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A Small Molecule Antagonist of the Chemokine Receptors CCR1 and CCR3 : Potent Inhibition of

Eosinophil Function and CCR3-Mediated HIV-1 Entry

Ian Sabroe1, Michael J. Peck2, Berend Jan Van Keulen2, Annelies Jorritsma1, Graham Simmons3, Paul R.

Clapham3, Timothy J. Williams1 and James E. Pease1.

1Leukocyte Biology Section, Biomedical Sciences Division, Sir Alexander Fleming Building, Imperial

College School of Medicine, South Kensington, London SW7 2AZ, U.K.

2UCB Pharma R & D, B-1420 Braine l'Alleud, Belgium.

3Wohl Virion Centre, Department of Molecular Pathology and Clinical Biochemistry, University College

Medical School, London W1P 6DB, U.K.

Address correspondence to James Pease, Leukocyte Biology Section, Biomedical Sciences Division, Sir

Alexander Fleming Building, Imperial College School of Medicine, South Kensington, London SW7 2AZ.

Email: j.pease@ic.ac.uk. Phone: +44 020 7594 3162; Fax: +44 020 7594 3119

Running title

A small molecule antagonist of CCR1 and CCR3.

JBC Papers in Press. Published on June 14, 2000 as Manuscript M908864199 by guest on A

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Summary

We describe a small molecule chemokine receptor antagonist, UCB35625, (the

trans-isomer J113863 published by BANYU Pharmaceutical Company, patent

W098/04554), which is a potent, selective inhibitor of CCR1 and CCR3. Nanomolar

concentrations of UCB35625 were sufficient to inhibit eosinophil shape change responses

to MIP-1α , MCP-4 and Eotaxin, whilst greater concentrations could inhibit the

chemokine-induced internalisation of both CCR1 and CCR3. UCB35625 also inhibited

the CCR3-mediated entry of the HIV-1 primary isolate 89.6 into the glial cell line, NP-2

(IC50=57nM). Chemotaxis of transfected cells expressing either CCR1 or CCR3 was

inhibited by nanomolar concentrations of the compound (IC50 values of CCR1:MIP-1α

=9.6 nM , CCR3:Eotaxin= 93.7 nM). However, competitive ligand binding assays on

the same transfectants revealed that considerably larger concentrations of UCB35625

were needed for effective ligand displacement than were needed for the inhibition of

receptor function. Thus, it appears that the compound may interact with a region present

in both receptors that inhibits the conformational change necessary to initiate intracellular

signalling.

By virtue of its potency at the two major eosinophil chemokine receptors,

UCB35625 is a prototypic therapy for the treatment of eosinophil-mediated inflammatory

disorders, such as asthma and as an inhibitor of CCR3 mediated HIV-1 entry.

Keywords

Eosinophil, chemokine receptors, inflammation, antagonist, asthma

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Introduction

A characteristic feature of asthma is a leukocyte infiltrate in the bronchial wall in which eosinophils

predominate (1). Within the bronchial mucosa, eosinophils degranulate, releasing toxic proteins such as major

basic protein, eosinophil cationic protein and eosinophil peroxidase that cause tissue damage, for example,

epithelial injury and shedding (1, 2). Eosinophil activation is thought to play a significant role in the underlying

bronchial hyperreactivity that is a hallmark of human asthma (3,4).

The chemokine family of proteins is central to the regulation of selective leukocyte recruitment (5). A

chemokine with high selectivity for eosinophils was isolated by protein purification of bronchoalveolar lavage

fluid taken from allergen challenged, sensitised guinea pigs, sequenced and named “eotaxin” (6). The cDNA

was subsequently cloned (7). Using this sequence, eotaxin homologues have been identified in several species

including man (8), mouse (9) and rat (10). Eotaxin is expressed at both the mRNA and protein level at sites of

allergic inflammation such as in asthma (11). In models of pulmonary allergic inflammation, eosinophil

accumulation correlates with local eotaxin generation (12, 13). Eotaxin acts both to cause the local recruitment

of eosinophils from the microcirculation and also to enhance the rapid mobilisation of bone marrow eosinophils,

in synergy with IL-5 (14-16). Eotaxin generation is T lymphocyte dependent (17) and recent work has

demonstrated that the Th2 type T cell-derived cytokine, IL-13, which has a significant role in the aetiology of

bronchial hyperreactivity, acts in part by promoting eotaxin generation (18-20).

The eotaxin receptor, CCR3, is a member of the G protein coupled 7-transmembrane receptor family, with

significant sequence homology conserved across several species including human, mouse, and guinea pig (21-

25). Eotaxin and the more recently discovered eotaxin-2 and eotaxin-3 (26, 27) signal exclusively via CCR3.

The CC chemokines MCP-3, MCP-4 and RANTES can also signal via CCR3 but are not selective and can

signal via additional receptors (28). CCR3 expression is highly restricted and is present on cells involved in

allergic inflammation including eosinophils (28), basophils (29) and Th2 type T lymphocytes (30). Eosinophils

express at least three chemokine receptors including CCR3, CCR1 and CXCR2 (31). Of these, CCR3 achieves

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by far the highest expression levels and is thought to be the major eosinophil chemokine receptor. Eosinophils

express lower levels of the closely related receptor CCR1 (21), which binds and signals in response to MIP-1α,

MCP-3 and RANTES.

Little attention has been given to the ability of MIP-1α to mediate eosinophil recruitment, although this

chemokine is also found in the bronchoalveolar lavage fluid of asthmatic subjects (32). However, we have

recently shown that eosinophils from a subgroup of the normal population respond to MIP-1α with similar

potency to eotaxin in an assay of leukocyte shape change measured by flow cytometry (31). Furthermore,

eosinophils from almost all individuals may be readily induced to respond to MIP-1α in vitro (31).

The central role of CCR3 in allergic inflammation has made this receptor a major target for drug

development. It is, however, possible that CCR1 in addition to CCR3, is involved in eosinophil recruitment

into the lungs of asthmatic patients. We report here the effects of a small molecule antagonist of CCR1 and

CCR3, compound UCB35625, which we have used to explore the roles of both receptors in eosinophil

stimulation.

Experimental Procedures

Materials

All chemokines were obtained from Peprotech EC (London UK). Cell culture reagents were purchased

from Life Technologies (Paisley, UK) and general laboratory reagents from Sigma Chemical Co. (Poole, UK).

