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J. Biol. Chem., Vol. 275, Issue 47, 36626-36631, November 24, 2000
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From the Departments of
Received for publication, July 25, 2000, and in revised form, August 31, 2000
Eosinophils have been implicated in the
pathogenesis of asthma and other allergic diseases. Several CC
chemokines including eotaxin (CCL-11), eotaxin-2 (CCL-24), RANTES
(CCL-5), and monocyte chemotactic protein-3 (MCP-3, CCL-7) and 4 (MCP-4, CCL-13) are potent eosinophil chemotactic and activating
peptides acting through CC chemokine receptor-3 (CCR3). Thus,
antagonism of CCR3 could have a therapeutic role in asthma and other
eosinophil-mediated diseases. A high throughput, cellular
functional screen was configured using RBL-2H3 cells stably expressing
CCR3 (RBL-2H3-CCR3) to identify non-peptide receptor antagonists. A
small molecule CCR3 antagonist was identified, SK&F 45523, and chemical
optimization led to the generation of a number of highly potent,
selective CCR3 antagonists including SB-297006 and SB-328437. These
compounds were further characterized in vitro and
demonstrated high affinity, competitive inhibition of
125I-eotaxin and 125I-MCP-4 binding to
human eosinophils. The compounds were potent inhibitors of eotaxin- and
MCP-4-induced Ca2+ mobilization in RBL-2H3-CCR3 cells and
eosinophils. Additionally, SB-328437 inhibited eosinophil chemotaxis
induced by three ligands that activate CCR3 with similar potencies.
Selectivity was affirmed using a panel of 10 seven-transmembrane
receptors. This is the first description of a non-peptide CCR3
antagonist, which should be useful in further elucidating the
pathophysiological role of CCR3 in allergic inflammatory diseases.
The recruitment of inflammatory cells into sites of inflammation
is a normal physiological response designed to fight infection, remove
damaged cells, and stimulate healing. However, the excessive recruitment of such cells often exacerbates tissue damage, slows healing, and in some cases leads to host death. Therefore, inhibition of inflammatory cell recruitment may be an appropriate therapeutic strategy in a number of inflammatory diseases, such as asthma, reperfusion injury, arthritis, and inflammatory bowel disease.
Chemokines are a superfamily of approximately 30 distinct small
secreted proteins, and additional members continue to be identified (1,
2). They are classified into two major groups, CXC and CC, based on the
position of the first two of their four invariant cysteines (3). The
actions of chemokines are mediated via interactions with
7-TM1 G protein-coupled
receptors on the surface of immune and inflammatory cells. To date, 18 unique chemokine receptors, including 11 CC chemokine receptors, have
been cloned (4, 5).
The properties of the chemokines suggest that they are essential for
leukocyte trafficking and inflammatory processes and thus are important
components in a number of disease states (6, 7). Eosinophils are
proinflammatory granulocytes that play a major role in allergic
diseases, such as bronchial asthma (8), allergic rhinitis (9), atopic
dermatitis (10), and eosinophilic gastroenteritis (11). Upon
activation, eosinophils release lipid mediators, cytotoxic proteins,
oxygen metabolites, and cytokines, all of which have the potential to
produce pathophysiology. Recent studies have clearly demonstrated the
presence of eosinophils or eosinophil-specific products in inflamed
lung biopsy tissues in human asthma (10).
Although the molecular mechanism responsible for the selective
infiltration of eosinophils into inflamed tissue has not been elucidated, recently the CC chemokine eotaxin was identified in guinea
pig lung following antigen challenge in sensitized guinea pigs (12,
13). Furthermore, neutralizing antibodies to eotaxin in a mouse model
of allergy demonstrated inhibition of eosinophil recruitment when
administered before the antigen challenge (14). Five CCR3 ligands have
been shown to induce eosinophil transendothelial migration using human
umbilical vein endothelial cells. This migration is inhibited by
pretreatment with anti-CCR3. In addition, a human lung epithelial cell
line (BEAS-2B), stimulated with proinflammatory cytokines, has been
shown to produce eotaxin and MCP-4 (15, 16). In humans, biopsies
obtained from asthmatic lung have shown increased levels of CCR3 and
its ligands, eotaxin, eotaxin-2, RANTES, and MCP-4, both at the
mRNA and protein levels (17). Other data are more controversial,
including targeted deletion of the eotaxin gene, which results in
either a reduction (18) or no effect on eosinophil recruitment
(19).
