BrianFKingandAndreaTownsend- P2XReceptors Nicholson P2Xreceptors(P2XRs)areligand-gatedion-channels (LGICs)widelydistributedamongstvarioustissue UniversityCollegeLondon,GowerStreet, types,includingautonomic,central,entericand London,WC1E6BT,UK. sensoryneurons,cochlearandretinalcells, endotheliumandepithelium,vascularandvisceral BrianKingisaSeniorLecturerinthe smoothmuscle,heartanddevelopingskeletal DepartmentofPhysiology,andAndrea muscle,bone,haemopoieticcells.TheseLGICsserve Townsend-NicholsonaLecturerinthe tocontroltheexcitabilityoftheirhostcellsthrough DepartmentofBiochemistryandMolecular twoprocesses:i)influxofextracellularsodiumionsto elicitdepolarisation,andii)influxofextracellular Biology.Theyshareacommoninterestin calciumionstoactivateinternalenzymes. purinesignallingatrecombinantandnative P1andP2receptorsandhaveworked P2XRassembly togetheroverthelastsevenyearsonthe Sevenbuildingblocks, orsubunitproteins,are isolationandcharacterisationofnucleotide involvedintheconstructionofP2XRs.Theseseven receptors. buildingblocks–theP2X17toP2Xsubunits–have beenclonedfromhumanandratcDNAlibrariesand, insomecases,alsofrommouse,guinea-pig,chick andzebrafishlibraries.1,2 Fewstructuraldifferences havebeennotedbetweenspeciesorthologuesofthe Introduction sameP2Xsubunit.However,thedegreeofhomology canbeaslittleas27%betweendifferentsubunitsof Extracellularnucleotidesignallingmolecules,suchas thesamespecies.1 Furthermore,theP2Xgenesare ATP,ADP,UTPandUDP,workthroughspecific complexand,duringtranscription,canundergo ionotropic(P2X)ormetabotropic(P2Y)cell-surface alternativesplicingwhich,subsequently,generates receptors.Theuseofanyligandasasignalling variantsubunitproteinsthatareeithernon-functional, moleculenecessitatesamechanismforits dominantnegativeormodulatory. inactivationandso,purinenucleotidesarehydrolysed toadenosine,anucleosidethatactsthrough Eachofthesubunitproteinscanformhomomeric metabotropicadenosine(P1)receptorsubtypes,to P2XRassemblies,althoughthereappeartobe exertphysiologicaleffectsinalmostallorgan difficultiesformingP2Xreceptors3 andP2X systems.,formedbythedeaminationof 56 receptors.4 Forsuchdifficulties,post-translational ,hasalsobeenshowntohaveagonist modificationofsubunitproteins–particularly activityatnucleosidereceptors.Furthermore, glycosylation–hasastronginfluenceonthe dinucleotidesaresignallingmoleculesintheirown functionalityofmembrane-traffickedP2XRs.5,6,7 Itis right–notonlyatP2receptors,butalsoatspecific currentlybelievedthateitherthreeorsixglycosylated ApAreceptors–and,throughdegradation,can n subunitsmakeafunctionalP2XR,byformingeithera generatenucleosidesandnucleotidestobroaden stretchedtrimerorahexamerofconjoinedtrimers.2,8 theirextracellulartargets. Inadditiontohomomericassembliesofidentical subunits,P2Xproteinsalsoformheteromeric SevenP2X,eightP2Yandfouradenosinereceptor assembliesbyusingtwoorthreebuildingblocks. proteinshavebeenclonedandcharacterised.Despite WheretwoP2Xsubunitsareinvolved, theubiquityofbothpurinergicligandsand immunoprecipitationstudieshavepredictedupto11 purinoceptors,thedifferentialexpressionof heteromericreceptorsubtypes.9 Here,aninteresting purinoceptorsubtypesandligand-metabolising patternhasemerged–P2Xsubunitsaremost enzymesallowsforthegenerationofveryspecific 7 discerningandonlywillformhomomericreceptors, physiologicalresponsesinparticularcelltypesor whereasP2Xsubunitsaremostpromiscuousand tissues.Itisforthisreasonthatnucleotideand 5 willheteropolymerizewithallP2Xsubunits(except nucleosidereceptorshavenowbecometherapeutic P2X).OtherP2Xsubunitscanpolymerisewithsome, targetsforthetreatmentofconditionsasdiverseas 7 butnotall,P2Xsubunits.Asyet,noonehas pain,urinaryincontinence,strokeanddepression, undertakenasystematicstudyofheteromeric andthefieldofpurinoceptorresearchhasexpanded assembliesinvolvingthreedifferentsubunits, significantlyinthelasttenyears.Inthisbriefreview, althoughsuchassembliesmustsurelyoccurinnative weexamineanumberofthepharmacologicaltools tissues,sinceanumberoftissues(e.g.dorsalroot currentlyavailableforthestudyofnucleotideand ganglia,autonomicgangliaandsmoothmusclecells) nucleosidereceptors. possessinabundancetranscriptsandproteinsfor severalP2Xsubunits.

TocrisCooksonLtd.,UK TocrisCooksonInc.,USA Tel:+44(0)1179826551 Tel:(800)421-3701 Fax:+44(0)1179826552 Fax:(800)483-1993 [email protected] [email protected] [email protected] www.tocris.com [email protected] Figure1.Thepurinoceptorfamilyofcell-surfacereceptors Thepurinoceptorfamily,atthispointintime,comprises5structurallyandpharmacologicallydistinctgroups–the adeninereceptorgroup(AdenR),adenosinereceptors(ARs),metabotropicnucleotidereceptors(P2YRs), ionotropicnucleotidereceptors(P2XRs)intheirhomomericandheteromericforms,anddinucleotidereceptors (ApnnAR).Thelastgroup(ApARs)mighteventuallybesubdividedintoionotropicandmetabotropicsubgroups,for whichthereispharmacological,butnotyetstructural,evidence.Finally,membersoftheARandP2YRgroups appeartoheterodimerise,althoughonlytheA11/Ycomplexhasbeenstudiedfunctionally. The“purinergic”family

AdenRARsP2YRsP2XRsApnARs

1R

A1AAA2A2B 3 ?