UCB35625 was reconstituted as a 10 mM stock solution in DMSO and stored at –20˚C. In all experiments,

UCB35625 was diluted further as required in the relevant assay buffer. Where serial dilutions were required,

these were performed in assay buffer containing DMSO to maintain a constant concentration of DMSO

throughout the experiment. FITC-labeled goat anti-mouse polyclonal F(ab')2 were obtained from DAKO (High

Wycombe, UK). IgG1 (clone MOPC 21) and IgG2a (clone UPC 10) control antibodies were obtained from

Sigma Chemical Co. (Poole, UK). Mouse anti-human CD49d (αVLA4-RPE) were obtained from Serotec

(Oxford, UK). Cellfix was obtained from Becton Dickinson (Mountain View, CA). Anti-human CCR1 mAb

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and anti-human CCR3 mAb (7B11) were generous gifts of LeukoSite (Boston, MA; now part of Millenium

Pharmaceuticals). The CXCR4 antagonist AMD3100 was kindly provided by Eriq De Clercq (Leuven,

Belgium).

Stable Transfected cell lines

CCR1 and CCR3 transfectants used the murine pre-B cell lymphoma cell line 4DE4 as a background and were

generated and maintained as previously described (33). Transfectants were cultured with 5mM n-butyric acid

for 18 h before experimentation as previously described (8). The glial cell line, NP2/CD4, stably expressing

CCR3 was kindly provided by Hiroo Hoshino (Gunma, Japan) (34).

Radioligand binding assays

125I-MIP-1α and 125I-eotaxin were purchased from Amersham Pharmacia Biotech with a specific activity

of ~ 2000 Ci/mmol. The cells (2 x106 cells in a final volume of 50 µl) were incubated with 0.1 nM 125I-MIP-

1α or 0.1 nM 125I-eotaxin in buffer (RPMI-1640 + 25 mM HEPES + 0.1% BSA, 0.05% NaN3, pH 7.4) and

varying concentrations of unlabelled chemokines or UCB35625 at room temperature for 60 minutes. The same

buffer containing NaCl was added to a final concentration of 0.5 M NaCl, the samples mixed, and layered onto

silicone oil. The cells were pelleted through the oil by centrifugation (13,000g for 5 minutes) and counted in a

Canberra Packard Cobra 5010 gamma counter (Canberra Packard, Pangebourne, UK). Data are presented

without the subtraction of non-specific binding. Curve-fitting of this and subsquent data was carried out using

the program PRISM and IC50 values obtained by non-linear regression analysis (GraphPad Software, Inc, San

Diego, CA, USA).

Chemotaxis of receptor transfectants

CCR1 or CCR3 transfectants were resuspended in buffer (RPMI-1640 + 25 mM HEPES + 0.1% BSA) at

a concentration of 2.5 x106 cells/ml. 200µl of this suspension (containing 500,000 cells) was placed into

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Transwell inserts (Nunc AS, Roskilde, Denmark) within 24-well tissue culture plates containing 300 µl of

buffer (RPMI-1640 + 25 mM HEPES + 0.1% BSA +/- chemokine), in the presence of varying concentrations of

UCB35625. A concentration of 20 nM eotaxin which gave a maximal response was employed for CCR3-

mediated chemotaxis (typical chemotactic index or C.I. of 20-30). For the CCR1 transfectants, 50 nM MIP-1α

was chosen for CCR1-mediated chemotaxis which, whilst giving a smaller response (typical C.I. 6-8), avoided

the chemotactic response that we previously observed in naïve 4DE4 cells at MIP-1α concentrations of 100 nM

and above (33). The plates were incubated for 5 hours at 37 ˚C, and the chemotactic responses determined

using FACS analysis of cells migrating into the lower chamber as described previously (28).

Leukocyte Gated Autofluorescence/ Forward Scatter (GAFS) assays

GAFS assays were performed as described previously (31). Briefly, polymorphonuclear leukocytes

(PMNL, comprising eosinophils and neutrophils) and peripheral blood mononuclear cells (PBMC) were

purified from citrated blood, freshly isolated from healthy volunteers, over discontinuous platelet-poor plasma/

Percoll gradients (35). For PMNL, the leukocytes were pre-incubated at a concentration of 5 x106 cells/ml for

30 minutes at 37 ˚C in filtered GAFS buffer (PBS containing 0.9 mM CaCl 2, 0.5mM MgCl 2, 10 mM HEPES,

10 mM glucose + 0.1% BSA). The cells were washed and aliquots (5 x105 cells in 40 µl) incubated with 10 µl

of GAFS buffer or varying concentrations of UCB35625 for 5 minutes at room temperature, following which

chemokines or buffer were added (in 50 µl) and the cells incubated for 4 minutes at 37 ˚C in a shaking

waterbath. The cells were placed on ice and rapidly cooled by the addition of 250 µl of ice-cold optimised

fixative and analysed on a FACSCalibur flow cytometer (Becton-Dickinson, Mountain View, CA, USA) as

described previously (31). In some experiments, eosinophil responses to MIP-1α and MCP-4 were upregulated

by pre-incubation of the PMNL in GAFS buffer without Ca2+ and Mg2+ for 90 minutes at 37 ˚C as described

previously. The cells were washed in GAFS buffer containing Ca2+ and Mg2+, and the GAFS assays performed

in the presence of these cations as for the cells pre-incubated for 30 minutes alone. The pA2 value for the

competitive inhibition of eotaxin induced shape change by UCB35625 was calculated using Schild regression

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analysis (36). Calculations of statistical significance were performed using the program PRISM (GraphPad

Software, Inc, San Diego, CA, USA), using ANOVA.

To measure monocyte shape change, PBMC were incubated for 10 minutes at room temperature with a

1:166 dilution of a FITC-labelled anti-CD14 mAb1 (Pharmingen, San Diego, CA, USA) as described previously

(31). Determination of monocyte shape change proceeded as for PMNL above, except that agonist stimulation

was for 10 minutes at 37 ˚C.

Intracellular calcium flux measurements

THP-1 cells were resuspended at 107 cells/ml in PBS without Ca2+ or Mg2+, and were loaded with 2.5 µM

Fura-2 (Molecular Probes, NL) for 30 min at 37°C in the dark. Cells were then washed twice in PBS without

Ca2+ or Mg2+ and resuspended at 1.5 x 106/ml. Cells were placed in a continuously stirred cuvette at 37 °C and

subjected to various concentrations of UCB 35625 followed 60 seconds later by stimulation with various

concentrations of MIP-1α. Data were recorded every 200 ms using a fluorimeter (LS-50B, Perkin Elmer, UK)

as the relative ratio of fluorescence emitted at 510 nm after sequential stimulation at 340 and 380 nm as

previously described (37).

FACS analysis of Chemokine Receptor Internalisation

Freshly isolated PMNL were washed in assay buffer (10 mM PBS with Ca2+ and Mg2+, 10 mM HEPES,

10 mM glucose and 0.1 % BSA) and resuspended in the same buffer to a concentration of 1 x 107 cells/ml.