CCR3 was cloned from a human monocyte (20) or an eosinophil library by
two groups (21, 22) and subsequently shown to bind eotaxin, RANTES, and
MCP-3 (21-23). More recently, eotaxin-2 and MCP-4 (24-26) were also
reported as functional ligands for CCR3. Monoclonal antibodies raised
to CCR3 demonstrate that the receptor is primarily localized to
eosinophils and a subset of Th2 T-cells (21, 22, 27). This restricted
expression pattern may be responsible for the selective recruitment of
eosinophils and Th2 T-cells in allergic inflammatory diseases. In
addition to antibodies, a dual CCR1/CCR3 low molecular weight
compound (28) and a number of proteins (Met-RANTES, vMIP-II,
and MIP-4) have also been reported as antagonists of CCR3 (29-31).
Some of these proteins appear to be specific (30, 31), while others bind multiple chemokine receptors (29).
The above delineated studies provided a rationale to identify selective
antagonists of CCR3, which could be therapeutically beneficial in the
treatment of eosinophil-mediated diseases. Here, the identification and
initial characterization of the first potent and selective non-peptide
small molecule antagonists of CCR3 is described. These compounds were
utilized to demonstrate that inhibition of CCR3 is sufficient to
prevent eotaxin-, eotaxin-2-, and MCP-4-induced eosinophil chemotaxis
in vitro. Potent and selective CCR3 antagonists, like
SB-328437, should be useful to help define the pathophysiological role
of CCR3 and eosinophils in allergic diseases.
Cloning of CCR3
CCR3, which was initially identified from an expressed sequence
tag library, and was obtained from Human Genome Sciences (Rockville, MD). The expressed sequence tag was converted to a full-length cDNA
and subcloned into a mammalian expression vector, pCDN, and stably
expressed in rat basophilic leukemia (RBL-2H3) cells (ATCC, Manassas,
VA). Stable clonal lines, produced by serial dilution in
selection medium containing G418 (400 µg/ml), were screened for
eotaxin-induced Ca2+ mobilization to identify the best
responding cell line.
FLIPR Calcium Mobilization Assay
CCR3 Ca2+ mobilization studies were carried out
using Fluo 3-loaded RBL-2H3-CCR3 cells and a microtiter plate-based
assay using a fluorescent imaging plate reader (FLIPR; Molecular
Devices, Sunnyvale, CA). RBL-2H3-CCR3 cells were grown to confluence in RPMI 1640 medium containing 10% fetal calf serum (Hyclone, Logan, Utah) in T-150 flasks with 5% CO2 at 37 °C. Cells were
removed from the T-150 flask by removing the medium and then treating the cells with 5 ml of Versene (Life Technologies, Inc., Rockville, MD)
for 5 min at room temperature. Cells were washed once in RPMI 1640 medium, 10% fetal calf serum and then plated into sterile 96-well
black ViewPlatesTM (Packard, Meriden, CT) at 40,000 cells/well and incubated for 18-24 h. On the day of assay, the medium
was aspirated and replaced with 100 µl of Earle's mimimal essential
medium with Earle's salts containing L-glutamine, 0.1%
bovine serum albumin, 4 µM Fluo-3 acetoxymethyl ester
(Fluo-3/AM; Molecular Probes, Inc., Eugene, OR), and 1.5 mM
sulfinpyrazone. Plates were incubated for 60 min at 37 °C, medium
was aspirated and replaced with the same medium without Fluo-3/AM, and
plates were incubated for 10 min at 37 °C. Cells were washed three
times and incubated at 37 °C in 100 µl of assay buffer (120 mM NaCl, 4.6 mM KCl, 1.03 mM
KH2PO4, 25 mM NaHCO3,
1.0 mM CaCl2, 11 mM glucose, 20 mM HEPES (pH 7.4) with 1.5 mM sulfinpyrazone.
