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• W2134866739 abstract "The chemokines CCL3 and CCL5, as well as their shared receptor CCR1, are believed to play a role in the pathogenesis of several inflammatory diseases including rheumatoid arthritis, multiple sclerosis, and transplant rejection. In this study we describe the pharmacological properties of a novel small molecular weight CCR1 antagonist, CP-481,715 (quinoxaline-2-carboxylic acid [4(R)-carbamoyl-1(S)-(3-fluorobenzyl)-2(S),7-dihydroxy-7-methyloctyl]amide). Radiolabeled binding studies indicate that CP-481,715 binds to human CCR1 with a K d of 9.2 nm and displaces 125I-labeled CCL3 from CCR1-transfected cells with an IC50 of 74 nm. CP-481,715 lacks intrinsic agonist activity but fully blocks the ability of CCL3 and CCL5 to stimulate receptor signaling (guanosine 5′-O-(thiotriphosphate) incorporation; IC50 = 210 nm), calcium mobilization (IC50 = 71 nm), monocyte chemotaxis (IC50 = 55 nm), and matrix metalloproteinase 9 release (IC50 = 54 nm). CP-481,715 retains activity in human whole blood, inhibiting CCL3-induced CD11b up-regulation and actin polymerization (IC50 = 165 and 57 nm, respectively) on monocytes. Furthermore, it behaves as a competitive and reversible antagonist. CP-481,715 is >100-fold selective for CCR1 as compared with a panel of G-protein-coupled receptors including related chemokine receptors. Evidence for its potential use in human disease is suggested by its ability to inhibit 90% of the monocyte chemotactic activity present in 11/15 rheumatoid arthritis synovial fluid samples. These data illustrate that CP-481,715 is a potent and selective antagonist for CCR1 with therapeutic potential for rheumatoid arthritis and other inflammatory diseases. The chemokines CCL3 and CCL5, as well as their shared receptor CCR1, are believed to play a role in the pathogenesis of several inflammatory diseases including rheumatoid arthritis, multiple sclerosis, and transplant rejection. In this study we describe the pharmacological properties of a novel small molecular weight CCR1 antagonist, CP-481,715 (quinoxaline-2-carboxylic acid [4(R)-carbamoyl-1(S)-(3-fluorobenzyl)-2(S),7-dihydroxy-7-methyloctyl]amide). Radiolabeled binding studies indicate that CP-481,715 binds to human CCR1 with a K d of 9.2 nm and displaces 125I-labeled CCL3 from CCR1-transfected cells with an IC50 of 74 nm. CP-481,715 lacks intrinsic agonist activity but fully blocks the ability of CCL3 and CCL5 to stimulate receptor signaling (guanosine 5′-O-(thiotriphosphate) incorporation; IC50 = 210 nm), calcium mobilization (IC50 = 71 nm), monocyte chemotaxis (IC50 = 55 nm), and matrix metalloproteinase 9 release (IC50 = 54 nm). CP-481,715 retains activity in human whole blood, inhibiting CCL3-induced CD11b up-regulation and actin polymerization (IC50 = 165 and 57 nm, respectively) on monocytes. Furthermore, it behaves as a competitive and reversible antagonist. CP-481,715 is >100-fold selective for CCR1 as compared with a panel of G-protein-coupled receptors including related chemokine receptors. Evidence for its potential use in human disease is suggested by its ability to inhibit 90% of the monocyte chemotactic activity present in 11/15 rheumatoid arthritis synovial fluid samples. These data illustrate that CP-481,715 is a potent and selective antagonist for CCR1 with therapeutic potential for rheumatoid arthritis and other inflammatory diseases. Rheumatoid arthritis is a chronic inflammatory disease affecting 0.5–2% of the population in the Western world, the majority of whom are female. Central to the pathogenesis of this disease is the infiltration of monocytes into synovial tissue. This is supported by the predominance of monocytes in the joint during flare (1Cutolo M. Sulli A. Barone A. Seriolo B. Accardo S. Clin. Exp. Rheum. 