Intracellular Receptor Modulation: Novel Approach to Target GPCRs

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Article details

Ortiz Zacarías N.V., Lenselink E.B., IJzerman A.P., Handel T.M. & Heitman L.H. (2018),

Intracellular Receptor Modulation: Novel Approach to Target GPCRs, Trends in Pharmacological Sciences 39(6): 547-559.

Doi: 10.1016/j.tips.2018.03.002

Review

Intracellular Receptor Modulation: Novel Approach to Target GPCRs

Natalia V. Ortiz Zacarías,

Eelke B. Lenselink,

Adriaan P. IJzerman,

Tracy M. Handel,

and Laura H. Heitman

*

Recent crystal structures of multiple G protein-coupled receptors (GPCRs) have revealed a highly conserved intracellular pocket that can be used to modulatethesereceptorsfromtheinside.Thisnovelintracellularsitepartially overlapswiththeGproteinandb-arrestinbindingsite,providinganewmanner ofpharmacologicalintervention.Hereweprovideanupdateofthearchitecture andfunctionoftheintracellular region ofGPCRs,until nowportrayedas the signalingdomain.Wereviewtheavailableevidenceonthepresenceofintra- cellularbindingsitesamongchemokinereceptorsandotherclassAGPCRs,as wellasdifferentstrategiestotargetit,includingsmallmolecules, pepducins, and nanobodies. Finally, the potential advantages ofintracellular (allosteric) ligandsover orthostericligandsarealsodiscussed.

MultipleBindingSitestoTargeta GPCR

Gprotein-coupledreceptors(GPCRs,seeGlossary)compriseoneofthelargestfamiliesofdrug targets,withapproximately34%ofthecurrentlymarketeddrugstargetingthisreceptorclass[1].As lack of efficacycontinuestobethemainreasonoffailureinPhaseIIandPhaseIIIclinicaltrials[2], novel approachestosuccessfullytargetthesereceptorsarestillnecessary.Asitisapparentfrommost GPCRcrystalstructuresreportedsofar,smallmoleculesoftenoccupyabindingsiteexposedtothe extracellularsolvent–theso-calledorthostericbindingsitewhichisusedbyendogenousligands [3](Figure 1A). However, targeting GPCRs has proved to be challenging, especially when drugs need tocompetewithahigh(local)concentrationoftheendogenousligand,asisthecaseoftargeting chemokinereceptorsduringinflammatoryconditions[4].Hence,thedevelopmentofallosteric modulators(Box1)thatbindtospatiallydistinctbindingsites[5]hasemergedasapromising approachtoimprovenotonlydrug efficacy,butalsoselectivityandsafety[6–8].Avarietyofdifferent allostericbinding sites havealreadybeen identifiedin GPCRs, mostofthemclose to the orthostericbindingsite;yet,unexpectedligandbindingsiteshaverecentlybeenfoundincrystal structuresofclassAandclassBGPCRs[5].Inthisregard,therecentcrystalstructuresofCC chemokinereceptor 2(CCR2)[9],CCchemokinereceptor 9 (CCR9)[10],and b2-adrenergic receptor(b2AR)[11]haveforthefirsttimerevealedaspatiallyconservedintracellularbindingsite for small molecules in class A GPCRs (Figure 1A), providing a new avenue to inhibit or modulate these receptorsindifferentpathologies.

IntracellularRegionofGPCRs:BeyondSignaling

Ingeneral,GPCRsshareasimilarstructureconsistingofthreedifferentdomains(Figure1A):

theextracellulardomainthat includesthreeextracellularloops(ECLs)andthe Nterminus, whichvaryinlengthandstructuredependingontheGPCRsubfamily[12];thetransmem- brane(TM)domain,whichcomprisessevenTMhelices;andtheintracellulardomain,which includesthreeintracellularloops(ICLs),an amphipathichelix(H8),andthe Cterminus[3].

Traditionally,the upperTMsectionandthe extracellulardomainhavebeen consideredto

Highlights

Recent crystal structureshave sug- gested a high diversity ofallosteric bindingsites,includingnovelpockets intheintracellulardomainofGPCRs.

Theseintracellularsitescanpotentially betargetedwithsmallmolecules,pep- ducins,andnanobodies.

TherecentX-raystructuresofCCR2, CCR9, and b2AR have revealed a highly-conserved intracellular pocket forsmallmolecules,suggestingitspre- senceinmostchemokine receptors andotherclassAGPCRs.

Although manyallosteric ligands for GPCRs have been described, only fewallostericdrugshavereachedthe market.Yet,thenumberofallosteric modulators in development stages keepsincreasing,includingthenum- berofintracellularligandsin(pre)clin- icalstudies.

Thediscoveryofintracellularbinding sites,combinedwiththearrayofstra- tegiesfortargetingsuchsites,opens upnewapproaches tobetter study andtargetGPCRs.