Aliquots of 5 x 105 cells were mixed with either buffer or chemokine (to a final concentration of 10nM) in the

presence or absence of varying concentrations of UCB35625. Following incubation in a 37° C waterbath for 10

minutes, the cells were transferred to an ice-water bath prior to washing with ice-cold staining buffer (10 mM

PBS without Ca2+ and Mg2+, 10 mM HEPES, and 0.25% BSA) and staining with 50 µl of a primary antibody

(either 3 µg/ml anti-human CCR3 antibody, 10 µg/ml anti-human CCR1 antibody, 3 µg/ml mouse isotype-

matched control IgG2a (kappa UPC 10, Sigma) or 10 µg/ml mouse isotype-matched control IgG1).

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Following incubation for 45 minutes at 4° C, cells were washed once and stained with a FITC (fluorescein

isothiocyanate)-conjugated secondary antibody. Isotype matched control (UPC-10) at a concentration of

50µg/ml was then used in a blocking step, prior to staining with anti-VLA4 conjugated to RPE (rhodamine

phycoerythrin) to allow positive identification of eosinophils. Cells were then fixed using a 1/4 dilution of 1x

Cellfix in cold FACS buffer and the samples analysed on a FACSCalibur flow cytometer (Becton Dickinson,

Mountain View, CA). Data were plotted as the percentages of the basal chemokine receptor level following

incubation with the appropriate chemokine.

Virus Infection Assays

Virus infectivity assays were performed as previously described (38). Briefly, NP2/CD4/CCR3 cells were

cultured overnight in 48 well plates (Costar, U.K.) at a density of 1.5 x 104 cells per well. The following day

the cells were exposed to 75µl of serial dilutions of UCB35625 or AMD3100 (dissolved in DMSO) or medium

(containing an equivalent concentration of DMSO) for 30 minutes at 37°C. 75µl containing 100 focus forming

units of the multi-coreceptor tropic HIV-1 strain, 89.6, was added and the cells were incubated for a further 3

hours at 37°C. Following 3 washes with DMEM + 5% FCS, the cells were incubated for 3 days at 37°C in 0.5

ml of medium containing an appropriate concentration of either antagonist. The cells were fixed in cold

methanol:acetone (1:1) and immunostained with 2 monoclonal antibodies (38:96K and EF7) directed against

HIV-1 p24. Infected foci were visualised using a β-galactosidase conjugated goat anti-mouse antibody and

incubation in X-gal substrate. Focus-forming units per well were then counted using light microscopy.

Results

Eosinophil and Monocyte Shape Change in Response to Eotaxin, MCP-4 and MIP-1α, and its Inhibition

by UCB35625

UCB35625 was synthesised by UCB Pharma Chemistry Department and is the trans-isomer (J113863)

published in BANYU’s patent W098/04554. The structure of the compound is shown in Figure 1. Eosinophil

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shape change responses to a range of eotaxin concentrations as measured in mixed PMNL suspensions, were

dose-dependent and were inhibited by a 5 minute pre-treatment with UCB35625 in a manner characteristic of a

classical competitive antagonist (Figure 2A). An EC50 of 0.2 nM for the eotaxin response was reduced to 1.0

and 27.7 nM by concentrations of 10 and 100nM UCB35625 respectively (Schild analysis revealing a pA2 value

of 8.4). In further experiments, PMNL suspensions were pre-incubated for 90 minutes in the absence of Ca2+

and Mg2+ to up-regulate eosinophil shape change responses to MCP-4 (via CCR3) and MIP-1α via a CCR1

pathway (31). As shown in Figures 2B and 2C, MCP-4 and MIP-1α exhibited very similar activity to that of

eotaxin, inducing almost identical maximal responses in the eosinophils, over the same range of concentrations.

A 100 nM concentration of UCB35625 inhibited the responses of eosinophils to MCP-4, that was similar to the

inhibition of eosinophil responses to eotaxin (Figure 2B). The EC50 for the response to MCP-4 was reduced

from 0.2 nM to 20.5 nM by pre-treatment with 100nM UCB35625. UCB35625 was a more potent inhibitor of

shape change induced by MIP-1α than that induced by either eotaxin or MCP-4; a concentration of 100 nM

UCB35625 completely inhibited the responses of eosinophils to concentrations of 1-20 nM MIP-1α (Figure

2C). Titration of the UCB35625 compound in the same assay, revealed a reduced slope and maxima in

dose/response curves with increasing concentrations of the compound (Figure 2D). This is consistent with the

activity of UCB35625 at CCR1 being non-competitive, whilst its activity at CCR3 has the characteristics of a

competitive antagonist (i.e. parallel right shifts in dose /response curves with increasing compound

concentrations, and no changes in maximal responses).

MIP-1α is also an effective stimulator of peripheral blood monocytes. MIP-1α binds to two receptors

expressed on monocytes, CCR1 and CCR5 (39). Figures 3A and 3B illustrate that monocyte shape change

responses to MIP-1α, but not to the CCR2b-selective ligand MCP-1, were effectively inhibited by UCB35625.

Monocyte responses to a maximal concentration of 20 nM MIP-1α were completely inhibited by a 5 minute

pre-incubation of the leukocytes with both 10 and 100 nM concentrations of UCB35625. Using the monocytic

cell line THP-1, UCB35625 was also observed to be a potent inhibitor of intracellular calcium release in

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response to MIP-1α, being effective at low nanomolar concentrations (Figure 3C). Once more the dose

response curves exhibited a reduced slope and maxima, characteristic of a non-competitive antagonist.

Chemotaxis Mediated by MIP-1α and Eotaxin, and its Inhibition by UCB35625 at Concentrations Which

do not Effectively Compete for Ligand.

