aplaviroc.full

domain swap, as CCL2 and CCL7 activated the chimera withsimilar potencies as CCR2 WT, whereas CCL3 and CCL5 onlyactivated CCR5 WT (Fig. 1, DF). Additional chimeras withsingle exchanged extracellular domains were not activated bythe CCR2-specific chemokines indicating that more than onedomain was needed for proper binding of CCL2 and CCL7(supplemental Fig. 1). In summary, construction of the Trojanhorse CCR5-CCR2(all) successfully led to an active receptorwithmaintainedG protein coupling through the CCR5 scaffoldbut complementary chemokine specificity with high potencyactivation by CCR2 but not CCR5 chemokines.Identification of Small Molecule Agonists for CCR5 That Act

Independently of Extracellular Receptor RegionsBecause ofthe structural similarity and considerable overlap betweenendogenous ligands for CCR1 and CCR5 (both are activated byCCL3 andCCL5), we testedwhether previously identified smallmolecule agonists for CCR1 (17) also activated CCR5. Indeed,we found that the CCR1 agonists built over ametal ion chelatorcomplex also activated CCR5, but not CCR2, with equally highpotencies as observed for CCR1 (EC50 values from 4.5 to 23M;Fig. 2 and Table 1) (17). The interaction of these ligands withCCR5 seemed independent of any extracellular region, as theligands activated CCR5-CCR2(all) with similar or even higherpotencies (EC50 values ranging from 0.86 to 7.3 M) as com-pared with CCR5 WT (Fig. 2C). Similar to CCR1 (17), theiractivity was dependent on complex formation, as neithermetal ions nor chelators alone showed any activity on CCR5(data not shown). Conclusively, although the orthostericchemokine-binding site could be transferred from CCR2onto a CCR5 scaffold (and is thereby linked to the extracel-lular domains of CCR2), the small molecule activity wasretained within the transmembrane regions of CCR5 andwas unaffected by the exchange of the extracellular regionswith those of CCR2.Positive Cooperative Effects of Small Molecule Agonists in

CCR5 Could Not Be Transferred to CCR5-CCR2(all)In addi-tion to acting as agonists, the metal ion chelators were able toenhance the binding of 125I-CCL3 to CCR5 up to 7-fold above

maximum specific binding in the absence of unlabeled ligand,thus acting allosteric in relation to CCL3 similar to the previ-ously described action in CCR1 (17). The binding affinities ofmetal ion chelators as measured against 125I-CCL3 were in thesame rangewith their agonistic potencies (Ki from 7.3 to 93M,Fig. 3A), again as observed in CCR1 (17). However, comparedwith the high affinity displacement of CCL5 from CCR1 (17), a1030-fold lower affinity was observed for metal ion chelatorsin displacing 125I-CCL5 fromCCR5 (Ki from197 to 620M, Fig.3B). This indicates an allosteric binding mode in CCR5 relatedto CCL5, in contrast to a competition situation in CCR1 withoverlapping binding sites between metal ion chelators andCCL5. As only CCR2-specific chemokines bound to CCR5-CCR2(all), we tested whether the allosteric enhancing effect on125I-CCL3 binding to CCR5 could be transferred from theCCR5 transmembrane unit (binding the allosteric compounds)to the CCR2 extracellular interface. Despite the maintainedagonismof the smallmolecules in the chimeric CCR5-CCR2(all)(Fig. 2C and Table 1), no allosteric enhancement of 125I-CCL2or 125I-CCL7 binding was observed. In contrast, a low affinitydisplacement with Ki values from 162 to 528 M for 125I-CCL2and from 90 to 267 M for 125I-CCL7 was observed (Fig. 3, CandD), i.e.withKi values lying in the same range as observed for125I-CCL5 binding to CCR5 (Fig. 3B).Action of Small Molecule Agonists Depends upon Acidic Res-

idues in the Top of TM-VIITo investigate the extension andpossible overlap of the binding sites for chemokines and smallmolecules in CCR2, CCR5, and CCR5-CCR2(all), we initiallyfocused on two acidic residues in TM-VII, Asp in positionVII:-02 and Glu in position VII:06. Besides being anchor formany small molecule agonists and antagonists, the chemokinereceptor-conservedGluVII:06 is also the anchor point formanychemokines (7, 17). AspVII:-02 is also found predominantlyamong chemokine receptors (55% among these) as comparedwith 6% among nonchemokine class A 7TM receptors and

FIGURE 2. Activation of WT and chimeric receptors by small molecule ago-nists. CCR5 WT (A), CCR2 WT (B), and the chimeric CCR5-CCR2(all) (C) wereactivated by allosteric small molecule metal ion chelators ZnBip (), ZnPhe(F), CuBip (), and CuPhe (E). The activity was measured as IP turnover inCOS-7 cells as described in the legend to Fig. 1. The curves are normalized tothe maximal activation of the respective receptors by CCL5 (for WT CCR5) orCCL2 (for WT CCR2) and CCL7 (for CCR5-CCR2(all)). The structures of metal ionchelators ZnBip, ZnPhe, CuBip, and CuPhe (from left to right) are shown belowthe graphs (n 6 15).