1DivisionofDrugDiscoveryand Safety,LeidenAcademicCentrefor DrugResearch,LeidenUniversity, Einsteinweg55,2333CC,Leiden, TheNetherlands

2UniversityofCalifornia,SanDiego, SkaggsSchoolofPharmacyand PharmaceuticalSciences,LaJolla, CA92093,USA

*Correspondence:

l.h.heitman@lacdr.leidenuniv.nl (L.H.Heitman).

547

encompasstheligand-bindingdomain.Incontrast,thelowerTMsectionandtheintracellular domainhavebeenconsideredtobethesignalingdomain[3,13].Structurally,theintracellular domainis morehighly conservedand flexiblethan the extracellularregion containing the orthostericbindingsite[3,13],whichisprobablyrelatedtoacommonmechanismofreceptor activationandGprotein-coupling[14].Inthisregard,analysisofseveralactive-andinactive- statecrystalstructureshasrevealedaconservedrearrangementofresiduecontactsnearthe Gprotein-bindingsite,involvingresidues3x46inTM3,6x37inTM6,and7x53fromthehighly conservedNPxxY motif locatedin TM7(residues accordingtostructure-based Balles- teros-Weinsteinnumbering[15])[14].Inaddition,thisregionisalsoinvolvedinthecoupling andselectiverecognitionofdifferentGproteins[16,17]andothersignalingproteinssuchas b-arrestin[18],whichcanleadtoamultitudeofdifferentsignalingpathwaysuponactivationof aGPCR.Recently,thetraditionalviewofaseparateligand-bindingandsignalingdomainhas beenchallengedasmoreevidencesuggeststhattheintracellulardomainofGPCRscanalso beboundbyligandsandthusbeusedforreceptormodulation(Figure1) [5,9–11].

CommonIntracellularBindingSiteinClassAGPCRs

AmongGPCRsthereis nowmutational,pharmacologicalandstructuralevidenceofligand bindingsiteslocatedattheirintracellularinterface.Thisevidenceisparticularlyextensiveinthe caseofchemokinereceptors(Box2);thus,beforeextendingtootherclassAGPCRs,wewill firstreviewtheevidenceavailableforchemokinereceptors.

IntracellularBindingSiteatChemokineReceptors

TherecentX-raystructureofCCR2incomplexwithanorthostericantagonistandthenegative allostericmodulator(NAM,Box1)CCR2-RA-[R](PDB5T1A)[9],andofCCR9incomplexwith theNAMvercirnon(PDB5LWE)[10](Figure2)haveprovidedstructuralconfirmationofsuch intracellularbinding siteinchemokine receptors.Thetwo structures reportanoverlapping solvent-exposedbindingsiteintheintracellulardomainofthesereceptors,locatedmorethan 30ÅfromtheorthostericbindingsiteandenclosedbytheintracellularendsofTM1–TM3,TM6, TM7,andH8(Figure1A)[9,10].TheNAMsbindthisintracellularpocketwheretheyinteractwith severalconservedaminoacidresidues(Figure1B).

Interestingly,beforethesecrystalstructuresweresolved,intracellularligandbindingsites had already been suggested for chemokine receptors. In 2008, a putative intracellular bindingsiteforsmall-moleculecompoundshadbeenidentifiedinCCR4,CCR5,CXCR1, andCXCR2[19,20].Functional datafromthesestudiessuggestedthataseriesofcom- poundsrequiredintracellularaccessinordertoexerttheiractivity.Specifically,forCCR4it wasshown thatseveralcompoundssimilartocompound1(Figure2)exhibitedalackof correlationintheirpotencies whenmeasured inmembrane orcellularassays.However, afterpermeabilizationofthecellswithsaponinthepotenciesbecamecomparableinboth assays [19]. In CXCR2, the loss of cellular potency seemed to be dependent on the lipophilicity (logD) of the compounds. Lower lipophilicities resulted in a greater loss of potency,indicatingthatthesecompoundsneededacertainleveloflipophilicitytocrossthe cell membrane and reach the intracellular binding site [20]. A subsequent chimeric approach, with CCR4–CCR5 or CXCR1–CXCR2 chimeras, led to the suggestion that theC terminuswas partofthebinding siteforthesemolecules[19,20].In CXCR2, this intracellularbindingsitewasfurthermappedwithhelpofhomologymodelingandmuta- tionalstudies,whichresultedintheidentificationofseveralCterminalresiduesaspartof thisallostericbindingsite,includingD84^2x40,T83^2x39,A249^6x33,Y314^7x53,andK320^8x49 (Figure1B)[20,21].Thus,thesestudiesinCCR4andCXCR2providedthefirstbiochemical evidenceoftheexistenceofsuchbindingsite.

Glossary

Affinity:parameterthatdescribes howstrongaligandbindstoits target.

