolubleFe(III)inthecirculation

• Bindingtotransferrinistheprincipalmethodforsystemicirondistribution

• About3mgofironistransferrin-boundatanytime(±30%Tfsaturation),and30mgisturnedoverbyTfeachday

• HomozygousorcompoundheterozygousmutationsinTFresultintheraredisorderatransferrinemia;thisdiseaseisassociatedwithanaemiaandsecondaryironoverload,duetoaninsufficientironsupplytoimmatureerythroidcells

17,18,326

46

Transferrinreceptor1(TfR1,alsoknownasCD71)

TFRC • HomodimerictypeIItransmembraneglycoproteinubiquitouslyexpressedonthesurfaceofmostcells

• Anon-membranebound,truncatedmonomericform(sTfR)canbefoundintheserum

• EachmonomercanbindasingleTfwithnanomolaraffinityforbiferricTf.

• UponinternalizationoftheTf-TfRcomplexbyclathrin-mediatedendocytosis,ironisreleasedunderacidicconditionsinthelysosomeandtheapo-Tf/TfRcomplexisrecycled

• TfR1canalsobindandsupporttheinternalizationofh-ferritin

• ThefunctionofsTfRiscurrentlyunknown,butitservesasabiomarkerforanaemiaandsomemalignancies

• Completedeficiencyisembryoniclethalinmice

• Inhumans,ahomozygousmissensemutationinTFRCthataffectsitsinternalizationresultsincombinedimmunodeficiencyandmildanaemia;thelimitedimpactofthismutationonhaemoglobinlevelscouldberelatedtocompensationbySTEAP3

18,19,42,78,

327

Zrt-andIrt-likeprotein8(ZIP8)andZIP14

SLC39A8SLC39A14

• Transmembraneproteins,partofthezinctransporterfamily,highlyexpressedinliver,pancreas,brainandheart

• TakesupunboundFe(II)(aswellaszinc)intothecell

• ZIP14isfoundathighlevelsinliverandothertissuesthataccumulateironiniron-loadingdisorders

• ZIP8isinvolvedintheuptakeofunboundironinneurons

• Noknownmutationsinhumans• ZIP14-deficientmiceshowreducedcapacitytointernalizeironintheliverandpancreas,butserumironandHblevelsaresimilartowild-typemice

• ZIP14-deficientmicehaveskeletalunderdevelopmentduetoZndeficiency.

22,23,29,208

,328,329

Ironutilization

Ferritin(Ftn) FTH1(heavychain),FTL(lightchain)

• Ubiquitouslyexpressedproteinthatiscomposedof24subunitsandcomprisesa

• Storesiron:eachferritinmoleculecanbindupto4,500Fe(III)atoms

• AheterozygousmutationinFTLisassociatedwithneuroferritinopathy,a

46,50,51,128

,330

47

combinationofheavyandlightchains,whichtogetherformahollowshellofaround12nmindiameter

• EachH-subunitcomprisesaferroxidasecentrethatallowstheoxidationofFe2+intoFe3+,whiletheL-subunitspromoteironmineralnucleation.

• Extracellular(serum)ferritin,mostlyconsistingofL-ferritin,secretedbytheliverandmacrophages,servesasabiomarkerforsystemicironload.

neurodegenerativesyndrome• TheonlyknowncaseofcompleteFTLdeficiencyresultedinseizuresandrestlesslegsyndrome,likelyresultingfromirondeficiencyduetothehigheraffinityoftheheavychainforiron

• Fth1deficiencyinmiceisembryoniclethal

Haemoxygenase-1(HO-1)

HMOX1 • Highlyinduciblecatalyticenzyme,expressioncan,inprinciple,occurinmostcells

• Degradeshaemintoiron,carbonmonoxideandbiliverdin;thelattertwohaveanti-inflammatoryproperties

• Isimportantforironrecyclingfromerythrocytesbymacrophages

• Expressionislargelycontrolledbynuclearfactor-like2(Nrf2).