FIGURE 3. Competition binding between chemokines and small moleculeagonists in WT and chimeric receptors. The binding of 125I-CCL3 to WTCCR5 (A), 125I-CCL5 to WT CCR5 (B), 125I-CCL2 to CCR5-CCR2(all) (C), and

125I-CCL7 to CCR5-CCR2(all) (D) was tested in a heterologous binding experimentagainst the metal ion chelators ZnBip (), ZnPhe (F), CuBip (), and CuPhe(E).(n 3 6).

Interchange in 7TM Receptors, Implications for Future Drugs

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has been suggested as an interaction partner for theN terminusof CCL2 with CCR2 (12). Importantly, the recently publishedcrystal structure of CXCR4 uncovered that TM-VII is extendedon the extracellular side (36) and therefore includes residuesfrom the presumed ECL-3. A computational model of CCR5built over the crystal structure of CXCR4 (Fig. 4, C and F) illus-trated that AspVII:-02 (Asp276) in fact is part of TM-VII. Asseen in Fig. 4, A and B, Ala substitution of AspVII:-02 (D276A)impaired the activity of small molecule agonists as comparedwith CCR5 WT (2.713-fold decreased potencies), whereas asimilar substitution of GluVII:06 (E283A) completely abolishedthe activity (Fig. 4, A and B, and Table 2). In CCR5-CCR2(all), asimilar dependencewas observed for smallmolecule agonists ofthese acidic residues (Fig. 4,D and E). Thus, the action of smallmolecule agonists completely depends on GluVII:06, whereasAspVII:-02, located two helical turns above, contributes to alesser degree. Importantly, no difference was observed betweenCCR5 and CCR5-CCR2(all).

Different Interaction of CCL2 and CCL7 with CCR2 WT asCompared with Chimeric ReceptorContrary to the similardependence of the smallmolecule agonists on acidic residues inCCR5 WT and in CCR5-CCR2(all), the pattern differed for theCCR2 chemokine agonists (CCL2 and CCL7). Thus, CCL2 washighly dependent onGluVII:06 andAspVII:-02 in CCR2WT as13-fold decreased potency was observed for Ala substitutionof either of these residues (Fig. 5A and Table 2), as reported inearlier studies (12). For CCL7, however, GluVII:06 seemedmore important (63-fold decreased potency upon Ala substitu-tion) than AspVII:-02 (5.6-fold decreased potency) (Fig. 5B). InCCR5-CCR2(all), a much lower dependence was observed forboth acidic residues, as Ala substitution of GluVII:06 onlyresulted in 3.6-fold decreased CCL7 potency and 4.4-folddecreased CCL2 potency, whereas the impact of AspVII:-02was even smaller (1.13.2-fold decrease) (Fig. 5, C and D).Changes in receptor surface expression could not explain thechanges in potencies, as all Ala substitutions were found to be

FIGURE 4. Effect on small molecule agonists of the Ala substitutions of AspVII:-02 and GluVII:06 in CCR5 WT and in chimeric receptors. The activationof WT receptors and chimera (stippled) and the Ala substitutions of AspVII:-02 () and GluVII:06 (F) in WT CCR5 (A and B) and CCR5-CCR2(all) (D and E) was testedin an IP turnover assay in COS-7 cells as described in legend to Fig. 1. The activation curves are shown for ZnBip (left panel) and ZnPhe (middle panel) (n 315).In the right panel, a molecular model of CCR5 based on the crystal structure of CXCR4 is shown in view from the extracellular side (C) and from the side (F) withresidues AspVII:-02 and GluVII:06 highlighted as sticks.

TABLE 2Surface expression of mutations AspVII:-02Ala and GluVII:06Ala in CCR5, CCR2, and CCR5-CCR2(all) and their effect on chemokine and metal ionchelator-mediated receptor activationThe surface expression was determined by ELISA in COS-7 cells transiently transfected with FLAG-tagged receptors and is given as percent of the respective wild typereceptor. IP turnover was measured in COS-7 cells co-transfected with receptor and the promiscuous G protein Gqi4myr. EC50 values are given in log and nM/M. Fmutindicates the fold-decrease for the ligand potencies at the mutants as compared with the respectiveWT receptor. The number of experiments is shown in parentheses; NDmeans not determined.