1993; 11: 331-339PubMed Google Scholar, 2Tak P.P. Smeets T.J.M. Daha M.R. Kluin P.M Meijers K.A.E. Brand R. Meinders A.E. Breedveld F.C. Arth. Rheum. 1997; 40: 217-225Crossref PubMed Scopus (473) Google Scholar), the role of monocyte-derived proinflammatory mediators on disease progression (e.g. TNF, 1The abbreviations used are: TNF, tumor necrosis factor; PBS, Dulbecco's phosphate-buffered saline; PBMC, peripheral blood mononuclear cells; MMP, matrix metalloproteinase. interleukin 1) (3Dayer J.M. Joint, Bone, Spine: Revue du Rhumatisme. 2002; 69: 123-132Crossref PubMed Scopus (66) Google Scholar, 4Meyer O. Presse Medicale. 2000; 29: 463-468PubMed Google Scholar), and the ability of monocytes to secrete tissue-damaging proteolytic enzymes that participate in joint destruction (5Jenkins J.K. Hardy K.J. McMurray R.W. Am. J. Med. Sci. 2002; 323: 171-180Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Consequently, an agent that inhibits monocyte infiltration into synovial tissue has the potential to decrease tissue damage and joint destruction, thus, providing a novel therapy for rheumatoid arthritis. Leukocyte infiltration into inflammatory sites is believed to be regulated by small molecular weight cytokines known as chemokines. Chemokines are 7–10-kDa proteins that can be divided into four groups (CC, CXC, CX3C, and C) depending on the spacing of their N-terminal cysteine residues. Approximately 40 different chemokines have been identified. Some of these are constitutively expressed and play a crucial role in development and lymph node architecture. However, most chemokines are specifically induced at inflammatory sites during disease (6Proudfoot A.E. Nat. Rev. Immunol. 2002; 2: 106-115Crossref PubMed Scopus (613) Google Scholar). Chemokines are of interest as therapeutic targets because they exert their effects through seven-transmembrane G-protein-coupled receptors, well precedented drug targets. Although chemokines are best known for their ability to stimulate cell migration, they also have additional activities that can contribute to tissue damage and inflammation including enhancing T cell activation (7Ward S.G. Bacon K. Westwick J. Immunity. 1998; 9: 1-11Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar), regulating TH-1/TH-2 polarization (8Gao J.-L. Wynn T.A. Chang Y. Lee E.J. Broxmeyer H.E. Cooper S. Tiffany H.L. Westphal H. Kwon-Chung J. Murphy P.M. J. Exp. Med. 1997; 185: 1959-1968Crossref PubMed Scopus (387) Google Scholar, 9Colantonio L. Iellem A. Clissi B. Pardi R. Rogge L. Sinigaglia F. D'Ambrosio D. Blood. 1999; 94: 2981-2989Crossref PubMed Google Scholar, 10Weber C. Weber K.S.C. Klier C. Gu S. Wank R. Horuk R. Nelson P.J. Blood. 2001; 97: 1144-1146Crossref PubMed Scopus (210) Google Scholar), and stimulating macrophage function (11Fahey III, T.J. Tracey K.J. Tekamp-Olson P. Cousens L.S. Jones W.G. Shires G.T. Cerami A. Sherry B. J. Immunol. 1992; 148: 2764-2769PubMed Google Scholar) and protease secretion (12Klier C.M. Nelson E.L. Cohen C.D. Horuk R. Schlondorff D. Nelson P.J. Biol. Chem. Hoppe-Seyler. 2001; 382: 1405-1410Crossref PubMed Scopus (47) Google Scholar, 13Robinson S.C. Scott K.A. Balkwill F.R. Eur. J. Immunol. 2002; 32: 404-412Crossref PubMed Scopus (166) Google Scholar). In rheumatoid arthritis two chemokines believed to play a major role in regulating monocyte infiltration into synovial tissues are CCL3 (macrophage inflammatory protein-1α) and CCL5 (RANTES, regulated on activation, normal T-cell expressed and secreted). CCL3 and CCL5 have potent monocyte chemotactic activity both in vitro (14Uguccioni M. D'Apuzzo M. Loetscher M. Dewald B. Baggiolini M. Eur. J. Immunol. 