From the Departments of Immunology and
§ Pharmaceuticals Discovery, Berlex BioSciences, Richmond,
California 94806 and the ¶ Laboratory of Immunology, NIA, National
Institutes of Health, Baltimore, Maryland 21224
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ABSTRACT |
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The CC chemokines macrophage inflammatory
protein-1 (MIP-1
) and RANTES (regulated on activation normal T
cell expressed) have been implicated in rheumatoid arthritis and
multiple sclerosis. Since their effects are mediated through the CCR1
chemokine receptor, we set up a small molecule CCR1 antagonist program
to search for inhibitors. Through high capacity screening we discovered
a number of 4-hydroxypiperidine compounds with CCR1 antagonist activity and report their synthesis and in vitro pharmacology here.
Scatchard analysis of the competition binding data revealed that the
compounds had Ki values ranging from 40 to 4000 nM. The pharmacological profile of the most potent member
of this series, compound 1 (2-2-diphenyl-5-(4-chlorophenyl)piperidin-lyl)valeronitrite), was
further evaluated. Compound 1 showed
concentration-dependent inhibition of MIP-1
-induced
extracellular acidification and Ca2+ mobilization
demonstrating functional antagonism. When given alone, the compound did
not elicit any responses, indicating the absence of intrinsic agonist
activity. Compound 1 inhibited MIP-1
- and RANTES-induced migration
in peripheral blood mononuclear cells in a dose-responsive manner.
Selectivity testing against a panel of seven transmembrane domain
receptors indicated that compound 1 is inactive on a number of
receptors at concentrations up to 10 µM. This is the
first description of CCR1 receptor antagonists that may be useful in
the treatment of chronic inflammatory diseases involving
MIP-1
, RANTES, and CCR1.
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INTRODUCTION |
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The directed migration of select populations of leukocytes from the circulation to sites of inflammation is an integral part of the immune response. The chemokines are a diverse group of proteins that play an important role in this process (1). They are classified into two major groups, CXC and CC, based on the position of the first two of their four invariant cysteines (2). Each of the chemokines recognizes and induces the chemotaxis of a particular subset of leukocytes. For example, the CXC chemokines, like IL-81 and melonoma growth stimulatory activity, mainly chemoattract and activate neutrophils, in contrast to the CC chemokines, like RANTES and monocyte chemoattractant protein, which preferentially attract T lymphocytes and monocytes and induce their activation by producing changes in cellular morphology, transient increases in cellular calcium concentration, and the up-regulation of surface adhesion proteins.
The chemokines produce their biologic effects by interacting with specific receptors on the cell surface of their target cells (3). To date, 14 different chemokine receptors including eight CC chemokine receptors have been identified by cloning (4, 5). All of these receptors are characterized by a heptahelical structure and belong to a superfamily of serpentine receptors that are coupled to guanine nucleotide-binding proteins (G-proteins) (6).
Occasionally the immune system can turn upon its host, giving rise to
chronic inflammation and disease. Given their important role in this
process, chemokines have been implicated in the pathophysiology of
autoimmune diseases like multiple sclerosis and rheumatoid arthritis.
For example a recent study by Karpus et al. (7) provides
strong in vivo concept validation for a role of MIP-1 in
a mouse experimental autoimmune encephalomyelitis (EAE) model of
multiple sclerosis. These investigators were able to show that antibodies to MIP-1
prevented the development of both acute and relapsing paralytic disease as well as infiltration of mononuclear cells into the central nervous system. Treatment with MIP-1
antibody was also able to ameliorate the severity of ongoing clinical disease. These results led the authors to conclude that MIP-1
plays an important role in this T-cell mediated disease. In addition Godiska et al. (8) have shown an up-regulation of mRNA for a
number of chemokines, including MIP-1
, in EAE.
A number of reports have shown that both RANTES mRNA and protein appear to be up-regulated in rheumatoid arthritis (9, 10). For example, cultured synovial fibroblasts isolated from rheumatoid patients when treated with IL-1 were shown to up-regulate the expression of RANTES both at the mRNA and protein levels (9). Similarly Snowden et al. have used reverse transcriptase-polymerase chain reaction to detect RANTES mRNA in synovial tissue samples from patients with rheumatoid arthritis (10).
Based on these studies there is strong evidence in support of the
concept that the chemokines RANTES and MIP-1 play an important role
in the pathogenesis of multiple sclerosis and rheumatoid arthritis.