Allostericbindingsite:abinding sitenonoverlappingand topographicallydistinctfromthe orthostericbindingsite.

Allostericmodulator:anyligand thatbindstoanallostericbinding site,fromwhichtheycanmodulate theactivityoforthostericligands.

Constitutivelyactivemutant (CAM):mutationthatleadstoa permanentandagonist-independent activestateoftheGPCR,compared withthewild-typereceptor.

Efficacy:parameterthatdescribes thedegreeofeffectorresponse achievedbyaspecificligandupon bindingtoitstarget.

Gprotein-coupledreceptors (GPCRs):familyoftransmembrane proteinsthattransduceavarietyof extracellularsignalsintointracellular responsesviaGprotein-dependent or-independentsignalingpathways.

Invertebrates,GPCRsaredividedin fourdifferentclassesorsubfamilies:

classA(rhodopsin-like),classB (secretin),classC(metabotropic glutamate),andclassF(frizzled/

smoothened).

Intrabodies:intracellularly-expressed antibodyfragmentsaimedat intracellulartargets.

Nanobodies:recombinantsmallsize antibody(12–15kDa),containinga variable-domainfragmentderived fromcamelidheavy-chainantibodies.

Orthostericbindingsite:the bindingsiterecognizedandusedby theendogenousligandfora correspondingreceptor.

Orthostericligand:anyligandthat bindstotheorthostericbindingsite ofthereceptor.Orthostericligands includetheendogenousligandsand non-endogenousagonists, antagonists,orinverseagonists.

Pepducins:lipidatedpeptides derivedfromtheintracellularloopsor theCterminusofGPCRs,which specificallytargettheircognate receptorbyactingasallosteric agonistsorantagonists.

Polypharmacology:theabilityofa ligandto(purposelyand)effectively bindtoseveraltargets.

Potency:parameterthatdescribes theactivityofadrugbydefininghow muchofaligand(concentration)is

neededtoproduceahalf-maximal effect.

Structure-basedBallesteros- Weinsteinnumbering:numbering systemforaminoacidresidues,which takesintoaccountstructural informationtocorrectforbulgesand constrictions.Inthisnumbering schemethefirstnumberdenotesthe transmembranedomainandthe secondnumberdenotestheresidue positioninrelationtothemost conservedaminoacid,thelatter alwaysinposition50.E.g.Y^7x53 indicatesthatthistyrosineinlocatedin TM7,inposition53.Thisnumbering systemiscurrentlyusedbytheGPCR databaseAppendixAⁱⁱ[15].

Usingasimilarapproach,ahomologousbindingsitewasdiscoveredinCCR2,wheresmall molecules such as CCR2-RA-[R], JNJ-27141491and SD-24 can bind (Figure 2) [22,23].

Similar keyresidueswere identified, including V244^6x36,K311^8x49, Y305^7x53 andF312^8x50 (Figure1B)[22],whichhavenowbeenconfirmedbytheX-raystructure[9].Asimilarbindingsite hasalso been suggestedin CX3CR1after pharmacological characterization ofcompound AZD8797(Figure2),anoncompetitiveinhibitorofCX3CR1withstructuralsimilaritytoknown CXCR2intracellularligands[24].Inaddition,severalpepducinsderivedfromICL1ofCXCR4 have beenshown to interactselectively with CXCR4 ina noncompetitive manner [25,26].

Specifically, CXCR4 pepducin ATI-2341 has been predicted to interact with most of the residueslocatedinICL1–ICL3[27],indicatingthat thisreceptorcan alsobetargeted from theintracellularside.Finally,thestructureoftheviralchemokinereceptorUS28incomplexwith thechemokineligandCX3CL1andthenanobodyNb7(PDB4XT1)showsthatNb7bindsina similarsubpocketcomposedbytheintracellularendsofTM3,TM5,TM6andH8.Moreover, Nb7interactswithseveralresiduesalsoinvolvedinthebindingofsmall-moleculesorpepducins inhumanchemokinereceptors,orininteractionswithsignalingproteins[28,29].

