COMMUNICATION
Identification of a Potent, Selective Non-peptide CXCR2 Antagonist That Inhibits Interleukin-8-induced Neutrophil Migration*

John R. WhiteDagger §, Judithann M. LeeDagger , Peter R. YoungDagger , Robert P. Hertzberg, Anthony J. Jurewicz, Margery A. ChaikinDagger , Katherine Widdowsonpar , James J. Foley**, Lenox D. MartinDagger Dagger , Don E. GriswoldDagger Dagger , and Henry M. Sarau**

From the Departments of Dagger  Molecular Immunology,  Biomolecular Discovery, ** Pulmonary Pharmacology, par  Medicinal Chemistry, and Dagger Dagger  Immunopharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406

    ABSTRACT
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Abstract
Introduction
Procedures
Results & Discussion
References

Interleukin-8 (IL-8) and closely related Glu-Leu-Arg (ELR) containing CXC chemokines, including growth-related oncogene (GRO)alpha , GRObeta , GROgamma , and epithelial cell-derived neutrophil-activating peptide-78 (ENA-78), are potent neutrophil chemotactic and activating peptides, which are proposed to be major mediators of inflammation. IL-8 activates neutrophils by binding to two distinct seven-transmembrane (7-TMR) G-protein coupled receptors CXCR1 (IL-8RA) and CXCR2 (IL-8RB), while GROalpha , GRObeta , GROgamma , and ENA-78 bind to and activate only CXCR2. A chemical lead, which selectively inhibited CXCR2 was discovered by high throughput screening and chemically optimized. SB 225002 (N-(2-hydroxy-4-nitrophenyl)-N'-(2-bromophenyl)urea) is the first reported potent and selective non-peptide inhibitor of a chemokine receptor. It is an antagonist of 125I-IL-8 binding to CXCR2 with an IC50 = 22 nM. SB 225002 showed >150-fold selectivity over CXCR1 and four other 7-TMRs tested. In vitro, SB 225002 potently inhibited human and rabbit neutrophil chemotaxis induced by both IL-8 and GROalpha . In vivo, SB 225002 selectively blocked IL-8-induced neutrophil margination in rabbits. The present findings suggest that CXCR2 is responsible for neutrophil chemotaxis and margination induced by IL-8. This selective antagonist will be a useful tool compound to define the role of CXCR2 in inflammatory diseases where neutrophils play a major role.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results & Discussion
References

The recruitment of inflammatory cells into sites of tissue damage 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 reperfusion injury, arthritis, asthma, and inflammatory bowel disease.

The recruitment of neutrophils from post-capillary venules depends initially upon rolling of neutrophils via the interaction of neutrophil expressed sLex with endothelial expressed E-selectin, followed by attachment through the up-regulation of CD11b/CD18 (Mac-1), and diapedesis via a haptotactic gradient of IL-81 (1). The up-regulation of CD11b/CD18 on neutrophils appears to be mediated via IL-8 binding to neutrophil cell surface receptors (2).

IL-8 is a member of the super family of proinflammatory proteins known as chemokines, which are approximately 8 kDa in size. In human neutrophils, IL-8 binds with similar affinity to two distinct 7-TMRs, CXCR1 (3) and CXCR2 (4), whereas closely related chemokines containing a common amino-terminal Glu4-Leu5-Arg6 (ELR) amino acid sequence, including GROalpha , GRObeta , GROgamma , NAP-2, and ENA-78, bind only to CXCR2 (5). Both CXCR1 and CXCR2 are present on the surface of human neutrophils and a subset of T-cells (3, 4, 6, 7). Recent reports indicate that transendothelial migration of CLA+ T-cells is dependent on CXCR2 (8). In human neutrophils it is unclear whether chemotaxis is mediated by one or both receptors. In vitro studies using anti-receptor monoclonal antibodies, and cell lines stably expressing CXCR1 and CXCR2, have led to conflicting reports as to the importance of the two receptors in human neutrophil IL-8-induced chemotaxis (9-11).

Evidence in support of a role for ELR containing CXC chemokines in the pathogenesis of inflammation has resulted from studies manipulating the chemokine system in animal models. Neutralization of IL-8 with a monoclonal antibody in rabbits resulted in the suppression of a delayed type hypersensitivity reaction, which correlated with inhibition of both lymphocyte and neutrophil infiltration in the skin lesions (12). Additional studies with a monoclonal antibody demonstrated potent inhibition of neutrophil recruitment in a rabbit model of endotoxin-induced pleurisy (13) and in rabbit lung reperfusion injury (14). In mice, both injection of a monoclonal antibody to MIP-2, the mouse homologue of GRO, or targeted disruption of the IL-8 receptor resulted in decreased neutrophil-mediated inflammatory responses (14-17).

