Targeting VEGF signalling via the neuropilin co-receptor.

(1)

Targeting

VEGF

signalling

via

the

neuropilin

co-receptor

Snezana

Djordjevic

1

and

Paul

C. Driscoll

2

1_Department_of_Structural_and_Molecular_Biology,_Institute_of_Structural_and_Molecular_Biology,_University_College_London,_Gower_Street,_London_WC1E_6BT,_UK 2_Division_of_Molecular_Structure,_MRC_National_Institute_for_Medical_Research,_The_Ridgeway,_Mill_Hill,_London_NW7_1AA,_UK

The

blockade

of

tumour

vascularisation

and

angiogenesis

continues

to

be

a

focus

for

drug

development

in

oncology

and

other

pathologies.

Historically,

targeting

vascular

endothelial

growth

factor

(VEGF)

activity

and

its

association

with

VEGF

receptors

(VEGFRs)

has

represented

the

most

promising

line

of

attack.

More

recently,

the

recognition

that

VEGFR

co-receptors,

neuropilin-1

and

-2

(NRP1

and

NRP2),

are

also

engaged

by

specific

VEGF

isoforms

in

tandem

with

the

VEGFRs

has

expanded

the

landscape

for

the

development

of

modulators

of

VEGF-dependent

signalling.

Here,

we

review

the

recent

structural

characterisation

of

VEGF

interactions

with

NRP

subdomains

and

the

impact

this

has

had

on

drug

development

activity

in

this

area.

Introduction

Angiogenesis,thephysiologicalprocessofnewbloodvessel

for-mation,isanessentialcomponentoftumourprogression.Tumour

growthreliesonthedevelopmentofnewvasculaturetoprovide

oxygen and nutrients to the proliferating cellswhile removing

carbondioxideandmetabolicwaste.Targetingangiogenesishas

emergedasaprominentstrategytocomplement

chemotherapeu-tic approaches to treat cancer [1–3]. A pivotal pro-angiogenic

signalling molecule is vascular endothelial growth factor A

(VEGF-A)whichpromotesproliferation, survival,migrationand

permeabilityofendothelialcellslining theinnerlayerofblood

vessels[4].Thecreationandmaintenanceofthevasculaturenot

only supports rapid growth of malignant tumours but also

increasesmetastaticpotentialofcancerbyprovidingaroutefor

theescapeandtravelofmalignantcellstoremotesitesinthebody.

Anincreased levelofcirculating VEGFisdirectlycorrelatedtoa

poorpatientoutcome.Inothercontextsangiogenesisisan

instru-mental physiological response to inflammation, and it has a

centralroleineyediseasessuchasage-relatedmacular

degenera-tionanddiabeticretinopathy.Hereagain,bloodvesselformation,

maintenanceandpermeabilityareregulatedviaVEGFsignalling,

providingapotentialtargetfortherapeuticintervention.

VEGFs

and

VEGFRs

TheVEGFfamilyof secretedglycoproteins comprisesVEGF-A,

VEGF-B,VEGF-C,VEGF-D,VEGF-Eandplacentalgrowthfactor

(PGF)(Fig.1).Thebest-characterisedvariant,VEGF-Aislinkedto

importantphysiologicalprocessessuchaswoundhealing,

preg-nancy and maintenance of blood pressure, as well as various

pathologiesdependentuponangiogenesisincludingcancer[4–

6].AlternativesplicingoftheVEGF-Ageneproducesseveral

iso-formsof themature protein containingbetween121 and206

aminoacidresidues,withVEGF-A165beingthedominantisoform

responsibleforpathologicalangiogenesis[7,8].VEGFsexerttheir

activity through interactionwith a familyof three

transmem-brane tyrosine kinase receptors,the VEGF receptors (VEGFRs)

[9,10].TheindividualVEGF isoformsbindtotheVEGFRswith

differing affinityandthe combinatorial nature ofthe

growth-factor-receptorsignallingcomplexesmediatesarangeofcellular

responses(Fig.1).Intheendothelium,thepro-angiogenic

func-tion of VEGF-A is mediated primarily via interaction with

VEGFR2,alsoknownaskinaseinsertdomain-containingreceptor

(KDR)[11].VEGFbindingtoVEGFR2isassociatedwithreceptor

dimerisationandactivationthattriggersdownstreamsignalling

pathways including phosphatidylinositol 3-kinase (PI3-K)/Akt

and phospholipase C gamma/extracellular signal-regulated

kinase(PLCg/ERK)cascadesthat,together,supportcellsurvival

Reviews GENE T O SC REEN

(2)

and the stimulation of proliferation. Other manifestations of

VEGFsignalling such ascell migration, bloodvesselguidance

andbranchinginvolvetheformationofamorecomplexreceptor

assemblycontainingtheVEGFR2co-receptorneuropilin(NRP)

[12,13].

