Molecular Interactions of Cyclam and Bicyclam Non-peptide
Antagonists with the CXCR4 Chemokine Receptor*
Lars Ole
Gerlach
,
Renato T.
Skerlj§,
Gary J.
Bridger§, and
Thue W.
Schwartz
¶
From the
Laboratory for Molecular Pharmacology,
University of Copenhagen, Panum Institute, DK-2200 Copenhagen,
Denmark, § AnorMED Incorporated, Langley, British
Columbia, Canada V2Y 1N5, and ¶ 7TM Pharmaceuticals
A/S, Rønnegade 2, DK-2100 Copenhagen, Denmark
Received for publication, November 17, 2000
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ABSTRACT |
The non-peptide CXCR4 receptor antagonist
AMD3100, which is a potent blocker of human immunodeficiency virus cell
entry, is a symmetrical bicyclam composed of two identical
1,4,8,11-tetraazacyclotetradecane (cyclam) moieties connected by a
relatively rigid phenylenebismethylene linker. Based on the known
strong propensity of the cyclam moiety to bind carboxylic acid groups,
receptor mutagenesis identified Asp171 and
Asp262, located in transmembrane domain (TM) IV and TM-VI,
respectively, at each end of the main ligand-binding crevice of the
CXCR4 receptor, as being essential for the ability of AMD3100 to block
the binding of the chemokine ligand stromal cell-derived factor
(SDF)-1
as well as the binding of the receptor antibody 12G5. The
free cyclam moiety had no effect on 12G5 binding, but blocked SDF-1
binding with an affinity of 3 µM through interaction with
Asp171. The effect on SDF-1
binding of a series of
bicyclam analogs with variable chemical linkers was found to rely
either only on Asp171, i.e. the bicyclams acted
as the isolated cyclam, or on both Asp171 and
Asp262, i.e. they acted as AMD3100, depending
on the length and the chemical nature of the linker between the two
cyclam moieties. A positive correlation was found between the
dependence of these compounds on Asp262 for binding and
their potency as anti-human immunodeficiency virus agents. It is
concluded that AMD3100 acts on the CXCR4 receptor through binding to
Asp171 in TM-IV and Asp262 in TM-VI with each
of its cyclam moieties, and it is suggested that part of its function
is associated with a conformational constraint imposed upon the
receptor by the connecting phenylenebismethylene linker.
 |
INTRODUCTION |
Bicyclams derivatives composed of two cyclam
(1,4,8,11-tetraazacyclotetradecane) units linked by an aliphatic or
aromatic linker (see Fig. 1) have been identified as potent inhibitors of human immunodeficiency virus
(HIV)1 type 1 and type 2 replication (1). The lead compound of the bicyclam series was
discovered as an impurity being responsible for the antiviral effect in
a preparation of cyclam (2). The prototype bicyclam, AMD3100, in which
the two macrocyclic rings are connected by a phenylenebis(methylene)
linker (Fig. 1), has a potency against
HIV-1 and HIV-2 replication of 1-10 nM (3) and is
currently used as the first coreceptor antagonist in Phase II
clinical trials against AIDS (4).
The molecular mechanism of action of AMD3100 was initially believed to
be related directly to the viral gp120 protein (5). The problem was
that the bicyclams were discovered in 1989, i.e. long before
details of the molecular machinery on the target cell responsible for
HIV cell entry had been characterized. Shortly after the discovery of
the HIV coreceptors, it was shown that the bicyclams in fact inhibit
HIV replication by binding to the CXCR4 chemokine receptor, which is
the main coreceptor for gp120 used by X4, T-tropic strains of
HIV for membrane fusion and cell entry (6-8). Thus, AMD3100 was the
first non-peptide CXCR4 receptor antagonist discovered and optimized
long before the receptor was shown to bind the SDF-1
chemokine.
The structure-function relationship with respect to the antiviral
effect of the bicyclams has been worked out in great detail by
De Clercq et al. (1) and Bridger et al.