Plates were placed into FLIPR for analysis as described previously
(32). The maximal change in fluorescence after agonist addition was
quantitated. The percentage of maximal agonist-induced Ca2+
mobilization was determined for each concentration of antagonist, and
the IC50 is defined as the concentration of test compound that inhibits 50% of the maximal response induced by 33 nM
eotaxin (Peprotech, Rock Hill, NJ).
Calcium Mobilization Selectivity Assays
Calcium mobilization assays using freshly isolated monocytes
loaded with Fura-2 and stimulated with RANTES (10 nM),
MCP-1 (2 nM), and MIP-1 Eosinophil Isolation and 125I-Eotaxin and
125I-MCP-4 Binding
Human eosinophils were isolated from peripheral blood of
allergic volunteers (>5% eosinophils) as described previously (33, 34). Binding assays were performed in 96-well microtiter plates containing 1 × 105 human eosinophils/well in a final
volume of 0.1 ml. Assay buffer (RPMI 1640; 25 mM HEPES (pH
7.4), 0.1% gelatin, 0.1% sodium azide, and 0.08% CHAPS) and
compounds were added at the indicated concentrations in a volume of 25 µl; the final Me2SO concentration was <1%.
Binding was initiated by the addition (25 µl) of either
125I-eotaxin (Amersham Pharmacia Biotech; 2,200 Ci/mmol) or
125I-MCP-4 (PerkinElmer Life Sciences; 2,000 Ci/mmol), 0.2 nM final concentration. After a 1-h incubation at room
temperature, the plate was harvested using a 96-well filtermate
harvester (Packard) onto a Unifilter-96 GF/C (Packard) filtermate
blocked with 1% polyethylenimine (Sigma) and washed eight times (0.2 ml/wash) with 20 mM HEPES (pH 7.4) in 0.5 M
NaCl. The filter plate was dried, and the bottom was sealed.
Microscint-20 (Packard) (50 µl) was placed into each well, and the
plate was sealed with Topseal. The plate was counted using a Packard
TopCount NXT.
Other binding assays used to assess selectivity were performed
according to previously published reports (C5a (35) and
LTD4 (36)). The C3a binding was carried out as
described previously (37) except that RBL-2H3-C3a receptor cells were
used (4 × 106 cells/ml) with a final
125I-C3a concentration of 100 pM in a volume of
100 µl. MIP-1 Inhibition of Ca2+ Mobilization in Eosinophils
Human eosinophils, isolated as described above, or RBL-2H3-CCR3
cells were loaded with Fura-2/AM as previously described (40). For
antagonist studies, compounds were added at the indicated concentrations (final Me2SO <0.35%) to 1 × 106 eosinophils/ml in Krebs Ringer Henseleit buffer,
followed 15 s later by agonist at the designated concentration.
The maximal calcium concentration attained after agonist stimulation
was quantitated as described previously (40).