A11/Y

YYYYY124611Y12YY1314

X1XXXXXX234567

XXXXX1/22/32/61/54/6

Todate,sevenhomomericP2XRs(P2X17toP2X)and itself.NeitherADPnorAMP(whenpurifiedof fiveheteromericassemblies(P2X1/2,P2X1/5,P2X,2/3 contaminatingATP)willactivateP2Xreceptors, P2X2/6andP2X4/6)havebeencharacterisedbytheir exceptathomomericassembliesofP2X1 splice biophysicalandpharmacologicalproperties. variants(exon6deleted)whichareactivatedbyADP 14 OperationaldifferencesbetweenmanyoftheseP2XR andnotATP,andathomomericP2X7 receptors assembliesaresurprisinglysubtle.Also,mosttissues whichwillrespondtobothADPandAMP(withina expressbothhomomericandheteromericP2XRsofa periodoftime)afterbeingactivatedfirstbyATP.15 The similarpharmacologicalnature.Asaresult,itisan naturally-occurringdiadenosinepolyphosphates inordinatelydifficultprocesstodeterminetheidentity (ApnA,n=3-7)andclosely-relateddinucleotides ofnativeP2XRs.Atthispointintime,itisinadvisable (ApnG,n=3-6)alsoactivateP2XRs,butnonewith tocategorisenativeP2XRsasP2X,123P2X,P2X,etc. eitherthepotencyorefficacyofATP.Membersofthe and,instead,theyshouldbeassociatedwitha diadenosinepolyphosphatefamilyaresoclosein particularphenotype(e.g.P2X123-like,P2X-like,P2X- potencyandefficacyatP2Xassembliesthatthese like,etc.).TherolesofseveralP2Xreceptorsubunits substancescannotbeusedreliably(andalone)as innativechannelshavebeenexaminedbythe discriminatorsofP2Xsubtypes.Thediphosphates, generationofknockoutmice.Nullmutationsforthe Ap22AandApG,arenotagonistsatP2XRs,inkeeping 101112 P2X1,P2X37andP2Xsubunits,and withtherelativeinactivityofADP. 13 overexpressingP2X1 transgenicmice,havebeen described,implicatingP2Xreceptorsinprocessesas AmongstrecombinantP2XRs,thepotencyofATPcan diverseasfertility,thrombosis,painandcytokine varyenormously,withEC50 valuesrangingfrom50 production.invivo nMto300µM,dependingonsubunit composition.1,2,16-20 Althoughmutationalanalyses P2XRagonism havebeencarriedout,astructuralbasishasnotbeen AlloftheknownP2Xassembliesareactivatedby clearlyestablishedforthisobservedvariabilityin ATP,whichisthemostpotentofthenaturally- agonistpotency.AcommonATPrecognitionsitehas 2122 occurringnucleosidetriphosphates(NTPs).Other beenmappedforP2X12andP2Xsubunits,and NTPscanworkatcertainreceptorsubtypes(e.g.CTP thissiteissharedbytheremainingP2Xsubunits. atrecombinantP2X,1P2X4andP2X,55GTPatP2X, However,othermodulatoryfactorsmustclearlycome andUTPatP2X3),butnoneareaspotentasATP intoconsideration.Forinstance,agonistpotencyis

2 Table1.KeyligandsforhomomericP2Xreceptors

SubtypeAgonistsAntagonistsPotentiatorsInhibitorsReferences (pEC50)(pIC50)(pEC50/pKa)(pIC50i/pK)

+ rP2X1 ATP(7.0)NF449(9.5) –H(6.3)1,2,3,17-20, 2-MeSATP(7.0) NF279(7.7) Zn2+ (6.0)25-30,88,89 ATPγS(6.2)NF023(6.7) Gd3+ (6.5) Ap6A(6.0) (5.7) ApG65(5.7)IpI(8.5) αβ,-meATP(5.5)TNP-ATP(9.0) Bz-ATP(4.6)MRS2257(8.3) CTP(4.4)MRS2159(8.0) PPNDS(7.8) PPADS(6.9) RB-2(5.7)

(h*)A-317491(4.9)

+3+ rP2X2 ATP(5.3)RB-2(6.4)H(7.1)La(5.0)1,2,3,17-20, 2-MeSATP(5.1)NF279(6.4) Zn2+(5.0)Gd3+ (5.0)25-30,90-94 ATPγS(5.1)BBG(5.9)Cu2+(4.8)Ca2+ (1.1) 2+ (4.8)TNP-ATP(5.9)Cd(3.5) αβ,-meATP(3.0) PPADS(5.8) Suramin(5.0)

(h*)A-317491(4.33)

2++ rP2X3 2-MeSATP(6.7) A-317491(7.6)Zn(5.0)H(6.0)1,2,3,17-20, ATP(5.9)NF279(5.8) Ca2+ (1.8)88,89,95,96 ATPγS(5.9)NF449(5.6) (h*)RB-2(5.9) Ap5A(5.5) Suramin(5.4) ApG5 (5.6) NF023(5.0) αβ,-meATP(5.7)TNP-ATP(9.5) UTP(4.0)MRS2257(7.7) MRS2159(6.9) PPADS(6.7) IpI5 (5.5) RB-2(4.3)

+ rP2X4 ATP(5.4)TNP-ATP(4.8)(6.6) H(6.8)1,2,3,17-20, 2-MeSATP(3.6) BBG(3.9)Cibacronblue (5.5)25-30,97-103 CTP(3.5) Propofol(4.3) (h*)BBG(5.5)Zn2+ (5.9) partialagonist:(h*)PPADS(4.6) Cd2+ (5.0) Ap4A(5.5) αβ,-meATP(4.2)

2++ rP2X5 ATP(6.4)PPADS(6.7) Zn(4.4)H(5.5)1,2,3,17-20 2-MeSATP(6.4) TNP-ATP(6.3)Ca2+ (3.0) ATPγS(6.5) Suramin(5.8) Bz-ATP(5.9)RB-2(4.7) αβ,-meATP(6.0) Ap4A(6.6) GTP(4.6) CTP(4.3)

rP2X6 ATP(6.2)–––7

+ rP2X7 BzATP(5.2)BBG(8.0)H(6.1)1,2,3,15, 2-MeSATP(5.0)PPADS(4.3) Cu2+ (6.5)17-20,31-33, ATP(3.4)TNP-ATP(~4.3)Zn2+ (4.9)104 Mg2+ (3.3) (mh*)ADP(2.7)(*)KN-62(7.4) Ca2+ (2.5) (mh*)AMP(2.3)(*)KN-04(~6.0)Halideions

(BoldtextdenotescompoundsavailablefromTocris)

Potencyindicesforagonists,etc.givenas-log10EC50(pEC50)values.Dataforhuman(hm*)andmouse(*) isoformsaregivenforsomekeyligands.