(2, 9-11). The optimum structural features required for potent
antiviral activity of "bicyclam" analogs include the presence of
two azamacrocyclic rings containing 12-14 atoms/ring and a
meta- or para-substituted phenylenebismethylene
linker (para more active than meta) connecting the macrocyclic rings at nitrogen positions. The introduction of
electron-withdrawing or -donating groups to the aromatic ring did not
adversely affect antiviral activity; however, sterically hindering
groups that restrict rotation of the macrocyclic rings at the benzylic
position had a detrimental effect on antiviral potency.
The relatively simple, symmetric structure of AMD3100 combined with the
detailed knowledge of its structure-function relationship offer
particular opportunities for understanding the molecular interaction of
this non-peptide antagonist with its receptor target. It could, for
example, be envisioned that the cyclam moieties by themselves could
make strong enough interactions with the receptor for their binding
sites to be identified by mutagenesis. The isolated 1,4,8,11-tetrazacyclotetradecane ring has an overall charge of +2 at
physiological pH (12), and x-ray and neutron diffraction structures
have shown that the protonated cyclam ring has the propensity to form a
direct, hydrogen-bonded stabilized complex with carboxylic acid groups
(13). In accordance with this, initial mutagenesis studies have shown
that acidic residues in the second extracellular loop and in
transmembrane domain (TM) IV of the CXCR4 receptor apparently are
involved in the antiviral effect of AMD3100 (14). The cyclam rings are
also known to be able to chelate metal ions (15); and recently, it has
been shown that transition metal ions chelated by the two macrocyclic
rings of AMD3100 increase the CXCR4-binding affinity and potency of AMD3100 by 10-fold (16). This could indicate that, for example, metal
ion-binding His residues, of which there are several in the main
ligand-binding crevice of the CXCR4 receptor, could be involved in the
binding of the bicyclam compounds (Fig.
2).

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Fig. 2.
Helical wheel (A) and
serpentine diagram (B) of the CXCR4 receptor.
White letters in red circles represent residues
substituted with Ala or Asn probing the binding site of AMD3100.
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In this study, we have, based on the knowledge of the possible binding
mode of the cyclam as such, initially targeted aspartate and histidine
residues in the CXCR4 receptor for mutagenesis and combined this with
studies of a series of bicyclam analogs with variable chemical linkers.
The two major hits for AMD3100 binding (one of which was shown
to be involved in cyclam binding) were located at each end of the main
ligand-binding pocket, which we previously have analyzed in great
detail in several other 7TM receptors, for example, by metal ion
site engineering (17-19). In a molecular model of the CXCR4 receptor
built over the recently published x-ray structure of rhodopsin (20),
AMD3100 could be manually docked directly in between the two proposed
cyclam-binding sites.
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EXPERIMENTAL PROCEDURES |
Site-directed Mutagenesis--
Point mutations were introduced
in the receptor by the polymerase chain reaction overlap extension
technique (21) using the human wild-type CXCR4 receptor cDNA as
template. All reactions were carried out using the Pfu
polymerase (Stratagene) under conditions recommended by the
manufacturer. The generated fragments were subcloned into the pTEJ-8
eukaryotic expression vector (22) containing the wild-type CXCR4
receptor cDNA by substituting the wild-type cDNA fragment with
the mutant cDNA fragment. The mutations were verified by
restriction endonuclease digestion and DNA sequencing (ABI 310, PerkinElmer Life Sciences).
Expression of Mutant Receptors--
COS-7 cells were grown at
10% CO2 and 37 °C in Dulbecco's modified Eagle's
medium supplemented with 10% fetal calf serum, 2 mM
glutamine, and 10 µg/ml gentamycin. The wild-type and mutant CXCR4
receptors were transiently transfected into COS-7 cells by the calcium
phosphate precipitation method as described previously (23).
Ligands--
The human chemokine Met-SDF-1
was kindly
provided by Michael A. Luther (Glaxo Wellcome). This SDF-1
contains
an additional NH2-terminal methionine; however, the protein
shows the same binding properties as natural ligand SDF-1
(24, 25).