• OnlyonereportedcaseofHO-1deficiencyinhumans:patientwithcompoundheterozygousmutationspresentedwithanaemia,hemolysis,macrophagedysfunction,hyperlipidemiaandatherosclerosis,anddiedatage6

• MicelackingHMOX1developiron-deficiencyanaemia,aswellasironoverloadinliverandkidneys

331–333

Hypoxia-induciblefactor(HIF)

HIF1A(HIF1α)EPAS1(HIF2α)HIF3A(HIF3α)

• Oxygen-responsiveα-subunitthatdimerizeswithaβ-subunittoformanintracellularheterodimericprotein

• HIF1αisubiquitouslyexpressed,HIF2αispredominantlyexpressedbyrenal,hepatic,

• HIF2αinparticularmodulatestheexpressionofvariousproteinsinvolvedinironmetabolismbybindingtohypoxicresponseelements(HREs)ingenepromoterstoincreaseirontrafficking(includingHAMPsuppression)

• HIF2αtranslationisregulatedby

• Inhumans,loss-of-functionmutationsinHIF1orHIF2havenotbeendescribed.

• Gain-of-functionmutationsinHIF2havebeenfoundintwopatientswithpolycythaemia,andstabilizationofHIF1orHIF2hasbeenassociatedwithvariousformsofcancer

209–

213,334–336

48

macrophageal,glial,cardiacandendothelialcells

ironviathepresenceofanironresponseelement(IRE)elementinitsmRNA

• HIFsalsocontrolerythropoiesis,oxygentransport,andcellularglucosemetabolism

• Inmice,HIF1α-knockoutisembryoniclethal

• HIF2α-knockoutmiceareonlyviableinamixedgeneticbackground,andhavesevereabnormalitiesinnumerousorgans

Mitoferrin1andmitoferrin2

SLC25A37(mitoferrin1)andSLC25A28(mitoferrin2)

• Mitochondrialtransportersthatarepartofthemitochondrialcarrierproteins(SLC25)family

• Mitoferrin1isprimarilyexpressedinerythroidcellsandmitoferrin2iswidelyexpressed

• Importsironacrossthemitochondrialinnermembrane,supplyingironforhaemandFe-Sclustersynthesis

• Noknownmutationsinhumans• Inmice,knockoutofmitoferrin1isembryoniclethal

• SelectiveknockoutofSlc25a37inerythroidtissuesinmiceleadstosevereanaemia

45,337

Prolyl-hydroxylasedomain-containingprotein(PHD,alsoknownasHIF-PH)

ELGN2(PHD1)ELGN1(PHD2)ELGN3(PHD3)P4HTM(PHD4)

• Ubiquitouslyexpressedenzymefamily,localizedinthenucleus(PHD1andPHD3)andthecytosol(PHD2andPHD3)

• PHD4containsanendoplasmicreticulumtransmembranedomain

• Theoxygen-sensitivePHDs,especiallyPHD2,hydroxylatetheα-subunitofHIFtopromoteitsubiquitylationanddegradation,andtherebylimitirontrafficking

• EspeciallyPHD2fulfilsacrucialrole.

• Fe(II)isrequiredasacofactor

• Inhumans,heterozygousmutationsinELGN1resultinlossoffunction,whichincreaseslevelsofEPOandHb

• Elgn1knockoutisembryoniclethalinmice;Elgn2andElgn3knockoutmicearephenotypicallynormal

216,217,219,

338,339

Ironefflux

Ceruloplasmin(Cp)

CP • Glycoproteinmemberofthemulti-copperoxidasefamily,eachproteincontains6copperions

• OxidizesFe(II)toFe(III)viaconcurrentreductionofdioxygentowater

• EnablesbindingofFe(III)to

• DeficiencyofceruloplasminduetomutationsinCpareassociatedwithcognitiveandneurologicaldegeneration,typeIIdiabetes,