3 stronglyaffectedinonewayoranotherby P2X47andP2X,showtime-dependentchangesin extracellularH+ions(pH)andextracellularZn2+ ions channelpermeabilityandshiftsinreversal 23,24 atalltheknownP2XRsubtypes.Otherextracellular potential.ThiseffectismostprofoundforP2X7 ions,includingNa+,Ba2+,Ca2+,Cd2+,Cu2+,Mg,2+ receptors,whichcanbecomemorepermeableto Mn2+,Al3+,Gd3+,La3+ andhalideanions,alsocan largercations(e.g.NMDG+)and,later,undergoa affectATPpotencytodifferentdegreesat channel-to-poreconversiontoallowthepassageof recombinantP2XRs.Somemodulatorswillwork largedyemolecules(e.g.ethidiumorYO-PRO).In throughkeystructuralelementsinP2Xsubunits, contrasttopermeabilitychanges,somedrugscan othersthroughtheionizationstateofATP,andsome causeanapparentgain-of-functionatP2XRs,by throughthebiophysicalandbiochemicalpropertiesof increasingthenumberofion-channelsavailablefor thecellmembraneitself. activation.ThisismostobviousforhomomericP2X4 andheteromericP2X4/6 receptors,whicharemodified InadditiontoNTPsanddinucleotides,therearea allostericallybyivermectin.Furthermore,humanP2X3 numberofsyntheticanaloguesthatwillactivate andratP2X4 receptorsaremodifiedinasimilarway recognizedP2XRs.Bestknownarethe2-thioalkyl byCibacronblue(oneoftwoisomersofReactive derivatives(e.g.2-MeSATP),methylene-phosphonate blue2).Generalanaesthetics(propofol)andethanol derivatives(e.g.αβ,-meATP)andmodifiedribose canalsomodifysomeP2XRsubtypes. derivatives(e.g.2´,3´-BzATP).Noneofthese compoundsismorepotentthanATP,andnoneclearly P2XRantagonism discriminatesbetweenP2Xsubtypes.However,their Significantadvanceshavebeenmadeinthe potenciesrelativetoATPandothernaturally- identificationofP2Xsubtype-selectiveantagonists. occurringnucleotides(seeabove)helpestablish Twolinesofchemistryhavedominated,basedon agonistprofilesforeachrecombinantP2XR.This eithersuraminorPPADSasthetemplateforhighly knowledge,inturn,canhelptodeterminethe potentderivatives.25 Themajorsuraminanalogues dominantpopulationofnativeP2XRsinwhole includeNF023,NF279andNF449,thelastshowing tissues. subnanomolarblockingactivityattheP2X1 subtype (IC50,0.3nM)andbeing1000-foldmorepotenthere 25,26 Thechannelpropertiesareclearly differentforP2XR thanatP2X3 receptors.ThemajorPPADS subtypes.2 InthepresenceofATPorotheragonist, analoguesincludeisoPPADS,PPNDS,MRS2159and someactivateandinactivatewithinseveralseconds MRS2257.20,25 Thephosphonateanalogue, (e.g.P2X,135P2X,P2X,P2X1/2,P2X1/5),someover MRS2257,is14-foldmorepotentthanPPADSat tensofseconds(P2X,246P2X,P2X,P2X2/3,P2X,2/6 P2X1(IC50,5nM)and10-foldmorepotentthan 27 P2X4/6)and,onceactivated,theP2X7 receptordoes PPADSatP2X3(IC50,22nM).Bothkeycompounds notinactivateatall.Thereversalpotentialforion –NF449andMRS2257–arerelativelyinactiveatthe currentsfallsintheregionof-5to0millivoltsforall otherP2XR(andP2YR)subtypes.Furthermore, P2XRs,butsomesubtypes,particularlyP2X,2 P2X,2/3 thesepotentderivativesnolongershowtheworst side-effectsofsuramin(e.g.G-proteininhibition)and ofPPADS(e.g.ATPaseinhibition).Otherlinesof chemistry,involvingthenucleotidetemplate,also Figure2.Chemicalstructuresofsome haveprovenfruitful.Theribose-modifiedderivative, purinoceptoragonistsandmodulators TNP-ATP,showsnanomolarblockingactivityatP2X1 (IC50,1nM)andP2X3(IC50,0.3nM)andalmostas potentatheteromericP2X2/3 (IC50,11nM),yet1000- foldlesspotentatotherP2XRsubtypes(when 28 comparedtoP2X3 activity).Thenewerdinucleotide antagonist,Ip5I,providesgreaterdiscriminationandis apotentblockeronlyatP2X1(IC50,3nM),notas 29 potentatP2X3(IC50,3µM)andisinactiveatP2X.2/3 IpI5isnotanantagonistofP2X,2P2X45andP2X receptors.

ThemostrecentadvancesinP2XRdrugdesign 2-Methylthioadenosinetriphosphate includethecompetitiveantagonist,A-317491,which tetrasodium(Cat.No.1062) blocksP2X3(IC50,22nM)andP2X2/3 (IC50,92nM; 30 pA,2 6.63).Thisnon-nucleotidicdrugshows analgesicpropertiesinvivo,presumablythroughthe blockadeofnativeP2X3-andP2X2/3-likereceptorson primaryafferentfibres.A-317491ismarkedlyless potentatP2X1(IC50,10µµM)andP2X2(IC50,47M), whilstitisinactiveatP2X4 receptorsandP2YR subtypes.

AnothernotableantagonistisCoomassieBrilliantblue G(BBG),whichblocksratP2X7(IC50,10nM)and 31 humanP2X7(IC50,265nM).Thisdrugalsoblocks humanP2X4(IC50,3µM)and,therefore,isthemost Ivermectin(Cat.No.1260) potentantagonistknownforthisreceptorsubtype. Unfortunately,BBGisalsoactiveatP2X2 receptors (IC50,1µM)and,so,itcannotreadilydiscriminate betweenP2X24andP2Xreceptorswhenusedalone. However,abroadrangeofantagonistsworkatP2X2 receptors(e.g.suramin,PPADS,Reactiveblue2, TNP-ATP)which,otherwise,arelargelyineffectiveat P2X4 receptors. 2-Methylthio-ADP Theisoquinolinederivative,KN-62,isapotentblocker ofhumanP2X7(IC50,34nM)butisinactiveattherat (BoldtextdenotescompoundsavailablefromTocris)