Fmut (n) Fmut (n) Fmut (n) Fmut (n) Fmut (n) Fmut (n) Fmut (n) Fmut (n)Mean SEM (n) EC50 SEM EC50 EC50 SEM EC50 EC50 SEM EC50 EC50 SEM EC50 EC50 SEM EC50 EC50 SEM EC50 EC50 SEM EC50 EC50 SEM EC50

)Mn()dlof()Mn()dlof()Mn()%( (fold) (nM) (fold) (M) (fold) (M) (fold) (M) (fold) (M) (fold)

)5()62(0,19,150,07,8-)42(0,17160,08,7-)3(91001tw (4) -4,6 0,05 23 1,0 (15) -5,3 0,09 4,5 1,0 (14) -4,7 0,06 20 1,0 (10) -5,3 0,08 4,9 1,0 (10)D276A VII:-02 109 19 (3) -7,7 0,14 20 1,1 (3) -7,3 0,70 55 29 (3) -4,1 0,04 82 3,6 (3) -4,4 0,34 39 8,8 (3) -4,3 0,08 55 2,7 (3) -4,2 0,09 62 13 (3)E283A VII:06 109 23 (3) -8,0 0,03 10 0,6 (3) -8,0 0,08 10 5,3 (3) >1000 >44 (3) >1000 >222 (3) >1000 >50 (3) >1000 >206 (3)

1(0,16,180,08,8-)51(19,761,01,8-)5()6()3(12001tw )7()7()6()7()3D284A VII:-02 103 18 (3) )3()3()3()3()3(6,59,871,01,8-)3(31>001E291A VII:06 98 24 (3) )3()3()3()3()3(36>001>)3(31>001

,153,090,05,9-)51(0,19,470,03,8-)7()7()3(61001tw 0 (14) -5,1 0,05 7,3 1,0 (14) -6,1 0,06 0,86 1,0 (15) -5,2 0,06 6,5 1,0 (13) -5,9 0,08 1,4 1,0 (13)D284A VII:-02 144 24 (3) -7,8 0,17 15 3,2 (3) -9,4 0,01 0,37 1,1 (2) -4,4 0,05 38 5,3 (3) -5,4 0,22 4,4 5,1 (3) -4,5 0,03 33 5,1 (3) -4,5 0,36 29 21 (3)E291A VII:06 149 14 (3) -7,7 0,29 21 4,4 (4) -8,9 0,18 1,2 3,6 (3) >1000 >138 (3) >1000 >1161 (3) >1000 >153 (5) >1000 >716 (3)

(log))gol()gol()gol()gol()gol(

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PotencyCuPheCuBipZnPheZnBipCCL7CCL2CCL5CCL3Surface Expression

no activationno activationno activation

> -3,0

> -3,0

0,3- >0,3- >

0,3- >0,3- >



n.d.n.d.

> -7,0

> -3,0

> -3,0


> -7,0> -7,0

no activationn.d.n.d.

no activation

no activation

n.d.n.d.

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PotencyPotency Potency


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equal to or even higher expressed than the respective WTreceptors as determined by ELISA (Table 2). Furthermore,homologous competition binding experiments uncoveredunchanged CCR2 WT-like affinities for CCL2 and CCL7 onboth Ala substitutions in CCR2 (Table 3), despite the decreasesin potencies (Table 2). In contrast, the two Ala substitutions inthe CCR5-CCR2(all) background resulted in minor decreases inaffinities (1.94.0-fold, see Table 3), which for both ligandswere similar to the observed decreases in potencies (1.14.4-fold, see Table 2). Thus, it is concluded that the two acidicresidues at the top of TMVII (Asp284 and Glu291) are of hugeimportance for the activation but not the binding of CCL2 andCCL7 in CCR2WT, although this dependence for chemokine-mediated receptor activation is weakened in the CCR5-CCR2(all) chimera.Allosteric Mechanisms for the Small Molecule Agonists in

CCR5 WT and CCR5-CCR2(all)To further investigate theaction of the small molecule agonists, their effects on chemo-kine potency and affinity were assessed. Although only smalldecreases in potencies were observed for CCL3 and CCL5 acti-vating CCR5 WT in the presence of small molecule agonists,the potencies of CCL2 and CCL7 at CCR5-CCR2(all) were up to100-fold reduced (Fig. 6). On the contrary, the affinities ofchemokine binding to CCR5 and CCR5-CCR2(all) wereunchanged in the presence of small molecule agonists, asobserved in competition binding experiments (Table 4).Conclusively, as ligands with overlapping binding sites (i.e.

competitive ligands) are expected to primarily affect the affinityof an orthosteric ligand, these ligand co-administration exper-iments clearly suggest an allosteric binding mode of the small

molecule agonists and thereby confirm the binding and func-tional data in Figs. 15.Retained Activity of CCR5 Nonpeptide Antagonists in the