Since MIP-1
and RANTES are ligands for CCR1, this receptor, located
on circulating mononuclear cells, is a prime therapeutic target for
these debilitating diseases. These studies then provided the rationale
for establishing a program to inhibit MIP-1
- and RANTES-mediated
biological activities by developing highly potent and specific
non-peptide CCR1 receptor antagonists. We developed an empirical
screening program to discover potential inhibitors of
125I-MIP-1
binding to the CCR1 receptor. We describe
here a series of 4-hydroxypiperidines that possess functional
antagonist activity at the CCR1 receptor. The compounds in this series
have Ki values of inhibition of chemokine binding
ranging from 40 to 4000 nM and are functional
antagonists.
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EXPERIMENTAL PROCEDURES |
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Materials-- Unlabeled chemokines were from Peprotech (Rocky Hill, NJ). 125I-Labeled chemokines were obtained from NEN Life Science Products.
Synthesis of Compounds-- Compounds 1-7 (see Fig. 1) were synthesized in a manner similar to that outlined as follows. To a solution of diphenylacetonitrile (2.0 g, 10.3 mmol) in 100 ml of N,N-dimethylformamide was carefully added sodium hydride (0.5 g, 12.4 mmol, 60% in oil) in small portions. The mixture was stirred at ambient temperature for 15 min, and then 1-chloro-3-iodopropane (3.1 g, 15 mmol) was added in one portion. The mixture was stirred at ambient temperature. After 20 h, the reaction was quenched with 200 ml of water and extracted with two portions of ethyl acetate (200 ml). The combined organic extracts were washed with water (2 × 400 ml) and then brine (400 ml), dried over MgSO4, filtered, and concentrated in vacuo to a yellow oil. Purification by flash column chromatography on silica gel afforded 2.0 g of 5-chloro-2,2-diphenylvaleronitrile as a white solid. This solid (136 mg, 0.48 mmol) was dissolved in 5 ml of N,N-dimethylformamide and 4-phenyl-4-hydroxypiperidine (86 mg, 0.48 mmol) was added, followed by potassium carbonate (330 mg, 2.4 mol, powdered). The resultant suspension was stirred at 45 °C for 20 h and then poured into 20 ml of water and 20 ml of ethyl acetate. The organic layer was separated, dried over MgSO4, filtered, and concentrated in vacuo to a yellow oil. Purification by flash column chromatography on silica gel afforded 140 mg of compound 3 as a clear, colorless oil. The structure and purity of the synthesized compounds were confirmed by proton magnetic resonance spectroscopy, electrospray ionization mass spectrometry, and combustion analysis.
Cell Lines-- The human monocytic cell line THP1 and the human embryonic kidney (HEK) 293 cell line were obtained from the American Type Culture Collection and were maintained in RPMI or Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, penicillin, and streptomycin in a 5% CO2 atmosphere at 37 °C. For binding assays, the cells were harvested and washed once with phosphate-buffered saline (PBS). Cell viability was assessed by trypan blue exclusion, and cell number was determined by counting the cells in a hemocytometer.
CCR1 Expression Vector-- CCR1 cDNA was obtained as described (11) and inserted into a mammalian expression vector containing the SV40 replication origin, the human cytomegaloviral enhancer with the puromycin N-acetyltransferase gene (puromycin resistance) and hygromycin B gene (hygromycin resistance), similar to that described previously (12, 13).
CCR1-expressing Cells--
HEK 293 cells were grown to confluent
monolayers in T flasks (225 cm2). CCR1 plasmid (10 µg)
was added to 125 mM Ca2+-containing Hanks'
balanced salts (final volume 500 µl) and incubated 15 min at room
temperature. Prior to transfection, cells were trypsinized and plated
(2-3 × 106 cells) in 100-mm tissue culture dishes in
growth medium and allowed to recover for 16 h. Growth medium was
then removed and replaced with 4 ml of calcium- and magnesium-free
Hanks' balanced salts (Life Technologies, Inc.), and cells were placed
back in a 37 °C incubator. The DNA mixture was immediately applied
to the HEK 293 cells and further incubated 3-4 h at 37 °C. The
Hanks' balanced salts/DNA mixture was then removed, and the cells were
placed back in growth media for 16 h. Hygromycin was added to
growth media at a final concentration of 150 µg/ml, and puromycin was added back at 5 µg/ml. Cells were grown for 3 weeks, and media were
replaced every 3 days. Once a pooled clone was established, cells were
sorted with an N-terminal CCR1 polyclonal antibody (14) by indirect
fluorescence-activated cell sorting. Cells sorted by
fluorescence-activated cell sorting were placed back in growth media
with hygromycin/puromycin and expanded. Cells were tested for their
ability to bind 125I-labeled MIP-1 and RANTES and for
biological responses by measuring changes in intracellular
Ca2+.