We further investigated the ability of UCB35625 to inhibit ligand binding and signalling functions at both

CCR1 and CCR3 using previously described stable transfectants treated with n-butyric acid to enhance receptor

expression (21). In this background, assays of shape change or intracellular calcium flux in response to

chemokines were not possible, but the cells did migrate in response to a chemotactic gradient. Figure 4A shows

that UCB35625 inhibited chemotaxis of the CCR1 transfectants in response to MIP-1α and chemotaxis of the

CCR3 transfectants in response to eotaxin, in a concentration-dependent manner . Chemotaxis mediated by

CCR1 (induced by MIP-1α), was more potently inhibited than that mediated by CCR3 (induced by eotaxin), the

IC50 values being 9.57nM ± 1.31 and 93.8 nM respectively. The naïve 4DE4 murine pre-B cell line, in which

the receptor transfectants were established, exhibited a robust chemotactic response to the human CXCR4-

selective ligand SDF-1α (chemotactic index of 40-70 in response to 20 nM SDF-1α) which was not inhibited

by UCB35625. Figures 4B and 4C illustrate that UCB35625 competed, albeit weakly for the binding of 125I-

MIP-1α to the stable CCR1 transfectants and for the binding of 125I-eotaxin to the stable CCR3 transfectants. In

both cases the compound was unable to displace more than 50% of the labelled ligand at the concentrations

tested, making IC50 calculations impossible. In contrast, unlabelled MIP-1α and eotaxin were able to displace

more than 70% of their respective radioligands with IC50 values of 5.6 nM±1.0 (CCR1 transfectants) and 1.1

nM ±1.3 (CCR3 transfectants), consistent with previously reported data (21, 33, 40).

UCB35625 inhibits Chemokine-Induced Receptor Internalisation

Following ligation, chemoattractant receptors undergo desensitisation rendering them unresponsive to

subsequent stimulation. This is achieved by phosphorylation of the intracellular domains of the receptors by

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kinases such as Protein Kinase A and members of the G-protein receptor kinase (GRK) family. These

phosphorylation events render the receptors susceptible to sequestration by the arrestin proteins and

subsequently to internalisation into the cytoplasm, whereby the receptors are either degraded or recycled back to

the cell membrane (37, 41). Using a FACS based assay employing receptor-specific antibodies, we observed

that treatment of eosinophils with 10 nM of either MIP-1α or eotaxin resulted in the loss of 60-70% of the

resting cell surface levels of either CCR1 or CCR3 respectively (data not shown). We subsequently employed

this assay to assess the ability of UCB35625 to antagonise chemokine-induced receptor internalisation. As can

be seen in Figure 5A, UCB35625 was able to antagonise the internalisation of both CCR1 and CCR3 in

response to their native ligands, MIP-1α and eotaxin, with respective IC50 values of 19.8 nM ±1.7 and 410 nM

±1.65. The compound alone did not induce receptor internalisation (data not shown).

UCB35625 inhibits CCR3-Mediated Viral Entry of the Primary HIV-1 isolate 89.6

CCR3 has been shown by ourselves and others to be a broadly permissive co-receptor for HIV-1 when

expressed in the presence of CD4 (42-44). Using an immunoassay for HIV-1 p24gag previously described (45)

we assessed the ability of UCB35625 to antagonise entry of the HIV-1 primary isolate 89.6 via CCR3 (Figure

5B). This employed a glial cell line NP-2 which stably expresses both CCR3 and CD4. UCB35625 and eotaxin

were potent inhibitors of viral entry, acting in a dose dependent fashion, with IC50 values of 36.0 ng/ml (57.0

nM) and 211.9 ng/ml (25.5 nM) respectively. The CXCR4 antagonist, AMD3100 was used as a negative

control and was shown to be unable to antagonise the cellular entry of the isolate.

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Discussion

The superfamily of G-protein coupled receptors (GPCRs) has provided many key pharmacological targets

amenable to antagonism by small molecules. Amongst many examples are the α and β adrenergic receptors

(antagonists used in the treatment of angina, arrhythmias, and hypertension) and the H1 and H2-histamine

receptors (antagonists used in the treatment of allergic reactions and gastric ulceration). Chemokine receptors

are members of this superfamily which present themselves as exciting targets for the selective manipulation of

leukocyte recruitment in human inflammatory disease (46). Recent studies involving the antagonism of CCR3

in a guinea pig model of allergic airways inflammation, suggest that CCR3 blockade may be a useful

therapeutic strategy for the prevention of eosinophil recruitment e.g. in the context of asthma and allergy (25).

However, there is also some evidence that MIP-1α signalling through CCR1 may play a role in eosinophil

recruitment in man (47). We have recently shown that MIP-1α and eotaxin are equipotent in the induction of

eosinophil responses in a subgroup of individuals (31). In addition, eosinophils from most donors may be

induced to respond to MIP-1α in vitro by a simple pre-incubation in the absence of Ca2+ and Mg2+ (31). Here,

we have characterised the pharmacological activity of a compound, UCB35625, that is a potential inhibitor of

chemokine/eosinophil interactions. UCB35625 is a trans-isomer of a compound J113863, which was originally

identified by scientists at Banyu Pharmaceutical Company as a candidate small molecule chemokine receptor

antagonist.

Employing a sensitive assay of leukocyte stimulation, the GAFS assay, we discovered UCB35625 to be a

potent inhibitor of eosinophil shape change in response to eotaxin and MCP-4; agonists acting on eosinophils

exclusively via CCR3 (28). The responses of donor eosinophils to MIP-1α were up-regulated by pre-incubation

in the absence of Ca2+ and Mg2+. We observed that UCB35625 showed a greater efficacy when impeding the

actions of CCR1, with a 100 nM concentration of UCB35625 being sufficient to completely ablate eosinophil

shape change in response to concentrations of 1-20 nM MIP-1α (Figure 2C). By titration of the UCB35625

concentrations in the same assay, we were subsequently able to observe MIP-1α induced responses in the

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presence of antagonist, albeit with reduced slope and maxima, typical of a non-competitive mechanism of

action (Figure 2D).

Similar concentrations of the compound were used to inhibit MIP-1α induced shape change of human

monocytes. Although human monocytes express two receptors for MIP-1α, CCR1 and CCR5, in assays of

monocyte chemotaxis and enzyme release, MIP-1α is considerably more potent than MIP-1β, a selective CCR5

agonist (48). Shape change of the monocytes in response to the positive control MCP-1, which acts at the

receptor CCR2, was unaffected by 100nM UCB35625 (Figure 3A) showing that the compound exhibits

selectivity. However, shape change of monocytes in response to MIP-1α was ablated by the same

concentration of the compound (Figure 3B). This is consistent with the description of another antagonist of

CCR1, which has shown effective antagonism of monocyte chemotaxis to MIP-1α (49). Similarly, UCB35625

was observed to inhibit MIP-1α induced intracellular calcium release in the monocytic cell line THP-1 (Figure

3C). No intracellular calcium release was observed in response to 50 nM MIP-1β, suggesting that the response

to MIP-1α in THP-1 cells is mediated via CCR1 and hence, the antagonist activity of UCB35625 is directed at

CCR1.