IntracellularBindingSiteatOtherClassAGPCRs

This conserved intracellular binding site is not limited to chemokine receptors, as evidence for this site hasbeenfoundinotherclassAGPCRs.Inthisregard,thecrystalstructureofb2AR(PDB5X7D)has beensolvedwiththesmall-moleculeligand15PA(Figure2),apolyethyleneglycol-carboxylicacid derivativeofcompound15[30],cocrystalizedattheintracellularinterface[11].Compound15PA bindsinapocketformedbytheintracellularendsofTM1,TM2,TM6,TM7,H8,andICL1,whereit interactswithkeyresiduesalsoidentifiedinCCR2andCCR9[11](Figure3,KeyFigure).Moreover, thisbindingpocketpartiallyoverlapswiththebindingsiteofnanobodiesNb60andNb80inb2AR [31,32],Fab2838inAdenosineA2Areceptor(A2AAR)[33],Nb9-8inM2muscarinicacetylcholine receptor(M2R) [34]and Nb39in them-opioidreceptor(mOR) [35].Previous tothesecrystal structures,differentcomputationaltoolspredictedintracellularbindingpocketsinrhodopsinand M2R[36,37].Moleculardockingstudiesandvirtualscreeningidentifiedseveralrhodopsininhibitors thatbind attheinterface betweentheGPCRandGprotein[38,39],in anintracellularpocket resemblingthatidentifiedinchemokinereceptors.Moreevidenceforageneralizedintracellular pocketcomesfromtheproteinaseactivatedreceptor1(PAR1),whereaseriesofsmallmolecules suchascompound5-C(Figure2)andICL3-derivedpepducinswereshowntointeractwithresidues locatedinTM7andH8[40,41].SimilarICL-derivedpepducinshavealsobeendevelopedforPAR2 [42],PAR4[43],sphingosine-1-phosphatereceptor3(S1P3)[44]andformylpeptidereceptors1and 2(FPR1andFPR2)[45].Takentogether,thereismountingevidenceforthepresenceofaspatially conservedintracellularpocket,notonlyinchemokinereceptorsbutamongseveralclassAGPCRs.

StructuralFeaturesoftheIntracellularBindingSite

Therecent X-raystructuresofCCR2 [9],CCR9 [10]andb2AR[11] areprovidingstructural informationonthefeaturesthatdeterminebindingandselectivityinthisintracellularbindingsite (Figure3).Moreover,thesestructuresprovidenewopportunitiesfortheapplicationofstruc- ture-baseddrugdesign(SBDD)methods,suchasvirtualscreeningcampaigns,whichmight allow the identification and/or optimizationof novel intracellular ligands for these or other homologousreceptors [5,28]. Below,features of this site are discussedin termsof three componentparts:ahydrophobicsubpocketaboveH8,acentralTM7–H8bindingregion,anda regionformedbyTM3/6andTM2/ICL1.

HydrophobicSubpocket

AllligandsshareahighlyconservedhydrophobicsubpocketaboveH8.Threehighlyconserved residuesamongstclassAGPCRsformthebasisofthispocket:V^1x53(65%conserved),Y^7x53

(89%conserved)andF^8x50(65%conserved)(Figure3,upperpanel).Whilethereisonlysome evidencefortheroleofV^1x53inactivation[46],numerouspublicationshaveshowntheroleof the latter two residues in signaling and intracellular ligand binding at different GPCRs [14,47,48]. In termsof hydrophobicity,residues1x56 and1x57 arealso highly conserved

(A)

(B)

TM6

TM1

TM5 TM3 CCR2 H8 CCR9 β2AR

30 Å 26 Å

Extracellular

Intracellular

TM7

TM1

1x53 1x56 1x57 12x49 2x37 2x39 2x40 2x43 3x45 3x46 3x50 3x53 6x29 6x32 6x33 6x36 6x37 6x40 7x53 8x47 8x48 8x49 8x50 8x53

B&W Number

ICL1 TM2 TM3 TM6 TM7 H8

β2ARCCR2 CCR9 CCR1CCR3 CCR4CCR5 CCR6CCR7 CCR10CCR8 CXCR1 CXCR2 CXCR3 CXCR4 CXCR5 CXCR6 CX3CR1

ACKR1 ACKR2 ACKR3 ACKR4 CCRL2 XCR1

Figure1.NovelAllostericBindingSiteinClassAGPCRs.(A)EndogenousligandsbindclosetotheextracellularregionofGPCRs,intheso-calledorthosteric bindingsite.Mostoftheco-crystallizedsmallmoleculesalsobindinthisextracellularregion,suchasBMS-681inCCR2andcarazololinb2AR.Recently,thecrystal structuresofCCR2(purple,PDB5T1A),CCR9(green,PDB5LWE)andb²AR(yellow,PDB5X7D)haverevealedanallostericsolvent-exposedbindingsite,locatedinthe intracellularregionofGPCRs,around30Åawayfromtheorthostericbindingsite.Thisnovelbindingsitechallengesthetraditionalviewoftheupperseven- transmembrane(7TM)regionofGPCRsasligandbindingdomainandtheintracellularregionassignalingdomainonly.Asshowninthestructures,thisintracellular bindingsitecanalsobetargetedbysmallmoleculessuchasCCR2-RA-[R]inCCR2,vercirnoninCCR9and15PAinb2AR.Dottedlinesrepresenttheplaneofthe membrane.(B)Sequenceconservationamongchemokinereceptorsandb2AR,basedontheGPCRdatabaseAppendixAⁱⁱ(GPCRdb).Residuesshownareresidues involvedintheintracellularbindingsiteofCCR2,CCR9andb²AR(upperthreerows).Someoftheseresidueshavealsobeenfoundtobeimportantforligandbindingto otherclassAGPCRs,aswellasforGproteinandb-arrestinbinding.