4 32 P2X7 .Therelatedcompound,KN-04,also Theacceptedmonomericproteins(thatfunctionasa blockshumanP2X7 and,ofthetwo,isslightlyless nucleotidereceptor)includeP2Y,1246P2Y,P2Y,P2Y, 33 potent(pA:2 7.31vs8.10).KN-62,butnotKN-04,is P2Y11,,P2Y13andnewly-namedP2Y14 aninhibitorofCaMkinaseII.Sincebothanalogues (formerlycalledtheUDP-glucosereceptor).40 Missing actasnon-competitiveantagonistsatP2X,7 their receptorsintheabovesequencecanbeaccounted blockingactionshereareconsideredtobeCaMKII- forinthreeways:i)inclusionofnon-mammalian independent. P2YRs(e.g.chickP2Y,38andXenopus P2Y),ii) inclusionoforphanreceptors(e.g.P2Y,59P2Y, Theanthraquinonedye,Reactiveblue2,isaracemic P2Y10)whicharestructurallysimilartoknownP2YRs, mixtureofCibacronblueandBasilenblue.Each andiii)misidentificationofGPCRs(e.g.P2Y7 isa isomerhasbroadantagonistactivityatrecombinant leukotrieneB4 receptor).TheeightacceptedP2YRs P2XRs–alsosomeP2YRs–and,therefore,itsutility arediverseintheiroperationalandpharmacological todiscriminatebetweenP2XRandP2YRsubtypesis profiles–andevenshowmajordifferencesbetween questionable.Otherdyes,suchasAcidblue,Trypan speciesorthologues–makingtheidentificationof 41 blue,Uniblue,Evansblue,canblockP2X1-like nativeP2YRsaverydifficultprocess.However,in receptorsinratvasdeferensbuttheirselectivityfor thelast5years,anewgenerationofdrugs,notably P2X1 (overotherrecombinantP2XRsubtypes)has antagonists,hashelpedtoidentifysomeP2YR notbeenfullyinvestigated. subtypes,invivo.NullmutationsinP2Yreceptor subtypeshavealsohelpedidentifyrolesforknown P2YReceptors P2Ysubtypesinvivo,withknockoutmicehaving 42,434445 beengeneratedforP2Y1,P2Y24,P2Yand 46 P2Yreceptors(P2YRs)areGprotein-coupled P2Y.12 receptors(GPCRs)broadlydistributedamongst varioustissuetypesincludingautonomic,central, P2YRagonism entericandsensoryneurons,gliaandastrocytes, Awiderangeofnaturally-occurringnucleoside endotheliumandepithelium,vascularandvisceral triphosphates(NTPs)isactiveatP2YRs.Few smoothmuscle,heartanddevelopingskeletal receptors(onlyP2Y)1 areactivatedbyATPaloneand, muscle,softtissues(kidney,liver,lung,pancreas, evenhere,thisNTPcanbehaveaseitherapartial prostateandthymus),bone,haemopoieticcells.The agonistorantagonist.34 ATPfullyactivatesother P2YRsservemultiplefunctionsintheirhostcells, P2YRsincludingP2Y,24someisoformsofP2Yand workingthroughtwomajorprocesses:i)activationof P2Y11,buteachofthesesubtypesisactivated intracellularsignallingcascades,viathecatalyticG additionallybyotherNTPsinanequipotent proteinα-subunit,andii)modulationofmembrane manner.34,35 Inthisrespect,UTPwillfullyactivateall ion-channels,viaregulatoryGproteinβγ-subunits.A isoformsofP2Y24,allisoformsofP2Yand,asshown 47 third,lessunderstood,processinvolvesthephysical recently,humanP2Y116.UTPcanalsoactivateP2Y, interactionofP2YRswithmembraneproteinsintheir eitherpartiallyorfully,dependingonthereceptor closeproximity. reserve.CTP,GTPandITPwillfullyactivatemouse andratisoformsofP2Y.4 Furthermore, P2YRstructure diphosphates(NDPs)areimportantagonistsat TheP2YRsareheptahelicalproteinsofstructural P2YRs(insharpcontrasttoP2XRs).ADPisafull similaritytotherhodopsinGPCRtemplate.Fourteen agonistatP2Y,1dogisoformofP2Y11,P2Y12 and proteins–P2Y1toP2Y14 –havebeenlinkedtothe P2Y136,andapartialagonistatisoformsofP2Y.UDP P2YRfamily,andmosthavebeenclonedfromhuman isanagonistatallisoformsofP2Y,6 andbothIDP 34,35 cDNAlibraries.Insomecases,orthologuesof andUDPatisoformsofP2Y13.Amostunusual P2YRsubtypeshavebeenclonedfrombovine, agonist,UDP-glucose,isthepreferredandmain canine,chick,mouse,rat,skate,turkeyand Xenopus agonistofP2Y14 isoforms.Thenaturally-occurring 36 libraries.TheseP2Yproteinsrepresentsomeofthe diadenosinepolyphosphates(ApnA,n=2-6)activate shortestGPCRs(328to532residuesinlength)found anumberofP2YRs.48,49 Noneismorepotentthan invertebratespecies.P2Yproteinspossessseven eitherNTPsorNDPs,andnoneisclearly hydrophobicregions(TMI-VII)–believedtoform-α discriminatoryforP2YRsubtypes.48 helices–thatpassthroughthecellmembraneand areconnectedtoanextracellularN-terminus(21-51 ForrecombinantandnativeP2YRs,theabsolute residuesinlength)andintracellularC-terminus(16- potencyofNTPs,NDPsandApnAsisfirmly 217residuesinlength).Forthemembrane-spanning dependentonthelevelsofreceptorexpression. regions(TMI-VII),alignmentofproteinsequences Therefore,typicalEC50 valuesarenoteasilydefined reveals17to61%identityamongsttheP2YR forspecificagonistsatparticularP2YRsubtypes, family.34,35 TheP2YRgenes,unliketheirP2XR withoutpriorknowledgeofreceptornumber. counterparts,donotnormallyundergoalternative Furthermore,relativeagonistpotenciesat splicing.However,examplesofP2YRpolymorphisms recombinantP2YRscannotbeeasilyassimilatedwith havebeenreported,whereamutationhasoccurred agonistdatafromnativeP2YRs,since,atthelatter, inasinglecodon(e.g.anarginine-cysteinetransition thenaturallyoccurringagonistsaresusceptibleto 37 inP2Y2).Also,fusionproteinshavebeenidentified degradationandtransformationbylocalextracellular astheresultofintergenicsplicing(e.g.P2Y11 and .MostofthenaturallyoccurringP2YR SSF1genes).38 agonistsarerapidlybrokendownand,atthispointin time,therearefewinhibitorstochecktheactionsof EachP2Yproteinisbelievedtoexistinthecell theseextracellularenzymes,insitu.Forrecombinant membraneasamonomercoupledtoasingleG P2Yreceptors,littleinthewayofduplicatorywork protein.Suggestionsofhomodimericassembliesof hasbeencarriedoutontheknownmodulatorsof P2Yproteinshavebeenmooted,butthereisno agonistactivityatP2XRs(i.e.pH,extracellularZn,2+ concreteevidenceforsuch.However,compelling otherdivalentandtrivalentcationsetc.)tohelpwith evidencehasbeenpresentedforheterodimeric theproblemofnativeP2YRidentification. 39 assembliesofrecombinantP2Y11andAproteins. Theoccurrenceofsuchheterodimershasnotbeen Anumberofsyntheticnucleotidesanddinucleotides confirmedinwholetissues,althoughtheirlikely canactivatetherecombinantP2YRs.The existencecouldexplainthephenomenonofthenative phosphorothioates(ADPβS,UDPβγSandUTPS)are P3receptor(whereATPandadenosineare notablebecausetheyareresistanttobreakdownand equipotentagonists). showsomeselectivityforP2YRsubtypes.ADPβSis