Chemokine Binding Studies--
The high capacity screening
assays were performed in 96-well V-bottom microtiter plates (Nunc,
polypropylene) in a total volume of 100 µl. HEK 293 cells expressing
the human CCR1 receptor (KD = 1-3 nM
and Bmax = 2-3 × 106
sites/cell) were used as the source of the CCR1 receptor. The cells
were detached by trypsinization and plated into flasks at least 48 h prior to the experiment. HEK 293 cells expressing the CCR1 receptor
were detached by shaking, washed once in PBS, and resuspended in the
assay buffer (130 mM NaCl, 5 mM KCl, 1 mM MnCl2, 50 mM Tris, 30 µg/ml
bacitracin, 0.1% bovine serum albumin, pH 7.4) to about 1.1 × 105 cells/ml. Cells (about 8000 cells/well) were incubated
with 125I-MIP-1 (specific activity 2200 Ci/mmol; about
15,000-20,000 cpm/assay) in the presence and absence of varying
concentrations of compounds at room temperature for 30-40 min. The
reactions were terminated by harvesting through a GF/B filter plate
(Packard) presoaked with 0.3% PEI (Sigma catalog number P-3143) plus
0.5% bovine serum albumin and washing five times with cold PBS. The radioactivity in each well was determined by scintillation counting (Topcount Packard) following the addition of 50 µl of scintillation fluid, Scint-20. The nonspecific binding was defined by the binding in
the presence of 100 nM unlabeled MIP-1
. The dose curves
of each compound with six concentration points were generated, and IC50 values were determined by fitting the data to the
log-logit equation (linear) with an EXCEL spread-sheet. The
Ki values were then calculated by dividing the
IC50 by 1.025. For other binding assays, cells (5 × 105 cells/ml) were incubated in PBS with
125I-labeled ligands (0.2 nM) and varying
concentrations of unlabeled ligands or compounds at 4 °C for 1 h. The incubation was terminated by removing aliquots from the cell
suspension and separating cells from buffer by centrifugation
through a silicone/paraffin oil mixture as described previously
(15). Nonspecific binding was determined in the presence of 1 µM unlabeled ligand. The binding data were curve fit with
the computer program IGOR (Wavemetrics) to determine the affinity
(KD), number of receptor sites.
Measurement of Extracellular Acidification with the Microphysiometer-- THP-1 cells were washed once with a modified RPMI 1640 medium (containing 1 mM sodium phosphate, supplemented with 1 mg/ml bovine serum albumin) and resuspended in the same media at a density of 1 × 106 cells/0.1 ml. Cell suspension of 150 µl was mixed with 50 µl of agarose cell entrapment medium (Molecular Devices), and 7 µl of the cell-agarose mixture was spotted into the center of a disposable polycarbonate cell capsules (Molecular Devices) (16). The cell numbers in each well were about 300,000-600,000. To measure the rate of acidification, the assembled cell capsules with the agarose-entrapped cells were loaded into the chambers of the microphysiometer (Molecular Devices). The chambers were perfused with the low buffering media at a rate of 100 µl/min. For each cycle of operation, the cells were perfused with the media for 50 s, and then the flow was interrupted for 40 s, during which the rate of acidification of the media was measured and recorded. The flow was resumed, and the next cycle was begun. The chamber temperature was 37 °C. After stabilization, the cells were perfused with the indicated concentrations of chemokine and/or compound for about 100 s during which the rate of acidification was measured.
Cytosolic Ca2+ Measurements in HEK Cells--
HEK
293 cells expressing the CCR1 receptor were loaded by incubation with 5 µM Fura-2/AM (Molecular Probes, Inc., Eugene, OR) for 30 min at 37 °C in Hanks' balanced salts solution (Life Technologies,
Inc.) containing 3.2 mM calcium chloride. Cells were
pelleted, washed with Hanks' balanced salts solution, and resuspended
in the same medium at a density of 1 × 106 cells/ml.
