Modulation in selectivity and allosteric properties of small-molecule ligands for CC-chemokine receptors

Article abstract of DOI:10.1021/jm301121j

Among 18 human chemokine receptors, CCR1, CCR4, CCR5, and CCR8 were activated by metal ion Zn(II) or Cu(II) in complex with 2,2′-bipyridine or 1,10-phenanthroline with similar potencies (EC₅₀ from 3.9 to 172 μM). Besides being agonists, they acted as selective allosteric enhancers of CCL3. These actions were dependent on a conserved glutamic acid at TM-7 (VII:06/7.39). A screening of 20 chelator analogues in complex with Zn(II) identified compounds with increased potencies, with 7 reaching highest potency at CCR1 (EC₅₀ of 0.85 μM), 20 at CCR8 (0.39 μM), and 8 at CCR5 (1.0 μM). Altered selectivity for CCR1 and CCR8 over CCR5 (11, 12) and a receptor-dependent separation of allosteric from intrinsic properties were achieved (20). The pocket similarities of CCR1 and CCR8, contrary to CCR5 as proposed by the ligand screen, were elaborated by computational modeling. These studies facilitate exploration of chemokine receptors as possible targets for therapeutic intervention.

Full text of DOI:10.1021/jm301121j

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Article
Modulation in selectivity and allosteric properties of
small-molecule ligands for CC-chemokine receptors.
Stefanie Thiele, Mikkel Malmgaard-Clausen, Jens Engel-Andreasen, Anne Steen, Pia C. Rummel,
Mads C. Nielsen, David E. Gloriam, Thomas M. Frimurer, Trond Ulven, and Mette M. Rosenkilde
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm301121j • Publication Date (Web): 07 Sep 2012
Downloaded from http://pubs.acs.org on September 9, 2012
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Modulation in selectivity and allosteric properties of small-
molecule ligands for CC-chemokine receptors.
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a
a†
b
a
a
Stefanie Thiele , Mikkel Malmgaard-Clausen , Jens Engel-Andreasen , Anne Steen , Pia C. Rummel ,
b†
c
d
b
a
Mads C. Nielsen , David E. Gloriam , Thomas M. Frimurer , Trond Ulven , and Mette M. Rosenkilde *
a
Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, The Panum Institute,
University of Copenhagen, Blegdamsvej 3, Dk-2200 Copenhagen, Denmark
b
Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, Dk-5230
Odense, Denmark
c
Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenha-
gen, Universitetsparken 2, Dk-2100 Copenhagen, Denmark
d
The Novo Nordisk Foundation Center for Basic Metabolic Research, Faculty of Health and Medical Sciences,
University of Copenhagen, Blegdamsvej 3, Dk-2200 Copenhagen, Denmark
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ABSTRACT: Among 18 human chemokine receptors, CCR1, CCR4, CCR5 and CCR8 were activated by
metal ions Zn(II) or Cu(II) in complex with 2,2'-bipyridine or 1,10-phenanthroline with similar poten-
cies (EC50 from 3.9 to 172 µM). Besides being agonists, they acted as selective allosteric enhancers of
CCL3. These actions were dependent on a conserved glutamic acid in TM-7 (VII:06/7.39). A screening
of 20 chelator analogs in complex with Zn(II) identified compounds with increased potencies, with 7
reaching highest potency at CCR1 (EC50 of 0.85 µM), 20 at CCR8 (0.39 µM) and 8 at CCR5 (1.0 µM).
Altered selectivity for CCR1 and CCR8 over CCR5 (11, 12) and a receptor-dependent separation of allo-
steric from intrinsic properties was achieved (20). The pocket similarities of CCR1 and CCR8 contrary to
CCR5 as proposed by the ligand-screen were enlightened by computational modeling. These studies fa-
cilitate exploration of chemokine receptors as possible targets for therapeutic intervention.
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INTRODUCTION
Chemokine receptors belong to class A 7 transmembrane helix (7TM) receptors (also known as G pro-
tein-coupled receptors, GPCRs). The 7TM protein family is one of the largest families in the human ge-
nome and represents the one most extensively exploited by current drugs. 1,2 The involvement of the
chemokine system in numerous pathologies including inflammatory diseases, cancer development, pro-
gression and metastasis, and HIV-infection, emphasizes the importance of understanding the biology
and pharmacology of this system, and has lead to the development of numerous antagonists for chemo-
kine receptors (e.g. the marketed drugs Maraviroc and Mozobil). 3,4 Here agonists are characterized, but
as the path from an agonist to an antagonist could be as short as a methyl group, these data add im-
portant information to chemokine receptor ligand design.
The endogenous chemokine system comprises more than 50 ligands and 18 receptors and is character-
ized by redundancy and promiscuity. 5-7 Many receptors bind several chemokines, and the majority of
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chemokines interact with more than one receptor – an obviously challenging phenomenon in drug de-
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velopment. Most chemokines contain four conserved cysteines engaged in two disulfide bridges, a flex-
ible N-terminus before the first cysteine, an N-loop, a 310-helix, a three-stranded ß-sheet and a C-
terminal α-helix. It has repeatedly been reported that the chemokine N-terminus is involved in receptor
activation via interaction with the receptor transmembrane region, while the chemokine core interacts
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with the extracellular receptor domains. 7,9-11 The two first cysteine residues occur in four different se-
quential patterns that determine the nomenclature by being adjacent to each other (CCL1 to CCL28 12),
separated by one (CXCL1 to CXCL16) or three amino acid residues (CX CL1) or partially missing, i.e.