5 Table2.KeyligandsformonomericP2Yreceptors

SubtypeAgonists(pEC50)Antagonists(pIC50)References

hP2Y1 2-MeSADP(8.7-7.9)MRS2279(7.3)34,35,41,55-57, 2-MeSATP(8.5-6.9)MRS2179(6.5) 105-115 2-HT-AMP(6.4),ADP(8.0-6.6) A3P5P(6.0), PPADS(5.4) ATP(6.5-5.0),ATPγS(6.4)[BzATP(pKi,5.4), ADPβS(6.1-6.0) 2-MeSATP(pK,i 5.2), ATP(pK,i 5.3)] (r*)2-MeSADP(9.2), ADP(8.1) (tt*)2-MeSADP(7.6),ADP(6.3)(*) Suramin(pA,2 5.8)

hP2Y22UTP(7.7-6.2),UTPγS(6.6) Suramin(pA,4.9-4.3) 34,35,37,41,48, 5-BrUTP(5.7), ATP(7.1-6.6)52,53,107, ATPγS(6.2-5.8),GTP(4.9)116-118 UpU4 (7.0),dCpU4 (6.7) AP4A(6.6-6.1)

hP2Y4 UTP(7.6-5.6),UpU4 (6.4) PPADS(4.8) 34,35,41,48,52, dCpU4 (6.1),UTPγS(5.8) ATP(pK,b 6.1)53,60,107, 5-BrUTP(4.8-4.3)119-121 ATP(5.4-4.4),GTP(5.2) (r*)RB-2(4.7) ITP(5.1-4.0) (m*)RB-2(4.3)

(r*)ATP(5.9-5.7),Ap4A(5.5) (m*)ATP(6.4-6.2)

hP2Y6 UDPβS(7.6),UDP(7.0-6.5) RB-2(4.5)34,35,41,52, UpU3 (6.7), 5-BrUDP(6.1)122-127 UTP(5.2-5.0),IDP(4.5) ADP(4.5-4.2)

(r*)UDP(8.2-6.7) (m*)ADP(7.4)

hP2Y11 AR-C67085(5.8),ATPγS(5.5-4.6) Suramin(4.8) 34,35,50,51, (cAMPassay)dATP(5.1-5.0),BzATP(5.1-4.2) AMPS(3.5-2.5)128-134 ATP(4.9-4.2) 2-MeSATP(4.6-4.3) αβ,-meATP(4.1-3.9) βγ,-meATP(3.7)

(c*)2-MeSADP(6.8)

hP2Y12 2-MeSATP(10-8.5) AR-C69931(7.6)34,35,135-138 2-MeSADP(10-7.9)C-1330-7(7.4), RB-2(5.9) ADP(7.4-6.5), ADPβS(7.0-6.4)2-MeSAMP(5.9-5.3) ATP(6.2-5.9) Suramin(5.4)

(rr*)2-MeSADP(8.7)(*)AR-C67085 ADP(7.1)(r*)BzATP(5.3-5.0)

hP2Y13 ADP(8.0),2-MeSADP(7.9) –139,140 2-MeSATP(7.1), ADPβS(7.4) ATP(6.6),IDP(6.3)

(m*)ADP(8.4) (m*)IDP(8.0)

hP2Y14 UDP-glucose(7.1)–141,142 UDP-galactose(6.4) UDP-N-acetylglucosamine(6.1)

(r*)UDP-glucose(7.6) (m*)UDP-glucose(7.7)

(BoldtextdenotescompoundsavailablefromTocris)

Potencyindicesforagonists,etc.givenas-log10EC50(pEC50)values.Dataaregivenasarangeofreported values,tomakeallowancefortheeffectsofreceptorreserveondrugpotency.Dataforcanine(cm*),mouse(*),rat (rt*)andturkey(*)isoformsaregivenforkeyligands.Someligands(e.g.ATP)havebeenreportedaseither agonistsorantagonistsatsomeP2Ysubtypes(e.g.P2Y1 andP2Y4).

6 apotentagonistatP2Y1 and,whilstalsoactiveata notedthattheP2Y1 receptorfailedtoelevatecAMP 18 fewP2XRsubtypes,canstillbeusedjudiciously. levelsinthesameexpressionsystem.TheP2Y12,13 UDPβγSisapotentagonistatP2Y,6 whereasUTPS and14 receptors,whichsharecommonstructural 40 isusefulasastableagonistofP2Y24andP2Y motifs,coupletoGi tolowercAMPlevels.Thus,the receptors.However,atthispointintime,neither patternofGPCRsignallingandrangeofagonists UDPβγSnorUTPSiscommerciallyavailable.The affectingaparticularP2YRareseenaskeyelements 2-thioalkylderivatives(2-MeSADPand2-MeSATP) intheidentificationofnativeP2YRs.Thisis arepotentagonistsattheP2Y1 subtype,butarenot particularlytruefornativeP2Y12 which,otherwise, selectiveandalsoactivatetheknownisoformsof hasbeencalledtheGi-coupledP2YADP receptorof P2Y11,P2Y12andP2Y13.Theribose-modified bloodplatelets. derivative,BzATP,isactiveatP2Y2andP2Y11,the latteralsobeingpotentlyactivatedbyAR-C67085 P2YRantagonism (2-propylthio-,βγ-dichloromethylene-D-ATP)50 and ForonlyafewP2YRsubtypes,significantadvances βγ,-methyleneATP.51 Thesyntheticdinucleotides, havebeenmadeintheidentificationofselective CpUn andUpnU,arepotentagonistsatmostofthe antagonists.ThemostpotentP2Y1 antagonistsare -activatedP2YRs(P2Y,2P2Y46andP2Y) structuralanaloguesofadenosinebis-phosphates and,importantly,aremuchmorestablethanUTPand (e.g.MRS2179andMRS2279)55 withMRS2279(and UDPinvivo.52,53 Theseuridine-baseddinucleotides [3H]-MRS2279)showingnanomolarblockingactivity 56,57 arenotyetcommerciallyavailable. atbothnativeandrecombinantP2Y1 receptors. ForP2Y12,structuralanaloguesofATP(AR-C67085 Regardlessofhowtheyareactivated,the andAR-C69931)shownanomolarblockingactivity.58 recombinantP2YRsshowspecificpatternsof ,oncerenderedactivebyliver intracellularsignalling.P2Y1,2,4,6 and11 receptors metabolism,isalsoaneffectiveblockerofnativeand 59 couplestronglytoGq3andactivatethePLCβ/IP recombinantP2Y121receptors.SinceP2Yand 2+ pathwaytoreleaseintracellularCaincommon P2Y12 receptorsco-existinbloodplatelets,the heterologousexpressionsystems.34,35 However, identificationofP2Ysubtype-selectiveantagonists P2Y2,4 and6 willcouplesecondarilyto pertussistoxin- hashelpedconsiderablyinunderstandingthe sensitiveGi/o toliberateregulatoryβγ,-subunitsand processesofbloodclotformationandthrombolysis. inactivatecationchannelsinspecialisedexpression systems(e.g.mammaliansympatheticneurons). Therearenopotentandselectiveantagonistsforthe P2Y11 willalsocoupletoGs toraiseintracellular pyrimidine-activatedP2YRs,althoughsuramin(P2Y2 cAMPlevelsinvariousexpressionsystemsandsome andP2Y11),Reactiveblue2(RB-2,P2Y46andP2Y) celllines(e.g.HL-60).Arecentreportdescribesa andPPADS(P2Y,4P2Y6andP2Y11)showblocking 54 34 P2Y6 receptor-mediatedelevationofcAMP. activityatmicromolarconcentrationsandhigher.At Whetherthisisachieved,asforP2Y11,bydirect humanP2Y4,ATPitselfisconsideredtobea 60 couplingthroughGs isunclear,althoughitshouldbe competitiveantagonist(pA,2 6.15).Noantagonists