2 ml of this suspension, or 2 × 106 cells, were used
for each experiment. Changes in the cytosolic free Ca2+
concentration were measured with an SLM/Aminco model AB2 fluorescence spectrophotometer. To determine the reversibility of the CCR1 antagonist, HEK 293 cells expressing CCR1 were loaded with Fura-2/AM as
above and preincubated with 1000 nM compound 1 for 15 min. The cells were then washed twice with 100-fold PBS buffer for 15 min at
37 °C and stimulated with 10 nM MIP-1.
Chemotaxis-- Cell migration was examined using a 48-well microchemotaxis assay as described previously (17). Briefly, various concentrations of chemokines (1.25, 12.5, and 125 nM) were placed in the lower wells of a 48-well microchemotaxis chamber. PBMCs (2 × 106 cells/ml) derived from a normal healthy leukophoresis donor (CIS0003, age 27, male, Caucasian) treated with or without compound 1 at concentrations of 20, 200, and 2000 nM for 30 min at 37 °C were then placed in the upper compartment of the chamber. The upper and lower wells of the chamber were separated by a 5-µm polycarbonate filter coated with laminin (Sigma). The chambers were incubated for 4 h at 37 °C (a time period over which chemokine equilibrium between the upper and lower chambers is optimally achieved) after which the filters were scraped, washed, fixed with methanol, and stained with Diff-Quik. Cell migration was measured by counting the number of cells attached to the lower surface of the filter in six high power fields, and each concentration of chemokine was tested in either triplicate or sets of six wells. The results were expressed as the number of migrating cells per six high power fields (±S.D.).
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RESULTS AND DISCUSSION |
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Since the CCR1 receptor ligands RANTES and MIP-1 appear to play
an important role in the pathogenesis of multiple sclerosis and
rheumatoid arthritis, this receptor is a prime therapeutic target.
Although peptide antagonists of chemokine receptors have been
described, they suffer from poor metabolic stability and oral
bioavailability, thereby limiting their therapeutic utility. For this
reason, we set out to discover potent, selective, small molecule
antagonists of the CCR1 receptor for use as potential therapeutics in
autoimmune diseases like multiple sclerosis and rheumatoid
arthritis.
Seven-transmembrane domain G-protein-coupled receptors have been an
extremely fertile source of biological targets in the pharmaceutical
industry. The actual drugs that interact with this receptor superfamily
are too numerous to detail but include antihistamines, antipsychotics,
antidepressants, antimuscarinics, - and
-adrenoreceptor agonist
and antagonists, opiates, prostanoids, etc. More recently, compound
library screening has proven successful in the discovery of antagonists
of neuropeptide receptors, i.e. Pfizer's substance P
antagonist (18), Merck's CCK antagonists (19), Sterling's bradykinin
antagonist (20), and Sanofi's neurotensin antagonist (21). Thus, based
on these and numerous other additional (endothelin, vasopressin,
angiotensin II, etc.) past successes using an empirical file screening
approach, it seemed reasonable to pursue leads for the CCR1 receptor in
this manner.
The initial approach involved empirical screening of our available
compound libraries to discover potential CCR1 receptor antagonists.
Initial screening of 125I-MIP-1 binding to the CCR1
receptor transfected into the HEK 293 cell line yielded 258 compounds
that demonstrated >50% inhibition of binding at a concentration of 5 µM. Several of these active compounds belonged to the
same structural family, the 4-hydroxypiperidines (Fig.
1) and were investigated in greater
detail.
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The Ki values of these compounds were determined
from concentration-response curves based on their ability to displace the specific binding of 125I-labeled MIP-1 in HEK 293 cells stably expressing the CCR1 receptor. Competition binding studies
for the 4-hydroxypiperidine compounds 1 and 2 (Fig. 1) are shown in
Fig. 2A. Compounds 1 and 2 were able to competitively displace radiolabeled MIP-1
with an
IC50 of 40 and 347 nM, respectively. In
addition, the 4-hydroxypiperidines were shown to displace radiolabeled
RANTES binding from the same transfected cells with similar
IC50 values (Fig. 2B and Table I). These data demonstrate that templates
possessing the 4-hydroxypiperidine moiety are potent inhibitors of
RANTES and MIP-1
binding to the CCR1 receptor.