49,340

49

• Abundantlyavailableintheblood;producedintheliver

• Membrane-anchoredCpisfoundinastrocytesandretinalcells

transferrintofacilitatecellularironexportbyferroportin

retinaldegradationandhepaticironoverload

Felineleukaemiavirus,subgroupC,receptor1(FLVCR1aandFLVCR1b,alsoknownasSLC49A1)

FLVCR1 • Transmembraneproteinisoformswithubiquitousexpressioninplacenta,uterus,duodenum,liver,kidney,lung,brainandbonemarrow

• Exportsofhaemfromcells(FLVCR1a),especiallyerythroidprogenitorsandmacrophages

• Mayfunctionasan‘emergencyvalve’,preventingoxidativedamageincaseofexcessiron

• Mightdirectlysupplyhaemtodevelopingerythroblastsfrommacrophages

• Exportshaemfrommitochondria(FLVCR1b)inhaem-producingcells

• EnhancedalternativesplicingofFLVCR1hasbeenobservedinpatientswithDiamondBlackfananaemia,arareformofanaemiacharacterizedbyareductionoferythroidprogenitors

• SimilaranaemiaisobservedinFLVCR1-deficientmice,whichalsohaveimpairedT-celldevelopment

53,341–345

Ferroportin SeeTable1

Hephaestin(Hp) HEPH • Transmembraneproteinthatcontains6copperionspermolecule;memberofthemulti-copperoxidasefamily

• Mainlyexpressedintheintestine,butalsoinplacenta,heart,brainandpancreaticβ-cells

• OxidizesFe(II)toFe(III)viaconcurrentreductionofdioxygentowater

• EnablesbindingofFe(III)totransferrintofacilitatecellularironexportbyferroportin

• Noknownmuationsinhumans• Hephaestin-deficientmicehaveiron-deficiencyanaemiawithenterocyticironoverload

346–348

50

Table3–Therapeuticagentstargetingironmetabolismundercommercialdevelopment

Target Name Company Type Mechanism Indication Developmentstatus

Trialidentifiersa

BMP6receptorpathway

LY3113593 EliLillyandCompany

Humanizedantibody

CaptureofBMP6tolimithepcidinexpression

Anaemia Phase2(lostthroughattrition2016;seeFurtherinformation)

NCT02604160NCT02144285

FMX-8 FerruMaxPharmaceuticals,Inc

HJV-Fcfusionprotein

CaptureofBMP6tolimithepcidinexpression

Anaemia Phase2(terminatedtrials2016)

NCT02228655NCT01873534

Ferroportin LY2928057 EliLillyandCompany

HumanizedIgG4antibody

Bindingofferroportintopreventitsdegradationbyhepcidin,therebystabilizingferroportinexpression

Anaemia Phase2(removedfrompipeline2016)

NCT01991483NCT01330953

Hepcidin M012 MerganserBiotechAustraliaPtyLtd.

Peptide Hepcidinmimeticpeptidethatfunctionsasahepcidinagonist

β-thalassemia,lowriskmyelodysplasticsyndrome,polycythaemiavera

Phase1 ACTRN12616000093482

LJPC-401 LaJollaPharmaceuticalCompany

Peptide Hepcidinmimeticpeptidethatfunctionsasahepcidinagonist

Ironoverload Phase1 ACTRN12616000132448

51

PTG-300 ProtagonistTherapeutics

Peptide Hepcidinmimeticpeptidethatfunctionsasahepcidinagonist

Ironoverload preclinical n.a.

PRS-080 PierisPharmaceuticalsGmbH

Anticalin Bioengineeredlipocalinthatcaptureshepcidintopreventferroportindegradation

Anaemia,functionalirondeficiency

Phase1b NCT02754167NCT02340572

NOX-H94(lexaptepidpegol)

NOXXONPharmaAG

L-RNAspiegelmer L-stereoisomericRNAaptamerthatcaptureshepcidintopreventferroportindegradation

Anaemia Phase2 NCT02079896NCT01691040NCT01522794NCT01372137

12B9m AmgenInc HumanIgG2antibody

Captureofhepcidintopreventferroportindegradation

Anaemia Preclinical n.a.