125I-Labeled Met-SDF-1
was prepared by oxidative
iodination using IODO-GEN (Pierce), followed by high pressure liquid
chromatography purification to separate unlabeled from labeled
compound. The monoclonal antibody 12G5 was kindly provided by Jim Hoxie
(University of Pennsylvania, Philadelphia, PA). 12G5 was
125I-labeled using Bolton-Hunter reagent (Amersham
Pharmacia Biotech) as described (26). AMD3100, AMD3106, AMD3108,
AMD2763, AMD2849, AMD3389, and AMD2936 were synthesized as described
(9, 11). Cyclam (1,4,8,11-tetraazacyclotetradecane) was purchased from Aldrich.
Receptor Binding Assays--
The transfected COS-7 cells were
transferred to culture plates 1 day after transfection. The number of
cells seeded per well was determined by the apparent expression
efficiency of the individual clones; the number of cells/well was
adjusted aiming at 5-10% binding of the added radioligand. Two days
after transfection, cells were assayed by competition binding performed
on whole cells for 3 h at 4 °C using 12 pM
125I-Met-SDF-1
or 32 pM
125I-12G5 plus variable amounts of unlabeled peptide or
non-peptide compounds in 400 µl of 50 mM HEPES (pH 7.7)
supplemented with 1 mM CaCl2, 5 mM
MgCl2, and 0.5% (w/v) bovine serum albumin (Sigma). After
incubation, cells were washed quickly four times in 4 °C binding
buffer supplemented with 0.5 M NaCl. Determinations were made in duplicate.
Calculations--
IC50 values were determined by
nonlinear regression using Prism 3.0 (GraphPAD Software, San Diego, CA).
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RESULTS |
Binding of Cyclam Versus Bicyclam to CXCR4--
In COS-7 cells
transiently expressing the human CXCR4 chemokine receptor, cyclam
(1,4,8,11-tetraazacyclotetradecane) competed for
125I-Met-SDF-1
binding with an affinity
(Ki) of 13 µM, whereas it was unable
to displace the radiolabeled monoclonal receptor antibody
(125I-12G5) from CXCR4 even at millimolar concentrations
(Fig. 3). In contrast, the bicyclam
compound AMD3100, in which two identical cyclam rings have been joined
by a phenylenebis(methylene) linker, showed a 200-fold higher affinity
compared with the cyclam moiety (Ki = 74 nM) in competition against 125I-Met-SDF-1
,
and the bicyclam could compete against 125I-12G5 binding to
CXCR4 with an affinity that was <10-fold lower than against the
chemokine ligand (Fig. 3).

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Fig. 3.
Competition binding experiments using AMD3100
(A) or the isolated 1,4,8,11-tetraazacyclotetradecane
(cyclam) (B). Whole cell competition binding was
performed on the wild-type CXCR4 receptor expressed in COS-7 cells
using either 125I-Met-SDF-1 (open symbols) or
125I-12G5 monoclonal receptor antibody (closed
symbols) as radioligand. Data are shown as means ± S.E.
(n = 4-11).
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Effect of Asp and His Mutations on Cyclam Binding--
Asp and His
residues in the CXCR4 receptor were targeted for mutagenesis based on
the assumption that each cyclam ring of AMD3100 and analogs would be
doubly protonated at physiological pH or conceivably would be able to
complex transition metal ions such as zinc in vitro to give
a metal complex with an overall charge of +2. In either structural
scenario, the Asp and His residues of the CXCR4 receptor could be
considered partners for complexation. Thus, all His residues located in
the extracellular loops or in the transmembrane domains
(i.e. His113, His203,
His281, and His294) were individually mutated
to Ala residues (Fig. 2). In addition, four Asp residues
(Asp171 (located in TM-IV), Asp182 and
Asp193 (located in extracellular loop 2), and
Asp262 (located in TM-VI)) were mutated to Asn residues
(Fig. 2).
As shown in Table I, all of these single
mutations were expressed well in the COS-7 cells, and all bound
the chemokine ligand Met-SDF-1
with similar affinity as the
wild-type CXCR4 receptor. The affinity of the cyclam as determined in
competition for 125I-Met-SDF-1
binding was also
unaffected by all these mutations, except for the Asn substitution of
Asp171 located in the extracellular part of TM-IV, which
impaired the cyclam binding by 30-fold (Fig.