1995; 25: 64-68Crossref PubMed Scopus (330) Google Scholar) and in vivo, as demonstrated in human subjects injected intradermally with these agents (15Lee S.C. Brummet M.E. Shahabuddin S. Woodworth T.G. Georas S.N. Leiferman K.M. Gilman S.C. Stellato C. Gladue R.P. Schleimer R.P. Beck L.A. J. Immunol. 2000; 164: 3392-3401Crossref PubMed Scopus (91) Google Scholar, 16Beck L.A. Dalke S. Leiferman K.M. Bickel C.A. Hamilton R. Rosen H. Bochner B.S. Schleimer R.P. J. Immunol. 1997; 159: 2962-2972PubMed Google Scholar). Furthermore, several studies have demonstrated an elevation of CCL3 and CCL5 in the synovial tissue and fluid of rheumatoid arthritis patients (17Katrib A. Tak P.P. Bertouch J.V. Cuello C. McNeil H.P. Smeets T.J.M. Kraan M.C. Youssef P.P. Rheumatology. 2001; 40: 988-994Crossref PubMed Scopus (69) Google Scholar, 18Koch A.E. Kunkel S.L. Harlow L.A. Mazarakis D.D. Haines G.K. Burdick M.D. Pope R.M. Strieter R.M. J. Clin. Invest. 1994; 93: 921-928Crossref PubMed Google Scholar, 19Ellingsen T. Buus A. Moller B.K. Stengaard-Pedersen K. Scand. J. Rheumatol. 2000; 29: 216-221Crossref PubMed Scopus (21) Google Scholar, 20Violin M.V. Shah M.R. Tokuhira M. Haines G.K. Woods J.M. Koch A.E. Clin. Immunol. Immunopathol. 1998; 89: 44-53Crossref PubMed Scopus (117) Google Scholar, 21al-Mughales J. Blyth T.H. Hunter J.A. Wilkinson P.C. Clin. Exp. Immunol. 1996; 106: 230-236Crossref PubMed Scopus (59) Google Scholar, 22Robinson E. Keystone E.C. Schall T.J. Gillett N. Fish E.N. Clin. Exp. Immunol. 1995; 101: 398-407Crossref PubMed Scopus (163) Google Scholar) coincident with the infiltration of monocytes expressing their shared receptor, CCR1 (23Katschke Jr., K.J. Rottman J.B. Ruth J.H. Qin S. Wu L. LaRosa G. Ponath P. Park C.C. Pope R.M. Koch A.E. Arthritis Rheum. 2001; 44: 1022-1032Crossref PubMed Scopus (254) Google Scholar). In these studies, the levels of CCL3 and monocytes in synovial tissue were directly proportional to the magnitude of joint pain (2Tak P.P. Smeets T.J.M. Daha M.R. Kluin P.M Meijers K.A.E. Brand R. Meinders A.E. Breedveld F.C. Arth. Rheum. 1997; 40: 217-225Crossref PubMed Scopus (473) Google Scholar). The role of these chemokines in the pathogenesis of arthritis is also supported by human genetic association studies (24Makki R.F. al Sharif F. Gonzalez-Gay M.A. Garcia-Porrua C. Ollier W.E. Hajeer A.H. Clin. Exp. Rheumatol. 2000; 18: 391-393PubMed Google Scholar) and by the beneficial effects observed in animal models of arthritis using neutralizing antibodies and receptor antagonists (25Plater-Zyberk C. Hoogewerf A.J. Proudfoot A.E. Power C.A. Wells T.N. Immunol. Lett. 1997; 57: 117-120Crossref PubMed Scopus (208) Google Scholar, 26Barnes D.A. Tse J. Kaufhold M. Owen M. Hesselgesser J. Strieter R. Horuk R. Perez H.D. J. Clin. Invest. 1998; 101: 2910-2919Crossref PubMed Scopus (178) Google Scholar, 27Kasama T. Strieter R.M. Lukacs N.W. Lincoln P.M. Burdick M.D. Kunkel S.L. J. Clin. Invest. 1995; 95: 2868-2876Crossref PubMed Scopus (260) Google Scholar). The potential importance of CCR1 in the pathogenesis of rheumatoid arthritis coupled with the well known success in identifying antagonists of G-protein-coupled receptors prompted us to initiate a search for small molecular weight antagonists of CCR1. These efforts led to the identification of the novel antagonist, CP-481,715. In this study we describe the in vitro biological properties of CP-481,715 and demonstrate its ability to dose-dependently inhibit CCL3- and CCL5-induced chemotaxis, integrin up-regulation, intracellular calcium mobilization, and matrix metalloprotease production. Furthermore, CP-481,715 retains activity in whole blood, and as evidence for its potential therapeutic utility, we demonstrate its ability to inhibit the monocyte chemotactic activity induced by synovial fluid from rheumatoid arthritis patients. These studies suggest that blocking CCR1 may represent a novel therapeutic approach to prevent monocyte infiltration into inflammatory sites such as the synovial tissue. Materials—The CCR1 antagonist CP-481,715 (quinoxaline-2-carboxylic acid [4(R)-carbamoyl-1(S)-(3-fluorobenzyl)-2(S),7-dihydroxy-7-methyloctyl]amide; see Fig. 1) and [3H]CP-481,715 were prepared by the Pfizer Medicinal chemistry group. 