Inprinciple,interferenceofVEGFsignalling,arguablythemost

directandpracticalwaytoinhibitangiogenesis,canbecarriedout

eitherthroughsuppressionoftheactivityofVEGFitselforthrough

theblockadeofVEGFRfunction.Bothofthesestrategieshavebeen

exploredexperimentally.Forexample,removalofVEGFfromthe

circulationisthemechanismofactionofthefirstcommercially

available angiogenesis inhibitor bevacizumab, a specific

huma-nised monoclonal antibody [14,15]. Bevacizumab(Avastin1

) is

approved for the treatment of certain metastatic cancers and,

despite some reservations regarding safety and effectiveness

[16,17],further clinicaltrials areunderwayto investigateother

potentialusesofthisantibodyincancerandineyedisease.

RemovalofVEGFsfromthecirculationhasalsobeenachieved

withso-called‘VEGFtraps’comprisingengineeredsolubleVEGFR

fragments.Oneofthese,aflibercept,recentlygainedFDAapproval

for the treatment of age-related macular degeneration [18,19].

Similarly,itwasreportedthatamutatedsolubleNRP2canreduce

VEGFbioactivity[20].Inaddition,sometherapeuticsuccesshas

beenachievedbytargetingVEGFreceptorfunctionthroughthe

developmentofantibodiesthatblock ligandbindingorVEGFR

dimerisation,andwithsmallmoleculeinhibitorsofthe

intracel-lularreceptortyrosinekinase activity[21–27].Forexample,

sor-afenib[23,24]andsunitinib[25–27]havebeenapprovedforuse

againstadvancedstagerenalcellcarcinoma.However,these

mole-cules exhibit limited specificity: they not only interact with

VEGFR2butalsowiththeplatelet-derivedgrowthfactor(PDGF)

receptorkinaseandRafkinase[28].

EversinceFolkman’shypothesisthatinhibitionofangiogenesis

insolidtumourscouldbeusedtotreatcancer[2],manypotential

inhibitors have been tested and currently there are numerous

antiangiogenictherapeuticsindevelopmentorundergoing

clin-ical trials [1,15,29]. Unfortunately, many of the VEGF and/or

VEGFR inhibitors tested so far have been reported to exhibit

limitedefficacyandhightoxicitywithproblemsincluding

hyper-tension,proteininurineandarterialbloodclotsthatcanleadto

strokeorheartattack,andresultinonlyamildimprovementin

patient survival [3,16,17,30–32]. New approaches to confront

cancer-associatedangiogenesisarebeingexplored[33,34].In

par-ticular,itwouldbedesirabletoachievehigherselectivityof

VEGF-signallinginhibitorsto eliminateoff-targetactivityandthereby

reducetoxicity.Inthesearchforrelevantnewapproaches,NRP1

hasemergedasanattractivetargetowingtoitsroleasaco-receptor

forVEGF-AalongsideVEGFR2. Here,we describethemolecular

D1 Exons: (a) (b) (c) 2-5 6a 6b 7a 7b 8a 8b VEGF-A206 VEGF-A189 VEGF-A183 VEGF-A₁₆₅ VEGF-A148 VEGF-A145 VEGF-A121 VEGF-A165b VEGFR1 (Flt-1) VEGFR2 (KDR) VEGFR3 (Flt-4) a1 a2 b1 b2 c/MAM NRP-1 NRP-2 Tyrosine kinase D2 D3 D4 D5 D6 D7

Drug Discovery Today

FIGURE1

(a)SchematicrepresentationofthefamilyofVEGF-Aspliceisoformsindicatingtheexon-basedoriginofthedomainorganisation.TheVEGFRbindingdomain derivesfromexons2–5,thetwo-moduleheparinbindingdomainfromexons6and7andtheextremeC-terminaltailregion(sixaminoacidresidues)fromexon 8.(b)VEGFproteinsaredisulphide-crosslinked(red)antiparallelhomodimers,indicatedhereforVEGF-A165.(c)OutlinedomainstructureofVEGFRand NRPisoforms,drawnastransmembranemonomers(left).Cartoonrepresentation(right)illustratinghowVEGF-A165mightcrosslinkVEGFR2andNRP1toeffect signalling,principallythroughtrans-autophosphorylationoftheVEGFR2cytoplasmicdomain.Thepositionofglycosaminoglycans,implicatedincomplex formation,isnotshown,asistheengagementoftheNRP1C-terminalregionwithputativebindingpartners,orthepotentialfordirectcontactsbetweenthe ectodomainsofVEGFR2andNRP1.

Reviews GENE T O SCREEN

(3)

biology ofNRP1, its3D structureand potential fortherapeutic exploitation.