(Figure3,upperpanel).However,inCCR9Y^1x57adoptsanorientationthatfurtheropensupthe pocket,allowingthelarge4-tert-butylsubstituentoftheligandtoreachdeeperintothispocket, indicatingaroleinconferringligandselectivity.

CentralTM7-H8BindingRegion

ThecentralpartofthepocketconsistsofthekinkbetweenH8andTM7,formedbyeitherP^8x48 (b2AR)orG^8x47(chemokinereceptors).Thissubpocketincludesresidues8x47to8x49,which

Box1.AllostericModulationinGPCRs

Gprotein-coupledreceptors(GPCRs)areconsiderednaturalallosteric proteins,asthesiteofinteractionofthe endogenousligand–theorthostericbindingsite–differsfromthesiteofthesignalingeffectors,suchasGproteins andb-arrestins[74].Inadditiontotheorthostericsite,GPCRspossessavarietyoftopologicallydistinctallosteric bindingsiteswhereligandscanbind[5].Whenallostericmodulatorsbind,theymodulatetheactivityoforthosteric ligandsbyinducingconformationalchangesinthereceptor.

Orthostericligandsarecompetitiveandthus,theyreplacetheendogenousligandresultinginasinglepharmacological state.Incontrast,bymodulatingtheactivityofanotherligand,allostericligandshavethepotentialforfine-tuninga receptorresponse,maximizingtheefficacyinsometherapeuticcontexts[6,7],and/orminimizingthepotentialside effectsandotherliabilities[6,8].Dependingontheireffect,allostericmodulatorscanbedividedin[6,7,75]:

 Positiveallostericmodulators(PAMs):enhancetheaffinityand/orefficacyoftheendogenousororthostericligand.

 Negativeallostericmodulators(NAMs):decreasetheaffinityand/orefficacyoftheendogenousororthostericligand.

 Ago-PAMs:PAMswithsomeinherentlevelofagonistactivityontheirown.

 Silentallostericmodulators(SAMs):havenoeffectontheaffinityorefficacyoftheendogenousororthostericligand.

Theirpresencemayleadto,forinstance,enhancedthermostabilityofthereceptorandincreasedsignalinglifetime.

Somekeypharmacologicalpropertiesofallostericmodulatorsare:

 Insurmountability:the abilityofallosteric ligandstocause adecrease inthe potencyand/orefficacyofthe endogenousagonist,evenwhentheendogenousligandispresentathighconcentrations.

 Selectivity:generally,allostericbindingsitesshowlessevolutionarypressureleadingtoaless-conservedaminoacid sequenceandthus,higherligandselectivitythantheorthostericbindingsite.Ifanallostericsiteishighlyconserved, selectivitycanbeachievedviaoptimizationofcooperativitywiththeorthostericligandorbytargetingspecificnon- conservedaminoacids.

 Saturabilityorceilingeffect:thelimitofthepharmacologicaleffectproducedbytheallostericligandduetosaturation oftheeffectafterfulloccupancyoftheallostericsite.

 Probe-dependence:boththemagnitudeanddirectionoftheallostericeffectachievedbytheallostericmodulatorare dependentontheorthostericligandusedasa‘probe’.

 Biasedsignaling:theabilityofaligandtopreferentiallystabilizeaconformationthatleadstotheselectiveactivationof asignalingpathway.

Box2.ChemokineReceptors

ChemokinereceptorsrepresentoneofthelargestsubfamilieswithinclassAGprotein-coupledreceptors(GPCRs).So far,23chemokinereceptorshavebeenidentifiedthatcanbeactivatedbymorethan45chemokineligands(IUPHAR/

BPSGuidetoPharmacologyAppendixAⁱ).Chemokinesandchemokinereceptorsaresubdividedinfourdifferent families,accordingtothenumberandarrangementofconservedcysteineresiduesintheN-terminusofthechemokine ligands:C,withonlyoneconservedcysteinepresent;CC,CXCandCX3C,withzero,oneandthreeextraresidues betweentwoconservedcysteineresidues[76].

Bothchemokinesandchemokinereceptorscomprisetheso-calledchemokinesystem,whichplaysanimportantrolein themigrationandpositioningofimmunecellsinhomeostaticorpathologicalconditions[77].Accordingtotheirimmune function,chemokinereceptorscanbeclassifiedashomeostatic,ordualinflammatory/homeostatic[78].Thechemokine systemisacomplex,seeminglyredundantsysteminwhichonechemokineligandisabletoactivatemultiplechemokine receptors,andonechemokinereceptorcanbeactivatedbymultiplechemokineligands.Yet,evidencesuggestsitisa highlyfine-tunedsystemasitistightlyregulatedbyspecificspatialandtemporalcontrolofchemokineexpression [79,80].