4B and Table I). The binding of the bicyclam AMD3100 was also affected by this mutation and to a
similar extent (Fig. 4A). However, in contrast to the free cyclam, the binding of the bicyclam AMD3100 was also impaired by the
Asn substitution of Asp262, located at the extracellular
end of TM-VI (Fig. 4A), but not by any of the other Asp or
His substitutions (Table I). In the D171N and the D262N mutants, the
competition curve of AMD3100 against 125I-Met-SDF-1
binding was shifted almost to the position of the cyclam in the
wild-type receptor (Fig. 4, A and B). Moreover, just as cyclam was unable to compete for 125I-12G5 binding
in the wild-type CXCR4 receptor (Fig. 3), AMD3100 was also unable to
compete for 125I-12G5 binding
in the D171N and D262N mutants (Fig. 5
and Table II). The double mutant
D171N/D262N unfortunately had an expression level that was too
low to verify an expected complete loss of binding of AMD3100.
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Table I
Affinity of Met-SDF-1 , AMD3100, and cyclam for the wild-type CXCR4
receptor and His- and Asp-substituted CXCR4 receptor mutants
The data were obtained from competition binding on COS-7 cells
expressing the wild-type and mutant receptors using
125I-Met-SDF-1 as radioligand. Values in parentheses
represent number of experiments (n). ND, not determined. The
D171N/D262N construct showed too low expression to be investigated.
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Fig. 4.
Effect of Asp-to-Asn substitution at
positions 171 and 262 in the CXCR4 receptor on AMD3100
(A) and cyclam (B) competition for
SDF-1 binding. Whole cell competition binding was
performed on wild-type CXCR4 (WT, - - -),
D171N CXCR4 ( ), and D262N CXCR4 ( ) receptors expressed in COS-7
cells using 125I-Met-SDF-1 as radioligand. Data are
shown as means ± S.E. (n = 3-5).
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Fig. 5.
Effect of Asp-to-Asn substitution at
positions 171 and 262 in the CXCR4 receptor on competition for 12G5
antibody binding. Whole cell competition binding was performed on
wild-type CXCR4 (WT; ), D171N CXCR4 ( ), and D262N
CXCR4 ( ) receptors expressed in COS-7 cells using
125I-12G5 as radioligand. Data are shown as means ± S.E. (n = 3-6).
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Table II
Affinity of 12G5 and AMD3100 for the wild-type CXCR4 receptor and His-
and Asp-substituted CXCR4 receptor mutants
The data were obtained from competition binding on COS-7 cells
expressing the wild-type and mutant receptors using 125I-12G5
as radioligand. Values in parentheses represent number of experiments
(n).
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Based on these mutagenesis results and on the assumption that the
cyclam moiety could directly interact with the carboxylic acid group of
Asp residues (2), it was presumed that AMD3100 would bind in between
the transmembrane segments, with one cyclam moiety toward
Asp171 in TM-IV and the other interacting with
Asp262 in TM-VI. This binding mode for AMD3100 was also
compatible with molecular models of the CXCR4 receptor (see
"Discussion"). To probe this binding mode further, a series of
analogs of AMD3100 with different linkers between the two cyclam
moieties were tested in the Asp171 and Asp262 mutants.
Binding Mode of Bicyclam Analogs--
In AMD2763 and AMD2849, the
aromatic phenylenebis(methylene) linker of AMD3100 was substituted with
an aliphatic linker consisting of either three or six methylene groups.
Of these, AMD2849 with the long aliphatic linker behaved almost like
two "free" cyclam molecules, whereas AMD2763 with the short linker
behaved more like the bicyclam AMD3100. Thus, the affinity of AMD2763
was only 3-fold lower than that of AMD3100 as monitored in competition with 125I-Met-SDF-1
binding (Table II), and like
AMD3100, the high affinity binding of AMD2763 was dependent on both
Asp171 and Asp262 (Fig.
6A), although the substitution
at position 171 affected its binding more than the substitution at
position 262. In AMD2849, the extension of the linker between the two
cyclam moieties with three more methylene groups compared with AMD2763
decreased the affinity almost to that observed with cyclam,
i.e. Ki = 2.3 versus 13 µM (these figures are even closer when it is taken in
account that AMD2849 in fact holds two cyclam moieties). Importantly, AMD2849 binding was (again like cyclam) dependent only on
Asp171 and was not affected by the substitution of
Asp262 (Fig. 6B).