3
exist as one single cysteine (XCL1 and XCL2). 6
Metal ions are important for the function of numerous proteins. Zn(II) for instance serves as a part of
the active site in metalloenzymes and acts as stabilizer of transcription factors in Zn(II) finger binding
motifs. 13 Moreover, zinc-ions are high-affinity modulators of membrane protein function, as shown for
the dopamine transporters 14 as well as tachykinin NK3, 15 the nicotinic acetylcholine 16 and ionotropic
glutamate receptors. 17 Due to their well-described interaction with proteins, metal ions alone or in com-
plex with chelators have proven very useful in 7TM receptor research during the last decades. 18 The in-
troduction of metal ion sites in for instance the ß -adrenergic receptor and in CXCR3 have contributed
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to the understanding of the molecular requirements for receptor activation, and have suggested that an
inward movement of the extracellular part of TM-6 towards TM-3 is necessary for agonist-mediated re-
ceptor activation. 18-20 The inward movement of helices on the extracellular side comes along with an
outward movement of the intracellular parts, 21,22 exposing motifs important for interaction with down-
stream signaling molecules, such as G proteins. This understanding of receptor activation has indeed
been confirmed by the several crystal structures of 7TM receptors in their active and inactive states re-
leased during the last years. 23-26
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Although metal ions alone can induce activation in certain wildtype and modified receptors, 15,27-34 link-
ing a hydrophobic molecule such as a chelator to the metal ion potentially improves ligand activity by
adding interactions with additional residues (second-site interactions). For instance in CXCR3, an ago-
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nistic metal ion chelator site could be built by introduction of a His in TM-3 (position III:05/3.29 ) that,
together with an endogenous Asp in TM-4 (position IV:20/4.60), constituted the metal ion binding site,
while the chelator interacted with a Tyr in TM-6 (TyrVI:16/6.51). Importantly, this chelator site was fur-
ther stabilized by an aromatic zipper consisting of PheIII:08/3.32 and TyrVII:10/7.43, that together with
TyrVI:16/6.51 stabilized the inward movement of the extracellular part of TM-6 (with the chelator site)
towards TM-3 (with the metal ion binding site) and thus the active receptor conformation. 20
Interestingly, the existence of naturally occurring agonistic metal ion chelator sites, more precisely
Zn(II) or Cu(II) in complex with 2,2'-bipyridine (Bip) or 1,10-phenanthroline (Phe), has been shown in
CCR1 and CCR5. 11,39 In these two receptors, the metal ion chelator activities were found to be highly de-
pendent on a Glu located in the top of TM-7 (position TM-VII:06/7.39) between the major (delineated
by TM-3 to -7) and the minor binding pocket (delineated by TM-1 to -3 and -7) of the main binding
crevice. 40 This Glu is highly conserved among chemokine receptors (74%) while it is found in < 1% of
non-chemokine class A 7TM receptors. 41 Importantly, the metal ion chelator complex may emulate oth-
er reported structures for chemokine receptor small molecule ligands that, independently of their effica-
cy, share the motif of one or two centrally located positive charges linked to aromatic/hydrophobic ter-
minals. In most cases, the positively charged groups of the ligands are anchored to the conserved Glu-
VII:06/7.39. 11,39,41-44
Here the entire group of endogenous chemokine receptors with G protein-mediated signaling includ-
ing 10 CC-chemokine receptors (CCR1-CCR10), 6 CXC-chemokine receptors (CXCR1-CXCR6) as well
as CX
3
CR1 and XCR1 were tested for their ability to be activated by the metal ions Zn(II) or Cu(II) in
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complex with the chelators 2,2'-bipyridine or 1,10-phenanthroline. CXCR7 was excluded due to no
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known independent signaling but only CXCR4 modulating effects. CCR1 and CCR5 share chemokine-
binding profiles, as both receptors are activated by CCL3 and CCL5, and have both been shown previ-
ously to bind metal ion chelator complexes as agonists and positive allosteric enhancers. 11,39 In addition
to CCR1 and 5, the metal ion chelators activated CCR8 and CCR4, despite no overlap in chemokine-
binding between CCR1/5 and CCR4 or 8, but none of the remaining chemokine receptors. The activity
of metal ion chelators was dependent on GluVII:06/7.39 in all four receptors (CCR1, 4, 5 and 8). Further
screening of four bipyridine analogs, 13 phenanthroline analogs and three chelators with a new 2,2':6',2''-
terpyridine-scaffold in complex with Zn(II) showed that the agonistic potencies of metal ion chelator
complexes could be enhanced up to 106-fold as compared to ZnBip and that selectivity could be intro-
duced for CCR1 and CCR8 over CCR5. Intriguingly, a separation between the allosteric and the agonis-
tic properties could be achieved in the new class of 2,2':6',2''-terpyridines, which were pure agonists on
CCR1 and pure allosteric modulators of CCR5, a property of special interest for drugs targeting the
highly promiscuous chemokine-system. 46,47
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RESULTS
Several CC-chemokine receptors are activated by metal ion chelator complexes. All endogenous
chemokine receptors were screened for activation by four selected metal ion chelators Zn(II)- or Cu(II)-
ions in complex with 2,2'-bipyridine (Bip) or 1,10-phenanthroline (Phe), abbreviated as ZnBip, ZnPhe,
CuBip, CuPhe, by using inositoltriphosphate (IP )-turnover measurements in COS-7 cells transiently
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transfected with the receptor of interest and the promiscuous G protein Gqi4myr. In addition to CCR1 and
5, 11,39 also CCR8 was activated with micromolar potencies and efficacies reaching almost the level of the
endogenous ligand CCL1 (up to 92%) (Figure 1). As for CCR1 and 5, the phenanthroline complexes dis-
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played highest potencies at CCR8 (3.9 µM for ZnPhe and 4.7 µM for CuPhe) as compared to the bipyri-
dine complexes (42 µM for ZnBip and 50 µM for CuBip). Less efficacious (< 50% of endogenous chemo-
kine), but still potent activation was observed in CCR4, where ZnPhe and CuPhe had potencies of 24 µM
and 20 µM, respectively, whereas ZnBip and CuBip displayed lower potencies (172 µM and 74 µM, re-
spectively) (Figure 1). Control experiments showing activation of receptors by their endogenous ligands,
as well as lack of activity in untransfected cells are provided in Supporting Information 1.
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Figure 1. Agonistic activity of the four metal ion chelators ZnBip, ZnPhe, CuBip and CuPhe on all hu-
man chemokine receptors (CCR1-10, CXCR1-6, XCR1 and CX CR1).
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As in CCR1 and 5, 11,39 complex formation between metal ions and chelators was essential for activation
in CCR4 and 8 as neither metal ions nor chelators alone could activate the receptors (data not shown).
Activation of CXCR1 and CX CR1 also appeared, yet with very low potency (Figure 1). In contrast to
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CCR1 and 5, where the four metal ion chelator complexes acted as allosteric enhancers of 125I-CCL3
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binding, 11,39 no enhancement, but low affinity displacement was observed of 125I-CCL1 from CCR8
(Supporting Information 2). 48
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The activity of metal ion chelators depends on a glutamate in TM-7 (VII:06/7.39). As many small
molecule ligands for CC-chemokine receptors contain a centrally located positive charge that interacts
with GluVII:06/7.39, 41 the role of this residue in CCR8 and CCR4 was explored by Ala-substitution:
[E286A]-CCR8 and [E290A]-CCR4. The activities of ZnBip, ZnPhe, CuBip and CuPhe completely de-
pended on this residue in both receptors (as exemplified by CuPhe in Figure 2), in consistency with pre-
vious data for CCR1 and 5. 11,39 Despite very low surface expression of [E286A]-CCR8, CCL1 induced a
robust activation of this mutant with wt-like potency, while the activity of CCL22 at the well-expressed
mutant [E290A]-CCR4 was completely abolished (Supporting Information 3). This indicates that metal
ion chelator activity was crucially dependent on GluVII:06/7.39 in all four CC-chemokine receptors
(CCR1, 4, 5 and 8).
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Figure 2. Mutation of GluVII:06/7.39 abrogates metal ion chelator activity.
Phylogenetic clustering of chemokine receptors with an agonistic metal ion chelator site. A phylo-
genetic analysis based on the entire receptor sequence was performed to investigate the correlation of
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receptor primary structure with activation by metal ion chelators (Figure 3A). All four receptors with the
agonistic metal ion chelator site (CCR1, 4, 5 and 8) clustered in the same area with the highest similarity
between CCR1 and 5. However, also CCR3 and CCR2, which are not activated by these metal ion chela-
tors, were located in the same cluster. A similar correlation with a closer relation of CCR1, 8 and 4 was
found when only residues lining the main ligand-binding crevice were analyzed (highlighted in grey in
Figure 2C), while the branch-length to CCR5 was longer, indicating structural differences of the ligand-
binding site in CCR5 (Figure 3B). Furthermore, although GluVII:06/7.39 is required for activation by
metal ion chelators (Figure 2), 11,39 it is not sufficient, as it is present in most chemokine receptors (except
for CCR7, 9, 10, CXCR3 and XCR1), and also in CCR2 and CCR3 where no metal ion chelator activities
were observed despite close similarity to CCR5 and CCR1.
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Figure 3. Phylogenetic and chemogenomic analysis of chemokine receptors.
Large variation in Zn(II)-affinities among 22 different metal ion chelators. To improve the ligands
activities, a series of chelator analogs were introduced comprising five bipyridines (Chart 1A, Bip and 1-
4
), 14 phenanthrolines (Chart 1B, Phe and 5-17) and three terpyridines (Chart 1C, 2,2':6',2''-terpyridine
(18) and analogs 19 and 20). Substitutions comprised methyl-, methoxy-, hydroxyl-, formyl-groups and
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halogens. First, the ability of the chelators to bind Zn(II) was tested in a fluorescence-based assay with
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the Zn(II)-sensitive fluorophore Fluozin3 under the same conditions as the functional IP -turnover
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measurements (Figure 4, Table 1). 49
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Chart 1. Structures of the chelator analogs.