Dysregulationofthiscomplexsystemhasbeenimplicatedinavarietyofinflammatoryandimmunediseases,including arthritis,diabetes,inflammatoryboweldiseaseandcancer[81].Threedrugstargetingchemokinereceptorshave alreadygainedmarketapproval:maraviroc,asmall-moleculetargetingCCR5;plerixafor,asmall-moleculetargeting CXCR4;andmogamulizumab,ananti-CCR4antibody[76].

areconservedintermsofpolarity,andresidue6x36(Figure3,centralpanel).Forchemokine receptorsthiskinkallowsligandstointeractwiththebackboneofresiduesK/R^8x49andF^8x50.In CCR2,thespecificconformationofthissubpocketallowstheligandtobindclosertoH8,where thenegativelychargedoxygenoftheligandisalsoabletointeractwiththebackboneofE^8x48.In b2AR,P^8x48forcesS^8x47inwards,allowingittointeractwiththeoxygenoftheamideinthe ligand,whileasecondinteractionisformedbetweenthenitrogenofanotheramideandD^8x49. Noteworthyisposition6x36whichisnotstronglyconserved(59%intermsofhydrophobicity) amongGPCRs.Thisresidueiskeyforligandbindinginbothb2ARandCCR2:inb2AR,T^6x36 forms a hydrogen bond with an amide of 15PA; in CCR2, V^6x36 makes a hydrophobic interactionwiththecyclohexylsubstituentoftheligand.However,differenteffectshavebeen reporteduponmutationofthisresidue.WhilethemutationV^6x36Aabolishedligandbindingin CCR2[22],itincreasedthestabilityofCCR9,facilitatingitscrystallization[10].InCXCR4,a T^6x36Pmutationabolishedsignaling[49],whereasM^6x36Tmadethedeltaopioidreceptora constitutivelyactivemutant(CAM)[50].Finally,intheAdenosineA_2Breceptor(A_2BAR)this residueactsasadeterminantforGproteinselectivity[51],indicatingthatthispositionmightbe crucialfortargetselectivityofintracellularligandsaswell.

RegionFormedbyTM3/6andTM2/ICL1

Thelargestdifferencesareobservedinthisregionofthebindingsite;residuesfoundinTM3 includeR^3x50fromthehighlyconservedDRYmotif,andresidue6x40conservedintermsof

CCR2

Vercirnon

JNJ-27141491 CCR4

Compound 1 GSK2239633 AZD8797 Compound 5-C

CX₃CR1 PAR1

SD-24 SCH 527123

CXCR2 15PA CCR2-RA-[R]

CCR9 β2AR

Figure2. ChemicalStructures of Selected IntracellularSmall-Molecule LigandsforDifferent Class A GPCRs.Upperrowshowsthechemicalstructures ofcocrystallized intracellularligandswith theircorresponding receptor: CCR2-RA-[R] with CCR2, vercirnon with CCR9, and 15PA with b²AR. Vercirnon, SCH 527123 and GSK2239633areexamplesofintracellularligandsthathaveprogressedtoclinicaltrials.

KeyFigure

Overview of Structural Features of the Intracellular Binding Site

(Seefigurelegendonthebottomofthenextpage.)

hydrophobicity(Figure3,lowerpanel).Residue6x37seemstobeimportantforselectivity,as exemplifiedbyT^6x37inCCR9thatallowsthechlorosubstituentofvercirnontogodeeperinto thispocket.Interestingly,mutationofthisresiduehasbeenimplicatedinalteredsignaling[51]

andimprovedstabilityofA_2AARtofacilitatecrystallization[52].PolarresiduesfoundattheTM2/

ICL1interfaceinteractwithboththeCCR9andb2ARligand.Forexample,R^12x49(ICL1)formsa cation-piinteractionwiththeb2ARligandwhileinCCR9itinteractswithbothD^2x40andthenitro groupof the ligand.However, thesepolar residuesdo notinteractwith theCCR2 ligand, indicatingadifferentbindingmode.

StrategiesforIntracellularModulation

Ingeneral,threemainstrategieshavebeenusedtotargettheintracellularsideofGPCRssofar:

smallmolecules,pepducinsandnanobodiesor‘intrabodies’.

SmallMolecules

Smallmolecules currently account for the majority of drugtypes in clinical trials targeting GPCRs [1]. Althoughmost of these smallmolecules are presumedto be orthosteric, the numberofconfirmedallostericmodulatorstargetingGPCRsisincreasinginclinicaltrials[1].In thisregard,severalintracellularsmallmoleculeshavealreadybeenidentifiedforanumberof GPCRs,butfewofthesehaveprogressedtoclinicaltrialsandnonehasmadeittothemarket.