CCR5-CCR2(all) ChimeraAs a third pharmacological aspect,we investigated the effect of the domain swapping in the CCR5-CCR2(all) chimera on the CCR5 antagonists SCH-C and aplavi-roc and the dual CCR5/CCR2 antagonist TAK-779. Asexpected from the dual nature of TAK-779, the antagonisticproperty was retained in the chimera. Interestingly, the activityof SCH-C was also retained and thereby independent of theextracellular CCR2 domains. This was not observed for aplavi-roc, which completely lost its antagonistic functionality inCCR5-CCR2(all) (Fig. 7 and Table 5). However, as the CCR5-binding site of aplaviroc has been suggested to include residuesSer180 and Lys191 in ECL-2b (14), the lack of aplaviroc activity inthe CCR5-CCR2(all) chimera could be due to the absence ofthese particular residues in ECL-2b of CCR2 and consequentlyin CCR5-CCR2(all).Rescue of Aplaviroc Activity by Reintroducing ECL-2b from

CCR5 into CCR5-CCR2(all)Reintroduction of ECL-2b fromCCR5 into CCR5-CCR2(all), creating the new chimera CCR5-CCR2(all2b), indeed recovered the activity of aplaviroc. Thus,in this new chimera, aplaviroc antagonized CCL2 and CCL7activity with an IC50 of 10 and 29 nM, respectively, i.e. with

FIGURE 5. Effect on chemokine ligands of the Ala substitutions ofAspVII:-02 and GluVII:06 in CCR2 WT and in the chimeric receptor. Theactivation of WT receptors and chimera (stippled), and the Ala substitutionsof AspVII:-02 () and GluVII:06 (F) in WT CCR2 (A and B) and CCR5-CCR2(all) (Dand E) was tested in an IP turnover assay in COS-7 cells as described in legendto Fig. 1. The activation curves are shown for CCL2 (left panel) and CCL7 (rightpanel). (n 215).

TABLE 3Homologous chemokine binding to CCR2, CCR5, and CCR5-CCR2(all)with or without Ala substitutions of acidic residues in TMVIICompetition binding experimentswere performed in transiently transfectedCOS-7cells using 125I-CCL3, 125I-CCL2, and 125I-CCL7 as radioligands. The IC50 values aregiven as log and in nM, and the impact of the mutants is given as fold-decreasecompared with the affinities in background receptor (WT or CCR5-CCR2(all)). Thenumber of experiments (n) is given.

IC50 SEM IC50(nM) (fold) (n)

wt -8,4 0,05 4,1 1,0 (3)D276A VII:-02 -7,8 0,17 14 3,5 (3)E283A VII:06 -8,5 0,16 3,1 0,8 (3)

wt -8,2 0,14 7 1,0 (5)D284A VII:-02 -7,8 0,17 17 2,4 (4)E291A VII:06 -7,9 0,39 12,3 1,8 (4)

wt -9,1 0,10 0,89 1,0 (5)D284A VII:-02 -8,8 0,16 1,7 1,9 (5)E291A VII:06 -8,5 0,18 3,5 4,0 (5)

wt -8,0 0,16 10 1,0 (7)D284A VII:-02 -7,9 0,25 13 1,2 (5)E291A VII:06 -8,0 0,19 9,9 1,0 (6)

wt -8,8 0,21 1,6 1,0 (5)D284A VII:-02 -9,2 0,17 0,59 0,38 (5)E291A VII:06 -8,3 0,33 4,8 3,1 (4)

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potencies in the same range as inCCR5WT (Fig. 8 andTable 5).As in the CCR5-CCR2(all) chimera, the activity of the otherantagonists was retained in the CCR5-CCR2(all2b) construct(Table 5). In addition, the potencies of the various chemokine(and small molecule) agonists were unaltered in CCR5-CCR2(all2b) as compared with CCR5-CCR2(all). Thus, CCL2and CCL7 could activate CCR5-CCR2(all2b) with potencies of4.4 and 1.0 nM, respectively. Also the affinities of CCL2 andCCL7 toCCR5-CCR2(all2b) were determined to be in nanomo-lar range (logKd(CCL2) 9.7 0.43 (n 2); logKd(CCL7) 9.0 0.16 (n 3)). The potencies of the metal ion chelatorswere ranging from1.3M forZnPhe, over 3.1M forCuPhe and11 M for ZnBip to 29 M for CuBip. This indicates that the

absence of ECL-2b from CCR2 makes no difference for properchemokine or small molecule-based activation.