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9
0
Figure 4. The chelators affinities for Zn(II).
Focusing only on the chelator scaffold, it was found that 2,2':6',2''-terpyridine (18) had a higher Zn(II)-
affinity than phenanthroline and bipyridine, in agreement with the higher number of coordinating ni-
trogens in terpyridines. Furthermore, phenanthrolines display higher Zn(II)-affinities as compared to
bipyridines. Different chemical substituents had different effects on the Zn(II)-affinity of the chelators.
Electron-donating groups, such as methyl- (2, 3, 6, 9, 7, 10, 11 and 8) or methoxy-groups (4), led to a
higher electron density at the ring-nitrogens and consequently to a higher Zn(II)-affinity. Thus, the me-
thyl-substituted phenanthroline analogs 8, 11, 10, 7 and 9 were among the compounds with highest
Zn(II)-affinities, only exceeded by the two terpyridine compounds 18 and 19. Also substitution of Phe
with bromomethyl-groups in the ortho-position (17) resulted in an increased Zn(II)-affinity, possibly
owing to a direct interaction of the bromines with the metal ion. For other ortho-methyl substituted che-
lators, decreased Zn(II)-affinities were observed (1, 5), as in fact also for ortho-substitutions with formyl-
(16) or hydroxymethyl-groups (15). As expected, para-methyl-substituted chelators (3 and 7) displayed a
higher Zn(II)-affinity than meta-methyl substituted chelators (2 and 6) and 3,4,7,8-tetramethyl-
phenanthroline (8) displayed the highest affinity among all tested phenanthrolines. There was no ex-
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pected increase in Zn(II)-affinity by addition of hydroxyl-groups in para-position of Phe (13), instead
this compound was found to have a 5.4-fold decreased Zn(II)-affinity as compared to Phe. The electron-
donating nature of hydroxyl-groups was however confirmed by 4'-hydroxy-terpyridine (19) which had a
Zn(II)-affinity that was ~ 2-fold higher compared to the unsubstituted terpyridine (18). It is suggested
that this very high affinity of 83 nM originated from the deprotonation of the hydroxyl-group, in its ke-
to-tautomeric form resulting in a formal negative charge at the central ring-nitrogen.
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Finally, the electron-withdrawing formyl (16), chloro (12, 20) or hydroxymethyl substitutents (15) re-
sulted in compounds with lower Zn(II)-affinities than methyl-substituted chelators (Table 1). A Zn(II)-
coordinating role might be expected for the ortho-hydroxymethyl groups in 15 (similarly to 17). Howev-
er, a 23-fold lower Zn(II)-affinity was observed as compared to Phe.
Table 1. The chelators affinity to Zn(II) and their potency at activating CCR1, CCR8 and CCR5.
Affinity to Zn(II)
CCR1
CCR8
CCR5
Potency
F
lig
Efficacy
(n)
Potency
F
lig
Efficacy
SEM
(n)
Potency
F
lig
Efficacy
max
E SEM
(n)
Scaffold Pos.
Subst.
Full name
IC50
log)
±
SEM IC50
(n)
EC50
±
SEM
E
max
±
SEM
EC50
±
SEM
E
max
EC50
±
SEM
(
(µM)
(log)
(µM)
(fold)
(log)
(µM)
(fold)
(log)
(µM)
(fold)
Bip
2,2'-Bipyridine
-4.3
-5.9
±
±
0.05
0.07
54
1.1
(4)
(4)
-4.5
-5.2
±
±
0.05
0.07
34
1.0
1.0
5.2
5.5
±
±
0.20 (34)
0.32 (30)
-4.4
-5.4
±
±
0.03
0.05
42
1.0
1.0
3.3
3.5
±
±
0.14 (39)
0.13 (37)
-4.7
-5.3
±
±
0.05
0.08
19
1.0
1.0
5.8
5.0
±
±
0.33 (20)
0.35 (20)
Phe
1,10-Phenanthroline
5.9
3.9
4.9
2
1
2
3
4
o
-CH
-CH
-CH
3
3
3
6,6'-Dimethyl-2,2'-bipyridine
5,5'-Dimethyl-2,2'-bipyridine
4,4'-Dimethyl-2,2'-bipyridine
4,4'-Dimethoxy-2,2'-bipyridine
-4.0
-5.3
-5.5
-5.7
±
±
±
±
0.09
0.06
0.08
0.08
110 (4)
-4.9
-4.8
-5.4
-5.0
±
±
±
±
0.33
0.08
0.07
0.14
12
17
2.8
2.0
9.4
3.3
3.2
4.9
5.1
5.5
±
±
±
±
0.76 (4)
0.45 (7)
0.41 (6)
0.34 (7)
-5.3
-5.1
-5.5
-5.3
±
±
±
±
0.24
0.06
0.06
0.03
4.8
7.5
3.3
5.4
8.7
5.6
13
1.3
2.9
3.1
3.0
±
±
±
±
0.06 (4)
0.20 (5)
0.29 (4)
0.19 (3)
> -3
-5.6
-4.9
-4.7
> 1000
2.3
(4)
0.83 (3)
0.70 (3)
0.32 (3)
2
m
4.5
2.9
2.0
(4)
(4)
(4)
±
±
±
0.05
0.04
0.13
8.2
1.7
6.4
5.1
4.7
±
±
±
2
p
p
3.6
10
11
2
-O-CH
3
7.7
22 0.87
2
5
6
7
8
9
o
-CH
-CH
-CH
-CH
-CH
-CH
-CH
-Cl
3
3
3
3
3
3
3
2,9-Dimethyl-1,10-phenanthroline
3,8-Dimethyl-1,10-phenanthroline
4,7-Dimethyl-1,10-phenanthroline
3,4,7,8-Tetramethyl-1,10-phenanthroline
3-Methyl-1,10-phenanthroline
-5.1
-6.1
-6.5
-6.8
-6.4
-6.6
-6.7
-6.0
-5.2
±
±
±
±
±
±
±
±
±
0.02
0.01
0.03
0.04
0.03
0.07
0.01
0.01
0.03
8.4
(3)
-5.8*
-5.3
-6.1
-5.8
-5.9
-6.0
-5.7
-5.7
> -4
-5.3
-5.0
-4.9
-4.8
±
±
±
±
±
±
±
±
0.09
0.13
0.14
0.09
0.09
0.03
0.04
0.02
1.6
4.9
3.8
1.2
7.0
3.9
4.5
6.2
3.3
3.0
3.7
6.1
4.9
4.3
6.8
4.9
4.7
4.6
±
±
±
±
±
±
±
±
0.67 (4)
0.30 (4)
0.06 (3)
0.33 (4)
0.37 (4)
0.43 (3)
0.46 (3)
0.32 (3)
(3)
> -3
-5.2
-6.3
-6.3
-6.2
-6.2
-5.7
-5.5
-4.1
-4.8
> -3
> -3
> -3
> 1000
(4)
0.22 (3)
0.30 (4)
0.42 (4)
0.08 (3)
0.43 (4)
0.20 (4)
0.39 (4)
0.06 (3)
0.07 (3)
(4)
-4.2*
-5.7
-5.6
-6.0
-5.7
-5.7
> -3
> -3
> -3
-5.0
> -3
> -3
> -3
±
±
±
±
±
±
0.13
0.05
0.06
0.04
0.18
0.03
67 0.07
6.4
6.7
5.2
6.3
6.5
5.5
±
±
±
±
±
±
1.2
(3)
2
m
0.87 (3)
0.31 (3)
0.16 (4)
0.43 (3)
0.27 (4)
0.21 (3)
±
±
±
±
±
±
±
±
±
0.12
7.0 0.56
3.5
2.6
3.1
3.8
3.4
2.6
2.7
1.9
3.0
±
±
±
±
±
±
±
±
±
1.9
2.6
2.7
1.9
5.0
2.2
2.7
0.14 (4)
0.87 (3)
0.41 (3)
0.23 (4)
0.89 (3)
(3)
2
p
0.85
1.5
0.13 0.52
0.14 0.48
0.32 0.67
0.13 0.67
7.5
8.2
5.9
5.9
1.9
1.3
2
2
m
p
1.0
m
p
1.3
2.2
1
0
4-Methyl-1,10-phenanthroline
1.0
1.8
11
5
5-Methyl-1,10-phenanthroline
1.8
0.11
0.06
0.16
0.11
2.0
3.0
> 1000
> 1000
> 1000
1
2
5
5-Chloro-1,10-phenanthroline
1.0
5.9
(3)
(3)
2.0
(3)
2
13
p
p
o
o
o
-OH
-CH=O
-CH -OH
-CH=O
-CH -Br
4,7-Dihydroxy-1,10-phenanthroline
4,7-Diformyl-1,10-phenanthroline
2,9-(Bishydroxymethyl)-1,10-phenanthroline
1,10-Phenanthroline-2,9-dicarbaldehyde
2,9-(Bisbromomethyl)-1,10-phenanthroline
> 100
5.0
86 0.48
15 0.26
(3)
2
2
2
2
1
4
not tested
0.03
±
±
±
±
0.10
0.05
0.36
0.18
1.2
4.5
3.2
2.2
3.9
±
±
±
±
0.16 (4)
0.34 (5)
0.30 (3)
0.40 (4)
±
±
0.09
0.06
10 0.48
> 1000
2.5
4.9
±
±
0.23 (4)
(4)
15
2
-4.6
-4.2
-6.5
±
±
±
25
64
(3)
(3)
10 0.57
13 0.47
15 0.39
> 1000
1
1
6
7
0.07
> 1000
> 1000
(3)
> 1000
> 1000
(3)
2
0.09
0.33 (3)
(3)
(4)
1
1
8
9
2,2':6',2''-Terpyridine
-6.8
-7.1
-6.2
±
±
±
0.08
0.12
0.29
0.15 (3)
0.08 (3)
0.59 (3)
-4.2
> -3
-5.9
±
±
0.15
0.09
64 0.53
4.7
5.7
±
±
0.65 (6)
(3)
-5.3
> -3
-6.4
±
±
0.11
4.9
8.4
2.7
3.4
±
±
0.24 (3)
(3)
-4.5
> -3
> -3
29 0.67