NRP:

history

and

target

validation

TheNRPs,single-passtransmembranereceptors,originally

discov-eredin the Xenopusnervous system[35],arehighly conserved

amongvertebrates.Inhumans,thetworelatedproteinsNRP1and

NRP2 exhibit 44% sequence identity. NRPs are differentially

expressedwithNRP1foundprimarilyinarterialendothelialcells,

whereasNRP2 expressionis localised to venous and lymphatic

endothelium.Inaddition,thetwoproteinsexhibitdifferencesin

thesubsetofligandsthattheyrecognise;forexample,asidefrom

VEGFs,NRP1isareceptorforsemaphorin-3A,-3Cand-3F,whereas

NRP2preferentiallybindssemaphorin-3B,-3C,-3Dand-3F[36–

39].Class3semaphorinsaremembersofafamilyofaxonguidance

moleculesthatsignalbyinteractionwithtransmembranereceptor

complexes that incorporate NRPs as co-receptors to plexins –

major receptorsfor all semaphorin familymembers. NRPs also

recognisevariousVEGFsthat,comparedtosemaphorins,represent

a rather distinct set of ligands. Similar to the situation with

semaphorins,thereis distinctpreference betweenthe NRPsfor

differentsubsetsofVEGFs.NRP1interactswithheparin-binding

isoformsofVEGF-A,-B,-EandPGF,whereasNRP2interactswith

VEGF-A, -C and -D [40–42]. VEGF–NRP binding is manifested

primarily in the context of the NRP having a co-receptor role

alongsidetheVEGFreceptor.

Thedifference inexpressionpatterns along withthe distinct

agonistspecificityarereflectedintheseparatephysiologicalroles

thatNRP1andNRP2haveindevelopmentanddisease.NRP1gene

deletion in mice results in embryonic lethality with embryos

exhibitingabnormalitiesinheart,vasculatureandneuronal

gui-dance [43–45]. By contrast, NRP2-deficient mice are viable,

althoughsmallerinsizethanwild-type,andtheydisplayminor

abnormalities in the lymphatic system [46]. Double knockout

miceshow aneven more severe phenotype and diein uteroat

dayE8.5[47].Mostoftheeffectsfromthemutantmousemodels

pointtowardinteractionoftheVEGF-A165isoformwithNRP1in

endothelialcellsandsemaphorin-3Aand/orsemaphorin-3Fwith

NRP1and/orNRP2inthenervoussystem[43–48].

ThepresenceofNRPshasalsobeendemonstratedincancercell

linesaswellasinvariousprimarytumours.NRP1participatesinan

autocrine VEGF165-dependent signalling mechanism that

pro-motesbreastcancer[49,50].Preclinicalstudiessupportarolefor

tumourcellNRP1in lungandrenalcancer cell migration,

pro-liferationandinvasion[51,52].Recently,itwasshownthatNRP1

isessentialinskintumourigenesis,becauseNRP1deletion

abro-gatedthe responseofcancer stemcellstoautocrine VEGF[53].

NRP1alsoappearstosupportproliferationofhumanglioma

stem-likecellsinglioblastomamultiforme[54].

Manyresearchgroups haveinvestigated NRPexpression

pat-ternsandgeneratedevidenceforNRPsinendothelialandtumour

cells[39,49,55–60].Recently,acomprehensiveevaluationofNRP1

expressionwascarriedoutforbreast,colorectalandlungcancer

[61].Inthisstudy,avalidated,highlyspecificmonoclonal

anti-bodywasusedforinsituanalysisofNRP1expressionincancerous

tissueandduringnormaldevelopmentalangiogenesis.NRP1was

detected in more than 98% of blood vessels associated with

primaryand metastaticlung,colorectaland breasttumours.By

contrast,thepatternofNRP1expressionontumourcells

them-selvesismuchmorevaried:NRP1wasdetectedon6%ofprimary

breastcarcinomas,14%ofsecondarybreastcarcinomas,36%of

primary non-small-celllung cancers(NSCLCs) and 50% of

sec-ondary NSCLCs; there was no NRP1 detectable on colorectal

cancer cells.Whenthesameantibodywasusedforthe analysis

ofNRP1expressionduringmousedevelopment,NRP1wasfound

throughouttheendocardialendothelium,whereasexpressionin

thevasculatureofothertissueswasonlydetectedinlocalisedareas

[61]. In addition, NRP1 was detected in the nervous system,

smooth muscle cells and pericytes. Furthermore, blockade of

NRP1signallingresultedindefectiveVEGF-dependent

angiogen-esisinthepostnatalmousetracheaprovidingfurthersupportfor

the hypothesis that NRP1 is a valid antiangiogenic target and

potentialantitumourtargetinatleastasubsetofcancers.