Thelargestnumberofsmall-moleculeintracellularligandsreportedsofartargetchemokine receptors,includingCCR2[9,22],CCR4[19,53,54],CCR9[10],CXCR1andCXCR2[20,21].

These intracellularligands share similar chemical features such as the presence of acidic groupsactingashydrogen-bondacceptorswheninteractingwiththetarget(Figure2).Agood balance of hydrophobic and polar residues make this binding site highly druggable, as described in a previous section [9]. However, intracellular ligands must cross the cellular membrane in order to exert their effect; therefore attention must be paid to the overall physicochemicalproperties of theseintracellular smallmolecules, suchas lipophilicity and molecularweighttoensuregoodpermeability.Mostoftheseintracellularligandshavebeen foundusingatraditionalmedicinal-chemistryapproach.However,inthecaseoftheb2AR,the co-crystallizedcompound15PA(Figure2)wasderivedfromanovelb2ARNAM(compound15) identifiedinascreeningcampaignusingDNA-encodedsmall-moleculelibraries,suggestinga novelapproachtodiscoverintracellularmodulatorsinGPCRs[30].

Oneof the suggestedintracellular ligands,the CCR2 antagonistCCX140-B from Chemo- centryx(structureundisclosed)[55],hasrecentlydemonstratedpositiveresultsinaPhaseII clinical trial in patients with type 2 diabetes and diabetic nephropathy [56]. The CCR9 intracellularantagonist,vercirnon(Figure2) [10],also showedpromisingresultsinPhaseII clinicaltrialsinpatientswithCrohn’s disease[57];however, itdid notdemonstrateclinical efficacyinthelastPhaseIIIstudy[58].IncaseofCCR4,GlaxoSmithKline(GSK)hasidentified more than three different chemical scaffolds for intracellular antagonists –termed ‘site2’ antagonistsbyGSK[54].Yet,onlyoneoftheseligands,GSK2239633(Figure2),progressedto

Figure3.CommonfeaturesinintracellularligandbindingderivedfromthecrystalstructuresofCCR2(PDB5T1A),CCR9 (PDB5LWE)andb²AR(PDB5X7D).Residuesarenumberedusingstructure-basedBallesteros-Weinsteinnumbers[15].

ResidueconservationamongallclassAGPCRsisshowninthefollowingway;residuesthatareoverallconserved (identical)inclassA(>50%)areshownfirst(*);forresiduesthatarenotconservedweshowhowconservedtheyarein termsofpolarity(^)orhydrophobicity(@).Thethreedifferentboxesrepresentthreedifferentsectionsoftheintracellular bindingsites,intheupperpanelallreceptorsaresuperimposedwhileinthelowertwoboxesthereceptorsareshown separately.CCR2iscoloredblue,CCR9iscoloredgreenandb²ARiscoloredorange.

PhaseIclinicaltrials,beforefailingduetolackofefficacy[59].DevelopmentofCXCR1–CXCR2 intracellularligandssuchasSCH527123(Figure2)hasalsoresultedinseveralclinicaltrialsfor thetreatmentofchronicobstructivepulmonarydisease(COPD)andasthma[60].Although noneoftheseligandshasbeenapprovedyet,thisstrategyhasledtoseveralclinicalstudiesthat mightultimatelyleadtoanewmarketedtherapeuticagent.

PepducinsandNanobodies

AnotherstrategyforintracellulartargetingofGPCRsistheuseofpepducins,peptidesderived fromtheICLsofthetargetreceptor,ornanobodies.Astheuseandpharmacologyofseveral pepducins[61,62]andnanobodies[63,64]havebeenrecentlyreviewedelsewhere,wewillonly brieflydiscussthemhere.Thepepducinapproach hasbeenexploredwithseveralGPCRs, includingCXCR1,CXCR2[65],CXCR4[26,66],PAR1[41],andb2AR[67].Althoughinmany casespepducinshavebeenemployedaspharmacologicaltools,severalinvitroandinvivo preclinicalstudiessupportthe roleofpepducinsas therapeuticagents [62].In thecaseof PAR1,arecentclinicaltrialinvolvingpepducinPZ-128demonstratedpositiveresultsinpatients withcoronaryarterydisease[68].Finally,theintracellulardomaincanalsobetargetedwith intracellularnanobodiesor‘intrabodies’,as exemplifiedbyUS28[29],b2AR [16,31,32,69], A_2AAR[33],M2R[34],andmOR[35].Althoughmostoftheseintrabodieshavebeenusedtoaid GPCRcrystallizationandunderstandreceptorfunction,theirtherapeuticpotential hasalso beenhighlighted[70].