In this paper, the identification of the first potent and selective non-peptide antagonist of a chemokine receptor is described. Using this small molecule antagonist it appears that inhibition of CXCR2 is sufficient to prevent IL-8-induced neutrophil chemotaxis in vitro and sequestration in vivo. SB 225002 should be a useful tool compound to define the pathophysiological role of CXCR2.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results & Discussion
References

Cloning of CXCR1 and CXCR2-- The two IL-8 receptors were cloned by reverse transcription polymerase chain reaction. Total RNA from human neutrophils was prepared using a GuSCN/CsCl gradient and cDNA synthesis was primed with oligo(dT). Sequence specific primers (CXCR1 (5'-3'), CTAGAATTCCCTGGCCGGTGCTTCAGTTAGACTAAACC, GGTAGCTACTTCCTTATAGAGAGATCTCCTTCG; CXCR2 (5'-3'), GATGAATTCGTCAGGATTTAAGTTTACCTCAAAAATGG, GGAGGACGGATTCACGTCGGGAGATCTTAC) were used to amplify CXCR1 and CXCR2 cDNAs. Receptor sequences were identical to those published (3, 4), except for a conservative Thr to Ser change at residue 276 of CXCR1. These receptors were cloned into a mammalian expression vector containing a cytomegalovirus promoter and dihydrofolate reductase selection. Chinese hamster ovary (CHO) cell lines expressing either CXCR1 (CHO-CXCR1) or CXCR2 (CHO-CXCR2) were generated by single cell cloning following electroporation of the expression vector and selection by growth in nucleoside-free medium followed by amplification in 80 nM methotrexate. High expressing clones were identified using 125I-IL-8 binding to cells.

Radioligand Binding Experiments-- CHO-CXCR1 and CHO-CXCR2 membranes were prepared according to Kraft and Anderson (18). Assays were performed in 96-well microtiter plates where the reaction mixture contained 1.0 µg/ml membrane protein in 20 mM Bis-Tris-propane, pH 8.0, with 1.2 mM MgSO4, 0.1 mM EDTA, 25 mM NaCl, and 0.03% CHAPS and SB 225002 (10 mM stock in Me2SO) added at the indicated concentrations, the final Me2SO concentration was <1% under standard binding conditions. Binding was initiated by addition of 0.25 nM 125I-IL-8 (Amersham Pharmacia Biotech, 2,200 Ci/mmol). After 1-h incubation at room temperature the plate was harvested using a Tomtec 96-well harvester onto a glass fiber filtermat blocked with 1% polyethyleneimine, 0.5% BSA and washed three times with 25 mM NaCl, 10 mM Tris·HCl, 1 mM MgSO4, 0.5 mM EDTA, 0.03% CHAPS, pH 7.4. The filter was dried, sealed in a sample bag containing 10 ml of Wallac 205 Betaplate liquid scintillation fluid, and counted with a Wallac 1205 Betaplate liquid scintillation counter. Other binding assays were performed according to previously published reports: C5a (19), fMLP (20), LTB4 (21), and LTD4 (22).

Synthesis of SB 225002-- A solution of 2-hydroxy-4-nitroaniline (500 mg, 3.24 mmol) in N,N-dimethylformamide (1 ml) was treated with 2-bromophenylisocyanate (3.24 mmol) for 16 h at 80 °C. The product was purified by dilution with methylene chloride and precipitation with hexanes. Filtering afforded the title compound (530 mg, 47%), m.p. = 193-195 °C. 1H NMR (Me2SO): delta  11.05 (s), 9.49 (s), 9.12 (s), 8.47 (d), 7.93 (d), 7.74 (d), 7.68 (s), 7.34 (t), 7.00 (t); electron ionization-mass spectroscopy m/z 350(M - H)-; analysis (C15H10BrN3O4·1N,N-dimethylformamide) C,H,N (23).

Inhibition of Ca2+ Mobilization-- Human neutrophils were separated from whole blood of healthy volunteers by the one-step Hypaque-Ficoll method (24). HL60 cells were differentiated, under incubation conditions, with Me2SO (0.5%) for 3 days. Cells (PMN, HL60, CXCR1-RBL-2H3, or 3ASubE) were loaded with Fura-2AM as described previously (25). For antagonist studies, SB 225002 (final Me2SO < 0.35%) was added at the indicated concentrations, to 106 cells/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 (25).