Additional

NRP1

functions

ThereisagrowingamountofevidenceavailablethatsuggestsNRP1

mightdisplayseparatefunctionsthroughmechanismsthatmight

notinvolveVEGFR2.Forexample, suppressionofNRP1 protein

levels via siRNA resultsin changes in endothelial cell adhesion

properties, whereas the sameeffect isnot reproduced by siRNA

knockdownofVEGFR2.ItappearsthatNRP1supportsendothelial

cellmatrixadhesionthroughinteractionwithintegrinsina

VEGF-dependent manner [62–64]. Additionally, several studies have

shown that NRP1 might signal through otherreceptor tyrosine

kinases inresponseto ligandssuch ashepatocytegrowthfactor

(HGF)andPDGF.ItwasreportedthatNRP1andNRP2mediate

HGF-activatedendothelialcellmigrationandproliferation[65]andthat

NRP1 interaction with PDGF-BB (the dimeric B-form of PDGF)

stimulatesmigrationofsmoothmusclecells[66].Anincreasein

tyrosinephosphorylationofascaffoldingproteinp130Cas,

down-streamofthekinasePyk2,appearstobethemajoroutputofNRP1

signallinginresponsetoHGFandPDGFinU87gliomacells[67,68]

andinhumancoronaryarteryvascularsmoothmusclecells[69].

p130Castyrosinephosphorylationislinkedtocontrolofcell

migra-tionandisreduceduponNRP1knockdownbysiRNA.The

activa-tionofp130Casseemstobedependentuponthecytoplasmicregion

of NRP1 because inhibition of VEGF-inducedp130Cas tyrosine

phosphorylationwasalsoobservedinexperimentsinvolving

over-expressionofNRP1lackingitsentirecytoplasmicregion[67].In

addition,thecytoplasmicdomainwasimplicatedina

VEGF-depen-dentregulationofspatialseparationofarteriesandveins[70].A

knockin mouse modelexpressing NRP1 lackingthe cytoplasmic

domainshowedatypicallyfrequentoccurrenceofcrossoverofveins

andarterieswithnoabnormalitiesinvasculogenesisand

angiogen-esis,suggestingthatthecytoplasmicdomainofNRP1isrequiredfor

normal arteriovenous patterning [70]. Furthermore, it was

sug-gestedthattheNRP1hasVEGF165-and/or

semaphorin-3A-indepen-dentactivityinregulatinga5b1-integrintrafficand downstream

signalling[63].NRP1hasalsobeen implicatedinsignallingthat

leadstoPyk2-dependentphosphorylationattheTyr407siteoffocal

adhesionkinase(FAK)[67]andactivationofp38mitogen-activated

proteinkinase(MAPK)whichisinvolvedinformationof

pericyte-associatedvessels[71].Thesestudiesarehoweveratanearlystage;

furtherinvestigationofthesignallingpathwaysthatoperate

down-streamofNRP1andthepreciseco-receptorcontextfortheseevents

isrequiredtoresolvetheapparentcomplexity.

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NRP

structure

NRPsaretype1,single-pass,transmembraneproteinswithalarge

(>920 aminoacid residues)extracellular regioncomprisingfive

modulardomainsnameda1,a2,b1,b2and c,joinedtoa

trans-membranehelicalregionand ashort(44residue)cytoplasmic

domain [37] (Fig. 1). The different extracellular domains each

share similaritytofunctionallydiverse structuralmodules

com-monly foundin cell-surface receptorsand proteinsinvolved in

mediating cellular interactions (Figs 1 and 2a). The tandem a

domains(a1a2)belongtothestructuralfamilyof

C1r/C1s-Uegf-BMp1(CUB)domainshomologoustocomplementbindingfactors

C1randC1s.Theb1andb2domainsarehomologoustotheC1

andC2(discoidin)domainsofcoagulationfactorsFVandFVIII.

Themembrane-proximalcdomainofNRPbelongstothefamilyof

MAMdomains(meprin,A-5proteinandreceptorproteintyrosine

phosphatasem)thathavebeenimplicatedinprotein

homodimer-isation.Asingletransmembranehelixthatisreportedtomediate

NRP1 dimerisation [72] links the modular ectodomain to the

intracellular region, the structure ofwhich is unknown. It has

beenshownthattheextremeC-terminaltripeptide

(-Ser-Glu-Ala-COOH)isrequiredforNRP1interactionwiththePDZdomainof

synectin(alsoknownasNIPorGIPC),aproteinthatisreportedto

playapartintheregulationofarterialbranching[73–75].Several

reportsconfirmtheimportantcontributionoftheNRP1

cytoplas-micregioninvariouscontexts,althoughitisnotclearwhether

synectinisalwaysinvolved.