Inhibition of Eosinophil Chemotaxis
Eosinophil motility was determined using a modified Boyden
chamber procedure as described (40). Briefly, for the measurement of
chemotaxis, lower chambers were filled with 30 µl of eotaxin (3.3 nM), MCP-4 (10 nM), or C5a (10 nM)
separated from the upper chamber by a 5-µm polycarbonate filter
(Poretics, Livermore, CA). Into the upper chambers was placed 50 µl
of an eosinophil suspension (3 × 106 cells/ml) in
PAGCM buffer (147 mM NaCl, 5 mM KCl, 20 mM PIPES (pH 7.4) with 0.03% human serum albumin, 0.1%
p-glucose, 1 mM CaCl2, 1 mM MgCl2), with vehicle (control) or in the
presence of antagonist, at the indicated concentrations. Antagonists
were dissolved at 10 mM in Me2SO and diluted in
PAGCM to the desired concentration; the final Me2SO
concentration was <0.1%. Eosinophil migration proceeded for 30 min at
37 °C in a 5% CO2 incubator, after which the chamber
was disassembled. Following fixation of the filter (75% methanol) and
staining (Diff-Quick), the migrated cells were counted in four
successive high power fields.
Synthesis of SK&F-L-45523, SB-297006, and
SB-328437
SK&F-L-45523:
(S)-Ethyl-2-benzoylamino-3-(3,5-dii-odo-4- hydroxyphenyl)propionate
Bis(pyridine)iodonium(I) tetrafluoroborate (41) (0.24 g, 0.64 mmol) was taken up in CH2Cl2 (3.5 ml).
N-Benzoyl-L-tyrosine ethyl ester (0.10 g, 0.32 mmol) was then added, immediately causing the color of the solution to
turn from pale pink to pale yellow. The solution was stirred at room
temperature under nitrogen for 1 h. Aqueous saturated
Na2S2O3 was poured into the flask,
and the product was extracted with CH2Cl2. The
organic portion was dried over MgSO4, filtered, and
concentrated to a yellow oil. Trituration with diethyl ether induced
precipitation of a white solid (0.11 g, 61%) which was filtered,
washed with diethyl ether, and dried. MS (ES+)
m/e 566 [M + H]+, 588.
SB-297006:
(S)-ethyl-2-benzoylamino-3-(4-nitrophenyl)propionate
(S)-Ethyl-2-amino-3-(4-nitrophenyl)propionate
Hydrochloride--
4-Nitro-L-phenylalanine monohydrate
(1.02 g, 4.47 mmol) was suspended in ethanol (20 ml), and HCl gas was
bubbled into the suspension until a clear solution formed. The mixture
was refluxed for 6 h, cooled to room temperature, and concentrated
to yield the title compound as a white solid (1.23 g, 100%). MS (ES+)
m/e 239 [M + H].
(S)-Ethyl-2-benzoylamino-3-(4-nitrophenyl)propionate--
(S)-Ethyl-2-amino-3-(4-nitrophenyl)propionate
hydrochloride (1.23 g, 4.47 mmol) was suspended in dichloromethane (25 ml) and treated with benzoyl chloride (0.628 g, 4.47 mmol) and
triethylamine (1.36 g, 13.4 mmol). The mixture was stirred overnight at
room temperature under nitrogen. The crude reaction was washed with 5%
HCl, water, brine; dried (Na2SO4);
concentrated; and purified by flash chromatography (silica gel, 2:1
hexanes/ethyl acetate) to afford SB-297006 as a white powder (0.865 g,
57%). MS (ES+) m/e 343 [M + H]; melting point
99-101 °C.
SB-328437:
(S)-Methyl-2-napthoylamino-3-(4-nitrophenyl)propionate
(S)-2-Napthoylamino-3-(4-nitrophenyl)propionic Acid--
A
suspension of 4-nitro-L-phenylalanine (5.0 g, 22 mmol) in
water (45 ml) was treated with a solution of sodium hydroxide (0.9 g,
20 mmol) in water (5 ml). 1-Napthoyl chloride (3.8 g, 20 mmol) in
toluene (10 ml) was added followed by a solution of sodium hydroxide
(0.9 g, 20 mmol) in water (5 ml). The mixture was stirred at room
temperature for 30 min and acidified with 1 N sulfuric
acid. The resulting solid was isolated; washed with water, diethyl
ether; and dried to a white powder (5.5 g, 75%).