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Fig. 6.
Competition binding experiments using AMD2763
(A) or AMD2849 (B) in the wild-type
CXCR4 receptor compared with the D171N and D262N mutants.
125I-Met-SDF-1 competition binding was performed on
wild-type CXCR4 (WT; ), D171N CXCR4 ( ), and D262N
CXCR4 ( ) receptors expressed in COS-7 cells. Data are shown as
means ± S.E. (n = 3).
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In AMD3106 and AMD3108, the meta-disubstituted phenyl group
of the linker of AMD3100 was exchanged with either a 2,6-disubstituted pyridine (AMD3106) or with a 2,4-disubstituted pyridine (AMD3108) (Fig.
1). Of these, the pyridine nitrogen of the 2,4-disubstituted analog
(AMD3108) has a strong tendency to form pendant interactions with the
adjacent macrocyclic (cyclam) ring, which apparently constrains the
conformation of the compound in a way that impairs its function (2,
11). For sterical reasons, this pendant interaction is not possible in
the 2,6-disubstituted analog (AMD3106), which therefore should be able
to function in a manner very similar to AMD3100 (2). In accordance with
this, AMD3106 in the transfected COS-7 cells bound with an affinity
almost identical to that of AMD3100, and its binding was affected by
the Asp171 and Asp262 substitutions in a manner
almost identical to that which was observed for AMD3100 (Fig.
7A). In contrast, the affinity
of the AMD3106 isomer (AMD3108) was decreased 10-fold compared with
AMD3100 and AMD3106, and importantly, the binding of AMD3108 was
dependent only on Asp171 and not on Asp262
(Fig. 7B). Thus, although AMD3108 binds with a higher
affinity than cyclam (albeit still reduces compared with AMD3100), the binding mode of the 2,4-disubstitutted pyridine derivative appears to
be similar to that of a cyclam, i.e. being independent of
Asp262.

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Fig. 7.
Competition binding experiments using AMD3106
(A) or AMD3108 (B) in the wild-type
CXCR4 receptor compared with the D171N and D262N mutants.
125I-Met-SDF-1 competition binding was performed on
wild-type CXCR4 (WT; ), D171N CXCR4 ( ), and
D262N CXCR4 ( ) receptors expressed in COS-7 cells. Data are shown as
means ± S.E. (n = 3).
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A High Affinity Monocyclam Analog--
Deletion of one of the
macrocyclic rings of AMD3100 resulted in a monocyclam analog (AMD3389),
which bound with surprisingly high affinity. AMD3389, which is a cyclam
with the attached para-xylylene group (the "linker" in
AMD3100), displaced 125I-Met-SDF-1
from the wild-type
CXCR4 receptor with an affinity ~100-fold higher than that of the
free tetraazacyclotetradecane moiety, i.e. with an affinity
almost as high as that of the intact AMD3100 molecule
(Ki = 170 versus 74 nM) (Fig.
8A and Table
III). Importantly, the binding
mode of AMD3389 was similar to that of the cyclam, as it was highly
dependent on Asp171 and not impaired by substitution of
Asp262 (Fig. 8B). As the binding of AMD3389 was
not affected by any of the other mutants tested in this study (data not
shown), the structural basis for its high affinity binding compared
with free cyclam could not be further determined here. However, the
data do indicate that the para-phenylenebis(methylene)
linker could contribute substantially to the binding of not only
AMD3389, but possibly also AMD3100. In contrast to the bicyclam
AMD3100, which potently inhibited the binding of the receptor antibody
12G5, and in contrast to the free monocyclam, which did not affect 12G5 binding, AMD3389 surprisingly potentiated the binding of the
125I-12G5 receptor antibody in wild-type CXCR4 with an
EC50 similar to its IC50 for inhibition of
125I-Met-SDF-1
binding (Fig. 8B). This
ability of AMD3389 to potentiate 12G5 binding was impaired in the D171N
mutant and almost eliminated in the D262N mutant.

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Fig. 8.