ThereareseveralX-raycrystalstructuresavailablethatdescribe

eithertheisolatedNRP1b1domainor variouscombinationsof

tandemNRP1(andNRP2)domains(i.e.b1b2,a1a2anda1a2b1)

[76–78](Fig.2a).Thesestructureshaveprovidedvaluable

informa-tionthathasdirectedthedevelopmentofNRP-targeted

therapeu-tics.SeveralstructuresdescribecomplexesofNRPdomainswith

anti-NRPFabfragmentsthatinterferewitheithersemaphorinor

VEGFbinding.Althoughbiochemicalevidencehadalready

indi-cated that VEGF165 bindsto the b1 domain ofNRP1via its

C-terminaltailregion,thefirstatomicmodelforthisligand–receptor

interactionwasderived fromthecrystal structureofa complex

betweenNRP1tandemb1b2domainsandTuftsin–an

immunor-eactivetetrapeptidethatisproducedbyproteolysisofIgGheavy

chainFcfragments[77,79].TheTuftsinaminoacidsequence,

Thr-Lys-Pro-Arg,issimilartotheC-terminaltailofVEGF165,

Lys-Pro-Arg-Arg.TheNRP1–Tuftsinstructureenablesthepredictionofthe

modeofVEGFbindingvia the conservedcarboxy-terminalArg

residuewhich,withspecificelectrostatic,H-bondingandvander

Waalscontacts,nestlesinashallowbindingsiteontheb1domain

surface(Fig. 3a).The restofthepeptide extendsawayfromthe

grooveformedbytheb-strand-connectingloopsoftheb1domain

b-sandwichfold.Thelocationoftheinteractionsiteisanalogous

to the ligand-binding region for other examples of discoidin

domains including those found in coagulation factors

[77,80,81].Aninteresting featureofthe NRP1–Tuftsinstructure

is an extensiveinterface between the b1 and b2 domains that

suggestsa‘stable’relativeorientationofthesemodulesintheNRP

extracellularregion.Inallsubsequentlydeterminedcrystal

struc-turesofNRP1andNRP2constructstheorganizationofthis

inter-face isconserved(Figs2aand 3). The presenceof anextensive

a1 a2 (a) (b) VEGF165-HBD b1 b2 c

FIGURE2

TopologyanddomainorganisationoftheNRPectodomain.(a)Aribbondiagramofdomainsa1a2b1b2isshownwitheachdomaincolouredseparately.Thefigure isbasedontheNRP2chainfromthecrystalstructureofNRP2incomplexwithasemaphorin-blockingFab(notshown;PDB2QQL).AsimilarstructureforNRP1has notyetbeenobtained.Nocrystalstructureofthecdomainisavailableandaputativetopologyofthisdomainisshowninparenthesesbasedonthecrystal structureoftheMAMdomainfromproteinphosphatasem(PDB2C9A).(b)VEGF165-HBD(magenta)andtheb1domainfromthefusionproteinstructureare superimposedovertheb1domainfromthea1a2b1b2structureshownin(a).

(5)

interdomain interface was also observed for the tandem a2b1

domaincombination[76].Ingeneral,thepackingofalldomains

within the context of longer polypeptide NRP constructs (i.e.

a1a2b1ora2b1b2)isconservedirrespectiveofcrystalsymmetry,

implyingthateventhoughtheNRPspossess,insequenceterms,a

modular organisation the tertiary domain architecture is well

definedandprobablypresentsdefinedorientationsofouterfaces

forinteractionwithproteinpartners(Fig.3).Withexceptionofthe

relativelysmall domain interface betweendomains a1 and a2,

everyconformational changeinvolvingdisruption ofthe other

interdomain contacts would probably incur a large energetic

penalty.

BindingofVEGF165toNRPsisdependentuponthepresenceof

theVEGFheparin-bindingdomain(HBD)encodedbyexons7and

8 ofthe VEGF gene [77,82,83]. Bycontrast, the VEGFR2 binds

VEGF165atasitethatspansaregionencodedbyexons2to5[84–

86].Aprevalentmodelforthetertiarycomplexformedbetween

VEGF165, VEGFR2 and NRP1 unites the two binding modesby

suggestingthatVEGF165actsasabridgebetweenthetworeceptor

ectodomains(Fig.1).However,themodeldoesnotdifferentiate

betweenscenarioswhereNRPandVEGFR2arepresentonthesame

or on neighbouring cells (Fig. 1). Furthermore, it appearsthat

binding of VEGF165 to NRP1 is required for the formation of

detectablelevelsofcomplexesbetweenVEGFR2and NRP1[87–

90]andthatthePDZ-bindingdomainofNRP1isindispensablefor

NRP1–VEGFR2complexformation[74].