Figure3.Chemicalstructuresofsomepurinoceptorantagonists

NF023hexasodiumsalt(Cat.No.1240)

Suraminhexasodiumsalt(Cat.No.1472)

PPADStetrasodiumsalt(Cat.No.0625)

NF279hexasodiumsalt (Cat.No.1199)

iso-PPADStetrasodiumsalt(Cat.No.0683)

NF449octasodiumsalt(Cat.No.1391)

(BoldtextdenotescompoundsavailablefromTocris)

7 havebeenreportedforP2Y13andP2Y14 receptors. ARAgonism Oneofthebiggestchallengescurrentlyfacingthe PharmacologicaltoolsforthestudyofARsaremuch P2YRfieldistheidentificationoffurtherselective moreprecisethanthosecurrentlyavailableforthe blockersforP2Y2,4,6,11,13 and14 subtypes. studyofP2receptors.Whilstanumberofsubtype- selectiveagonistsareknown,caremustbetaken AdenosineReceptors whenusingtheseathighconcentrationsastheir selectivityislimited.Theaffinityofadenosineatthe Theidentificationandcharacterisationofadenosine differenthumanARshasbeendeterminedin 79 receptors(ARs)precededthatofthenucleotide functionalassayswiththefollowingEC50 values receptors.Thediscoverythatadenosinecouldaffect obtained:A,10.31µM;A2A,0.7µµM;A2B3,24M;A, numerousphysiologicalsystemswasmadeasearly 0.29µM.ThesevaluesindicatethattheA1,A2A3andA as1929,61 althoughthesuggestionthatadenosine receptorscanbeactivatedbyphysiological actsthroughspecificmembranereceptorscamefifty concentrationsofadenosine,whereas yearslater,62 withtwodifferentformsofreceptor pathophysiologicalconcentrationsofadenosineare 63 beingidentifiedfiveyearsafterthat.In1989,cDNAs requiredtoactivatetheA2B receptor.Inosine,another encodingtwodifferentARsubtypeswereisolated64 endogenousligandofARs,activatesrodentreceptors 65 and,shortlyafter,theA3 subtypewasidentified. withKi valuesof15-25µM,butisaweakpartial 80 AlthoughtheA1,A2AandA2B subtypeshadbeen agonistatthehumanA3 receptor. identifiedthroughpharmacologicalandfunctional studiespriortotheirmolecularcloning,theexistence Figure4.Chemicalstructuresofsomekey oftheA3 receptorhadnot.ARsareoccasionally adenosinereceptorligands referredtoasP1receptors,mosttypicallywhenbeing compareddirectlywiththeirnucleotidereceptor cousins,theP2receptors. DPCPX(Cat.No.0439) ARstructure FourdifferentARsubtypes,A1,A2A,A2B3andA,have beenidentified.Allaremembersoftherhodopsin-like familyofGPCR,andallfourARsubtypeshavebeen clonedfrom alargenumberofdifferentspecies.At 318aminoacidsinlength,theA3 subtypeisthe shortestwhilstA2A isthelongest(412residues).Their N-terminiarerelativelyshort(7-13residuesinlength), asaretheirC-termini(32-120residues).Inthe transmembranedomains(TMI-TMVII),humanARs MRS1706(Cat.No.1584) share39-61%sequenceidentitywitheachotherand 11-18%identitywithP2YRs.Itisinterestingtonote thatP2Y11 isascloselyrelated,intermsofsequence identity,totheA2Breceptor(18%)asitistoP2Y12 (17%),P2Y13(17%)orP2Y14 (17%)receptors.Each ofthefourhumanARgenescontainsanintronwithin thecodingregion–locatedimmediatelyaftertheend ofthethirdtransmembranedomain–although alternativesplicingwithinthecodingregionhasnot [3H]-MRS1754(Cat.No.R1585) beenobserved.Polymorphismshavebeenobserved 6667 intheA1andtheA2Areceptors;polymorphicA2A receptorshavebeenimplicatedinpanicdisorders.68