0.48 (3)
central
-OH
-Cl
4'-Hydroxy-2,2':6',2''-terpyridine
4'-Chloro-2,2':6',2''-terpyridine
> 1000
1.2
> 1000
> 1000
> 1000
(4)
(5)
20 central
28
0.40 (8)
0.18 0.39
106
0.22 (5)
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1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Improvement of intrinsic activity and allosteric properties. The complete series of chelator analogs
(Chart 1) in complex with Zn(II) was tested for functional properties in CCR1, 5 and 8 (CCR4 was ex-
cluded due to the lower potencies and efficacies (Figure 1)). Comparing the potencies for the Zn(II)-
chelator complexes on the three receptors with the Zn(II)-affinities of the chelators revealed a positive
correlation (Figure 5 and Supporting Information 4).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The methyl-substituted phenanthroline analogs that were among the compounds with highest Zn(II)-
affinities (8, 11, 10, 7, 9 and 6, ranked after affinities) (Figure 4, light grey columns), also displayed the
highest potencies for all three receptors (Figure 5), except for 11 that was inactive in CCR5. In fact all
methyl-substituted phenanthroline analogs activated the receptors with high potencies (in contrast to 19)
showing that no saturation of the metal ion occurred. This might be attributed to the lower number of
coordinating nitrogens of phenanthrolines compared to terpyridines. Similarly, addition of methyl- (3,
2) or methoxy-groups (4) to bipyridine led to increased potencies as compared to the unsubstituted bi-
pyridine.
Chelators with substitutions in the ortho-position (17, 5, 15, 16 and 1) had lowered Zn(II)-affinities
(
except for 17, see above), and were impaired in activity with no action on CCR5. Only 1 activated CCR1
and CCR8, whereas 16 and 15 activated CCR1, yet with low efficacy, thus being somewhat affected by
the ortho-substitutions (Figure 5, Table 1).
The terpyridines 18 and 19 displayed low potencies (18) or a complete inability (19) to activate the re-
ceptors, despite very high Zn(II)-affinities. Introduction of the electron-withdrawing 4-chloro substitu-
ent on terpyridine (20) resulted in high Zn(II)-affinity, albeit lower than 18 and 19 (Figure 4), and high
potency in CCR1 and CCR8 (EC50 values of 1.2 µM and 0.39 µM, respectively). Interestingly, this com-
pound had no activity on CCR5 (see below).
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Finally, 4,7-diformyl-1,10-phenanthroline (14) activated all three receptors similarly to Phe. Addition
of hydroxyl-groups at the same positions (13) abolished all activity, which might be explained by the de-
creased Zn(II)-affinity of the chelator, but could also be caused by the hydrophilic character of the sub-
stituent and disfavored interaction with the receptor. As mentioned in more details below, similarly to
the 5-methyl-substituted Phe (11), 5-chloro-substituted Phe (12) was able to activate CCR1 and 8, but
not CCR5.
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5. Zn(II)-affinities and potencies of the chelators in complex with Zn(II) are positively correlated.
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
CCR5 has a differently shaped binding pocket than CCR1 and 8. The broad analysis of all Zn(II)-
chelated complexes uncovered that the top-five most potent compounds were identical in CCR1 and 8:
4
,7-dimethyl-phenanthroline (7), 4-methyl-phenanthroline (10), 4'-chloro-terpyridine (20), 3-methyl-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
phenanthroline (9) and 3,4,7,8-tetramethyl-phenanthroline (8) with EC50 values from 0.85 µM to 1.5 µM
in CCR1 with 7 being most potent, and EC50 values from 0.39 µM to 0.67 µM in CCR8 with 20 having
highest potency (Table 1). In contrast, the picture was somewhat different in CCR5, where the five most
potent compounds were 8, 10, 6, 9 and 2 with EC50 values from 1.0 µM to 2.3 µM, and 8 being most po-
tent (Table 1).
This was a general trait, as comparison of potencies and efficacies not only of the top five, but of all
Zn(II)-chelator complexes uncovered a close correlation of CCR1 and 8 contrary to CCR5. Both in re-
spect of potency and efficacy, CCR1 and 8 followed each other, with only few exceptions as for instance
the unsubstituted terpyridine (18) that had an 8.6-fold increased potency as compared to ZnBip at CCR8
while its potency was unchanged (or in fact slightly decreased) at CCR1 (Figure 5). In comparison to
CCR5 remarkable differences were found. 4,4'-Dimethyl-bipyridine (3) with methyl-substitutions in pa-
ra-position had potencies that were 9.4- and 13-fold higher than that of ZnBip at CCR1 and CCR8, re-
spectively, while it had a potency similar to ZnBip at CCR5. The opposite was found for meta-substituted
5
,5'-dimethyl-bipyridine (2), which was 8.2-fold more potent at CCR5, but less improved at CCR1 and
CCR8 (2.0- and 5.6-fold increased EC50, respectively) (Figure 6A-C). Phenanthrolines with substitutions
at position 5 (5-methyl-phenanthroline (11) and 5-chloro-phenanthroline (12)) lacked activity on CCR5,
but activated CCR1 and 8 with potencies similar to or slightly better than ZnPhe (Figure 6E-G). Finally,
terpyridine compound 20 did not activate CCR5, but CCR1 and 8 with very high potencies. A similar
trait was observed for 18 (Figure 6I-K, Table 1). Importantly, non of the metal ion chelator complexes
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could activate untransfected cells as presented by ZnBip, ZnPhe, Zn-2, -3, -11, -12, -18 and -20 in Figure
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
6 D, H and L.
In summary, chelators with central steric bulk, i.e. large chelators such as terpyridines or phenanthro-
lines with substitutions at the central ring do not activate CCR5, whereas the elongated (e.g. meta-
substituted bipyridines as compared to para-substituted) chelators are superior at CCR5 activation as
compared to CCR1 and 8. The observed similarity between CCR1 and 8 was in line with the phylogenetic
analysis based on residues lining the cavity around GluVII:06/7.39, where these two receptors were locat-
ed closer to each other than to CCR5 (Figure 3B).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 6. CCR1 and CCR8 are similar, but different from CCR5.