Recently,inanattempttogainaninsightintotheNRP–VEGF

interaction the crystal structure of a fusion protein was

deter-mined, where the polypeptide sequence corresponding to

VEGF165–HBD was appended to the C-terminus of the NRP1

b1domain[91].Thefusionproteinformsahomodimersuchthat

the C-terminus of VEGF165–HBD, comprising two Cys-bridged

modules, bindstothe ligand-binding grooveof the b1domain

oftheneighbouringmoleculeintheasymmetricunit.The

struc-tureofthischimericproteinprovidessomeadditionalinformation

with respectto themodeofVEGF165–HBDbindingtoNRP1b1

domain.However,itispossiblethattheorientationoftheHBD

withrespecttotheb1domainmightberestrictedbythecovalent

linkofHBDtotheC-terminusoftheneighbouringb1domain,and

influencedbycrystalpackingconstraints(Fig.2b).Basedonthis

structure,andintheabsenceofdefinitiveinformationaboutthe

segmentalflexibilityofVEGF165,itisdifficulttoextrapolatefrom

thisresultexactlyhowthefull-lengthVEGF165homodimer

inter-actswithintactNRP1.

So far, verylittle is knownabout the structureof the NRP c

(MAM)and intracellulardomains.Presently,thereareonlytwo

MAMdomainstructuresdepositedintheProteinDataBank(PDB),

both of which are common to the receptor protein tyrosine

phosphatasem[92].Althoughinthelattercasetheall-bdomain

isheavilyglycosylated,therearenorecognisablesequencemotifs

withintheNRP1andNRP2MAMdomainsthatsuggestthe

pre-sence ofglycosylationsites.However,ithasbeendemonstrated

thatNRP1hasanappendedglycosaminoglycan–dominatedby

either heparan sulphate or chondroitin sulphate – attached at

Ser612 which resides between the b2 and c domains [68,93].

Interestingly,this NRP1glycosylation, that asmuchasdoubles

themolecularmassofthepolypeptide,hasaprofoundeffecton

signalling.Althoughtheeffectofglycosylationon

VEGF-depen-dent signalling remains to be investigated in vivo, it has

beenshownthatthepresenceofchondroitinsulphateonNRP1

b1 b2 a1 a2 (a) (b) (c)

FIGURE3

(a)BindingofTuftsintoNRP1b1b2domains(PDB2ORZ).Thepeptide(ball-and-stickrepresentation)sitsinagrooveonthetopoftheb1domain.Theribbon diagramofNRP1iscolouredasrainbowfromtheN(blue)totheC(red)terminusofthepolypeptide.(b)SurfacerepresentationoftheNRP2a1a2b1b2monomer. (c)PutativedimeroftheNRP2a1a2b1b2structurebasedonthecrystallographicsymmetrycontacts.Thecrystallographicinterfacethatresultsinadimer formationwasobservedintwodifferentcrystalformsofNRP2suggestingthatthistypeofinteractionmightexistinasolutionbeforecrystallisation;however, thereisnodirectconfirmationthatthisdimerisation,mediatedbyreciprocalcontactsbetweena1domains,isthebiologicallyrelevantformofNRP.Greycircles indicateareasengagedinbindingtotheC-terminusoftheVEGF165-HBD.

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modulatesp130CastyrosinephosphorylationinU87MGhuman

gliomacells[68]andthatthedifferentcompositionoftheNRP1

glycosaminoglycaninendothelialandsmoothmusclecellsleads

toopposingresponsestoVEGF[93].Furthermore,ithasalsobeen

observed that the presence of heparin in NRP1 binding assays

resultsinincreasedaffinityforVEGF165[94,95].Themechanistic

basis for this enhancement and, in particular, whether this is

differentforcovalentlyversusexogenousnon-covalentlyattached

GAGsiscurrentlyunclear.

AsimilarlyunresolvedissueisthestructuralnatureoftheNRP

cytoplasmic domain. Standard secondary structure prediction

algorithmssuggestanabsenceofasignificantquotientofregular

structure. It is therefore tempting to assign this region as an

intrinsicallydisorderedpolypeptidethatperhapsadoptsaspecific

conformationonlyinacontextofacomplexwithaninteraction

partner.Todate,theonlyproteinidentifiedtointeractwiththe

cytoplasmicNRPdomainissynectinwhich,asdescribedabove,

binds to the NRP C-terminal Ser-Glu-Ala tail through its PDZ

domain[73,74].AlthoughthestructureoftheNRPintracellular

domainisnotknown,theC-terminalSer-Glu-Alasequencedoes

havethetypicalsequencecharacteristicsofaPDZdomain-binding

motif. In other cases PDZ domains bind to their partners by

extensionofab-sheetthroughtheadditionofanantiparallel

b-strandconstitutingtheC-terminaltailoftheproteinligand[96].

WhethersynectinisconstitutivelyboundtoNRPsinthe cellor

whetheradditionalproteinscontributetoNRP-dependent

mem-brane-associatedsignallingcomplexesisnotcurrentlyknown.

NRP-targeting

strategies

The demonstrated involvement of NRP in the pathogenesis of

cancerhascatalysedinterestintargetingthismoleculetocombat

thedisease.Themodularorganisationoftheproteinoffersseveral

avenuesforattack,suchastheblockingofagonist–NRP

interac-tion,interferenceofNRPassociationwithpartnerreceptors(e.g.