ARscoupleprincipallytoadenylatecyclase.A1 and A3 arenegativelycoupledtoadenylatecyclase throughtheGi/o proteinα-subunits,whereasA2A and ZM241385(Cat.No.1036) A2B arepositivelycoupledtoadenylatecyclase throughG.sThehumanA2B receptorhasalsobeen observedtocouplethroughGq/11 toregulate (BoldtextdenotescompoundsavailablefromTocris) 69 phospholipaseCactivityandtheA3 receptormay 70 interactdirectlywithGs. Forthemostpart,the AlloftheknownARagonistsarecloselyrelatedto pharmacologyofARorthologuesisquitesimilar, adenosineinstructure,withfewmodifications 81 exceptforA3 receptorsubtypes,wheresignificant permitted. Alterationoropeningoftheribosering differencesinpharmacologyanddistributionhave drasticallyreducesaffinity.Thehydroxylgroupatthe beenobserved.71 2´-positionisneededforbothaffinityandactivity, whilstremovalofthe3´-and5´-hydroxylgroupsleads Thediversephysiologicaleffectsmediatedbythe topartialagonistswithreasonablyhighaffinity. differentARsubtypes,particularlymodulationofthe Substitutionatthe5´-positionoftheriboseis cardiovascular,immuneandcentralnervoussystems, permittedandNECA(5´-N-ethylcarboxamido- havebeenconfirmedbytransgenicknockoutmice. adenosine)isapotent,non-selectiveARagonist. NullmicehavebeengeneratedforeachoftheA,A12A NECAisalsothemostpotentA2B receptoragonist 72-75 andA3 receptorsand,inallknockoutanimals withaKiofapproximately300nMatthehumanA2B generated,theARsinquestiondonotappeartoplay receptor,comparedwithKi1’sof14nMatA,20nMat a criticalroleduringdevelopment.Knockoutmice A2A3and6.2nMattheAreceptor.Theaminogroup haveyettobedescribedfortheA2B receptorsubtype. attachedtotheC6-positionofthepurineringis Incontrasttoknockoutstudies,overexpressionof essentialforagonistactivity,butsubstitutionofoneof eitherA13orAsubtypesintransgenicmicehasa thetwohydrogenatomsinthisgrouptendstogive 76,77 cardioprotectiveeffect,althoughitwouldappear risetoA1-selectiveagonistsofhighpotency.Themost that,undercertainconditions,overexpressionofthe selectiveagonistfortheA1 subtypeisCCPA(2- 6 A3 subtypecangiverisetoanembryoniclethal chloro-N -cyclopentyladenosine),whichis phenotype.78 approximately50-foldselectiveinhumanandover 600-foldselectiveinratthanattheA3 receptor subtype.ModificationattheC2-positionofthepurine

8 Table3.Keyligandsforadenosinereceptors

SubtypeAgonistsAntagonistsReferences

hA1 CCPA(9.1,9.4-9.6*)DPCPX(9.1,9.5-9.7*) 79,82,83 (R)-PIA(8.7,8.9-9.3*) CGS15943(8.5,7.7-8.2*) IAB-MECA(8.1,7.7*)XAC(7.5,8.4-8.6*) NECA(7.9,7.9-8.4*)ZM241385(6.6,5.7*) Cl-IB-MECA(6.9,6.1*) SCH58261(6.5,6.9*) CGS21680(6.5,5.5*) MRS1754(6.4,7.8*) Adenosine(6.51)MRE3008F20(6.0,5.0*)

hA2A NECA(7.7,7.7-8.0*) SCH58261(9.2,8.6*)79,82 CGS21680(7.6,7.7*)ZM241385(9.1,9.5*) IAB-MECA(6.3,6.7*)XAC(9.0,7.3-7.6*) Adenosine(6.2)1 CGS15943(8.4,8.5-8.9*) ()R -PIA(6.1,6.1-6.9*) DPCPX(6.9,6.5*) Cl-IB-MECA(5.7,6.3*) MRE3008F20(6.9,5.7*) CCPA(5.6,5.4*) MRS1754(6.3,6.2*)

hA2B NECA(6.4-6.5) MRS1754(8.7)79,82 Adenosine(4.61)XAC(7.9) ()R -PIA(4.5-5.4) CGS15943(7.8) CCPA(4.4)ZM241385(7.5) CGS21680(3.4)DPCPX(7.3) MRE3008F20(5.7)

hA3 IAB-MECA(9.2,8.9*)MRE3008F20(9.5,>5.0*)79,82 NECA(8.2,6.6*)CGS15943(7.3,>4.0*) Cl-IB-MECA(8.0,9.5*) XAC(7.0,>4.0*) ()R -PIA(7.8,6.7-6.8*)MRS1754(6.2) CCPA(7.4,6.6*)DPCPX(5.4,>5.0*) CGS21680(7.2,6.2*) SCH58261(5.0) Adenosine(6.54)1 ZM241385(5.0,3.8*)

(BoldtextdenotescompoundsavailablefromTocris) 1 Potencyindicesforagonists,etc.aregivenaseither-log10iK(pK)ior-log10EC50(pEC50)values.Dataforrat(r*) receptorsaregivenforsomekeyligands.

ringcangivesomeselectivityforA2 receptors onexperimentalconditions.Asaconsequence,the dependingonthesizeofthesubstituent.CGS21680, useofselectiveantagonistsisafarmorereliable themostselectiveA2A agonist,issubstitutedbothat meansofcharacterisingendogenousARresponses. the5´-positionoftheriboseandtheC2-positionofthe purinering.AlthoughCGS21680isapproximately ARantagonists 26-foldselectivefortheratA2A receptor,itislessthan Caffeineisthemostfrequentlyconsumed 3-foldselectiveforthehumanA2A receptor,compared psychotropicdrugintheworld,andtheconsumption toitsactionsattheA3 subtype.WhilstIAB-MECAis ofcoffeeandteabeveragesleadstosignificant approximately13-foldselectiveforboththehuman plasmaconcentrationsofcaffeine,, andratA3 receptors,Cl-IB-MECAis11-foldselective theobromineandothermethylxanthinecompounds.85 forthehumanand~1400-foldselectivefortheratA3 Thesemethylxanthineslookverymuchlike receptor.Cl-IB-MECA issubstitutedatthe5´-,C2- adenosine,butwithouttheribosemoiety.Ingeneral, 6 andN -positions.Asmentionedpreviously,thereisno methylxanthinesareweakARantagonistsyetalso selectiveA2B agonistavailable. canactthroughothermechanismssuchas phosphodiesteraseinhibition.Foradenylatecyclase Radiolabelledagonistsusedforthecharacterisation assays,IBMX(3-isobutyl-1-methylxanthine)is 3 3 ofARsinbindingassaysinclude:[H]-CCPA,[H]- frequentlyincludedasaphosphodiesteraseinhibitor 3333 NECA,[H]-CHA,[H]-PIA,[H]-CPA,[H]-CGS innon-adenosinergicreceptorsystems(suchasthe 125125 21680,[I]-I-AB-MECAand[I]-APNEA.Affinities P2Yfamily).ARsandP2YRsarefrequently (Ki values)forARagonistshavebeendetermined expressedinthesamecells;thefunctional usingbothwhole-cellassaysandmembrane characterisationofheterodimericassembliesbetween preparations,andincompetitionagainsteither thesetwofamiliesofGPCRs39 mayhavebeen 82 radiolabelledagonistsorantagonists.Twoaffinity impededbytheuseofARantagonistsinassaysof states,onehighandonelow,areobservedfor P2YRfunction. agonistswhenbindingassaysareperformedusing membranepreparations,whereasasingleKi value, Modificationofmethylxanthines,atcertainpositions, correspondingtothelowaffinitysiteobservedin hasledtosomesubtype-selectiveARantagonists.81 membranebindingassays,isdeterminedinwhole- DPCPX(8-cyclopentyl-1,3-dipropylxanthine;also cellassays.83,84 Thisdifferenceismostlikelydueto referredtoasCPX)isanA1 receptorantagonistwith highintracellularlevelsofGTPpresentinintactcells. subnanomolaraffinity(Kd1=0.8nMatthehumanA Agonistaffinitiesobservedinfunctionalassays, 83 receptor).Unfortunately,theKi ofDPCPXatthe representedastheEC50 values,varywidelyasa humanAreceptorisapproximately50nM,which functionofexpressionlevelsandcouplingefficiency 2B makesthefold-selectivityofthiscompoundfortheA1 toGproteins.Theaffinityofagivenagonistata receptorlessthantwoordersofmagnitude.Themost particularreceptorsubtypecanvarywidelydepending selectiveA2B receptorantagonistisMRS1754(~200-