125I-Labeled CCL3 was purchased from PerkinElmer Life Sciences. All unlabeled chemokines were obtained from Peprotech (Rocky Hill, NJ) unless otherwise indicated and checked for purity by high performance liquid chromatography. Human recombinant interleukin-2 and TNFα were obtained from R&D Systems (Minneapolis, MN). Reagents—Bovine serum albumin, fluorescein diacetate, and Accuspin™ System-Histopaque-1077 tubes were purchased from Sigma. Heparin was purchased from American Pharmaceutical Partners Inc. (Los Angeles, CA). Dulbecco's phosphate-buffered saline (PBS) without calcium chloride and magnesium chloride, Hanks' balanced salt solution, and Geneticin were obtained from Invitrogen. Fetal bovine serum was purchased from Hyclone, Logan, Utah. RPMI 1640, HEPES, glutamine, and penicillin/streptomycin were all obtained from BioWhittaker (Walkersville, MD). Tissue culture medium for cell cultures consisted of RPMI 1640 containing fetal bovine serum (10%), l-glutamine (2 mm), HEPES (10 mm), penicillin (100 units/ml), and streptomycin (50 μg/ml). Cell Lines—CCR1-transfected HEK 293 cells (ATCC CRL-1573) were prepared as previously described (28Neote K. DiGregorio D. Mak J.Y. Horuk R. Schall T.J. Cell. 1993; 72: 415-425Abstract Full Text PDF PubMed Scopus (700) Google Scholar). CCR1-, CCR2-, CCR5-, CXCR1-, CXCR2-, and CXCR4-transfected 300-19 cells were obtained from Dr. Israel Charo at the Gladstone Institute. Cells were maintained in Geneticin for selection, split twice weekly, and discarded after passage 12. The human monocytic cell line, THP-1 (ATCC TIB 202), was maintained in RPMI medium and split 24 h before use. Chemokine and CP-481,715 Binding Studies—Binding assays were conducted with CCR1-transfected HEK 293 cells as previously described (28Neote K. DiGregorio D. Mak J.Y. Horuk R. Schall T.J. Cell. 1993; 72: 415-425Abstract Full Text PDF PubMed Scopus (700) Google Scholar). The number of CCR1 receptors on the cells was calculated to be 75,000/cell. Cells were typically grown to 80% confluence, removed by trypsinization and plated into flasks at least 48 h before the experiment. On the day of the experiment, cells were collected, washed in Hanks' balanced salt solution, and resuspended in the assay buffer (50 mm HEPES, 1 mm CaCl2, 5 mm MgCl2, 0.5% bovine serum albumin, pH 7.4) at a concentration of 2.5 × 105 cells/ml. Cells were then placed into a 96-well plate (25,000 cells/well) and incubated with 50 pm125I-labeled CCL3 or 125I-labeled CCL5 (specific activity 2200 Ci/mmol) in the presence or absence of varying concentrations of CP-481,715 at 4 °C for 60 min. The reaction was terminated by harvesting through a GF/B filter plate (Packard) presoaked with 0.3% polyethyleneimine (Sigma) and washed 3 times with cold wash buffer (10 mm HEPES, 0.5 m NaCl, 0.5% bovine serum albumin, pH 7.4). The radioactivity in each well was determined using a scintillation counter (PerkinElmer Life Sciences) after the addition of 50 μl of Scint-20 scintillation fluid. Nonspecific binding was assessed in competition studies using unlabeled CCL3 (100 nm). The K d for 125I-labeled CCL3 was calculated to be 3 nm. Similar experiments on CCR1-transfected cells were done using 3H-CP-481,715. Assessment of G-protein Signaling Using [35]GTPγS Incorporation— THP-1 cells were freeze-thawed three times to create membranes that were then utilized for the assay. 