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The 4-hydroxypiperidine moiety was optimized by synthesizing a number
of analogs (Fig. 1) and examining their ability to inhibit MIP-1
binding to CCR1 (Table I). Several interesting points are worth noting
from this study. First, substitution of the piperidinol phenyl segment
by a halogen (compound 1) improves activity over compound 3, which does
not have a halogen. Other groups such as OMe or CF3 (compounds 2 and 4)
appeared to be less favorable. In addition, the phenyl ring appears to
be important for activity, since replacement by a naphthyl group
(compound 5) reduced potency almost 20-fold, while replacement by a
butyl group (compound 6) lead to an almost total loss of activity.
Interestingly, we were recently made aware of a patent filed by Takeda
Chemical Industries, Ltd. in which they describe a urea piperidine
derivative of the nitrile group (compound 7), which had a reported
IC50 for MIP-1 binding of 5000 nM and a reported IC50 for RANTES binding of 6 nM
(22).
The medicinal chemistry outlined above identified a number of small
molecules as potent inhibitors of chemokine binding to the CCR1
receptor. To determine whether these molecules were also functional
antagonists of the CCR1 receptor, we measured their ability to inhibit
MIP-1- and RANTES-induced biological responses by microphysiometry
(16). The biological activity of MIP-1
was initially assessed by
stimulating THP-1 cells, which have been shown to express CCR1 (23),
with the chemokine and measuring their increase in extracellular
acidification rate. As shown in Fig. 3,
MIP-1
induced a rapid increase in the extracellular acidification rate, reaching a maximum after about 2 min and returning close to
base-line levels within 8-10 min. The MIP-1
effect on THP-1 cells
was dose-dependent, and at 1 nM it produced an
approximately 50-60% increase in extracellular acidification. These
kinetics are quite similar to those reported for chemokines in human
monocytes and in transfected cell lines (24, 25). Pretreatment of THP-1 cells with increasing concentrations of compound 1 dose-responsively inhibited the ability of MIP-1
to induce changes in the metabolic activity of the cells (Fig. 3). The concentration of compound 1 required to half-maximally inhibit 1 nM MIP-1
was 124 nM, about 6-fold higher than that predicted from the
binding constants for compound 1 and MIP-1
, which were 40 and 2 nM, respectively (Fig. 2). Compound 1 was also able to
inhibit RANTES-induced changes in THP-1 cells, similar to the
inhibition demonstrated for MIP-1
(data not shown).
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MIP-1 or RANTES activation of the CCR1 receptor elicits an increase
in the concentration of cytosolic free Ca2+ (11), which was
examined to further evaluate the pharmacologic effects of the CCR1
antagonists in vitro. The change in intracellular Ca2+ concentration in response to a single concentration of
chemokine was measured by fluorimetry using HEK 293 cells transfected
with CCR1 receptors loaded with the indicator Fura-2/AM. As shown in Fig. 4, MIP-1
(10 nM)
produced a transient rise in intracellular Ca2+. When
compound 1 was added to the cells, at concentrations up to 1 µM, in the absence of MIP-1
, no stimulation in
Ca2+ was observed (data not shown), indicating that the
compound had no intrinsic agonist activity. However, preincubation of
compound 1 with CCR1-transfected HEK 293 cells prior to the addition of MIP-1
dose-responsively inhibited the increase in Ca2+
elicited by the chemokine (Fig. 4). The concentration of compound 1 required to half-maximally inhibit 10 nM MIP-1
was
around 200 nM, well in line with that predicted from the
binding constants for compound 1 and MIP-1
(Fig. 2). Compound 1 was also able to effectively inhibit the RANTES-induced
increase in Ca2+ (data not shown). To determine the
reversibility of this effect, cells incubated with 1000 nM
compound 1 were washed twice in PBS to remove the inhibitor and then
stimulated with 10 nM MIP-1
. The washed cells were able
to respond to the chemokine in a manner similar to that of cells
untreated with the antagonist (Fig. 4).
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Further functional characterization of the antagonism by compound 1 was
investigated in cells transfected with the chemokine receptors CCR5 and
CXCR2. CCR5 is a related chemokine receptor that responds to MIP-1,
MIP-1
, and RANTES, while CXCR2 responds to IL-8 and melanoma growth
stimulatory activity as agonists. Importantly, while compound 1 antagonized the effects of RANTES or MIP-1
on the Ca2+
response in cells expressing CCR1, no such attenuation of
MIP-1
- or IL-8-stimulated Ca2+ flux was evident in cells
expressing CCR5 and CXCR2, respectively (Fig.