9 fold).ThemostselectiveA2A3andAantagonistsare greatlyexceedthatpredictedbypharmacologicaland notxanthinederivatives.SCH58261andZM241385 functionalstudies.Thishasbeentrueinthecaseof aresubnanomolarA2A antagonistsinhuman,but theP2Yreceptors,whereeighthumansubtypeshave ZM241385ismoreselectiveattheratA2A receptor. alreadybeenidentifiedatthemolecularleveland MRE3008F20isthemostselectivehumanA3 probablymoreremaintobecloned.Thishasalso receptorantagonist,althoughthiscompoundis beentrueinthecaseoftheP2Xreceptors,wherethe essentiallyinactiveattheratreceptor.MRS1523is complexityofsubunitassemblyhasrevealedatleast 80 reasonablyselectivefortheA3 receptorinratand 12subtypesofATP-gatedion-channelsandmore alsoblocksthisreceptorsubtypeinfrog.70 remaintobecharacterised.Additionally,the Radiolabelledantagonistsusedforthe molecularcloningofareceptorforadenine87 addsa characterisationofARsubtypesinbindingassays furtherdimensiontothepurinoceptorfamily (Figure include:[3H]-DPCPX,[33H]-SCH58261,[H]- 1). ZM241385,[1253I]-ZM241385,[H]-MRS1754,[125I]- ABOand[3H]-XAC.XAC(xanthineaminecongener) Theuseofrecombinantsystems,particularly isagoodnon-selectiveantagonistathumanARs(A,1 Xenopuslaevis oocytes(theonlyexpressionsystem 29nM;A2A,1nM;A2B3,7.3nM;A,92nM),asisthe knowntobedevoidoffunctionalP1andP2 non-xanthinecompoundCGS15943(A1,3.5nM;A,2A receptors),hasenabledtheoperationalprofileof 4.2nM;A2B3,16nM;A,51nM).Theuseof individualreceptorsubtypestobedetermined.Such methylxanthinesasARantagonistshasalwaysbeen informationisofgreatutilityinattemptingtounravel plaguedbytheiractivityatothersites. thewaysnucleosideandnucleotidereceptorsinteract MethylxanthinesarealsodirectinhibitorsofPI3Klipid witheachotherandwithotherproteinsinvivo.There kinase(p110deltainparticular)activity,asisthenon- isstillmuchtodointhesearchforsubtypeselective xanthineadenosinereceptorantagonistCGS15943 agonistsandantagonists,butligandselectivityisnow 86 (IC50 <10µMatp110delta).Careshouldbe achievableforsomesubtypes–withthepromiseof exercisedintheuseofARantagonistsathigh moretofollow.Wehopethattheligandsidentifiedin concentrations. thisreviewwillhelpinvestigatorstoexplore nucleotideandnucleosidesignallinginvivo,with Closingremarks greaterconfidence.

Theidentification,in1992,oftheA3 receptorwasthe firsthintthatthenumberofmolecularentities encodingnucleosideandnucleotidereceptorswould

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11

Agonists 1063CGS21680HCl*...... A2A agonist 6 17052-Chloro-N -cyclopentyladenosine...... Potent,selectiveA1 agonist 11042-CI-IB-MECA...... HighlyselectiveA3 agonist 6 1702N -Cyclopentyladenosine...... Potent,selectiveA1 agonist 1579HEMADO...... HighaffinityselectiveA3 agonist 1066IB-MECA...... A3 selectiveagonist 1691NECA...... Adenosinereceptoragonist

Antagonists 1699CGS15943...... Potentadenosinereceptorantagonist 04861,3-Dipropyl-8-phenylxanthine...... A1 selectiveantagonist 0439DPCPX...... A1 selectiveantagonist R439[3H]-DPCPXb ...... Radiolabelledformof(0439) 1217MRS1220...... Highlypotent,selectivehA3 antagonist 1584MRS1706...... PotentandselectiveA2B antagonist 3 R1585[H]-MRS1754b ...... SelectivehA2B antagonistradioligand(analogueof1584) 1359VUF5574...... Potent,selectivehA3 antagonist 1036ZM241385...... Potent,highlyselectiveA2A antagonist R1036[3H]-ZM241385b ...... Radiolabelledformof(1036)

Other 0481Dilazep2HCl...... Adenosineuptakeinhibitor 0691Dipyridamole...... Adenosinetransportinhibitor 1363PD81723...... Allostericpotentiator ofA1 receptors

Purinergics

0845EvansBluetetrasodiumsalt...... SelectiveP2Xpurinergicantagonist 1260Ivermectin...... Modulatesglutamate/GABA-activatedCl- channels 1277KN-62...... Non-competitiveP2X7 antagonist 10622-Methylthioadenosinetriphosphatetetrasodiumsalt....P2purinergicagonist 1388MRS2159...... Potent,highlyselectiveP2X1 antagonist 0900MRS2179tetraammoniumsalt...... SelectiveP2Y1 antagonist 1203MRS2219...... PotentiatesP2X1-mediatedresponses 1204MRS2220...... P2X1 antagonist 1240NF023hexasodiumsalt...... Selective,competitiveP2Xantagonist 1199NF279hexasodiumsalt...... PotentselectiveP2X1 antagonist 1391NF449octasodiumsalt...... HighlyselectiveP2X1 antagonist 1682PIT...... P2Yligand;displaysmixedantagonism/potentiation 0625PPADStetrasodiumsalt...... P2purinergicantagonist 0683iso-PPADStetrasodiumsalt...... P2Xpurinergicantagonist 1309PPNDS...... Potent,selectiveP2X1 antagonist 1472Suraminhexasodiumsalt...... Non-selectiveP2antagonist

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NucleotideandNucleosideReceptors, TocrisReviewsNo.23, March2003

©2003TocrisCookson PublishedanddistributedbyTocrisCookson,Bristol,UK

Editors:SamanthaManley,Ph.D.,NatalieBarker,B.Sc. DesignandProduction:JaneChampness

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