DISCUSSION

Construction of Chimeric Receptors, Trojan Horse or CentaurPrincipleA chimeric receptor was constructed to investigatethe behavior of allosteric and orthosteric sites in CCR5 andCCR2. Importantly, the chimeric setup in CCR5-CCR2(all) isdomain-related (Trojan horse), rather than sequence-related(centaur), meaning that the sites of interchange between thetwo receptors were not placed randomly and consecutivelybetween the N and C terminus but precisely where the TMhelices turn to extracellular loops. In the case of CCR5 andCCR2, which originate from one precursor by gene duplicationand therefore are highly homologous (20), similar sequenceswere found in these areas. This allowed us to match the exactposition betweenhelix and loopwhen constructing the chimera(Fig. 1). Thus, although CCR5-CCR2(all) looks like CCR2 fromthe outside (confirmed by the high affinity binding of CCR2chemokines CCL2 and CCL7), the chimera resembles CCR5 inits core and transduces signals in aCCR5-specificmanner (con-firmed by the binding and action of CCR5-specific small mole-cules). So far, the majority of chemokine receptor chimerashave been constructed in a centaur way (2730). However, theTrojan horse chimeras have also beenmade previously to studyCCR1 and CCR6 ligands (chimeras with CCR3 and CCR5,respectively) (25, 37). Other 7TMchimeras have focused on theintracellular domains with a local or global domain exchange,designed to characterize and eventually shift theG protein cou-pling and signaling pathways (3840) and to be used in thedeorphanization of receptors with unknown coupling specific-ity (41).Chemokine Binding Depends Solely on the Extracellular

Receptor RegionsIt is noticeable that the construction of ourchimera actually leads to a functional receptor withmaintainedligand binding (Tables 3 and 4) and action (Fig. 1). PreviousTrojan horse chimeras, for instance between CCR6 and CCR5

FIGURE 6. Effect of small molecule agonists on the potency of chemokinesat CCR5 and CCR5-CCR2(all). The activation of CCR5 by CCL3 and CCL5 (A)and CCR5-CCR2(all) by CCL2 and CCL7 (B) in the absence and presence ofZnBip, ZnPhe, CuBip, and CuPhe was determined in an IP turnover assay inCOS-7 cells. The concentrations of the small molecule agonists were chosento reach 25 and 65% of maximal chemokine activation and were as follows:CCR5, ZnBip/CuBip 10 and 32 M and ZnPhe/CuPhe 3.2 and 10 M; CCR5-CCR2(all), ZnBip/CuBip 3.2 and 10 M and ZnPhe/CuPhe 1.0 and 3.2 M. Thefold decrease of potency as compared with the absence of small moleculeagonist is shown. w/o indicates without small molecule (n 35).

TABLE 4Chemokine binding to CCR5, CCR5-CCR2(all), and CCR2 in the absence and presence of small molecule agonistsThe competition binding experimentswere performed in transiently transfectedCOS-7 cells using 125I-CCL3, 125I-CCL2, and 125I-CCL7 as radioligands. The log IC50 valuesfor the homologous binding curves in the absence and presence of the smallmolecule agonists are shown togetherwith the average fold increase inBmax calculated fromeachassay. The number of experiments (n) is given. MC stands for metal ion chelator.

IC50 SEM BMAX SEM (n) IC50 SEM BMAX SEM (n) IC50 SEM BMAX SEM (n) IC50 SEM BMAX SEM (n) IC50 SEM BMAX SEM (n)

MC -8,1 0,10 1,0 0,0 (8) -8,9 0,17 1,0 0,0 (6) -9,0 0,17 1,0 0,0 (6) -8,2 0,15 1,0 0,0 (6) -8,0 0,05 1,0 0,0 (4)

+ 3,2 M ZnBip -9,0 0,18 0,93 0,10 (6) -9,1 0,28 0,56 0,28 (2) -8,3 0,20 1,5 0,08 (5) -8,3 0,18 0,55 0,28 (3) + 10 M ZnBip -8,3 0,15 0,72 0,10 (4) -9,0 0,19 0,91 0,15 (6) -8,9 0,28 1,6 0,41 (3) -8,4 0,20 1,3 0,27 (6) -8,5 0,24 0,31 0,17 (3) + 32 M ZnBip -8,3 0,13 1,0 0,21 (4) -8,8 0,21 1,3 0,18 (4) -8,9 0,32 1,5 0,45 (3) -8,5 0,26 1,1 0,24 (5) -7,9 0,09 1,2 0,33 (3) + 100 M ZnBip -8,2 0,11 8,2 2,0 (6)

+ 3,2 M ZnPhe -8,5 0,13 0,44 0,13 (4) -9,0 0,24 1,0 0,18 (6) -8,8 0,05 1,0 0,55 (2) -8,3 0,19 1,4 0,18 (5) -8,7 0,09 0,13 0,03 (2) + 10 M ZnPhe -8,3 0,15 0,93 0,15 (4) -8,9 0,20 1,2 0,19 (6) -8,9 0,34 1,5 0,34 (3) -8,4 0,21 1,3 0,30 (6) -8,1 0,13 0,92 0,27 (3) + 32 M ZnPhe -8,4 0,13 1,8 0,17 (4) -8,7 0,21 1,5 0,29 (4) -9,4 0,22 0,33 0,02 (3) -8,3 0,20 1,7 0,23 (5) -8,1 0,11 0,93 0,22 (3)