Iron Deferoxamine(Desferal)

Novartis smallmolecule Chelationofiron Ironintoxicationandoverload

Marketed

Deferasirox(Exjade)

Novartis Smallmolecule Chelationofiron Ironoverload Marketed

Deferiprone(Ferriprox)

ApoPharmaInc. Smallmolecule Chelationofiron Ironoverload Marketed

DpC OncochelTherapeutics

Smallmolecule Chelationofiron Advancedsolidtumours

Phase1 NCT02688101

52

Matriptase-2

IONIS-TMPRSS6-LRx

IonisPharmaceuticals

AntisenseDNAoligo-nucleotide

InhibitionofMT-2expressiontopromotehepcidintranscription

β-thalassemia Preclinical n.a.

ALN-TMP Alnylam siRNA InhibitionofMT-2expressiontopromotehepcidintranscription

β-thalassemia,ironoverload

Preclinical n.a.

Prolylhydroxylase

FG-4592/ASP1517(Roxadustat)

FibroGen

AstellasPharma

AstraZeneca

Smallmolecule InhibitionofprolylhydroxylasestostabilizeHIFs

Anaemiainchronickidneydiseaseandend-stagerenaldisease

Phase3 NCT02805374NCT02780726NCT02779764NCT02780141

GSK1278863/GSK1278863A(Daprodustat)

GlaxoSmithKline Smallmolecule InhibitionofprolylhydroxylasestostabilizeHIFs

AnaemiainchronickidneydiseaseWoundhealing

Phase3 NCT02879305NCT02876835NCT02829320NCT02791763

AKB-6548(Vadadustat)

Procter&Gamble

AkebiaTherapeutics

MitsubishiTanabePharma

Smallmolecule InhibitionofprolylhydroxylasestostabilizeHIFs

Anaemiainchronickidneydisease

Phase3 NCT02892149NCT02865850NCT02680574NCT02648347

BAY85-3934(Molidustat)

BayerHealthCareAG

Smallmolecule InhibitionofprolylhydroxylasestostabilizeHIFs

Anaemiainchronickidneydisease

Phase2 NCT02312973NCT02064426NCT01975818NCT02021409

53

JTZ-951 JapanTobaccoInc.

Smallmolecule InhibitionofprolylhydroxylasestostabilizeHIFs

Anaemiainchronickidneydisease

Phase2 NCT02805244JPRN-JapicCTI-152892JPRN-JapicCTI-152891JPRN-JapicCTI-152881

ZYAN1 CadilaHealthcareLtd

Smallmolecule InhibitionofprolylhydroxylasestostabilizeHIFs

Anaemiainchronickidneydisease

Phase1 CTRI/2016/02/006665

JNJ-42905343 Johnson&Johnson

Smallmolecule InhibitionofprolylhydroxylasestostabilizeHIFs

Anaemia,functionalirondeficiency

Preclinical n.a.

Transferrinreceptor1

MBP-426 MebiopharmCo.,Ltd

Liposomaloxaliplatinfunctionalizedwithtransferrin

DrugtargetingtohighlyTfR1-expressingtumours

Gastric,gastro-oesophageal,andoesophagealadenocarcinoma

Phase2(lasttrialinitiated2009)

NCT00964080NCT00355888

SGT-53 SynerGeneTherapeutics,Inc.

LiposomalplasmidDNAencodingp53functionalizedwithTfR1-targetingsingle-chainvariablefragment

GenetherapytohighlyTfR1-expressingtumoursandthatcrossestheblood-brainbarrier

Solidtumours Phase2 NCT02354547NCT02340156NCT02340117NCT00470613

54

SGT-94 SynerGeneTherapeutics,Inc.