AdvantagesandTherapeuticImplicationsofIntracellularLigands

Asaconsequence oftheirability tobindtodistinct sitesonaGPCR,intracellularallosteric modulatorscanhaveuniquepropertiescomparedtocompoundsthattargetthe(orthosteric) bindingsiteofendogenousligands[8].Someofthesekeypropertiesincludethemodulationof affinityand/orefficacyoforthostericligands,improvedselectivity,polypharmacology,or biasedsignaling(Box1,Figure4).

ModulationofAffinityandEfficacyofOrthostericLigands

Inb2AR,twoallostericintrabodies,aNAMandaPAM,wereabletomodulatetheaffinityofthe orthostericagonistisoprenaline bymore than15000-fold, anunexpectedly large dynamic range(Figure4A)[32].AlthoughbothintrabodiesinsertintothepocketwheretheGprotein binds,they modulatethefunctionalstateofthereceptordifferentlybyengaging withother residueswithinthebindingpocket.TheimpactofthePAMandNAMintrabodiesonapanelof orthostericligandsofdifferentefficacieswasalsoshowntobeconsistentwiththepresenceof multiplereceptorstates.Theconceptofmorethantwofunctionalstates–inactiveandactive– mayallow forfiner controlof functionalresponsesthan previouslythought.Inaddition,the demonstrated ability of allosteric ligands to differentially modulate the activity of distinct orthosteric ligands (referred to as probe dependence, Box 1) has important implications regardingtheselectivity ofdrugs forreceptors thatare activated bymultipleligands,asis the case for chemokinereceptors. Intracellular NAMsof CCR2 and CCR9 are thoughtto functionbydirectlyinhibitingtheinteractionwithintracellularsignalingproteins,whileatthe sametimeblockingtheoutwardmotionofTM6andtheupwardmotionofTM3,requiredfor receptoractivation[9,10].BypreventingGproteincouplingandstabilizinganinactivestate, theyalsopresumablyreducetheaffinityoftheendogenousagonists.Inaddition,intracellular NAMsinhibitthe receptorinan insurmountablemanner(Figure4B).Aspreviouslydemon- stratedinCCR2,CCR2-RA-[R]wasabletodecreasethemaximumeffectoftheendogenous chemokineCCL2,evenatthehighestCCL2concentrationtested[23].Anotheradvantageof allostericoverorthostericinhibitorsistheirsaturabilityortheso-called‘ceilingeffect’, which limitsthe allostericactivity to a certainlevel,despite further increments inthe doseofthe

modulator[6–8].Whethercompoundstargetingthissitecanbeappropriatelydesignedwiththe rightlevelofsaturabilitywillbecomeclearwithmoreintracellularcompoundsinclinicalstudies.

SelectivityversusPolypharmacology

Asthisintracellularbindingsiteislikelypresentinmostchemokinereceptors,itmaybeauseful site for simultaneously blocking multiple chemokine receptors in disease contexts where polypharmacologyhasbeendeemeduseful(Figure4C).Thismayholdtrueinmultiplesclerosis orrheumatoidarthritiswheremultiplechemokinereceptorshavebeenfoundtoplayarole[71].

Asallostericmodulators,pepducinsmayproveusefulforpolypharmacologybecausetheyare derivedfromtheintracellularloopsofGPCRs,whichoftendisplayahighdegreeofsequence similarityamongstrelatedreceptors[61].Forexample,thepepducinP4pal-10wasshownto inhibitdiverseGq-coupledreceptorswithoutaffectingb2AR(Gs)orCXCR4(Gi)signaling[72].Its broad spectrum inhibition profile was exploited to investigate the effect of blocking Gq- mediatedsignaling fromanumberofreceptorsforthetreatmentofasthma,whichinvolves Figure4.PotentialAdvantagesofIntracellularAllostericModulators.(A)Intracellularallostericmodulators(small molecules,pepducinsorintrabodies,showninorange)havethepotentialtopositivelyornegativelymodulatetheaffinity and/ortheefficacyoftheendogenousligand(showningreenorred)oranyorthostericligand.Theultimateresponse dependsonthelevelofpositiveornegativecooperativitybetweenthetwoligands.(B)Intracellularligandscandisplay insurmountability,astheycaninhibitthereceptor(showninblue)evenwhenhighconcentrationsofendogenousligandare present.(C)Ahighly-conservedintracellularbindingsiteprovidesthepossibilityofdesigningintracellularligandsthatbind andexerttheireffectinmultiplereceptors(receptorAinblueandreceptorBinpurple).Thesepharmacologicalligands,as opposedtoselectiveligands,mightbe advantageousindiseaseswheremore thanonereceptorisinvolved.(D) Intracellularligandscanalsopromotebiasedsignaling,bypreferentiallymodulatingonesignalingpathwayoveranother uponactivationbytheendogenousligand.Forinstance,theycanstabilizeGproteinsignalingoverb-arrestinsignaling.

Sourceofcellularbiologyillustrations:ServierMedicalArtbyServier,availablefromhttps://smart.servier.com/.