100 nm CCL3 was added to 100 μg of the membrane preparation in the presence of various concentrations of CP-481,715 and 10 mm GDP. [35S]GTPγS incorporation was measured using an SPA assay (Amersham Bioscience). To assess the potential for inverse agonist activity, GTPγS incorporation was also measured on membranes in the presence of CP-481,715 without added CCL3. Isolation of Human Peripheral Blood Mononuclear Cells (PBMCs)— Human blood was collected from normal volunteers in heparinized syringes (30 units/ml) and then immediately transferred to Accuspin tubes and centrifuged at 800 × g. PBMCs were collected from the interface and washed twice with PBS. Any remaining red blood cells were removed by hypotonic lysis. Cells were then subjected to slow speed centrifugation (100 × g) to remove any platelets. Chemotaxis Assays—PBMC chemotaxis was conducted in 48-well chemotaxis chambers purchased from NeuroProbe, Inc. (Gaithersburg, MD) as previously described (29Cole K.E. Strick C.A. Paradis T.J. Ogborne K.T. Loetscher M. Gladue R.P. Lin W. Boyd J.G. Moser B. Wood D.E. Sahagan B.G. Neote K. J. Exp. Med. 1998; 187: 2009-2021Crossref PubMed Scopus (734) Google Scholar). Briefly, agonists were diluted in RPMI 1640 containing 0.1% bovine serum albumin then added to the bottom wells of the chamber. A polyvinylpyrrolidone-free filter with 5-μm pores (Neuroprobe) was placed between the upper and lower wells of the chamber. PBMCs were then added to the top chamber (2 × 105) in the presence or absence of various concentrations of CP-481,715, and the apparatus was incubated for 60 min in a 5% CO2-humidified incubator at 37 °C. After the incubation period, the non-migrating cells were removed from the upper chamber, and the top of the filter was wiped. The bottom portion of the filter was stained with Diff-Quik (Dade Behring, Newark, DE), and the number of migrating cells in six random fields were enumerated with a microscope. A similar procedure was employed for THP-1 cells except a 96-well chemotaxis chamber was utilized, 8 × 105 cells were added to the upper wells, and the chamber was incubated for 3 h rather than 60 min. In this case, after incubation and the removal of non-migrating cells as above, 2 mm cold EDTA was added to the upper wells, and the chemotaxis chamber was incubated at 4 °C for 20 min. The EDTA was then removed, and the 96-well microtiter plate was centrifuged at 800 × g for 10 min. The filter was then removed, the culture medium was discarded, and 0.2% fluorescein diacetate was added to the plates. The plates were then incubated for 1.5 h at 37 °C until a yellow color developed. The number of migrating cells was quantitated by reading the intensity of the color on a microtiter plate reader at 490 nm. Calcium Measurements—Human CCR1-transfected 300-19 cells were harvested, centrifuged, and resuspended at 2 × 106 cells/ml in Hanks' balanced salt solution containing 1.6 mm CaCl2. The cells were loaded with indo-1 AM (2 μm; Molecular Probes, Eugene, OR) and then washed twice with buffer and suspended at 1 × 107/ml. Calcium mobilization was assessed using a fluorometer (Photon Technology Corp. International, Lawrenceville, NJ) after the addition of CP-481,715 or diluent and CCL3 or CCL5. Matrix Metalloproteinase 9 (MMP9) Generation—PBMCs were placed in 24-well plates (VWR, Boston, MA) at 2 × 106/ml in tissue culture medium. 