VEGFR2) independent of ligand binding and inhibition of the

functionoftheintracellularNRP-signallingdomain.Inadditionto

experimental approaches basedupon theseconcepts, computer

simulationshavealsobeenemployedtopredictthemostefficient

methodfortargetingVEGF–receptorinteractions[97,98].Usingan

insilicomodelofVEGFinteractionswithendothelialcellreceptors

thatincludesexperimentalestimatesoftherateofVEGFR2–NRP

couplingbyVEGF165theauthorsconcludedthatblockadeofNRP–

VEGFRinteractionmightprovidethemosteffectivedecreasein

VEGF–VEGFR2signalling[98].

Current efforts to target NRPs,and NRP1in particular,have

focused on the specific interactionwith the exon 7-8-encoded

regionofVEGF165.ResearchersatGenentech (http://www.gene.

com/gene/index.jsp) have developed a range of anti-NRP1 and

anti-NRP2 monoclonal antibodies that block interaction with

VEGF165andsemaphorins[12,76,99],andareundertakingclinical

trialstotesttheeffectivenessofthesemonoclonalantibodiesin

cancertreatmenteitherassingleagentsorcombinationtherapies.

AnantibodytoNRP2thatblocksVEGF-Cbindinghasalsobeen

reported[100].Othershaveidentifiedspecificpeptidesand

pepti-domimeticinhibitors of VEGF165–NRP binding with

antiangio-genicactivity[101–105].Allofthesemolecules arecompetitive

inhibitorsofVEGF165bindingandwereabletoreducedownstream

VEGF signalling as demonstrated by reduced VEGFR2 tyrosine

phosphorylation. Although peptides are not considered to be

viable drug candidates they provide a starting point for

struc-ture-baseddesignofpeptidomimeticsandsmallmolecule

inhibi-tors. Giordano et al. employed a method of amino acid

retroinversionthatinvolves substitutionofD-forL-aminoacids

andsequencereversal,togeneratethepotentpeptidomimeticD

-(Leu-Pro-Arg)[105].IninitialstudiesthisD-tripeptidewasresistant

to proteolysis and exhibited antiangiogenic activity mediated

through NRP1 (and VEGFR1) based on in vivo assays in three

animalmodelsofcancerandretinopathy.Inaddition,asynthetic

peptide targeting the transmembrane domainof NRP1showed

antitumouractivity[106].Asyntheticoligonucleotide(G18) has

alsobeenreportedtobindNRP1resultinginreceptor

internalisa-tionandinhibitionofangiogenesis[107].

Thefirstnon-peptidicsmallmoleculeantagonistofNRP1

func-tionhasrecentlybeenreportedbyresearchersatArkTherapeutics

(http://www.arktherapeutics.com/main/index.php)[108](Fig.4).

StartingfromthepreviouslycharacterisedbicyclicpeptideEG3287

EG00229

Y297

Y353

D320

(a) (b)

FIGURE4

Ligand-bindingpocketoftheNRP1b1domain.(a)StickrepresentationofsmallmoleculeVEGFantagonistEG00229(grey).Keyresiduesinthebindingpocketare labelled.(b)SuperpositionofTuftsinmolecule(sticks,green)andVEGF165-HBD(magenta)onthestructureoftheNRP1b1/EG00229(PDB3I97)complex.Theb1 domainsfromthecorrespondingstructureswerenotincludedinthefigureforclarity.Thedifferencesintheside-chainconformationsoftheb1domainresidues amongthreestructuresarenegligible.

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[103]that corresponds tothe C-terminal28-residue segmentof

VEGF-A165,thesmallmoleculeinhibitorEG00229wasdeveloped.

Mutagenesis,X-raycrystallographyand NMRspectroscopywere

usedtoshowthatEG00229bindstothetargetedpocketontheb1

domainofNRP1(Fig.4).Thecompoundexhibitsactivity

consis-tentwithinhibitionofVEGF-A165bindingtoNRP1anddecreases

VEGFR2phosphorylationandcellmigrationinvitro.EG00229is

also reported to demonstrate activity against tumour cells by

enhancing the cytotoxic effect of the chemotherapeutic drugs

paclitaxel and 5-fluorouracil. Currently, EG00229 is a valuable

toolforprobingthe molecularbiologyofNRP1function;other

morepotentcompoundswithasuperiorpharmacokineticprofile

arebeingdevelopedbythesameteam.