To characterise further the receptor specificity and mechanisms of action of UCB35625, we employed

clones of the murine pre-B cell line 4DE4 which had been stably transfected with either CCR1 or CCR3. These

lines demonstrate a chemotactic response to MIP-1α and eotaxin respectively (33). Increasing concentrations of

UCB35625 readily inhibited chemotaxis of both CCR1- and CCR3-bearing cell lines in response to their

respective ligands, with CCR1 mediated chemotaxis being approximately ten-fold more sensitive to the

compound; (IC50 values of 9.57 nM and 93.70 nM respectively, Figure 4A). In both cell lines the maximal 1µM

concentration of UCB35625 was able to reduce the levels of migrating cells to those attracted with buffer alone.

Chemotaxis of the naïve 4DE4 cells to SDF-1α (the ligand for constitutively expressed CXCR4) was unaffected

by similar doses of the compound, again underscoring the selectivity of the compound. To investigate the nature

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of this inhibition further, we subjected the same transfectant cell lines to radioligand binding assays; cells which

we had previously shown bound their respective ligands with nanomolar affinity (33). Both labelled eotaxin

and labelled MIP-1α could be readily displaced from CCR1-and CCR3-bearing transfectants, respectively, by

increasing concentrations of the same unlabelled ligand (Figures 4B and 4C) with IC50 values in the low

nanomolar range for both ligands. UCB35625 was able to displace radiolabelled MIP-1α from CCR1 and

radiolabelled eotaxin from CCR3, albeit much less efficiently than the respective unlabelled ligand. Even at

concentrations of 10 µM, the compound could only displace approximately 40% of either radiolabelled ligand,

rendering calculation of an IC50 impossible. A previous study has suggested that buffer pH and ionic strength

can markedly influence chemokine binding and the subsequent biological activation of CCR3 (50). However,

since we employed identical buffers for both chemotaxis and binding experiments (with the addition of 0.05%

NaN3 in the binding buffer), it is unlikely that our observed dissolution of blockade of ligand binding from

antagonist activity is due to the physiological composition of the buffers. The patent W098/04554 cites the

actions of the Banyu compound J113863 as having affinities for human CCR1 and CCR3 at low- and sub-nM

concentrations respectively. It is possible that CCR1 and CCR3 as expressed in our transfectant cell lines, are

subtly different from their native conformations in leukocytes (or other transfectants described in the patent

literature), perhaps through variations in post-translational modifications such as glycosylation and sulfation

that are known to modulate chemokine binding (51). Such subtle differences may account for the differences in

the observed ability of UCB35625 to compete for ligand at CCR1 and CCR3. However we also observed

apparently non-competitive inhibition of MIP-1α signalling by UCB35625 in native leukocytes (Figure 2C &

2D). Taken together, these data are consistent with the hypothesis that the antagonist is acting at a location

within CCR1 that is distinct from the ligand binding site and is primarily an effective antagonist at both CCR1

and CCR3 by virtue of its ability to block receptor activation, rather than by simply antagonising ligand

binding.

UCB35625 was able to block chemokine-induced receptor internalisation (Figure 5A); again the

compound was more potent at CCR1 than CCR3. Interestingly, the concentrations of UCB35625 necessary to

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impede chemokine induced internalisation of either CCR1 or CCR3, correlates more closely with the

concentrations of compound necessary for the blockade of the respective ligand binding to each receptor and

not to the concentrations of UCB35625 required for the inhibition of biological responses. This suggests that a

low threshold of receptor signalling is required to initiate receptor internalisation and that only the almost

complete ablation of this signal results in inhibition of the receptor internalisation process. This is feasible

because the activation of one receptor kinase may ultimately lead to the phosphorylation and internalisation of

several receptors. UCB35625 alone was unable to induce either CCR1 or CCR3 internalisation at

concentrations of up to 100 nM (data not shown), in contrast to the high molecular weight antagonist AOP-

RANTES which at similar concentrations can induce the internalisation of both CCR1 and CCR3 (52).

Using the glial cell line NP-2 stably expressing both CCR3 and CD4, we were able to investigate the

ability of UCB35625 to inhibit entry of the HIV-1 primary isolate 89.6 (Figure 5B). Both UCB35625 and the

native ligand eotaxin were potent inhibitors of viral entry with EC50 values in the nanomolar range. UCB35625

inhibited the infection of the transfected cells over a concentration range similar to that effective against

chemokine-induced cell stimulation, rather than the higher concentration range necessary to observe ligand

displacement. This may be because intracellular signalling underlies the infection process in this system.

Studies of the CCR5:gp120 interaction suggest that the virus binds to the receptor in a two-step model, with the

initial step being gp120 binding to the amino-terminus, which can be blocked by CCR5 amino-terminal specific

antibodies (53). The second step appears to involve the second extracellular loop of CCR5. Monoclonal

antibodies specific for this region of CCR5 do not appear to block HIV-1 binding, but do inhibit infection,

suggesting that the latter interaction may involve either the induction of receptor signalling or a conformational

change in the virus envelope that allows fusion (54). These sites on CCR5 appear to partially overlap with the

chemokine binding sites of the receptor and their blockade by CCR5 -specific chemokine is effected by

inhibition of the initial step of gp120 binding to the receptor.

The data presented here suggest that whilst eotaxin appears to be a potent inhibitor of 89.6 entry,

(presumably by either removing CCR3 from the cell surface or by blocking gp120 binding), UCB35625 may

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block a second interaction by inhibiting a conformational change in the receptor necessary for fusion or for

receptor signalling. It is of note that whilst high nanomolar doses of eotaxin were able to completely inhibit

viral entry, even at micromolar concentrations of UCB35625, virus was still able to enter the cells, suggesting

that this second interaction is not an absolute requirement for 89.6 entry in this system.

In summary, using biological assays of cellular activation employing both primary cells and artificial

transfectants, we have shown that the inhibitory effects of UCB35625 at both CCR1 and CCR3 are specific and

potent. However, concentrations of UCB35625 that were inhibitory in functional assays were relatively poor at

displacing bound ligand from either receptor. These data suggest that UCB35625 acts upon a region present in

both receptors that only partially interferes with the ligand binding sites of CCR3, if at all with those of CCR1.

Data from a previous study employing chimeric CCR1 and CCR3 receptors to map ligand selectivity

determinants were consistent with a multi-site model for chemokine-chemokine receptor interactions, in which

one or more subsites determine chemokine selectivity, but others are needed for receptor activation (33). A

similar model has also been proposed for the interaction of MCP-1 with its receptor CCR2b (55) and also for

the interaction of the chemoattractant C5a and its receptor (56). CCR1 and CCR3 share significant homology

at the protein level (54% identity), with the majority of conservation occurring in the transmembrane helices. A

recent study has postulated a general switch mechanism for the signal transduction of GPCRs based on

mutagenesis studies of the C5aR transmembrane regions (57). Two distinct clusters of residues in the

transmembrane helices were identified that appear to be involved in the signalling mechanism, a ligand-binding

pocket at or near the extracellular membrane interface and a core cluster in the cytoplasmic bundles of the

receptor. This latter cluster is conserved in distantly-related GPCRs and is postulated to transmit the

conformational change induced upon ligand binding to the intracellular G proteins. Since it is likely that this

region is accessible to small molecules, one possibility is that the interaction of UCB35625 with this cluster in

both CCR1 and CCR3 interferes with the signal transduction process normally activated upon chemokine

binding.