Competition binding experiments using AMD3389
in the wild-type CXCR4 receptor compared with the D171N and D262N
mutants. Competition binding was performed on the wild-type CXCR4
(WT; ), D171N CXCR4 ( ), and D262N CXCR4 ( )
receptors expressed in COS-7 cells using either
125I-Met-SDF-1 (A) or 125I-12G5
(B) as radioligand. Data are shown as means ± S.E.
(n = 3).
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Table III
Binding of various ligands to the wild-type, D171N, and D262N CXCR4
receptors expressed in COS-7 cells using 125I-Met SDF-1 as
radioligand
Values in parentheses represent number of experiments (n).
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Correlation with Antiviral Activity--
Antiviral potency for the
analogs employed in this study has previously been reported (27),
and a good correlation was found between their antiviral potency and
the affinity measured in competition with the 12G5 receptor antibody
(Fig. 9A). Interestingly,
however, the monocyclam analog AMD3389 clearly fell outside this
correlation, as its high affinity in potentiating 12G5
binding (Fig. 8B) did not correlate with its relatively poor
ability to inhibit HIV-1 cell entry (Fig. 9A). A reasonable
correlation was found also between the antiviral potency of the cyclam
and bicyclam analogs and their affinity as antagonists of SDF-1
binding, r = 0.86 (Fig. 9B). However, in
absolute numbers, the correlation was not really good because the span
of antiviral potencies covered 5 orders of magnitude, i.e.
from 10
9 to 10
4
M, whereas the affinities for these compounds as
competitors of SDF-1
binding only covered 2 orders of magnitude,
from 10
7 to 10
5
M (Fig. 9B). An interesting highly significant
correlation was found between the potency of the compounds in HIV cell
entry assays and their dependence on Asp262 for binding,
again with ADM3389 being a clear exception (Fig. 9C).
However, it should be noted that concerning Asp262, the
affinity of AMD3389 was in fact increased by substitution of
this residue, whereas its ability to potentiate 12G5 binding was eliminated, indicating a complex interaction mode. Nevertheless, for the rest of the compounds, the very clear correlation shown in Fig.
9C indicates that interaction with Asp262 could
be particularly important for the ability of these compounds to
function as anti-HIV agents.

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Fig. 9.
Correlation of HIV-1 antiviral potency and
interaction with the CXCR4 receptor of the different bicyclam analogs
as assessed by linear regression analysis. HIV-1 antiviral potency
(data obtained from Bridger and Skerlj (2)) is plotted against
the binding affinity (Ki) measured either by
125I-12G5 (A) or 125I-Met-SDF-1
(B) or against the difference in binding affinity
(Ki) between the wild-type (WT) and D262N
mutant CXCR4 receptors measured by 125I-Met-SDF-1
(C).
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DISCUSSION |
In this study, the binding of the non-peptide CXCR4 receptor
antagonist AMD3100 has been characterized by combining receptor mutagenesis with studies of chemical analogs of the compound. In
respect to understanding ligand-receptor interactions, AMD3100 is
particularly interesting since it is a relatively simple, symmetric compound consisting of two identical azamacrocyclic structures combined
through a linker (Fig. 1). Thus, based on studies of the free cyclam
moiety and of bicyclam analogs with modified linkers, we propose a
molecular mode of action for AMD3100 in which the two cyclam moieties
bind to two Asp residues, Asp171 and Asp262,
located at the extracellular ends of TM-IV and TM-VI, respectively. This notion is supported by the fact that in a molecular model of the
CXCR4 receptor built over the newly published x-ray structure of
rhodopsin (20) by simple replacement of side chains, AMD3100 could be directly docked into an optimum interaction with these two Asp
residues (Fig. 10).

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Fig. 10.
Molecular model of the main ligand-binding
pocket of the CXCR4 receptor with either AMD3100 (A)
or two cyclam molecules manually docked into favorable interactions
with Asp171 in TM-IV and Asp262 (B)
in TM-VI. The receptor model is built over the rhodopsin model of
Palczewski et al. (20). The conformation of AMD3100 is based
on structural requirements of high antiviral effect of AMD3100 (9, 10)
and the crystallographic x-ray structure of
6,6'-spirobis(1,4,8,11-tetraazacyclotetradecane)-dinickel(II)
tetraperchlorate (30),obtained from the Cambridge Structural Data
Base.