Inhibition of Neutrophil Chemotaxis-- Neutrophils were washed twice with phosphate-buffered saline (PBS) and resuspended in PBS containing 1 mM MgCl2 and 1 mM CaCl2. Cell motility was determined using a modified Boyden chamber procedure as described (26). For measurement of chemotaxis, lower chambers were filled with 30 µl of IL-8 (1 nM) or GROalpha (10 nM), the empty upper chambers were lowered into place, and 50 µl of a PMN suspension (5 × 106 cells/ml), without (control) or with SB 225002, was added at the indicated concentrations. SB 225002, dissolved in Me2SO (100%) at 10 mg/ml, was diluted in PBS to the desired concentration; the final Me2SO concentration was <0.1%. Neutrophil migration proceeded for 60 min at 37 °C in the cell incubator, after which the chamber was disassembled. Following fixation (75% methanol) and staining (Diff-Quick) migrated cells were counted in four successive high power fields (HPF).

Inhibition of Neutrophil Sequestration in Vivo-- The in vivo neutrophil sequestration model was performed in rabbits as reported previously (27). Using sterile techniques, rabbits were surgically fitted with an implanted cannula in the external jugular vein. IL-8 (150 ng/kg/min) or fMLP (5 ng/kg/min) was directly infused into the blood in the absence or presence of SB 225002 (1.39-5.5 µg/kg/min) via the marginal ear vein. Blood samples were withdrawn at 2.5-5-min intervals via the vascular access port in the external jugular. White blood cell counts were determined with a Coulter counter, and differential counts were done using blood smears stained with Diff-Quick. Percent change of PMN count was determined relative to the base-line value.

    RESULTS AND DISCUSSION
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Abstract
Introduction
Procedures
Results & Discussion
References

To determine the feasibility of targeting individual IL-8 receptors with non-peptide, low molecular weight antagonists, a high throughput screen was configured using 125I-IL-8 binding to membranes of CHO-CXCR1 or CHO-CXCR2 cells. One compound identified from this screen was SK&F 83589 (Fig. 1A), which selectively inhibited 125I-IL-8 binding to CHO-CXCR2 with an IC50 of 500 nM. Chemical modification of SK&F 83589 led to SB 225002, N-(2-hydroxy-4-nitrophenyl)-N'-(2-bromophenyl)urea (Fig. 1B), which inhibited 125I-IL-8 binding to CHO-CXCR2 membranes with an IC50 = 22 nM (Fig. 2). SB 225002, at concentrations up to 3.3 µM (Fig. 2), failed to significantly inhibit the binding of 125I-IL-8 to CHO-CXCR1, or [3H]fMLP, [3H]LTB4, [3H]LTD4, or 125I-C5a to their cognate receptors. SB 225002 was, therefore, at leased >150-fold selective for CXCR2 over the other 7-TMRs tested.


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Fig. 1.   Structures of SK&F 83589 (A) and the high affinity analog SB 225002 (B).


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Fig. 2.   Competition binding of 125I-IL-8, [3H]FMLP, [3H]LTB4, [3H]LTD4, or 125I-C5a by SB 225002 to appropriate membranes expressing either cloned or primary receptors. Inhibition by SB 225002 of 125I-IL-8 binding to CHO-CXCR1 (open circle ) and CHO-CXCR2 (bullet ), 125I-C5a (triangle ) to RBL-2H3-C5a, [3H]fMLP (black-square) or [3H]-LTB4 (square ) to human PMNs or [3H]LTD4 to guinea pig lung membranes (black-down-triangle ). Results are expressed as percent of control specific binding and are the means of duplicate samples from a typical experiment performed three to five times.

To determine if SB 225002 was a functional CXCR2 antagonist, we monitored its effects on intracellular calcium mobilization stimulated by IL-8 or GROalpha . Cross-desensitization studies with Me2SO differentiated HL60 cells indicated that these cells predominantly express CXCR2 (~80%) with a smaller number of CXCR1 (~20%) receptors (Fig. 3A). In these cells, SB 225002 produced a concentration-dependent inhibition of both IL-8- and GROalpha -mediated calcium mobilization with IC50 values of 8 and 10 nM, respectively (Fig. 3B). Similarly, in 3ASubE (28) cells stably transfected with CXCR2, SB 225002 dose-dependently inhibited calcium mobilization induced by both GROalpha and IL-8, with IC50 values of 20 and 40 nM, respectively (Fig. 3B). In contrast to HL60 cells, human neutrophils express equal numbers of CXCR1 and CXCR2 on their cell surface. In these cells, SB 225002 inhibited GROalpha -, but not IL-8-, stimulated calcium mobilization (IC50 values = 30 nM and >10 µM, respectively, Fig. 3C). In addition, in RBL-2H3 cells, stably transfected with CXCR1, SB 225002 failed to inhibit calcium mobilization induced by either IL-8 or LTD4 (IC50 > 10 µM, Fig. 3C). The failure of SB 225002 to block IL-8-induced calcium mobilization in human neutrophils, presumably reflects the ability of IL-8 to circumvent the blockade of CXCR2 by activating CXCR1, which was not inhibited by this compound. SB 225002 also demonstrated functional selectivity, since it failed to inhibit calcium mobilization induced by optimal concentrations of LTB4 in PMNs (Fig. 3C), or RANTES, MIP-1alpha , or MCP-1 in monocytes (data not shown).