(S)-Methyl-2-napthoylamino-3-(4-nitrophenyl)propionate--
A
suspension of (S)-2-napthoylamino-3-(4-nitrophenyl)propionic acid (1.0 g, 2.8 mmol) in methanol (12 ml) and water (1 ml) was titrated to pH 7 with 20% aqueous cesium carbonate. The resulting mixture was
evaporated to dryness, and the residue was azeotroped twice from dry
N,N-dimethylformamide. The resulting solid was stirred with iodomethane (0.4 g, 3.1 mmol) in
N,N-dimethylformamide (5 ml) for 3 h before
the addition of water. The precipitated solid was isolated and dried to
a tan powder (0.97 g, 92%). MS (ES+) m/e 378 [M + H]+, 756 [2M + H]+.
A high throughput screen was configured using RBL-2H3-CCR3 cells
and a FLIPR-based 96-well calcium mobilization assay utilizing eotaxin
as the activating ligand (42, 43). One compound identified in this
screen was a diiodotyrosine ester, SK&F-L-45523 (Fig. 1A), which selectively
inhibited eotaxin-induced calcium mobilization in RBL-2H3-CCR3 cells
with an IC50 of 9 µM (data not shown). In the
125I-eotaxin radioligand binding assay, binding to human
eosinophils was inhibited in a concentration-dependent
manner with an IC50 of 800 ± 108 nM (Fig.
2A). The difference in potency
of SK&F-L-45523 between the binding and Ca2+
assay presumably reflects a difference in the configuration of the
assays and the concentrations of eotaxin used in the two assays. Replacement of the L- configuration of tyrosine in
SK&F-L-45523 with the corresponding nonnatural
D-amino acid led to a total loss of activity, suggesting
that the natural antipode was essential for compound interaction with
CCR3.
Chemical modifications to SK&F-L-45523 led to a number of
more potent derivatives including SB-297006 (Fig. 1B) and
the most potent CCR3 antagonist, SB-328437 (Fig. 1C), which
reversibly inhibited 125I-eotaxin binding to human
eosinophils with IC50 values of 60 ± 5.5 and 4.5 ± 1.6 nM, respectively (Fig. 2A). In addition,
all three antagonists inhibited binding of 125I-MCP-4 to
human eosinophils with IC50 values of 360 ± 64 nM, 44 ± 14.4, and 7 ± 0.5 nM for
SK&F-L-45523, SB-297006, and SB-328437, respectively (Fig.
2B).
To determine the specificity of these compounds for CCR3, we assessed
whether these compounds could inhibit the binding of a number of
agonists to their respective cognate 7-TM receptors. At concentrations
up to 33 µM, SB-297006 failed to significantly inhibit
the binding of 125I-IL-8 to CHO-CXCR1 cell or CHO-CXCR2
cell membranes, 125I-SLC (CCL-21) to HEK-293-CCR7
membranes, or [3H]LTD4 to guinea pig lung
membranes (Fig. 3). A number of other receptors were similarly insensitive at a single concentration (10 or
33 µM) of SB-297006 or SB-328437 (Table
I) in Ca2+ mobilization or
binding assays. SB-297006 and SB-328437 were therefore, at a minimum,
250-fold selective for CCR3 over the other 7-TM receptors
tested.
Identification of Potent, Selective Non-peptide CC Chemokine
Receptor-3 Antagonist That Inhibits Eotaxin-, Eotaxin-2-, and
Monocyte Chemotactic Protein-4-induced Eosinophil Migration*
§,,,,
,,, and
Immunology,
¶ Biomolecular Discovery, Pulmonary
Biology, Gene Expression Sciences, and ** Medicinal
Chemistry, SmithKline Beecham Pharmaceuticals,
King of Prussia, Pennsylvania 19406
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(CCL-3, 10 nM) were
utilized to assess functional selectivity of antagonists (33).
binding was carried out using HEK-293 cells
expressing CCR1 (Receptor Biology, Beltsville, MD), which were
reconstituted and used as described by the manufacturer (38). CXCR1 and
CXCR2 binding experiments were carried out as described previously
(39).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (12K):
[in a new window]
Fig. 1.