LiposomalplasmidDNAencodingRB94,functionalizedwithTfR1-targetingsingle-chainvariablefragment

GenetherapytohighlyTfR1-expressingtumours

Solidtumours Phase1 NCT01517464

aMostrecentclinicaltrials(4),orderedbydateofregistrationontheWHOInternationalClinicalTrialsRegistryPlatform(http://apps.who.int/trialsearch/Default.aspx)

55

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Authorbiographies

BartJ.Crielaard

BartCrielaardisanassistantprofessorattheZernikeInstituteforAdvanced

Materials,UniversityofGroningen,TheNetherlands.Afterfinishinghismedical

training,hefurtherspecializedinBiomedicalEngineeringandobtainedaPhD

degreeinPharmaceuticsfromUtrechtUniversity,TheNetherlands.Duringhis

PhDandhispostdoctoralfellowshipatWeillCornellMedicalCollege,U.S.A.,he

developedaparticularinterestinmacrophagebiologyandironmetabolism,

whichcouldbeexploitedfortherapeuticpurposes.Inhiscurrentroleasagroup

leaderattheUniversityofGroningen,heiscurrentlyinvestigatingthisandother

strategiesforthedevelopmentofnewtherapeutics.

TwanLammers

TwanLammersobtainedaDScdegreeinRadiationOncologyfromHeidelberg

University,Germany,in2008andaPhDdegreeinPharmaceuticsfromUtrecht

University,TheNetherlands,in2009.Inthesameyear,hestartedthe

NanomedicineandTheranosticsgroupattheInstituteforExperimental

MolecularImagingRWTHAachenUniversity,Germany.In2014,hewas

promotedtofullprofessoratRWTHAachen.Since2012,hehasalsoworkedasa

part-timeassistantprofessorattheDepartmentofTargetedTherapeuticsatthe

UniversityofTwente.Hisprimaryresearchinterestsincludedrugtargetingto

tumors,image-guideddrugdeliveryandtumor-targetedcombinationtherapies.

StefanoRivella

StefanoRivellaisProfessorofPediatricswithtenureattheChildren’sHospitalof

PhiladelphiaandUniversityofPennsylvania,U.S.A.,andholdstheKwameOhene-

FrempongChaironSickleCellAnemia.Hecharacterizedtheroleofseminal

94

factorscontributingtomorbidityandmortalityinvarioushaematological

disorders,includinghepcidinandferroportin,whichcontrolironabsorptionand

organirondistribution;andmacrophages,whichplayanimportantroleiniron

recycling,inflammationaswellaserythroidproliferationandmaturation.Based

onthesestudies,hecontributedtothedevelopmentofnoveltherapeutics

presentlyinclinicalandpreclinicaltrials.

Keypoints

- Ironmetabolismisatightlyregulatedphysiologicalprocessthathas

relativelylowredundancyanditsderegulationoftenleadstoirondeficiency

orironoverload.

- Irondeficiencyandironoverloadarehistoricallyassociatedwitherythroid

disorders,butaderegulatedironmetabolismhasalsobeenimplicatedin

numerousageing-related,non-haematologicaldisordersincluding

neurodegenerativedisorders,atherosclerosisandcancer.

- Intracellularironisdirectlyinvolvedintheformationofreactiveoxygen

speciesthatcancausecellularoxidativedamage,andwhichareimportant

forferroptosis,aformofnon-apoptoticcelldeath.

- Theinternalizationofironbymacrophagescanmodulatemacrophage

activitytowardsapro-inflammatoryphenotype,whichislikelyalso

dependentonthepathwayofironintake.

- Agentsthatinterferewithkeyregulatorsofironmetabolismandcellular

irontraffickingrepresentapromisingnewclassoftherapeuticagentsfor

variousdiseases,astheyexploitpathologicalpathwaysthatare

complementarytothosetargetedbyexistingtreatments.

- Targetingtherapeuticstodiseasedtissuesthatexpresshighlevelsof

transferrinreceptorisastrategythatisusedbyseveralagentsunderclinical

development,andextensionofthisstrategytowardsotherironmetabolism-

associatedcellulartransportersmaybeadvantageous.