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Fig. 3.   Effect of SB 225002 on agonist-induced calcium mobilization. A, cross-desensitization of differentiated HL60 cells by IL-8 and GROalpha . Stim 1 represents the addition of a 100 nM amount of either IL-8 or GROalpha as indicated for each individual trace. Stim 2 represents the addition of a second stimulus of agonist (100 nM) to the same cells that had also received the first stimulation. Quantitation of the calcium change indicates that CXCR2 represents ~80% of the IL-8 receptors on differentiated HL60 cells. B, inhibition of calcium mobilization in Me2SO-differentiated HL60 cells stimulated with 1 nM IL-8 (bullet ), 10 nM GROalpha (black-square), or 1 nM C5a (triangle  with solid lines) or in CXCR2-3ASubE cells stimulated with 2 nM IL-8 (bullet ) or 10 nM GROalpha (black-square with dotted lines). Calcium mobilized by HL60 cells in the absence of antagonist was 400-420, 385-410, and 410-425 nM for IL-8, GROalpha , and C5a, respectively, and in 3ASubE cells was 350-375 and 320-335 nM for IL-8 and GROalpha , respectively. C, isolated peripheral blood neutrophils were stimulated with 1 nM IL-8 (bullet ), 10 nM GROalpha (black-square), or 1 nM LTB4 (square  with solid line), or CXCR1-RBL cells were stimulated with 1 nM IL-8 (bullet ) or 1 µM LTD4 (black-diamond  with dotted line). Cells were pretreated for 15 s with the indicated concentrations of SB 225002 (1-10,000 nM) before the addition of agonist. Calcium mobilized by PMNs in the absence of antagonist was 1200-1400, 900-1100, and 1600-1800 nM for IL-8, GROalpha , or LTB4, respectively. Data in B and C are the average of duplicate points and are representative of three individual experiments.

Having identified SB 225002 as a potent and selective CXCR2 antagonist, the compound was evaluated for inhibition of human neutrophil chemotaxis in response to maximally effective concentrations of IL-8, GROalpha , or C5a. Using PMNs from nine individual subjects, SB 225002 inhibited both IL-8 (1 nM)- and GROalpha (10 nM)-mediated chemotaxis with similar IC50 values (20 and 60 nM, respectively) but did not affect chemotaxis induced by 50 nM C5a at concentrations up to 330 nM (Fig. 4). These data provide evidence that in vitro neutrophil chemotaxis is mediated predominantly by CXCR2.


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Fig. 4.   Effect of SB 225002 on IL-8-, GROalpha -, or C5a-induced human neutrophil chemotaxis. Chemotaxis was measured in 48-well Boyden chambers as described under "Experimental Procedures." The effect of SB 225002 on chemotaxis induced by 1 nM IL-8 (bullet ), 10 nM GROalpha (black-square), or 50 nM C5a (triangle ) was evaluated with PMNs. Control agonist responses were 269 ± 32.6, 265 ± 30.7, and 263 ± 40.9 neutrophils/HPF for IL-8, GROalpha , and C5a, respectively. The negative unstimulated response was 54.8 ± 4.0 neutrophils/HPF. Results are expressed as a percent of control cells responding to their respective ligands. Each point represents an average of nine determinations from individual donors ± S.E.

Rabbits, like humans, express both IL-8 receptors on their neutrophils (29). Therefore, this species was utilized to evaluate the in vivo effects of SB 225002. In vitro studies confirmed that SB 225002 is a potent antagonist of rabbit CXCR2, inhibiting rabbit PMN chemotaxis in response to optimal concentrations of human IL-8 or GROalpha (IC50 values of 30 and 70 nM, respectively).