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8
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Functional variations correspond to structural differences between CCR1 and 8 on one side and
CCR5 on the other. Receptor models of all three receptors were built based on the crystal structure of
CXCR4 50 but with the intracellular parts of TM-5 to -7 adjusted to an active conformation based on the
crystal structure of opsin, 23 and extracellular parts of helices aligned to active β
-adrenergic receptor
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
structures (see methods). 25,26 The shape of the main binding crevices in these models showed three major
differences between CCR1/8 and CCR5 (Figure 7). First, the major binding pocket of CCR5 was much
deeper (z-dimension) and wider (x/y-dimension) than in CCR1/8. Second, this part of the binding crev-
ice was in CCR5 extended to an area between TM-4 and TM-5, which appeared more closed in CCR1
and 8. Finally, also in the part of the binding crevice between TM-1 to -3 and -7 were found differences
in the pocket extension, with this pocket being narrower at the side of TM-1 and -2 in CCR5, but more
open between TM-1 and -7 as compared to the pockets of CCR1 and 8. These structural differences cor-
relate well with the functional differences between CCR1 and 8 in comparison to CCR5.
Figure 7. Models of receptors CCR1, CCR8 and CCR5 emphasize structural differences of these recep-
tors.
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4
Terpyridines display altered pharmacodynamic properties. Due to the positive allosteric effects of
ZnBip, ZnPhe, CuBip and CuPhe on CCL3 binding in CCR1 and CCR5, 11,39 a selection of chelators pre-
sented in this study was tested for their ability to enhance 125I-CCL3-binding to both receptors (Table 2).
While the affinities of the bipyridine and phenanthroline analogs in most cases were not increased as
compared to the control compounds ZnBip or ZnPhe, and were thus in striking contrast to the enhanced
potencies of most of these compounds (Figure 5, Table 1), an even more surprising pharmacology was
identified for the terpyridine compounds 18 and 20 (Figure 8). While in particular 20 acted as potent
agonist on CCR1, both compounds failed to show allosteric enhancement of 125I-CCL3 binding to CCR1
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
Figure 8A, Table 2). Consequently, with 18 and 20, two highly potent agonists without allosteric effects
were identified for CCR1. The opposite was seen for CCR5, where 18, with a potency similar to ZnBip,
enhanced 125I-CCL3 binding with an IC50 of 25 µM (3.4-fold increased affinity as compared to ZnBip)
and an efficacy similar to ZnBip. Compound 20, with absolutely no CCR5 activity, had an even higher
affinity of 8.1 µM (10-fold increased as compared to ZnBip) and enhanced 125I-CCL3 binding to ~ 60%
of ZnBip (Figure 8B). Therefore, with 20 allosteric activity was completely and receptor-dependently
separated from intrinsic agonistic activity.
Figure 8. Test of the new class of 2,2':6',2''-terpyridines in binding of 125I-CCL3 to CCR1 and CCR5.
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 2. Test of the chelator analogs in complex with Zn(II) in competition binding experiments of 125I-
CCL3 to CCR1 and CCR5.
CCR1
CCR5
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
IC50
over basal
IC50
over basal
log ± SEM
(µM)
% ± SEM (n)
log ± SEM
(µM)
% ± SEM (n)
CCL3
-8.1 ± 0.09
0.01
100 ± 0.0
(5)
-7.9 ± 0.17
0.01
100 ± 0.0
(5)
Bip
-3.9 ± 0.13
-4.1 ± 0.17
126
74
253 ± 46
335 ± 18
(4)
(3)
-4.1 ± 0.09
-4.3 ± 0.13
84
53
849 ± 130
698 ± 129
(4)
(4)
Phe
1
2
3
4
No effect
-3.7 ± 0.18
-4.0 ± 0.12
-3.7 ± 0.12
(3)
(3)
(4)
(3)
-3.3 ± 0.13
-3.7 ± 0.09
-3.5 ± 0.03
-2.8 ± 0.14
511 1000 ± 392
(3)
(4)
(4)
(3)
204
101
179
283 ± 5.5
243 ± 42
226 ± 54
206
337
1015 ± 308
826 ± 209
751 ± 226
1511
5
6
> -3
> 1000
(2)
-4.5 ± 0.25
not determined
-3.5 ± 0.04
No effect
30
573 ± 29
(2)
not determined
-4.0 ± 0.05
-3.8 ± 0.10
not determined
-3.6 ± 0.02
-3.9 ± 0.10
-5.3 ± 0.05
No effect
7
112
171
528 ± 215 (2)
319 ± 13 (2)
300
503 ± 122
(2)
(2)
8
9
not determined
-4.2 ± 0.18
No effect
10
261
119
5.1
526 ± 217 (2)
66
675 ± 260
(2)
(2)
(3)
(2)
(2)
(2)
(2)
(2)
11
319 ± 13
373 ± 34
(2)
(3)
(2)
(2)
(2)
1
1
1
1
1
1
2
3
4
5
6
7
No effect
> -3
> 1000
> 1000
587
> -3
> 1000
> 1000
4.8
> -3
> -3
-3.2 ± 0.02
-3.9 ± 0.17
-3.7 ± 0.72
692 ± 287
596 ± 217
613 ± 50
-5.3 ± 0.23
> -3
361 ± 151 (2)
(2)
128
> 1000
205
18
No effect
(5)
(2)
(4)
-4.6 ± 0.16
-3.3 ± 0.04
-5.1 ± 0.03
25
519
8.1
769 ± 143
775 ± 160
516 ± 102
(5)
(2)
(4)
1
2
9
0
No effect
No effect
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DISCUSSION AND CONCLUSION
3
4
5
6
7
8
9
1
1
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1
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1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
A broad screen for an agonistic metal ion chelator site among all endogenous chemokine receptors un-
covered such a site in CCR1, 4, 5 and 8. The metal ion chelator activity was dependent on GluVII:06/7.39
in the center of the main binding crevice. Further testing of 22 chelators, comprising three different che-
lator scaffolds (bipyridine, phenanthroline and terpyridine) and with variations in substituents, showed
that by rather simple chemical modifications the potencies of the Zn(II)-chelator complexes could be
increased up to 106-fold. This increase was in many cases related to an enhanced Zn(II)-affinity. It was
also possible to gain selectivity between CCR1 and 8 vs. CCR5 and to modulate the allosteric properties
of this class of ligands from being ago-allosteric modulators to being agonists without allosteric proper-
ties in CCR1 and simultaneously to being allosteric modulators with no intrinsic activity in CCR5.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Engineered and naturally occurring metal ion binding sites in 7TM receptors.