Structures. A,
SK&F-L-45523,
(S)-ethyl-2-benzoylamino-3-(3,5-diiodo-4-hydroxyphenyl)propionate;
B, SB-297006,
(S)-ethyl-2-benzoylamino-3-(4-nitrophenyl) propionate;
C, SB-328437,
(S)-methyl-2-napthoylamino-3-(4-nitrophenyl)propionate.
View larger version (22K):
[in a new window]
Fig. 2.
A, competition binding of
125I-eotaxin by SK&F-L-45523, SB-297006, and
SB-328437 in human eosinophils. SK&F-L-45523 ( 
),
SB-297006 (
), and SB-328437 (
) inhibit binding with
IC50 values of 800, 60, and 4.5 nM,
respectively. Results are expressed as a percentage of control specific
binding and are the mean ± S.E. of multiple experiments
(n = 3-4). B, competition binding of
[125I]MCP-4 by SK&F-L-45523, SB-297006, and
SB-328437 in human eosinophils. SK&F-L-45523 (),
SB-297006 (), and SB-328437 () inhibit binding with
IC50 values of 285, 44, and 7 nM, respectively.
Results are expressed as a percentage of control-specific binding ± S.E. (n = 3-4).
View larger version (18K):
[in a new window]
Fig. 3.
Competition binding of
125I-eotaxin, [3H]LTD4,
125I-IL-8, 125I-SLC,
125I-MIP-1 , or
125I-C5a by SB-297006 to membranes or whole cells
expressing either cloned or primary receptors. Inhibition of
125I-eotaxin () to human eosinophil,
[3H]LTD4 () to guinea pig trachea,
125I-SLC (
) to HEK-293-CCR7, 125I-MIP-1
() to HEK-293-CCR1, 125I-IL-8 to CHO-CXCR1 (
), and
125I-IL-8 to CHO-CXCR2 (
) membranes by SB-297006 is
shown. Results are expressed as a percentage of control-specific
binding and are the mean of duplicate samples from a typical experiment
of 3-5 performed.
SB-297006 and SB-328437 were tested for their ability to inhibit ligand
binding or function in either whole cell (whole) or membrane (membr.)
assays using the appropriate 125I-labeled (eotaxin, MIP-1,
IL-8, SLC, C3a), 3H-labeled (LTD4) or cold ligands in
functional assays
, and C3a binding assays were performed as
described under "Experimental Procedures." Whole cell calcium
mobilization assays used the ligand and cell types indicated. RANTES,
MCP-1, and MIP-1 were used in freshly isolated monocytes loaded with
Fura-2 (33), while C5a used HEK-293 cells transfected with the C5a
receptor.
To determine if SB-297006 and SB-328437 were functional CCR3
antagonists, we monitored their effects on intracellular calcium mobilization stimulated by eotaxin, eotaxin-2, or MCP-4. In
RBL-2H3-CCR3 cells, SB-297006 and SB-328437 concentration dependently
inhibited calcium mobilization induced by all three CCR3 agonists (Fig. 4, A and B). Thus,
these two antagonists inhibited calcium mobilization induced by
eotaxin, eotaxin-2, or MCP-4 with IC50 values for SB-297006 of 210, 90, and 80 nM and for SB-328437 with
IC50 values of 38, 35, and 20 nM, respectively.
Similarly, in Fura-2-loaded human eosinophils, SB-297006 (data not
shown) or SB-328437 inhibited calcium mobilization induced by eotaxin,
eotaxin-2, or MCP-4, in a concentration-dependent manner
with IC50 values of 35, 52, and 37 nM,
respectively. The compounds did not block the anaphylatoxin C5a-induced
Ca2+ response in eosinophils, IC50 > 1000 nM (Fig. 5).