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By virtue of its potent, specific inhibition of function of CCR1 and CCR3, the major eosinophil

chemokine receptors, UCB35625 shows promise as a lead compound for the design of future therapeutics,

which may be of use in the treatment of allergic inflammatory diseases such as asthma and also the blockade of

viral entry by HIV strains that utilise CCR3. Future mutagenesis studies of both receptors should pinpoint the

precise sites of action of this compound.

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Figure Legends

Figure 1. The structure of UCB35625. UCB35625 was synthesised in house at UCB Pharma SA, Research and

Development, Chemin du Foriest, Braine-l’Alleud, Belgium. It is the trans-isomer, J113863, patented by

BANYU Pharameutical Co, Japan.

Figure 2. Inhibition of eosinophil shape change responses to chemokines. Mixed PMNL comprising of

neutrophils and eosinophils were pretreated with control buffer or varying concentrations of UCB35625 at room

temperature, stimulated for 4 minutes at 37 °C with buffer or chemokines, and the resulting eosinophil shape

change measured by FACS as described. Panel A shows the eosinophil shape change in response to eotaxin

(closed circles, EC50=0.18nM ± 1.82) which was inhibited by pretreatment with 10 nM UCB35625 (open

circles, EC50=0.96 nM ± 1.42, p <0.01) and 100 nM UCB35625 (closed squares, EC50=27.71 nM ± 1.58,

p<0.01). In panels B and C, the PMNL were preincubated for 90 minutes in the absence of Ca2+ and Mg2+ to

upregulate their chemokine responsiveness as described, then re-equilibrated with standard buffer containing

Ca2+ and Mg2+ prior to determination of eosinophil shape change. Panel B shows the eosinophil shape change

response to MCP-4 (closed circles, EC50=0.19 nM ± 2.3) and its inhibition in the presence of 100 nM

UCB35625 (closed squares EC50=20.51 nM ± 1.34. p<0.05). Panel C shows the eosinophil shape change

response to MIP-1α (circles) and its complete inhibition in the presence of 100 nM UCB35625 (squares, p

<0.05). None of the chemokine or UCB35625 treatments caused detectable neutrophil shape change. Data

shown is mean ± SEM from separate donors, n=4 for panels A and C and n=3 for panel B. In Panel D,

illustrates eosinophil shape change in response to varying concentrations of MIP-1α (0.1 – 100 nM) in the

presence of buffer (diamonds, dotted line), 1 pM UCB35625 (filled squares), 10 pM UCB35625 (triangles), 100

pM UCB35625 (circles), and 1,000 pM UCB35625 (open squares). Data shown is from a single individual and

is representative of 5 separate experiments.

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Figure 3. Inhibition of monocyte responses to MIP-1α by UCB35625. Purified PBMCs were labelled with

FITC-conjugated anti-CD14 mAb to identify monocytes, pretreated with control buffer or varying

concentrations of UCB35625 at room temperature, stimulated for 10 minutes at 37 °C with buffer or

chemokines, and the resulting monocyte shape change measured by FACS as described. Panel A shows the

monocyte shape change in response to MCP-1 in the presence of buffer (filled circles) and 10 nM (open circles)

and 100 nM (filled squares) of UCB35625 (no significant difference). Panel B shows the monocyte shape

change in response to MIP-1α (filled circles) which was inhibited by pretreatment with 10 nM (open circles)

and 100 nM (filled squares) of UCB35625. Data shown is mean ± SEM from 3 separate donors, p<0.01. Panel

C shows intracellular calcium release by the monocytic cell line THP-1 in response to MIP-1α after 60 seconds

pretreatment with buffer (open circles), 1nM UCB35625 (closed circles), 5nM UCB35625 (open squares) and

20nM UCB35625 (closed squares). Data shown is from a single experiment, representative of three separate

experiments.

Figure 4. UCB35625 is a potent inhibitor of chemotaxis mediated via CCR1 and CCR3 but does not effectively

disrupt their ligand binding sites.

Panel A shows chemotaxis of 4DE4 cells to various chemokines. The open circles represent CCR3 expressing

4DE4 cells migrating in response to 20nM eotaxin (IC50=93. 75 nM ± 1.43) and the closed circles represent

CCR1 expressing 4DE4 cells migrating in response to 50nM MIP-1α (IC50=9.57 nM ± 1.31). The closed

triangles represent naïve 4DE4 cells migrating in response to 20nM SDF-1α which were unaffected by

UCB35625 treatment. Panel B shows the displacement of 0.1nM 125I-MIP-1α from CCR1 transfectants with

increasing concentrations of either cold MIP-1α (open circles, IC50=5.6 nM±1.0) or UCB35625 (closed

circles). Data shown are the compiled mean data ± SEM from 4 separate experiments. Panel C shows the

displacement of 0.1nM 125I-eotaxin from CCR3 transfectants with increasing concentrations of either cold

eotaxin (open circles, IC50= 1.1 nM ±1.3) or UCB35625 (closed circles).

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Figure 5. UCB35625 is an Inhibitor of Chemokine Receptor Internalisation and HIV-1 entry

Panel A shows the inhibition of both MIP-1α induced CCR1 internalisation (open circles) and eotaxin induced

CCR3 internalisation (closed circles) by UCB35625 as measured by FACS analysis on purified PMNL using

CCR1 and CCR3 specific antibodies in conjunction with a VLA-4 specific antibody to positively identify

eosinophils. IC50 values were 19.8 nM ±1.7 (MIP-1α induced CCR1 internalisation) and 410 nM ±1.6 (eotaxin

induced CCR3 internalisation). Data shown are from a typical experiment, representative of five separate

experiments. UCB35625 alone was unable to induce either CCR1 or CCR3 internalisation even at

concentrations of up to 100 nM (data not shown). Panel B demonstrates entry of the primary isolate 89.6 into

NP-2 glial cells expressing CCR3 and CD4 and its antagonism by UCB35625 (open circles), eotaxin (closed

circles) and the CXCR4 antagonist AMD3100 (open squares). IC50 values are UCB35625 36.0 ng/ml (57.0 nM)

and eotaxin 211.9 ng/ml (25.5 nM) respectively. Data shown are from a single experiment representative of 3

individual experiments.