Because of their well-defined size and coordination geometry, metal ions have proven to be useful
tools for structure-function studies. Initially, an antagonistic Zn(II)-site based on the binding site of the
antagonist CP96-345 was created in the NK1 receptor between the endogenous His197 in TMV:05/5.39
and introduced histidines at positions V:01/5.35 and VI:24/6.59. 27 This Zn(II)-site could also be intro-
duced in the κ-opioid receptor. 28 The first example of a site conferring intrinsic (agonist independent)
activity to a metal ion was in the CXC-chemokine receptor ORF74, where a Zn(II)-site was created be-
tween His residues introduced in the top of TM-5 (V:01/5.35 and V:05/5.39). Via this site, Zn(II) dis-
played inverse agonistic single digit micromolar potency and additionally gained positive allosteric ef-
fects on CXCL1 binding at higher concentrations. 29,33 At exactly the same, yet naturally occurring site in
7
15
the NK3 receptor, Zn(II) acted as an allosteric enhancer of the agonist (MePhe )neurokinin. Later, ag-
onistic metal ion (chelator) sites were constructed in the NK1 30 and β -adrenergic receptor 51 (between
2
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8
9
1
1
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1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
TM-3, -6 and -7), and in CXCR3 (between TM-3, -4 and -6), where an inward movement of the extracel-
lular part of TM-6 accounted for the formation of the metal ion binding site – findings that resulted in
the formulation of a model of class A 7TM receptor activation, which proposed a concerted inward- and
outward-movement of the extra- and intracellular parts of TM-6, respectively. 18-20 In addition to the
NK3 receptor, endogenous sites for metal ions alone have been identified in GPR39 34 and in the mela-
nocortin (MC)1 and MC4 receptors. 52 These sites were located in different areas, for instance between
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
15
34
271
the receptor N-terminus and ECL3 in GPR39 and between Cys in ECL2 and AspIII:05 in MC1 and 4.
5
2
In CCR1 and CCR5 GluVII:06/7.39 has been identified as essential for metal ion chelator mediated
activation. 11,39 The same dependency of GluVII:06/7.39 was found for the metal ion binding sites identi-
fied in CCR4 and 8 in the present study (Figure 1 and 2). However, in CXCR3, where GluVII:06 is miss-
ing, the metal ion site was constructed between residues in TM-3 and -4 (HisIII:05/3.29, substituted
from Gly, and AspIV:20/4.60). 20 Importantly, despite the different location of the metal ion binding site
in the four wt receptors CCR1, 4, 5, 8 and the engineered CXCR3, chelators complexed to the metal ions
were necessary for the agonistic action by providing second-site interactions to the otherwise silent metal
ion binding sites, and thereby constrain the receptor in active conformations. 11,20
Affinity of the chelators to Zn(II) is limiting for the potency of the metal ion chelator complex. The
affinity of the chelators to Zn(II) could be modulated by the introduction of electron-withdrawing or -
donating substituents (Figure 4). Generally, a positive correlation between Zn(II)-affinity and receptor
activating potency was observed (Supporting Information 4). However, in certain cases (3 and 2), the
potency of a ligand seemed limited by its Zn(II)-affinity (Figure 5). One strategy for improving the po-
tency of a Zn(II)-chelator complex therefore is to increase the strength of the chelator. It should however
be avoided to saturate the metal ions coordination sites by the chelator, which would prevent interaction
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with the receptor as observed for the chelators terpyridine (18) and 4'-hydroxy-terpyridine (19). Also the
cyclam-containing CXCR4-antagonists Mozobil (AMD3100) and analogs have the ability to chelate
metal ions, which increase their potency. As for the chelators presented here, the potency-increase of
Mozobil was directly correlated to the incorporated ion´s chelation enthalpy for acetate, which reflected
the affinity between chelator and ion. 53 That chelation of the metal ion via four nitrogens in such a
cyclam-ring did not “neutralize” the metal ion, as suggested for the three nitrogen-containing terpyri-
dines, might relate to the larger size and aliphatic character of the cyclam-ring which supposedly led to a
lower partial negative charge at each ring-nitrogen as compared to the pyridine ring. Furthermore, a di-
rect interaction of AspVI:23 in CXCR4 with a cyclam-nitrogen suggests that the chelator itself contrib-
utes to the affinity between ligand and receptor. 53
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Future chemokine receptor drugs designed for promiscuity, intrinsic activity and/or allosteric ac-
tion. The immense role of the chemokine system in inflammatory and allergic diseases has stimulated
numerous drug development programs, which so far resulted in two drugs on the market: the CCR5-
antagonist Maraviroc which inhibits HIV cell entry, and, as mentioned above, Mozobil (AMD3100),
which is used as stemcell mobilizer via CXCR4. This rather low number of successes could arise from the
difficulties concerning species differences and the promiscuity of the chemokine system. One way to
overcome the latter is to design promiscuous drugs that simultaneously block many chemokine receptors
that otherwise could substitute for each other. 46,47
By analyzing the chelators presented here, similar potency and efficacy-profiles were found for CCR1
and 8 whereas CCR5 was somewhat different (Figure 5), as also confirmed by the focused phylogenetic
analysis of residues in the main ligand binding crevice (Figure 3B), and by computational modeling
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1
2
3
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
Figure 7). Hereby, compounds 11, 12 and 20 were dual-tropic CCR1 and 8 agonists, while they would
discriminate against CCR5. This dual action has been reported previously for the small molecule agonist
LMD-559, which is a tetrazole-containing analog of earlier published selective CCR8-agonists, that was
found to activate both CCR1 and 8. 43,45 A similar restriction within homologous receptors has also been
observed in the MC1 and 4 receptors, where Zn(II) in complex with phenanthroline, 5-methyl- (11) or
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
-amino-phenanthroline, and bipyridine activated MC1, but not the MC4 receptor despite the presence
of a Zn(II)-site in both receptors. 52 In general, dual receptor targeting is suggested as solution to over-
come the promiscuity of the chemokine system, and was presented earlier for instance for CCR2 and 5
TAK-779 and TAK-652 54,55), CCR1 and 3 (UCB35625 ) CXCR1 and 2 (reparixin and SCH-527123 ),
56
57
58
(
5
9
but interestingly also for less related receptors such as CCR2 and CXCR2 (thiazolo(4,5-d) pyrimidines )
6
0
or CCR3 and the histamine H1-receptor (YM-344484 ). These examples emphasized the general possi-
bility to find dual ligands also for receptor pairs with low sequence homology, different endogenous lig-
ands and from different receptor subgroups.
Furthermore, most small molecule chemokine ligands are usually allosteric in respect to the endoge-
nous chemokine ligands constituting an extra level of complexity. 4,41,61 It is generally accepted that an
allosteric binding-mode provides higher specificity and safety, as the activity depends on the presence of
the endogenous ligand, and is limited by the efficacy of this ligand. 62 Interestingly, a study in CCR1
showed that a certain small molecule ligand may act differently on different endogenous ligands (for the
same receptor) by being an allosteric enhancer of CCL3 binding, but at the same time competing for
1
1
3
9
C
C
L
5
,
a
n
d
b
y
i
t
s
e
l
f
a
c
t
i
n
g
a
s
a
g
o
n
i
s
t
f
o
r
C
C
R
1
.
A
s
i
m
i
l
a
r
p
h
e
n
o
m
e
n
o
n
h
a
s
b
e
e
n
d
e
s
c
r
i
b
e
d
i
n
C
C
R
5
.
In the present manuscript a new class of chemokine-receptor ligands, the terpyridines, was identified of
2
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which the chloro-substituted analog (20) showed an interesting pharmacological phenomenon, as in this
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
ligand the allosteric enhancing properties were limited to CCR5, while it was a pure agonist on CCR1.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
In summary, by studying metal ions in complex with various chelators, it is illustrated how pharmaco-
logic properties can be manipulated. For instance, the ligands potencies could be increased, receptor
specificity could be modulated towards only one or two receptors (dual-tropic), and the intrinsic activity
could be separated from allosteric properties. The compounds included in the present study are all ago-
nists and/or positive allosteric modulators, however it might be speculated that by finding the right
chemical modifications and by substitution of the diffusible metal ion with a covalently linked positively
charged group, these compounds could be converted into antagonists of chemokine-action – as support-
ed by the notion that small molecule CC-chemokine receptor antagonists and agonists have similar
pharmacophores (centrally located positive charge and flanking aromatic moieties) and exploit the same
key-anchor points like GluVII:06/7.39. 44,63,64 Most importantly, the presented metal ion chelators prove
that compounds with chemokine- and receptor-dependent allosteric and intrinsic effects can be created
and thus illustrate the plurality of actions for small molecules in the chemokine system and the necessity
to screen for these various (inter)actions.