Althoughco-immunoprecipitationexperimentshave

demon-stratedassociationbetweenNRPandVEGFRs[87,88],thelackof

biophysicalandstructuraldatadescribingthemolecularbasisof

putativeprotein–proteininteractionsurfacesbetweenNRPand

VEGFR2,orotherreceptortyrosinekinases,createsanobstacleto

theformulationofstrategiestotargetNRPfunction.Forexample,

itwouldbehelpfultounderstandbettertheroleoftheNRPMAM

domainanditsputativeinteractionwiththemembrane-proximal

regionoftheVEGFR2ectodomain.Analysisofthedimeric

struc-ture of the membrane-proximal domain D7 of VEGFR2 [109]

suggests that this domainis crucial for receptor signalling. It

would be reasonable to suppose that the (agonist-dependent)

proximallocationoftheMAM domainofassociated NRP–in

additiontothepotentialroleofthelatterinNRPdimerisationon

thecellsurface[83,110]–couldpromoteinteractionwithVEGFR2

D7 or other domains and thereby bring the NRPcytoplasmic

regionwithintheorbitoftheVEGFRkinasedomain.Finally,a

betterunderstandingofthecontributionofNRPtointracellular

signaltransduction, throughcomplexformationwithproteins

thatassociatewiththeintracellulardomain,couldprovide

addi-tionalscopeforthedevelopmentoftherapeuticapproachesto

address the angiogenesis-related and -independent functions

ofNRPs.

Additional

application

of

NRP1-targeting

peptides

AnimportantpropertyofNRP1-bindingpeptideshasemergedthat

couldhavesignificant implicationsforcancerdrug targeting.A

series of investigations using phage particle libraries that were

initiatedtosearchfortissue-penetratingpeptidesledtothe

obser-vationthatshortpeptideswithbasic(ArgorLys)residuesatthe

C-terminusarereadilyinternalised.Toemphasisetherequirement

forthebasicresiduesbeinglocatedattheC-terminusthisactivity

wasnamedthe‘C-endrule’[111,112].Itwasshown that

inter-nalisation ofthese peptidesis mediated by NRP1and, because

NRP1isupregulatedinmanycancercelllines,thisactivityisbeing

exploredasamechanismforthetargeteddeliveryoftherapeutic

anddiagnosticagentstotumours[113,114].Forexample,when

pro-apoptopicaminoacidsequenceswerefusedtoNRP1-binding

peptides they were not only internalised but also exhibited a

potentantileukaemiacelleffect[114].Furthermore,arecentreport

showed that gold nanoparticles could be functionalised with

NRP1-targeting peptidesto effect internalisation [115].

Double-decoratedgoldnanoparticles,carryingatherapeutic

p53-stabilis-ingpeptidealongsidetheNRP-targetingpeptide,showed

promis-inginvitroanticanceractivity.Althoughthedevelopmentofthis

NRP1-dependent drug deliverysystem is at anearly stage, this

offerssignificant promisefor the targetedapplication of

multi-functionalagents withpotentialin areasasdiverse asimaging,

diagnosisandcombinationtherapy.

Concluding

remarks

Thisaccount introducedtheroleofVEGFsinsignallingvia the

principalclassofreceptor(theVEGFRs)–signallingeventsthat

providethemostdirectopportunityforthetherapeuticblockade

ofangiogenesis.Duringrecentyears,drugdiscoveryeffortsinthis

area have been substantial, with marketed drugs available for

oncology and ophthalmology indications, and are continuing

asevidencedbya numberofongoingclinicaltrials.Experience

hasshownthattheseagentsareassociatedwithvariableefficacy

andvariousside-effects.Wehavedescribedtheemergenceofthe

co-receptor role of the NRPs that also bind a subset of VEGF

isoforms. Thecontribution thatNRPsmaketoVEGFsignalling,

whether synergisticor otherwise,addscomplexity tothe VEGF

interactomeyet,atthesametime,providesasecondarymeansto

targetVEGFsignallingindisease.TheacknowledgedroleofNRPs

incellmigration,alongwiththeroleinVEGFsignalling,suggests

thatadifferentspectrumofresponsescouldemergebytargeting

thisaxis.Earlyeffortshavedemonstratedthe potentialfordrug

developmenttargetedtoVEGF–NRPinteractions,andthisislikely

toexpandasfurtherunderstandingofthestructuraland

biochem-icalaspectsoftheseinteractionsbecomesavailable.Moreover,new

discoveriesconcerningthefunctionofVEGFsandNRPs,suchas

thedeterminationofthe‘stemness’ofskincancercells[53]and

endotheliallipid transport[40,116] relevant totypeIIdiabetes,

willprobablyprovideadditionalavenuestoexploitdrugdiscovery

effortsinthisniche.

Disclosure

statement

S.D. hasbeen involved asconsultant to Ark Therapeuticson a

projectinvolvingsmallmoleculeinhibitorsofNRP1.Theworkwas

describedinpartinJarvisetal.[108].

Acknowledgements

S.D.acknowledgesthesupportoftheBritishHeartFoundation.

P.C.D.issupportedbytheMRC(filereferenceU117574559).

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