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Having characterized SB-297006 and SB-328437 as potent and selective
CCR3 antagonists, we evaluated the compounds for their ability to
inhibit human eosinophil chemotaxis in response to maximally effective
concentrations of three CCR3 ligands, eotaxin, eotaxin-2, and MCP-4, or
the anaphylatoxin C5a for selectivity. Using eosinophils from four
individual allergic donors, SB-328437 inhibited eotaxin (3.3 nM)-, eotaxin-2 (3.3 nM)-, and MCP-4 (10 nM)-mediated chemotaxis with similar potencies
(IC50 values of 32, 25, and 55 nM,
respectively) (Fig. 6). The CCR3
antagonist did not block chemotaxis induced by C5a (10 nM)
at concentrations up to 330 nM (Fig. 6).
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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CCR3 is a major chemokine receptor responsible for regulating
eosinophil trafficking. Since CCR3 and its cognate ligands, eotaxin,
eotaxin-2, MCP-4, and RANTES appear to play an important role in asthma
and other allergic diseases, we explored the feasibility of identifying
non-peptide, low molecular weight CCR3 antagonists. Although peptide
antagonists of 7-TM chemokine receptors have been reported (30, 44,
45), these molecules generally suffer from a number of disadvantages
including high metabolism and poor bioavailability. However, a recent
report has indicated that an amino-terminal alanine-methionine swap on
Ck
-7/PARC (CCL-18) resulted in a strong antagonistic chemokine that
specifically blocks CCR3 (30). To date, only five low molecular weight
chemokine receptor antagonists have been reported; these include
antagonists to CXCR2 (39), CXCR4 (46), CCR1 (47), and CCR5 (48) and recently a dual CCR1/3 receptor antagonist (28). Given the apparent central role of CCR3 and its chemokine agonists in eosinophil recruitment and the lack of specific CCR3 antagonists, we set out to
identify, via high throughput screening, a low molecular weight
antagonist of CCR3 that may be useful to define the pathophysiological role(s) of eosinophils. It is anticipated that such a molecule could be
a new therapeutic agent for several allergic eosinophil-mediated diseases.
A new technology has been developing over the last couple years to characterize 7-TM receptor calcium mobilization responses in a high throughput 96-well format. Several recent reports from our laboratories and others have shown the utility of this technology for identifying activating ligands of "orphan" 7-TM receptors (32, 49, 50). In addition, the FLIPR calcium assays have been used to pharmacologically characterize known 7-TM receptors using standard agonists and antagonists (51). In this study, we utilized this technique to configure a high throughput screen to identify novel 7-TM receptor antagonists from an in-house compound bank. To our knowledge, this is the first report utilizing the FLIPR technology and a cellular functional assay to identify selective 7-TM receptor antagonists.
The antagonists identified were capable of inhibiting the binding of eotaxin and MCP-4 to CCR3, suggesting that these antagonists inhibit the receptor-ligand interaction by binding to the chemokine receptor rather than binding to the chemokine itself. Thus, these compounds serve as a starting point to potentially inhibit binding and receptor activation by all CCR3 agonists.
These antagonists were able to inhibit a cellular calcium functional
response induced by three different CCR3 agonists with similar
potencies, to their inhibition of eotaxin binding. Again, this result
demonstrates that a single antagonist can bind the receptor in such a
way that it can inhibit a functional response induced by multiple CCR3
ligands. This would suggest that the CCR3 ligands, which vary in amino
acid identity between 34 and 64%, interact with the receptor in a
similar manner. Furthermore, the results support a pharmacological
approach that may have better disease-modifying activity than an
antibody to a single CCR3 ligand, which would inhibit only a single
agonist and may not deal with the redundant nature of the chemokine
family. Moreover, unlike the recently reported CCR1/3 antagonist (28),
these antagonists were highly selective and did not interact with
several other 7-TM receptors including other chemokine receptors,
indicating that their interaction is specific and presumably
represents a unique site within the receptor for antagonist binding.