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9
1
1
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1
1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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4
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4
4
4
4
4
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5
5
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5
5
5
5
5
5
5
6
EXPERIMENTAL SECTION
Materials. The human chemokines CCL1-3, -5, CCL11-22, -25, CXCL8, -11-13, -16, XCL1 and CX
3
CL1
were purchased from Peprotech (NJ, USA). The highest concentration of metal ion chelator complexes
ZnBip, ZnPhe, CuBip and CuPhe were made from 0.2 M ZnCl in water, 0.2 M CuSO in water, 0.4 M
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
4
phenanthroline in 70% ethanol, 0.4 M bipyridine in DMSO, and were supplemented with 10% DMSO,
water and 70% ethanol. Chelator analogs were dissolved in DMSO to 10 mM and prepared in similar
ways. Dilutions were made in water. Receptor CCR5 was cloned in house from a leukocyte cDNA li-
brary, CCR1-3, CXCR1, -2, -4 and CX CR1 cDNA was kindly provided by Tim Wells (GlaxoSmithKline,
3
UK), CXCR3 was kindly provided by Kuldeep Neote (Pfizer), and CCR4, -6-10, CXCR5, -6 and XCR1
were purchased from cDNA.org. The promiscuous G protein GαΔ6qi4myr (abbreviated as Gqi4myr) was kind-
3
ly provided by Evi Kostenis (University of Bonn, Germany). Myo-[ H]inositol (PT6-271), Bolton-
Hunter-Reagent and iodinated chemokine 125I-CCL1 were purchased from PerkinElmer (MA, USA). AG
1
-X8 anion exchange resin was from Bio-Rad (CA, USA).
Chemicals and synthetic procedures.
All commercially available starting materials and solvents were used without further purification, un-
less otherwise stated. THF was freshly distilled from sodium/benzophenone. DMF, Et
3
N and TMEDA
were dried over 4Å sieves. Purification by flash chromatography was carried out using silica gel 60
1
13
(0.040-0.063 mm, Merck). TLC analysis was performed on silica gel 60 F254 plates. H and C NMR spec-
tra were calibrated relative to TMS internal standard or residual solvent peak. High-resolution mass
spectra (HRMS) were obtained on Bruker micrOTOF-Q II (ESI) or IonSpec 4.7 T Ultima FTMS using
DHB matrix (MALDI). Electron ionization mass spectra were obtained on a Thermo Finnigan SSQ 710
(
EI). Purity was determined by HPLC and confirmed by inspection of NMR spectra. HPLC analysis was
2
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performed using a Dionex 120 C18 column (5µ, 4.6x150 mm) with 10% acetonitrile in water (0-1 min),
10-100% acetonitrile in water (1-10 min), 100% acetonitrile (11-15 min), both solvents containing 0.05
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
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4
4
4
4
4
4
4
4
5
5
5
5
5
5
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5
5
6
%
TFA as modifier, a flow of 1 mL/min and UV detection at 230 and 254 nm. All test compounds were
of ≥95% purity unless otherwise stated.
,6'-Dimethyl-2,2'-bipyridine (1), 5,5'-dimethyl-2,2'-bipyridine (2), 4,4'-dimethyl-2,2'-bipyridine (3),
0
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0
6
4,4'-dimethoxy-2,2'-bipyridine (4), 2,9-dimethyl-1,10-phenanthroline (5), 3,4,7,8-tetramethyl-1,10-
phenanthroline (8), 4-methyl-1,10-phenanthroline (10), 5-methyl-1,10-phenanthroline (11), 5-chloro-
1,10-phenanthroline (12), 2,2':6',2''-terpyridine (18), 4'-hydroxy-2,2':6',2''-terpyridine (19) and 4'-chloro-
2,2':6',2''-terpyridine (20) were purchased from Sigma-Aldrich. 4,7-Dimethyl-1,10-phenanthroline (7)
was purchased from Alfa Aesar.
3
,8-Dimethyl-1,10-phenanthroline (6). 2-Methylacrylaldehyde (1.4 g, 20 mmol) was added over 5 h
to a stirred solution of the o-phenylenediamine (650 mg, 6 mmol) and NaI (8.8 mg, 0.0575 mmol) in
0% H SO (2.2 mL, 25 mmol) at 110 °C. After 16 h at 110 °C the dark brown reaction mixture was
cooled to room temperature, poured into 1 M Na CO (25 mL) and extracted with CH Cl (3 x 25 mL).
The combined organic phases were extracted with 12 M HCl (5 x 10 mL) and the acidic solution is neu-
tralized with 3 M NaOH and 1 M Na CO and extracted with CH Cl (3 x 50 mL). The combined organic
phases were dried (MgSO ), concentrated and a part of the crude product (9.1 mg of 70 mg) was purified
7
2
4
2
3
2
2
2
3
2
2
4
by preparative HPLC to give 6 mg of a white solid (89% pure by HPLC). 65 1H NMR (400 MHz, CDCl
) δ
3
13
9
.23 (s, 2H), 8.27 (s, 2H), 7.87 (s, 2H), 2.68 (s, 6H); C NMR (101 MHz, CDCl
3
) δ 150.03, 138.37, 134.79,
+
128.91, 126.91, 19.01; HRMS (ESI) calcd for C14
H
13
N
2
(M + H ) 209.1079, found 209.1073.
3
-Methyl-1,10-phenanthroline (9). 2-Methylacrylaldehyde (700 mg, 10 mmol) was added over 5 h to
a stirred solution of the 8-aminoquinoline (865 mg, 6 mmol) and NaI (8.8 mg, 0.0575 mmol) in 70%
SO (2.2 mL, 25 mmol) at 110 °C. After 16 h at 110 °C the dark brown reaction mixture was cooled to
H
2
4
2
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room temperature, poured into 1 M Na
bined organic phases were extracted with 12M HCl (5 x 10 mL) and the acidic solution was neutralized
with 3M NaOH and 1M Na CO and extracted with CH Cl (3 x 50 mL). The combined organic phases
were dried (MgSO ), concentrated and purified by flash chromatography (SiO , CH Cl ) to give 138 mg
) δ 9.17 (dd, J =4.3, 1.7 Hz,
2
CO
3
(25 mL) and extracted with CH
2
Cl (3 x 25 mL). The com-
2
2
3
2
2
0
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2
3
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0
4
2
2
2
65
1
(
12%) of a white solid. R
f
= 0.23 (SiO
2
, CH
2
Cl
2
); H NMR (400 MHz, CDCl
3
1
7
1
1
H), 9.03 (d, J = 2.0 Hz, 1H), 8.22 (dd, J = 8.1, 1.7 Hz, 1H), 8.01 (d, J = 2.0 Hz, 1H), 7.81–7.68 (m, 2H),
13
.60 (dd, J = 8.1, 4.3 Hz, 1H), 2.60 (s, 3H); C NMR (101 MHz, CDCl
3
) δ 152.0, 150.4, 146.5, 144.4,
36.0, 135.3, 133.0, 128.5, 128.3, 126.6, 126.4, 122.8, 18.9; HRMS (ESI) calcd for C13
95.0922, found 195.0917.