10,072,010,03,9-)6(52,02,112,09,8-piBuCM2,3 + (2) -8,3 0,23 1,5 0,29 (5) -7,9 0,07 1,3 0,39 (3) + 10 M CuBip -8,2 0,15 1,0 0,08 (4) -8,9 0,21 1,4 0,29 (5) -9,0 0,43 1,5 0,58 (3) -8,4 0,16 1,1 0,15 (6) -8,1 0,12 0,66 0,09 (3) + 32 M CuBip -8,2 0,08 2,7 0,56 (4) -8,8 0,25 1,2 0,08 (4) -8,9 0,22 1,2 0,43 (3) -8,3 0,20 1,3 0,21 (5) -8,1 0,12 1,0 0,12 (3) + 100 M CuBip -8,0 0,08 12 3,1 (5)

+ 3,2 M CuPhe -8,3 0,14 0,80 0,13 (4) -9,0 0,26 1,2 0,27 (6) -8,8 0,01 0,98 0,43 (2) -8,3 0,20 1,2 0,10 (5) -8,2 0,26 1,0 0,61 (3) + 10 M CuPhe -8,1 0,12 3,1 0,80 (4) -8,8 0,19 1,3 0,25 (6) -9,1 0,22 0,87 0,36 (3) -8,1 0,12 1,6 0,47 (6) -8,0 0,09 1,3 0,29 (3) + 32 M CuPhe -8,2 0,09 5,3 0,48 (5) -8,7 0,19 1,3 0,36 (4) -9,4 0,20 0,41 0,18 (3) -8,2 0,14 1,2 0,15 (5) -7,8 0,28 1,9 0,71 (3)

Absence of

125I-CCL7/CCL7CCR5-CCR2(all)

Chemokine ligand 125I-CCL3/CCL3 125I-CCL2/CCL2125I-CCL2/CCL2

fold increase

125I-CCL7/CCL7

(log) (log)(log) (log)(log)fold increase fold increase fold increase fold increase

CCR5 wt CCR2 wt

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(CCR6-CCR5(all)), did not succeed in maintaining CCR5chemokine binding (37). This could be due to the low identitybetween these two receptors (33%) as compared with 74%between CCR5 and CCR2 and thereby a larger degree of struc-tural changes applied to the CCR6-CCR5(all) chimera. Impor-tantly, our data confirm the current understanding of chemo-kine binding to the extracellular receptor regions. A two-statemodel has been proposed for the action of CCL2 with CCR2,stating that an initial high affinity interaction of the chemokinecore with the extracellular domains of the receptor (especiallytheN terminus) is followed by a receptor-activating interactionof the chemokine N terminus with residues in the transmem-brane helices. Thus, CCL2 was shown to interact with a DYDYmotif in the CCR2 N terminus (42, 43), followed by interactionof the chemokine N terminus with AspVII:-02 (Asp284) andGluVII:06 (Glu291) in TM-VII (12). These data are indeed con-firmed here by the unchanged high affinity but very low poten-cies of CCL2 and CCL7 in the Ala substitutions of these twoacidic residues in CCR2 (Fig. 5 and Table 3). In addition, ECL-2has earlier been shown to be important for CCL2 activity (29),again in agreement with our finding that transfer of a singleregion from CCR2 to CCR5 (N terminus or any of the ECLs) isinsufficient for properCCL2 orCCL7 action (supplemental Fig.1). In CCR5, we confirm earlier studies showing that especiallyECL-2 (29) and, in the case of CCL3, also ECL-3 are highlyimportant, as we find that their replacement with CCR2 coun-terparts abolished CCL3 and CCL5 activity (supplemental Fig.1). Altogether, our chimera confirms that chemokines interactwith and recognize their receptors via the extracellular domains(Fig. 9).Identification of a Novel Class of Small Molecule Agonists for

CCR5Metal ion chelators (Zn2 or Cu2 ions in complexwith bipyridine or phenanthroline, Fig. 2) have earlier beenshown to be complex ago-allosteric modulators of CCR1 byacting as agonists with micromolar potencies and concomitantallosterically enhancing the binding of CCL3, although theydisplaced CCL5 with micromolar affinities indicating partly

overlapping binding sites in the area around GluVII:06, as Alasubstitution in this position resulted in huge and similardecreases in potencies for CCL5 and metal ion chelators (butnot CCL3) (17). In this study, we find that also CCR5 is acti-vated by thesemolecules that, as inCCR1, depend onGluVII:06(Table 2). Like in CCR1, the small molecule agonists enhancethe binding of CCL3 (act allosterically) but do not displaceCCL5 with the same high affinity as observed in CCR1. Thiscould be due to a lesser degree of overlap between smallmolecule agonists and CCL5-binding sites in CCR5 comparedwith CCR1, as clearly indicated by the lower impact of Ala sub-stitution ofGluVII:06 inCCR5 (5.1-fold decrease in potency forCCL5, Table 2) as compared with 26-fold decrease in CCL5potency in CCR1 (17). Our test of the impact of small moleculeagonists on the potency (Fig. 6) and affinity (Table 4) of thechemokine ligands confirms the allosteric mode of action as nochanges in the chemokine affinities were observed in the pres-ence of small molecule agonists, although their potencies wereslightly reduced at CCR5. In conclusion, CCL5 and the metalion chelator-based agonists do not overlap in CCR5, which inturn also confirms that CCL5 interacts differently with CCR5compared with CCR1, as suggested previously (27, 44).Separation of Small Molecule and Chemokine-binding Sites