20 nm CCL5 or 10 ng/ml TNFα was then added to the cells in the presence or absence of various concentrations of CP-481,715. Plates were incubated for 24 h at 37 °C and centrifuged, and the concentration of MMP9 in the supernatants was determined by enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN). Whole Blood Monocyte CD11b Up-regulation—CD11b up-regulation on monocytes in human whole blood was assessed as previously described (30Conklyn M.J. Neote K. Showell H.J. Cytokine. 1996; 8: 762-766Crossref PubMed Scopus (21) Google Scholar). Briefly, blood samples were preincubated at 37 °C for 5 min in the presence or absence of various concentrations of CP-481,715. CCL3 was then added to the blood at various concentrations. After 15 min, the assay tubes were placed in an ice-water bath to stop the reaction. Cells were then washed by adding 1 ml of PBS containing 2% fetal bovine serum and 0.2% sodium azide (wash solution). The cells were then pelleted at 200 × g for 10 min at 4 °C. CD11b expression on CD14+ monocytes was then determined by fluorescence-activated cell sorter analysis. Whole Blood Actin Polymerization—Human blood collected in EDTA was incubated with various dilutions of CP-481,715 or diluent for 5 min at room temperature. CCL3 (10 nm) was then added, and after 50 s the reaction was terminated by adding fluorescence-activated cell sorter lysing solution (BD Biosciences) containing paraformaldehyde (Electron Microscopy Sciences, Ft. Washington, PA). After 10 min, the cells were collected by centrifugation, washed with PBS, and stained for 1 h at room temperature in the dark with a solution containing lysophosphatidylcholine (Sigma), paraformaldehyde, and N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)) phallacidin (Molecular Probes). The cells were then washed with PBS containing 2% fetal calf serum, and the fluorescence was quantitated using a FACScan (BD Biosciences). Identification of CP-481,715 and Characterization of Its Effects on CCR1 Using Binding Assays—A binding assay using CCR1-transfected HEK 293 cells was used to screen our compound library for small molecular weight compounds that inhibit CCL3 binding to CCR1. Chemical optimization of active compounds from this screen led to the identification of quinoxaline-2-carboxylic acid [4(R)-carbamoyl-1(S)-(3-fluorobenzyl)-2(S)-7-dihydroxy-7-methyloctyl] amide (CP-481,715) as the lead compound (Fig. 1). As shown in Fig. 2A, competition binding experiments demonstrated that CP-481,715 displaces 125I-labeled CCL3 from CCR1-transfected HEK 293 cells in a concentration-dependent manner (IC50 = 74 nm). Similar results were observed with 125I-labeled CCL5 (data not shown). To better define its interaction, competition binding experiments were also conducted with radiolabeled CP-481,715. As shown in Fig. 2B, [3H]CP-481,715 binds to human CCR1-expressing HEK 293 cells with a K d of 9.2 nm. These data demonstrate that CP-481,715 binds to human CCR1 and prevents its interaction with CCL3. CP-481,715 Inhibits GTP Hydrolysis—Because interaction with the CCR1 receptor could impart both agonist and/or antagonist properties, we next sought to characterize this interaction from a functional perspective. During receptor activation, GDP bound to the α subunit of G-protein receptors is released, leading to the rapid binding of GTP and the subsequent dissociation of β/γ subunits. The release of these β/γ subunits initiates a cascade of intracellular events (i.e. calcium mobilization, adenylate cyclase activation, etc.) culminating in the hydrolysis of GTP to return the receptor to its resting state. The initial step in G-protein signaling can be quantitated by measuring the incorporation of the non-hydrolyzable form of GTP, [35S]GTPγS. As shown in Fig. 3, THP-1 cells had a basal level of receptor activity (35S-GTP) that was not significantly affected by CP-481,715, indicating that CP-481,715 was not acting as an inverse agonist (31Barr A.J. Manning