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Footnote

1The abbreviations used are mAb, monoclonal antibody; FACS, fluorescence activated cell sorting; GAFS,

gated autofluorescence/forward scatter; GPCR, G protein coupled receptor; PNML, polymorphonuclear

leukocyte.

Acknowledgements

We thank Professors J. Caldwell and Dr Y. Bakhle for their critical reading of the manuscript. We

gratefully acknowledge the financial support of the Imperial College School of Medicine (IS), The Wellcome

Trust (JEP, Programme Grant Number 038775/Z/96/A), The Medical Research Council (GS and PC) and The

National Asthma Campaign (TJW).

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Reference List

1. Spry, C.J., Kay, A.B., and Gleich, G.J. (1992) Immunol.Today 13, 384-387

2. Bousquet, J., Chanez, P., Lacoste, J.Y., Barneon, G., Ghavanian, M.N., Enander, I., Venge, P., Ahlstedt,S., Simony-Lafontaine, J., Godard, P., and Michel, P.-B. (1990) N.Engl.J.Med. 323, 1033-1039

3. Flavahan, N.A., Slifman, N.R., Gleich, G.J., and Vanhoutte, P.M. (1988) Am.Rev.Respir.Dis. 138, 685-688

4. Venge, P., Dahl, R., Fredens, K., and Peterson, C.G. (1988) Am.Rev.Respir.Dis. 138, S54-7

5. Luster, A.D. (1998) New Engl.J.Med. 338, 436-445

6. Jose, P.J., Griffiths-Johnson, D.A., Collins, P.D., Walsh, D.T., Moqbel, R., Totty, N.F., Truong, O.,Hsuan, J.J., and Williams, T.J. (1994) J.Exp.Med. 179, 881-887

7. Jose, P.J., Adcock, I.M., Griffiths-Johnson, D.A., Berkman, N., Wells, T.N.C., Williams, T.J., and Power,C.A. (1994) Biochem.Biophys.Res.Commun. 205, 788-794

8. Ponath, P.D., Qin, S., Ringler, D.J., Clark-Lewis, I., Wang, J., Kassam, N., Smith, H., Shi, X., Gonzalo, J.-A., Newman, W., Gutierrez-Ramos, J.-C., and Mackay, C.R. (1996) J.Clin.Invest. 97, 604-612

9. Rothenberg, M.E., Luster, A.D., and Leder, P. (1995) Proc.Natl.Acad.Sci.USA 92, 8960-8964

10. Williams, C.M.M., Newton, D.J., Wilson, S.A., Williams, T.J., Coleman, J.W., and Flanagan, B.F. (1998)Immunogenetics 47, 178-180

11. Ying, S., Robinson, D.S., Meng, Q., Rottman, J., Kennedy, R., Ringler, D.J., Mackay, C.R., Daugherty,B.L., Springer, M.S., Durham, S.R., Williams, T.J., and Kay, A.B. (1997) Eur.J.Immunol. 27,3507-3516

12. Humbles, A.A., Conroy, D.M., Marleau, S., Rankin, S.M., Palframan, R.T., Proudfoot, A.E., Wells, T.N.,Li, D., Jeffery, P.K., Griffiths-Johnson, D.A., Williams, T.J., and Jose, P.J. (1997) J Exp Med 186,601-612

13. Li, D., Wang, D., Griffiths-Johnson, D.A., Wells, T.N.C., Williams, T.J., Jose, P.J., and Jeffery, P.K.(1997) Eur.Respir.J. 10, 1946-1954

14. Collins, P.D., Marleau, S., Griffiths-Johnson, D.A., Jose, P.J., and Williams, T.J. (1995) J.Exp.Med. 182,1169-1174

15. Palframan, R.T., Collins, P.D., Severs, N.J., Rothery, S., Williams, T.J., and Rankin, S.M. (1998) J ExpMed 188, 1621-1632

16. Palframan, R.T., Collins, P.D., Williams, T.J., and Rankin, S.M. (1998) Blood 91, 2240-2248

17. MacLean, J.A., Ownbey, R., and Luster, A.D. (1996) J.Exp.Med. 184, 1461-1469

by guest on August 31, 2020

http://ww

w.jbc.org/

Dow

nloaded from

23

18. Wills-Karp, M., Luyimbazi, J., Xu, X., Schofield, B., Neben, T.Y., Karp, C.L., and Donaldson, D.D.(1998) Science 282, 2258-2261

19. Zhu, Z., Homer, R.J., Wang, Z., Chen, Q., Geba, G.P., Wang, J., Zhang, Y., and Elias, J.A. (1999) J ClinInvest 103, 779-788

20. Li, L., Xia, Y., Nguyen, A., Lai, Y.H., Feng, L., Mosmann, T.R., and Lo, D. (1999) J Immunol 162, 2477-2487

21. Ponath, P.D., Qin, S., Post, T.W., Wang, J., Wu, L., Gerard, N.P., Newman, W., Gerard, C., and Mackay,C.R. (1996) J.Exp.Med. 183, 2437-2448

22. Combadiere, C., Ahuja, S.K., and Murphy, P.M. (1995) J.Biol.Chem. 270 [Erratum: 30235], 16491-16494

23. Post, T.W., Bozic, C.R., Rothenberg, M.E., Luster, A.D., Gerard, N., and Gerard, C. (1995) J.Immunol.155, 5299-5306

24. Gao, J.-L., Sen, A.I., Kitaura, M., Yoshie, O., Rothenberg, M.E., Murphy, P.M., and Luster, A.D. (1996)Biochem.Biophys.Res.Commun. 223, 679-684

25. Sabroe, I., Conroy, D.M., Gerard, N.P., Li, Y., Collins, P.D., Post, T.W., Jose, P.J., Williams, T.J., Gerard,C., and Ponath, P.D. (1998) J Immunol 161, 6139-6147

26. Forssmann, U., Uguccioni, M., Loetscher, P., Dahinden, C.A., Langen, H., Thelen, M., and Baggiolini, M.(1997) J.Exp.Med. 185, 2171-2176

27. Shinkai, A., Yoshisue, H., Koike, M., Shoji, E., Nakagawa, S., Saito, A., Takeda, T., Imabeppu, S., Kato,Y., Hanai, N., Anazawa, H., Kuga, T., and Nishi, T. (1999) J Immunol 163, 1602-1610