Interestingly, the activity of small molecule agonists wasretained in CCR5-CCR2(all) and could therefore be assigned tothe transmembrane region of CCR5. However, as the loops ofCCR5 and CCR2 are similar to a certain extent, it cannot beexcluded that also residues in the extracellular domains con-tribute to the small molecule agonist site. Thus, in both recep-tors, ECL-2, which is linked to the top of TM-III via a conserveddisulfide bridge to CysIII:01, harbors a Glu in position Cys6 (6residues prior to the conserved Cys in ECL-2) and an aromaticresidue in position Cys4. Furthermore, ECL-2 varies greatly inlength and sequence throughout class A 7TM receptors, andwith its position on top of the major binding pocket, with a lidfunction in some receptors, ECL-2 contributes to ligand speci-ficity in some class A 7TM receptors (45). Indeed, we find that

FIGURE 7. Activity of small molecule antagonists on WT CCR5, WT CCR2, and CCR5-CCR2(all). The antagonistic properties of the antagonists TAK-779 (E),SCH-C (), and aplaviroc () on chemokine-activated WT CCR5 (A), WT CCR2 (B), and CCR5-CCR2(all) (C) were tested in an IP turnover assay in COS-7 cells. Thecurves are normalized to the maximum activation of CCL3 (for WT CCR5) and CCL7 (for WT CCR2 and chimeric CCR5-CCR2(all)) (n 3 6). Structures of TAK-779,SCH-C, and aplaviroc are shown below the graphs.




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aplaviroc loses its antagonistic property in CCR5-CCR2(all) butcould be rescued by reintroducing ECL-2b, which is in goodagreement with earlier studies (14). Interestingly, the presentlyavailable crystal structures of 7TM receptors have uncoveredseveral different conformations of ECL-2 ranging from-sheets with the receptor N terminus (rhodopsin) or ECL-1(adenosine 2A receptor) to small -helices (1- and 2-adreno-ceptors) (4654). In CXCR4, an anti-parallel-sheet is formed,which in one case is extended with another -strand from theco-crystallized peptide antagonist (36). In addition to the smallmolecule agonists, the activity of the antagonist SCH-C wasalso retained in the chimera. Thus, it can be concluded that wesuccessfully generated a receptor with two distinct sites thatbind ligands independently of each other (Fig. 9).As expected from studies in CCR1 (17), the small molecule

agonists anchored to GluVII:06 in CCR5-CCR2(all) and in WTCCR5 (Table 2). CCL2 and CCL7, however, have a reduced (oreven lost) dependence of both GluVII:06 and AspVII:-02 in thechimera as compared with CCR2. Thus, even though bothchemokines bind to the extracellular CCR2 domains in the chi-

mera, the interactionwithGluVII:06 andAspVII:-02wasweak-ened and was not sufficient to compete for the small moleculeagonists, presumably because of a slightly altered (although stillhigh affine) binding of the chemokines in the extracellulardomains (Fig. 9). Consequently, CCL2 and CCL7 are only dis-placed by the small molecule agonists from CCR5-CCR2(all)with rather low affinity, but neither is their binding enhanced,indicating that the positive allosteric cross-talk from the CCR5transmembrane region (binding the ago-allosteric molecules)with the CCR5 extracellular domains (binding CCL3), couldnot be established in CCR5-CCR2(all). Thus, positive allostericmodulation not only requires that similar active conformationsare stabilized but also maintained conformational interchangeand communication between domains, properties that couldnot be established in the chimera.Ago-allosteric Ligands in CCR5 and CCR5-CCR2(all)Allos-

teric modulators can be neutral, i.e. do not signal on their own,or they can be agonists or antagonists for a given receptor.However, independent of the efficacy, the binding of an allos-teric modulator leads to changes in the balance between differ-

TABLE 5Antagonistic activities of TAK-779, SCH-C, and aplaviroc in WT and chimeric receptorsIP turnover was measured in COS-7 cells co-transfected with receptor and the promiscuous G protein Gqi4myr. The receptors CCR5WT, CCR2WT, CCR5-CCR2(all), andCCR5-CCR2(all2b) were activated with chemokines or metal ion chelators ZnBip and ZnPhe. EC50 values are given in log and nM. The number of experiments is shown inparentheses; ND means not determined.