In rabbits, an intravenous infusion of LTB4, or other chemotactic factors, rapidly promotes neutrophil shape change and margination of neutrophils to the microcapillary endothelial cells of the lung (27, 30-32). Thus, this is the basis of a useful model to study the initial stages of neutrophil activation and attachment to the endothelium. As seen in Fig. 5, A and B, administration of IL-8 or fMLP resulted in rapid margination of neutrophils (62 and 68%, respectively), which lasted throughout the infusion period (30 min). Co-administration of SB 225002 (Fig. 5, A and B) inhibited, in a dose-dependent manner, IL-8-, but not fMLP-, mediated PMN sequestration (Fig. 5C).


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Fig. 5.   Effect of SB 225002 on IL-8- and fMLP-induced neutrophil margination in rabbits. A, effect of a 30-min infusion of IL-8 (150 ng/kg/min, triangle ) on the percent of neutrophils remaining in circulation. Co-administration of SB 225002 at 1.39 (square ), 2.78 (bullet ), or 5.5 (open circle ) µg/kg/min dose-dependently reversed the sequestration of neutrophils. Vehicle (10% Me2SO, 90% diphosphate-buffered saline, pH 7.4) alone produced a 20% increase in circulating neutrophils, which lasted for the entire infusion period (30 min) but had no effect on the ability of IL-8 or fMLP to sequester neutrophils. B, PMN sequestration by fMLP (5 ng/kg/min, open circle ) co-administered with vehicle or SB 225002 (5.5 µg/kg/min, bullet ). C, dose-dependent inhibition of PMN margination by SB 225002 induced by either fMLP or IL-8 taken (15 min) after addition of agonist. Each data point in A and B represent the average ± S.E. of three independent experiments. *, p < 0.01; **, p < 0.05; ***, p < 0.001 from control (IL-8) sequestration.

The in vitro and in vivo findings presented provide evidence that inhibition of CXCR2 is sufficient to prevent neutrophil margination and chemotaxis mediated by IL-8 and suggests that CXCR1 does not play a major role in neutrophil migration. The functional role of CXCR1 is not clear; however, a recent report, using modified antagonist forms of IL-8 and human neutrophils, demonstrated the importance of CXCR1 in IL-8-mediated superoxide generation and release of granular enzymes (33). This is consistent with the previous suggestion that CXCR1 on human neutrophils may require higher concentrations of IL-8 for chemotaxis and, therefore, may be involved in neutrophil activities closer to the site of injury and not in the early attachment and extravasation events (10). This hypothesis agrees with the finding in rodents, which appear to possess only the CXCR2 homologue, yet retain the ability to localize neutrophils to sites of inflammation (17).

The present study represents the first reported discovery of a potent and selective non-peptide antagonist of a chemokine receptor and the first low molecular weight inhibitor of a large (72 amino acid) agonist 7-TMR interaction. A number of small molecule antagonists have been reported for small peptide receptors, e.g. tachykinin (34), angiotensin (35), and endothelin (36), but to our knowledge the only antagonist reported for a large peptide receptor was a micromolar antagonist of the C5a receptor (37). As chemokine receptors are part of the 7-TMR family, which have traditionally been productive targets for drug discovery, it is anticipated that small molecule receptor antagonists may have potential as novel therapeutics. The availability of potent and selective non-peptide antagonists, such as SB 225002, will help define the apparent overlap in activities of the chemokines and their receptors and elucidate their relative importance. In particular, SB 225002 will be an important tool compound to assess the role of IL-8 and CXCR2 in neutrophil recruitment, a process that is thought to be important in several inflammatory diseases, including adult respiratory distress syndrome, chronic bronchitis, and asthma (38-41).

    ACKNOWLEDGEMENTS

We thank Dr. Robert Ames for providing 125I-C5a and membranes for the C5a binding assay, Dr. Douglas W. P. Hay for the critical reading of the manuscript, and Dr. Ann Richmond for the gift of CXCR2-transfected 3ASubE cells. All animal procedures were reviewed and approved by the SmithKline Beecham Animal Care and Use Committee.

    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. Molecular 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{at}sbphrd.com.

1 The abbreviations used are: IL-8, interleukin-8; GRO, growth-related oncogene; ENA-78, epithelial cell-derived neutrophil activating peptide; 7-TMR, seven-transmembrane domain G-protein-coupled receptor; ELR, Glu4-Leu5-Arg6; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline; HPF, high powered fields; LTB4, leukotriene B4; LTD4, leukotriene D4; fMLP, N-formyl-Met-Leu-Phe; C5a, serum complement fragment from C5; SB 225002, N-(2-hydroxy-4-nitrophenyl)-N'-(2-bromophenyl)urea; SK&F 83589, N-(2-hydroxy-4-nitrophenyl)-N'-phenylurea; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PMN(s), polymorphonuclear cell(s).

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Abstract
Introduction
Procedures
Results & Discussion
References

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