Interestingly, the compounds described in the present work are
structurally unrelated to the recently disclosed, nonselective,
aminopiperidine CCR1/3 antagonists (28). The lack of a basic center in
the CCR3-selective compounds reported here coupled with the apparent
requirement for a quaternary nitrogen in the nonselective compounds
suggests that the two series of antagonists may interact with distinct amino acid residue(s) in the receptor. In particular, to confirm selectivity, we studied the binding of 125I-MIP-1 to
CCR1 because CCR1 has the highest homology to CCR3 (62.5% identity)
and is therefore the most likely receptor to be inhibited by these
compounds. In this respect, none of these compounds inhibited
MIP-1
induced Ca2+ mobilization in human monocytes, nor
did they compete with 125I-MIP-1 binding to CCR1
membranes at concentrations up to 10 µM. In addition,
these compounds like UCB 35625 failed to antagonize the binding of
murine or guinea pig eotaxin to murine or guinea pig CCR3 at
concentrations up to 10,000 times the IC50 for inhibiting human CCR3. This indicates that although mouse and guinea pig CCR3 are
67 and 65% identical to human CCR3, respectively, there is still
sufficient divergence in the receptor sequence to prevent effective
binding of these compounds.
The present study represents the first report of a potent and selective
non-peptide functional antagonist of human CCR3 and is the sixth
reported small molecule antagonist of a chemokine receptor (28, 39,
46-48). Although small molecule antagonists of other 7-TM G
protein-coupled receptors including short peptide receptors
(e.g. tachykinin (52), angiotensin (53), and endothelin (54)) have been reported, chemokines and other large protein-agonist interactions with their receptors have been more difficult to antagonize. To our knowledge, the only antagonist reported for a large
peptide receptor other than the chemokine receptors was a micromolar
antagonist of the C5a receptor (55). Since chemokine receptors are part
of the 7-TM receptor family, which have traditionally been productive
targets for drug discovery, it is anticipated that small molecule CCR3
receptor antagonists may have potential as novel therapeutics. The
availability of potent and selective non-peptide antagonists, such as
SB-297006 and SB-328437, will help define the apparent overlap in
activities of the chemokines and their receptors and elucidate their
relative importance. In particular, SB-328437, the most potent CCR3
antagonist, will be an important tool compound to assess the role of
CCR3 in eosinophil recruitment, a process that is thought to be crucial
in the pathology of several inflammatory diseases including asthma,
allergic rhinitis, and eczema.
| |
FOOTNOTES |
|---|
* 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: Dept. of Immunology, SmithKline Beecham Pharmaceuticals, 709 Swedeland Rd., King of Prussia, PA 19406. Tel.: 610-270-4854; Fax: 610-270-5114; E-mail: John_R_White@SBPHRD.COM.
Published, JBC Papers in Press, August 31, 2000, DOI 10.1074/jbc.M006613200
| |
ABBREVIATIONS |
|---|
The abbreviations used are:
7-TM, seven-transmembrane;
MCP-3 and -4, monocyte chemotactic protein-3 and
-4, respectively;
CCR1, CCR3, CCR5, and CCR7, CC chemokine receptor-1,
-3, -5, and -7, respectively;
RANTES, regulated on activation of normal
T cell expressed and secreted;
RBL, rat basophilic leukemia;
MIP-1, macrophage inflammatory protein-1;
C3a and C5a, complement fragment
3a and 5a, respectively;
FLIPR, fluorescence imaging plate
reader;
MS, mass spectrometry;
ES, electrospray;
CHO, Chinese
hamster ovary;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
PIPES, 1,4-piperazinediethanesulfonic acid;
IL, interleukin;
LTD4, leukotriene D4.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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). B, inhibition of Ca2+ mobilization
in RBL-2H3-CCR3 cells by the higher affinity antagonist SB-328437,
stimulated by 3.3 nM of eotaxin (