H
11
N
(M + H+)
2
4,7-Dihydroxy-1,10-phenanthroline (13). The title compound was prepared as described previously.65
,7-Diformyl-1,10-phenanthroline (14). To a solution of 4,7-dimethyl-1,10-phenanthroline (1.093 g,
.25 mmol) in 1,4-dioxane (100 mL) at room temperature was added selenium dioxide (2.84 g, 25.6
4
5
mmol), and the mixture was heated to reflux for two hours. Additional selenium dioxide (3.03 g, 27.3
mmol) was added, and reflux was continued for 16 h. The hot reaction mixture was filtered through a
pad of Celite and cooled to room temperature, at which point the product started to precipitate. The so-
lution was put on ice bath to complete the precipitation. The solid was collected and recrystallized from
dry THF and dried to provide 530 mg (43%) of the title compound as light orange powder. Rf 0.46
); H NMR in correspondence to previously reported data; 66 13C NMR
1
(MeOH with 1% conc. aq. NH
3
) d 194.2, 151.3, 146.0, 136.7, 126.3, 124.6, 123.4; MS (MALDI) 237.1 (M + H+).
(
101 MHz, CDCl
3
2
,9-Bis(hydroxymethyl)-1,10-phenanthroline (15). The title compound was prepared according to a
previously described method. 67
1
,10-Phenanthroline-2,9-dicarbaldehyde (16). To a solution of 2,9-dimethyl-1,10-phenanthroline
(
1.95 g, 9.37 mmol) in 1,4-dioxane (140 mL) was added selenium dioxide (5.25 g, 47.3 mmol) at room
2
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temperature and the reaction mixture was heated to reflux for two hours. The yellow solution changed
color via orange and red to green-brown. The warm reaction mixture was filtered to remove a black pre-
cipitate and allowed to cool slowly to room temperature. A new precipitate formed, which was filtered
3
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off, washed several times with diethyl ether and dried under vacuum to give 1.62 g (73%) of a light grey
solid. NMR spectra were in accordance with previously published data. 67
2,9-Bis(bromomethyl)-1,10-phenanthroline (17). The title compound was prepared according to a
previously described method. 67
Zn(II)-affinity assay: 10 µl of the chelator were added to 90 µl of reaction buffer containing 20 nM
ZnCl, 1 µM Fluozin-3 (Invitrogen, UK), 10 mM LiCl in Hank´s balanced salt solution (Invitrogen, UK).
After two hours incubation at room temperature, fluorescence intensity (excitation at 485 nm and emis-
sion at 520 nm) was measured in a POLARstar Omega from BMG Labtech.
Site-directed Mutagenesis. Point mutations were introduced in the receptors by the polymerase chain
reaction overlap extension technique using wt CCR4 and CCR8 as templates. All reactions were carried
out using Pfu polymerase (Stratagene, CA, USA) under conditions recommended by the manufacturer.
The mutations were cloned into the eukaryotic expression vector pcDNA3.1+ and were verified by re-
striction endonuclease digestion and DNA sequencing (Eurofins MWG Operon, Germany).
Transfections and Tissue Culture. COS-7 cells were grown at 10% CO and 37 °C in Dulbecco´s modi-
2
fied Eagle´s medium with glutamax (Invitrogen, UK) adjusted with 10% fetal bovine serum, 180 units/ml
penicillin, and 45 µg/ml penicillin/streptomycin (PenStrep). Transfection of the COS-7 cells was per-
6
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formed by the calcium phosphate precipitation method.
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Inositol Phosphate Turnover (IP Turnover). COS-7 cells were transfected according to the procedure
6
mentioned above. Briefly, 6 x 10 cells were transfected with 20 µg of receptor cDNA in addition to 30 µg
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of the promiscuous chimeric G protein, Gαqi4myr, which turns the Gα -coupled signal, the most common
i
pathway for endogenous chemokine receptors, into the Gα
q
pathway (phospholipase C activation meas-
ured as IP
3
turnover). 68,69 From here two different assays differing in their scale, but which were found to
show the same result were carried out. “Traditional” IP
3
-assay: One day after transfection, COS-7 cells
5
3
(1.5 x 10 cells/well) were incubated for 24 h with 2 µCi of myo[ H]inositol in 0.3 ml of growth medium
per well in a 24-well plate. Cells were washed twice in PBS and were incubated in 0.2 ml Hanks´ Bal-
anced Salt Solution (Invitrogen, UK) supplemented with 10 mM LiCl at 37 °C in the presence of various
concentrations of ligands. Cells were extracted by addition of 1 ml of 10 mM formic acid to each well
3
followed by incubation on ice for 30 to 60 min. The generated [ H]inositolphosphates were purified on
AG 1-X8 anion exchange resin and after addition of Multi Purpose Liquid Scintillation Cocktail (Gold
Star, Triskem-International, France), γ-radiation was counted in a Beckman Coulter Counter LS6500.
Scintillation proximity assay (SPA)-IP
3
: One day after transfection, COS-7 cells (35.000 cells/well) were
3
incubated for 24 h with 0.5 µCi/ml myo[ H]inositol in 100 µl of growth medium per well in a 96-well
plate. Cells were thereafter treated as mentioned above with volumes adjusted as follows: 100 µl reaction
solution with LiCl, 50 µl formic acid. 20µl of the extract were transferred to a white 96-well plate and 80
µl of 1:8 diluted YSi Poly-D-lysine coated beads (Perkin Elmer, MA, USA) were added. Plates were
sealed and shaked at maximum speed for at least 30 min, centrifuged for 5 min at 1500 rpm and γ-
radiation was counted in a Packard Top Count NXT counter. In both assays, determinations were made
in duplicates. This general read-out has previously been used with success in other chemokine recep-
tors.11,43
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Binding Experiments. 6 x 10 COS-7 cells were transfected with 40 µg of receptor cDNA and transferred
to culture plates 1 day after transfection. The number of cells seeded per well was determined by the ap-
parent expression efficiency of the receptors and was aimed at obtaining 5-10% specific binding of the
added radioactive ligand. Two days after transfection, cells were assayed by competition binding for 3h
at 4 °C using 10-15 pM 125I-CCL1 or 125I-CCL3 plus unlabelled ligand in 0.2 ml 50 mM Hepesbuffer pH
6
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.4, supplemented with 1 mM CaCl
2
, 5 mM MgCl , and 0.5% (w/v) bovine serum albumin. After incuba-
2
tion, cells were washed quickly two times in 4 °C binding buffer supplemented with 0.5 M NaCl. Non-
specific binding was determined in the presence of 0.1 µM unlabeled CCL1. Determinations were made
in duplicates.
Receptor Surface Expression by Enzyme-Linked Immunosorbent Assay (ELISA). COS-7 cells were transi-
ently transfected with the N-terminal FLAG-tagged receptor variants. The cells were washed once in TBS
(
50 mM Tris-base, 150 mM NaCl, pH 7.6), fixed in 4% glutaraldehyde for 15 min following three washes
in TBS and incubation in blocking solution (2% bovine serum albumin in TBS) for 30 min at room tem-
perature. The cells were subsequently incubated two hours with anti-FLAG (M1) antibody (2 µg/ml)
(
Sigma-Aldrich, MO, USA) in TBS supplemented with 1% bovine serum albumin and 1 mM CaCl
room temperature. After three washes in TBS with 1 mM CaCl , the cells were incubated for 1 h with
goat anti-mouse horseradish peroxidase-conjugated antibody (Thermo Fisher Scientific Inc., IL, USA) in
the same buffer as the anti-FLAG antibody. After three washes in TBS supplemented with 1 mM CaCl
2
at
2
2
the immune reactivity was revealed by the addition of horseradish peroxidase substrate TMB Plus (Kem-
En-Tec Diagnostics A/S, Denmark) according to the manufacturer’s instruction.
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