5). This finding suggests selectivity of
antagonism, consistent with the selectivity of this compound in binding
studies (see below) and that the blockade of the Ca2+
signal is not due to a nonspecific effect.
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Chemokines were originally defined and classified as potent leukocyte
chemoattractants, mediating their effects through G-protein-coupled receptors (26). Thus, we assessed the ability of the CCR1 antagonist compound 1 to attenuate the directed migration of PBMCs stimulated with
a number of CC and CXC chemokines. As shown in Fig.
6, a 16-fold excess of compound 1 dose-responsively inhibited the MIP-1- and RANTES-mediated migration
of PBMCs but had no affect on the migration of PBMCs stimulated with
MIP-1
, monocyte chemoattractant protein, or stromal cell-derived
factor-1
. These data demonstrate that compound 1 is a potent
antagonist for CCR1 but has no effects on the related chemokine
receptors CCR5, CCR2, and CXCR4.
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The specificity of antagonism of the CCR1 receptor antagonists
versus other receptors or biological targets is a critical component to establishing a quality lead compound. This is particularly important, since the CCR1 receptor belongs to a superfamily of seven-transmembrane domain G-protein-coupled receptors that at the
present time numbers well over 450 members and responds to a diverse
array of ligands (27). Thus, the selectivity of the potential CCR1
antagonist compound 1 was tested in a panel of radioligand binding
assays for activity with members of this superfamily of receptors,
including related chemokine receptors and unrelated neurotransmitter
receptors. It is quite encouraging that while the activity of this
compound is under 40 nM at the CCR1 receptor, it is more
than 250-fold less active for most of these other receptors, including
the related chemokine receptor CCR5, which binds RANTES and MIP-1
with high affinity (Table II). The only
potential cross-reactivity of compound 1 was with several
biogenic amine neurotransmitter receptors, a result not surprising for
a structure reminiscent of the typical neuroleptic/antidepressant
structural motif (28, 29).
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Although we have not established the direct mechanism by which these
compounds are able to act as functional antagonists of the CCR1
receptor, we can rule out at least two formal possibilities. Since
MIP-1 and RANTES are able to bind with high affinity to CCR1 and
CCR5 but the CCR1 antagonist compound 1 only inhibited binding of the
ligands to CCR1, these data would tend to rule out inhibition of ligand
binding by direct interaction between the ligand and the compound.
Compound 1 was also unable to inhibit MIP-1
binding to CCR5, IL-8,
or melanoma growth stimulatory factor binding to CXCR2 and stromal
cell-derived factor-1
binding to CXCR4, thus further demonstrating
CCR1 receptor specificity. We can also rule out inhibition due to
toxicity of the compounds, since, in Ca2+ flux experiments,
the addition of the compound alone does not lead to an increase in
intracellular Ca2+ by increasing uptake from the
extracellular milieu as might be observed in cells that were
semipermeable. We favor the idea that our compounds are directly
competing for binding to specific sites on the CCR1 and thus
antagonize the ability of the chemokine ligands to transduce a biologic
effect by receptor blockade. Further studies will be carried out to
determine the nature and site of interaction of the receptor-antagonist
complex.
In summary, we have described here a class of highly potent nonpeptide antagonists of the CCR1 receptor. These compounds appear to be highly specific for the CCR1 receptor and are to our knowledge the first known examples of nonpeptide antagonists of a CC chemokine receptor. Classically, knowledge of the functional role of seven-transmembrane domain receptors has come from pharmacological studies using specific high affinity receptor antagonists. Thus, these compounds should also prove to be useful tools with which to evaluate the role of CCR1 receptors in normal physiology and in disease. Finally, this class of nonpeptide antagonists of the CCR1 receptor should hold promise for the future design of more potent, highly selective agents that could be useful therapeutically in treating autoimmune diseases like rheumatoid arthritis and multiple sclerosis.
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FOOTNOTES |
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* 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: Berlex
BioSciences, Dept. of Immunology, 15049 San Pablo Ave., Richmond, CA
94806. E-mail: Horuk{at}crl.com.
1 The abbreviations used are: IL, interleukin; RANTES, regulated on activation normal T cell expressed; MIP, macrophage inflammatory protein; HEK, human embryonic kidney; PBS, phosphate-buffered saline.
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REFERENCES |
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