D.R. J. Biol. Chem. 1997; 272: 32979-32987Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). In contrast, CP-481,715 was able to block CCL3 (100 nm)-induced [35S]GTPγS incorporation in a dose-dependent manner (IC50 of 210 nm). CP-481,715 Inhibits Calcium Mobilization but Has No Agonist Properties—We next characterized the effects of CP-481,715 on CCR1 function by measuring intracellular calcium mobilization in CCR1-transfected 300-19 cells. In these studies CP-481,715 did not directly induce calcium mobilization, supporting its lack of intrinsic agonist activity (data not shown). In contrast, as shown in Fig. 4, calcium mobilization induced by CCL3 was blocked by CP-481,715 in a concentration-dependent manner. The amount of CP-481,715 necessary to block this effect was directly proportional to the concentration of CCL3 utilized, demonstrating a 5–6-fold change in IC50 with each log change in CCL3 concentration. This data suggest that CP-481,715 is a competitive antagonist of CCR1. To provide evidence that CP-481,715 was not acting nonspecifically (e.g. by inhibiting downstream signaling), we performed experiments on THP-1 monocytes that express both CCR1 and CCR2. As shown in Fig. 5A, calcium mobilization was induced by THP-1 cells using either CCL3 or CCL2. CP-481,715 was able to inhibit CCL3- but not CCL2-induced intracellular calcium mobilization (Fig. 5B). Again, CP-481,715 did not directly induce any calcium mobilization on THP-1 cells. To further establish that the inhibitory effects of CP-481,715 were mediated through direct interaction with CCR1 and not through effects on its ligands, we also measured calcium mobilization on CCR5-transfected cells because both CCL3 and CCL5 also interact with this receptor. CP-481,715 did not inhibit calcium mobilization in CCR5 transfected cells in response to either ligand (data not shown). Collectively, these studies indicate that CP-481,715 completely antagonizes the CCR1 receptor, lacks intrinsic agonist activity, does not directly interfere with its ligands, and does not antagonize CCR5 or CCR2. CP-481,715 Inhibits Leukocyte Chemotaxis—A major property of chemokines is their ability to induce leukocyte migration and mediate the infiltration of these cells into sites of inflammation. As such, to better characterize the activity of CP-481,715 on CCR1 in a way that may be more relevant for disease, we performed a series of experiments examining the effects of CP-481,715 on chemotaxis. As shown in Fig. 6, CP-481,715 inhibited the chemotaxis of THP-1 cells and human peripheral blood monocytes to an optimal concentration of CCL3 (1.0 nm) with an IC50 of 110 and 55 nm, respectively. CP-481,715 also inhibited the chemotaxis of monocytes to other CCR1 ligands, including CCL5 and CCL14, and was able to partially inhibit the response to the shared ligand CCL7, which interacts with both CCR1 and CCR2 on monocytes (data not shown). Concentrations of CP-481,715 up to 25 μm did not inhibit the chemotaxis of THP-1 or primary human monocytes in response to the CCR2 ligand, CCL2, indicating that CP-481,715 did not exert nonspecific effects on cells that would generally interfere with migration. Because calcium mobilization studies demonstrated a proportional relationship between the amount of CP-481,715 necessary to inhibit activity and the concentration of CCL3, we next determined whether the same effects were observed on chemotaxis. As shown in Fig. 7, the concentration of CP-481,715 necessary to inhibit 50, 75, or 90% of the chemotactic response to CCL3 was directly dependent on the amount of agonist used to stimulate the response. For example, the IC50 for CP-481,715 was 5, 16, and 66 nm using CCL3 concentrations of 0.25, 0.5, and 1.0 nm, respectively. Similar