PotencyAgonist EC50 SEM (n) EC50 SEM (n) EC50 SEM (n) EC50 SEM (n)

(nM) (nM) (nM) (nM)

CCL3 -7,5 0,11 35 (6)CCL5 -7,5 0,08 32 (5)

)4(7190,08,7-)5(7231,06,7-)4(7,990,00,8-2LCC)4(1240,07,7-)5(7301,04,7-)4(4,961,00,8-7LCC

)3(7160,08,7-)3(9121,07,7-piBnZ)3(1170,09,7-)3(2141,09,7-ehPnZ

CCL3 -7,2 0,31 58 (6)CCL5 -7,6 0,20 27 (5)CCL2 (3) -6,6 0,22 275 (4) -7,9 0,64 12 (2)CCL7 (4) -6,4 0,27 356 (4) -8,4 0,02 3,7 (2)

)3(7280,06,7-)3(8141,07,7-piBnZ)3(1190,00,8-)3(4162,09,7-ehPnZ

CCL3 -7,3 0,32 51 (6)CCL5 -7,5 0,13 31 (6)CCL2 (4) (3) -8,0 0,18 10 (5)CCL7 (4) (3) -7,5 0,15 29 (5)ZnBip -7,6 0,11 24 (3) (3)ZnPhe -7,7 0,09 22 (3) (3)

CCR5 wt CCR2 wt CCR5-CCR2(all) CCR5-CCR2(all2b)Potency Potency Potency

(log) (log) (log) (log)

TAK

-779

n.d. n.d. n.d.n.d. n.d.

n.d. no antagonismn.d.

n.d.n.d.n.d.

n.d. n.d.n.d. n.d.

n.d. n.d.n.d. n.d.

Apl

aviro

c

n.d. n.d. n.d.n.d.

SC

H-C

n.d. n.d. n.d.n.d. n.d. n.d.

n.d. no antagonism n.d.n.d. no antagonism n.d.

n.d. n.d.n.d. no antagonism no antagonismn.d. no antagonism no antagonism

no antagonism




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ent receptor conformations, which in turn are likely to changethe affinity and/or function of the orthosteric ligands. In CCR5,we identified the metal ion chelator complexes to be agonistsand positive allosteric modulators toward CCL3 (ago-allostericligands). The allosteric nature is furthermore underlined by thefact that these compounds competed against 125I-CCL5 bind-ing to CCR5 and 125I-CCL2 and 125I-CCL7 binding to CCR5-

CCR2(all) with very low affinities (Fig. 3, BD), which impor-tantly were much lower compared with the affinities for theenhancement of 125I-CCL3 binding (Fig. 3A) and to the poten-cies as agonists (Fig. 2, A and C). These low affinity displace-ments and the decreased potencies of CCL2 and CCL7 atCCR5-CCR2(all) in the presence of smallmolecule agonists (Fig.6B) correlate well with the different dependence of chemokinesand small molecule ligands on Asp284 and Glu291 in CCR5-CCR2(all). Thus, because CCL2 and CCL7 do not compete withsmall molecule agonists for Asp284 and Glu291, higher concen-trations are needed to overcome the small molecule agonist-bound state to further activate the receptor, resulting in theobserved decrease in potencies in the presence of small mole-cule agonists. Different and equally complex kinds of allostericbehaviors have earlier been observed in e.g. the action ofL-692,429 at the ghrelin receptor (55) or the actions of Org-27569, Org-27759, and Org-29647 at the CB1 receptor (56).

We conclude that Trojan horse-based chimeric receptorsconstitute a powerful pharmacological tool to study allostericphenomena in 7TM receptors. The construction and combina-tion of receptors in the chimera can be chosen so that not onlya separation of the orthosteric and allosteric binding sites isachieved but also a separation between the ligand-binding andreceptor-activating domains. This also emphasizes themodule-like construction of 7TM receptors and confirms the under-standing that agonists may activate certain (or different) recep-tors in different ways (2). Furthermore, because of theiradvantages such as the higher specificity and dependence onthe orthosteric ligand, allosteric modulators and the under-standing of their action will become increasingly important indrug development.

AcknowledgmentsWe thank Thue W. Schwartz for fruitful discus-sions. We also thank Randi Thgersen, Inger Smith Simonson, andKristoffer Lihme Egerod for excellent technical assistance.

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FIGURE 8. Rescued aplaviroc activity in CCR5-CCR2(all2b). Schematicdrawings of CCR5 in black (left), CCR5-CCR2(all) (middle), and CCR5-CCR2(all2b)(right) with CCR2-derived parts shown in gray. The activity of aplaviroc on WTCCR5 (stippled), CCR5-CCR2(all) (), and CCR5-CCR2(all2b) (f) was tested in anIP turnover assay in COS-7 cells. A, antagonism against CCL2-activated chime-ras and CCL3-activated CCR5 (dashed line). B, antagonism against CCL7-acti-vated chimeras and CCL5-activated CCR5 (dashed line). All curves are normal-ized to the maximal chemokine activation of the respective curve (n 2 6).

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