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Proposed Binding Mode for the Bicyclam AMD3100--
The isolated
1,4,8,11-tetrazacyclotetradecane ring (cyclam) has an overall charge of
+2 at physiological pH (12), and x-ray and neutron diffraction
structures have shown that the protonated cyclam ring has the
propensity to form a direct, hydrogen-bonded stabilized complex with
carboxylic acid groups (13). Among the acidic residues located in the
main ligand-binding crevice of the CXCR4 receptor, Asp171
was identified as being important for the ability of the isolated cyclam moiety to compete for SDF-1
binding. Substitution of the other Asp residue in this main pocket, Asp262 in TM-VI, did
not affect binding of the free cyclam. Importantly, however, the
binding of the bicyclam AMD3100 was dependent not only on
Asp171, but also on Asp262. It is possible that
the free cyclam moiety also binds to Asp262, but that its
affinity for Asp262 is relatively low compared with the
binding to Asp171. If so, the binding to Asp262
will not be appreciated in the competition binding experiments. When
the supposedly main binding site for the free cyclam moiety is
eliminated in the D171N mutation, the affinity of the monocyclam was
determined to be ~400 µM. This could possibly represent
the affinity of the cyclam at the Asp262 site, which could
have been confirmed by the D171N/D262N double mutation. However, the
low expression level of this construct unfortunately prohibited this.
Another possibility is that although cyclam binds to
Asp262, this binding could be silent with respect to the
effect on SDF-1
binding. Nevertheless, the fact that the bicyclam
AMD3100 clearly is dependent on both Asp171 and
Asp262 and the fact that cyclam has a strong propensity to
interact with carboxylic acid groups indicate that each of the cyclam
moieties of the bicyclam binds to each of these two Asp residues
located in TM-IV and TM-VI, respectively.
The suggested binding mode for AMD3100 versus cyclam is
supported by the results obtained with analogs having different
linkers. When the aromatic, conformationally constraining
phenylenebismethylene linker of AMD3100 was exchanged with an aliphatic
propylene linker (AMD2763), the affinity of the compound was decreased,
and it became more dependent on Asp171 and less dependent
on Asp262 for competition against SDF-1
, i.e.
more "cyclam-like." When the aliphatic linker was extended by three
more methylene groups (AMD2849), the bicyclam behaved in the binding
assay almost exactly like two molecules of free cyclam (Fig. 6).
Furthermore, the disubstituted pyridine-linked analog, which has a
strong propensity to fold upon itself through pendant interaction of
the pyridine with the neighboring azamacrocyclic ring, was
dependent only on Asp171, i.e. it behaved like
the free cyclam, whereas the isomeric pyridine-linked analog lacking
this ability for pendant interaction bound just like AMD3100 (Fig.
7).
Molecular Model of AMD3100 Binding to the CXCR4
Receptor--
Recently, the first x-ray structure of a 7TM receptor,
bovine rhodopsin, was published by Palczewski et al. (20).
Based on this, residues in the rhodopsin structure located in the main ligand-binding pocket were substituted with the residues occurring in
the CXCR4 receptor. Moreover, we took the liberty of removing the loop
that connects TM-IV and TM-V and that also connects to the top of
TM-III through a disulfide bridge. This loop, which occupies basically
all of the space in the main ligand-binding pocket extracellular to the
retinal ligand in rhodopsin, varies tremendously between receptors and
is expected in "ordinary" 7TM receptors to be relatively moveable
and to give way for ligands. In Fig. 10, Asp171 and
Asp262, located at each "end" of the ligand-binding
pocket of the CXCR4 receptor, have been highlighted. AMD3100 in an
extended conformation corresponding to that previously suggested to be
essential for its antiviral activity (10) could be manually docked into
the main ligand-binding crevice of the CXCR4 receptor in a manner that
positioned each of the cyclam moieties in close proximity to
Asp171 in TM-IV and Asp262 in TM-VI,
respectively (Fig. 10A). If AMD3100 binds like this, how
does it then act as an antagonist? The x-ray structure is based on the
dark-state, inactive conformation of rhodopsin. Thus, conceivably
AMD3100 can interact directly with the corresponding inactive state of
the CXCR4 receptor, as indicated in Fig. 10A. Because of the
conformationally constraining spacer, AMD3100 could simply prevent the
receptor from changing into a yet unknown active conformation. The free
cyclam moiety conceivably can interact with both Asp171 and
Asp262 (Fig. 10B); however, the presumed
interaction with Asp262 is not detected in the binding assay.