28. Heath, H., Qin, S., Wu, L., LaRosa, G., Kassam, N., Ponath, P.D., and Mackay, C.R. (1997) J.Clin.Invest.99, 178-184

29. Uguccioni, M., Mackay, C.R., Ochensberger, B., Loetscher, P., Rhis, S., LaRosa, G.J., Rao, P., Ponath,P.D., Baggiolini, M., and Dahinden, C.A. (1997) J.Clin.Invest. 100, 1137-1143

30. Sallusto, F., Mackay, C.R., and Lanzavecchia, A. (1997) Science 277, 2005-2007

31. Sabroe, I., Hartnell, A., Jopling, L.A., Bel, S., Ponath, P.D., Pease, J.E., Collins, P.D., and Williams, T.J.(1999) J.Immunol 162, 2946-2955

32. Alam, R., York, J., Boyars, M., Stafford, S., Grant, J.A., Lee, J., Forsythe, P., Sim, T., and Ida, N. (1996)Am.J.Respir.Crit.Care Med. 153, 1398-1404

33. Pease, J.E., Wang, J., Ponath, P.D., and Murphy, P.M. (1998) J.Biol.Chem. 273, 19972-19976

34. Soda, Y., Shimizu, N., Jinno, A., Liu, H.Y., Kanbe, K., Kitamura, T., and Hoshino, H. (1999) BiochemBiophys Res Commun 258, 313-321

35. Haslett, C., Guthrie, L.A., Kopaniak, M.M., Johnston, R.B., and Henson, P.M. (1985) Am.J.Pathol. 119,101

by guest on August 31, 2020

http://ww

w.jbc.org/

Dow

nloaded from

24

36. Arunlakshana, O. and Schild, H. O. Some quantitive uses of drug antagonists. The British Journal ofPharmacology 14, 48-58. 59. (GENERIC)Ref Type: Generic

37. Sabroe, I., Williams, T.J., Hebert, C.A., and Collins, P.D. (1997) J.Immunol. 158, 1361-1369

38. Reeves, J.D. and Simmons, G. (2000) in Chemokine Protocols (AnonymousHumana Press, Towata, USA

39. Rollins, B.J. (1997) Blood 90, 909-928

40. Neote, K., DiGregorio, D., Mak, J.Y., Horuk, R., and Schall, T.J. (1993) Cell 72, 415-425

41. Ali, H., Richardson, R.M., Haribabu, B., and Snyderman, R. (1999) J Biol Chem 274, 6027-6030

42. Choe, H., Farzan, M., Sun, Y., Sullivan, N., Rollins, B., Ponath, P.D., Wu, L., Mackay, C.R., LaRosa, G.,Newman, W., Gerard, N., Gerard, C., and Sodroski, J. (1996) Cell 85, 1135-1148

43. He, J., Chen, Y., Farzan, M., Choe, H., Ohagen, A., Gartner, S., Busciglio, J., Yang, X., Hofmann, W.,Newman, W., Mackay, C.R., Sodroski, J., and Gabuzda, D. (1997) Nature 385, 645-649

44. Alkhatib, G., Berger, E.A., Murphy, P.M., and Pease, J.E. (1997) J Biol Chem 272, 20420-20426

45. Clapham, P.R., McKnight, A., and Weiss, R.A. (1992) J Virol 66, 3531-3537

46. Gura, T. (1996) Science 272, 954-956

47. Rot, A., Krieger, M., Brunner, T., Bischoff, S.C., Schall, T.J., and Dahinden, C.A. (1992) J.Exp.Med.176, 1489-1495

48. Uguccioni, M., D'Apuzzo, M., Loetscher, M., Dewald, B., and Baggiolini, M. (1995) Eur.J.Immunol. 25,64-68

49. Hesselgesser, J., Ng, H.P., Liang, M., Zheng, W., May, K., Bauman, J.G., Monahan, S., Islam, I., Wei,G.P., Ghannam, A., Taub, D.D., Rosser, M., Snider, R.M., Morrissey, M.M., Perez, H.D., andHoruk, R. (1998) J Biol Chem 273, 15687-15692

50. Dairaghi, D.J., Oldham, E.R., Bacon, K.B., and Schall, T.J. (1997) J.Biol.Chem. 272 , 28206-28209

51. Farzan, M., Mirzabekov, T., Kolchinsky, P., Wyatt, R., Cayabyab, M., Gerard, N.P., Gerard, C., Sodroski,J., and Choe, H. (1999) Cell 96, 667-676

52. Elsner, J., Mack, M., Bruhl, H., Dulkys, Y., Kimmig, D., Simmons, G., Clapham, P.R., Schlondorff, D.,Kapp, A., Wells, T.N., and Proudfoot, A.E. (2000) J Biol Chem 275, 7787-7794

53. Olson, W.C., Rabut, G.E., Nagashima, K.A., Tran, D.N., Anselma, D.J., Monard, S.P., Segal, J.P.,Thompson, D.A., Kajumo, F., Guo, Y., Moore, J.P., Maddon, P.J., and Dragic, T. (1999) J Virol73, 4145-4155

54. Wang, Z., Lee, B., Murray, J.L., Bonneau, F., Sun, Y., Schweickart, V., Zhang, T., and Peiper, S.C.(1999) J Biol Chem 274, 28413-28419

55. Monteclaro, F.S. and Charo, I.F. (1996) J.Biol.Chem. 271, 19084-19092

by guest on August 31, 2020

http://ww

w.jbc.org/

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nloaded from

25

56. Siciliano, S.J., Rollins, T.E., DeMartino, J., Konteatis, Z., Malkowitz, L., van Riper, G., Bondy, S., Rosen,H., and Springer, M.S. (1994) Proc.Natl.Acad.Sci.USA 91, 1214-1218

57. Baranski, T.J., Herzmark, P., Lichtarge, O., Gerber, B.O., Trueheart, J., Meng, E.C., Iiri, T., Sheikh, S.P.,and Bourne, H.R. (1999) J Biol Chem 274, 15757-15765

by guest on August 31, 2020

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Figure 1 Sabroe et al, (2000) A Small Molecule Antagonist of Chemokine Receptors CCR1 andCCR3

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Figure 4 Sabroe et al, (2000) A Small Molecule Antagonist of Chemokine Receptors CCR1 andCCR3

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Figure 5 Sabroe et al, (2000) A Small Molecule Antagonist of Chemokine Receptors CCR1 andCCR3

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Simmons, Paul R. Clapham, Timothy J. Williams and James E. PeaseIan Sabroe, Michael J. Peck, Berend Jan Van Keulen, Annelies Jorritsma, Graham

inhibition of Eosinophil function and CCR3-mediated HIV-1 entryA small molecule antagonist of the chemokine receptors CCR1 and CCR3 : Potent

published online June 14, 2000J. Biol. Chem. 

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