results were also observed when CCL5 was used as the agonist (data not shown). These studies further suggest that CP-481,715 is a competitive antagonist of CCR1. An important property for a receptor antagonist is reversibility. Non-reversibility could imply a covalent interaction of the antagonist with the receptor which, in long term use in patients, could lead to immunogenicity. To determine whether the effects of CP-481,715 were reversible, we exposed THP-1 cells to various concentrations of CP-481,715 (up to 10 μm) for 30 min. As expected, the migration of these cells was totally inhibited in response to CCL3. However washing CP-481,715-exposed cells restored their ability to respond to CCL3 (data not shown), indicating that the effects of CP-481,715 on CCR1 are fully reversible. Selectivity of CP-481,715 for CCR1—CP-481,715 was evaluated against a panel of G-protein-coupled receptors using binding assays (Table I). In all cases, <10% inhibitory activity was observed at a concentration of 3 μm. Furthermore, drug concentrations as high as 25 μm did not inhibit receptor binding of the appropriate ligand to cells expressing CCR2, CCR3, CCR5, CXCR1, and CXCR2, and did not inhibit the chemotaxis of human cells to the chemokines CCL2, CCL27, CCL20, CCL19, CXCL8, CXCL11, and CXCL12 (data not shown). These results indicate that CP-481,715 is >100-fold selective for CCR1. In addition to its selectivity as compared with other receptors, CP-481,715 is also selective for the human receptor and does not inhibit the effects of CCL3 on mouse, rat, guinea pig, dog, rabbit, or monkey leukocytes as demonstrated with binding, chemotaxis, and whole blood studies at concentrations up to 25 μm (data not shown).Table IPanel of G-protein-coupled receptors evaluated for activity with CP-481,715 GABA, γ-aminobutyric acid; AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; NMDA, N-methyl-d-asparate; PAF, platlet-activating factor.ClassReceptor evaluatedAdenosineA1, A2A, A3Adrenergicα1, non-selective, α2, non-selective, β1, β2Angiotensin IIAT1, AT2BradykininB1, B2DopamineD1, D2, D3, D4.4GABANon-selectiveGlutamateAMPA, Kainate, NMDAHistamineH1, central, H2, H3MelanocortinMC4MuscarinicM1, M2, M3, M4NeurokininNK1NicotinicNeuronal, muscle-typeOpiateδ, κ, μPlatelet-activating factorPAFSerotonin5-HT1A, 5-HT2A, 5-HT2C, 5-HT3, 5-HT4, 5-HT7 Open table in a new tab Effects of CP-481,715 on CCL3-mediated CD11b Up-regulation in Human Whole Blood—A crucial attribute for therapeutic agents is their ability to retain activity in the presence of high protein concentrations. This may be especially true for chemokine receptor antagonists since the cells migrating into inflammatory sites in response to a chemotactic gradient would come from the peripheral blood. To address this, we measured two components of CCR1 activity, integrin up-regulation and cytoskeletal rearrangement in whole blood. The up-regulation of integrins, such as CD11b, mediate the firm adherence of leukocytes to the endothelium, a prerequisite to cell infiltration. As shown in Fig. 8, CP-481,715 dose-dependently inhibited CD11b up-regulation on monocytes in human blood (average IC50 from 10 different donors = 160 nm). As an alternative assessment of whole blood activity that may be more indicative of cell migration, we also measured actin polymerization on monocytes in response to CCL3. CP-481,715 also inhibited CCL3-induced actin polymerization in human blood with an IC50 of 58 nm (Fig. 8). Schild Analysis of the Interaction of CP-481,715 in Whole Blood—To further characterize the antagonist properties of CP-481,715, we assessed the effect of multiple concentrations of CP-481,715 on" @default.
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