Differentiation between Inhibition of Binding of Various CXCR4
Ligands--
The three different ligands for CXCR4, i.e.
the endogenous chemokine SDF-1
, the monoclonal receptor antibody
12G5, and HIV virons or rather the gp120 envelope protein, are not
affected by AMD3100 in an identical manner. Importantly, receptor
mutations affect AMD3100 function differently for the different
ligands. In this study, the non-peptide compounds have mainly been
probed as non-peptide antagonists normally are, i.e. for
their effect on binding of the endogenous receptor ligand, in this
case, the chemokine SDF-1
. However, AMD3100 was originally developed
as a blocker of HIV replication, which turned out to be blockade of HIV
binding to the CXCR4 receptor. Blocking SDF-1
binding, antibody
binding, and binding of the HIV gp120 envelope protein is not
necessarily the same function. For example, in this study, we found
that mutations of Asp182 and Asp193 in
extracellular loop 2 (Fig. 2) have no effect on the ability of AMD3100
to antagonize SDF-1
binding, whereas these residues in an initial
mutational analysis of the CXCR4 receptor have been reported to be
partially important for the ability of AMD3100 to block HIV replication
(14). An interesting observation along these lines is that AMD3389
inhibition of SDF-1
binding is highly dependent on
Asp171, but independent of Asp262, whereas its
potentiation of 12G5 antibody binding is totally dependent
on Asp262 (Fig. 8). One explanation could be that the
monocyclam analog can in fact bind in two fashions (perhaps even at the
same time). In one binding mode, the compound binds mainly to
Asp171 and blocks SDF-1
binding. In the other binding
mode, AMD3389 interacts mainly with Asp262 and thereby
presumably presents the overlying extracellular loop 2, which holds
important epitopes for 12G5 (28) in a favorable way for antibody recognition.
Interaction with Asp262 in TM-VI appears to be an important
feature for this series of compounds with respect to their function as
anti-HIV agents. All of the bicyclam compounds were highly dependent on
interaction with Asp171, but varied in their dependence of
Asp262. Importantly, a strong correlation could be
demonstrated between the antiviral potency of the compounds and the
apparent loss of their binding energy observed when Asp262
was substituted with a non-charged Asn residue (Fig. 9C). It could be interesting to probe whether the good pharmacological properties of AMD3100 and close analogs and the apparent requirement for forming a connection across the main ligand-binding crevice can be
transferred to other classes of CXCR4 antagonists or other chemokine
receptor antagonists in general. It should be emphasized that one of
the hallmarks of AMD3100 is that it has a very broad specificity with
respect to blocking basically all types of X4 envelopes, which
obviously is a requirement for a good anti-HIV drug (29). This compound
has been optimized for its antiviral activity through the cumbersome
testing of every analog in HIV cell entry assays (2). For the moment,
this appears to be the safest approach. Although there is a correlation
between the potency of the compounds as anti-HIV agents and their
potency in inhibiting SDF-1
or 12G5 antibody binding, the
correlation is not close enough, and there are outliers that
deviate many orders of magnitude.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Michael A. Luther and colleagues
at Glaxo Wellcome for providing Met-SDF-1
.
 |
FOOTNOTES |
*
This work was supported by grants from the Danish Medical
Research Council and the 7TM Biotech Competence Center.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Lab. for Molecular
Pharmacology, Panum Institute 18.6.12, Blegdamsvej 3, DK-2200 Copenhagen, Denmark. Tel.: 45-3532-7603; Fax: 45-3535-2995; E-mail: schwartz@molpharm.dk.
Published, JBC Papers in Press, January 11, 2001, DOI 10.1074/jbc.M010429200
 |
ABBREVIATIONS |
The abbreviations used are:
HIV, human
immunodeficiency virus;
SDF, stromal cell-derived factor;
TM, transmembrane domain.
 |
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