The chemokine CXCL12 and the receptor CXCR4 play pivotal roles in normal vascular and neuronal development, in inflammatory responses, and in infectious diseases and cancer. For instance, CXCL12 has been shown to mediate human immunodeficiency virus-induced neurotoxicity, proliferative retinopathy and chronic inflammation, whereas its receptor CXCR4 is involved in human immunodeficiency virus infection, cancer metastasis and in the rare disease known as the warts, hypogammaglobulinemia, immunodeficiency, and myelokathexis (WHIM) syndrome. As we screened chemical libraries to find inhibitors of the interaction between CXCL12 and the receptor CXCR4, we identified synthetic compounds from the family of chalcones that reduce binding of CXCL12 to CXCR4, inhibit calcium responses mediated by the receptor, and prevent CXCR4 internalization in response to CXCL12. We found that the chemical compounds display an original mechanism of action as they bind to the chemokine but not to CXCR4. The highest affinity molecule blocked chemotaxis of human peripheral blood lymphocytes ex vivo. It was also active in vivo in a mouse model of allergic eosinophilic airway inflammation in which we detected inhibition of the inflammatory infiltrate. The compound showed selectivity for CXCL12 and not for CCL5 and CXCL8 chemokines and blocked CXCL12 binding to its second receptor, CXCR7. By analogy to the effect of neutralizing antibodies, this molecule behaves as a small organic neutralizing compound that may prove to have valuable pharmacological and therapeutic potential. The chemokine CXCL12 and the receptor CXCR4 play pivotal roles in normal vascular and neuronal development, in inflammatory responses, and in infectious diseases and cancer. For instance, CXCL12 has been shown to mediate human immunodeficiency virus-induced neurotoxicity, proliferative retinopathy and chronic inflammation, whereas its receptor CXCR4 is involved in human immunodeficiency virus infection, cancer metastasis and in the rare disease known as the warts, hypogammaglobulinemia, immunodeficiency, and myelokathexis (WHIM) syndrome. As we screened chemical libraries to find inhibitors of the interaction between CXCL12 and the receptor CXCR4, we identified synthetic compounds from the family of chalcones that reduce binding of CXCL12 to CXCR4, inhibit calcium responses mediated by the receptor, and prevent CXCR4 internalization in response to CXCL12. We found that the chemical compounds display an original mechanism of action as they bind to the chemokine but not to CXCR4. The highest affinity molecule blocked chemotaxis of human peripheral blood lymphocytes ex vivo. It was also active in vivo in a mouse model of allergic eosinophilic airway inflammation in which we detected inhibition of the inflammatory infiltrate. The compound showed selectivity for CXCL12 and not for CCL5 and CXCL8 chemokines and blocked CXCL12 binding to its second receptor, CXCR7. By analogy to the effect of neutralizing antibodies, this molecule behaves as a small organic neutralizing compound that may prove to have valuable pharmacological and therapeutic potential. Chemokines are small (8–10-kDa) secreted proteins that play roles in the normal physiology of the immune system as well as in orchestrating leukocyte recruitment and activation in the context of inflammatory and infectious diseases (1Fernandez E.J. Lolis E. Annu. Rev. Pharmacol. Toxicol. 2002; 42: 469-499Crossref PubMed Scopus (508) Google Scholar). Most of them belong to one of two major subfamilies: the β or CC chemokines in which two conserved cysteines from the amino terminus are adjacent to each other and the α or CXC chemokines in which these two cysteines are separated by one residue. Chemokine receptors are members of the superfamily of G protein-coupled receptors characterized by seven transmembrane-spanning regions and coupling to heterotrimeric G proteins. The CXC chemokine stromal cell-derived factor-1 (SDF1), 5The abbreviations used are:SDF1stromal cell-derived factor-1HIVhuman immunodeficiency virusEGFPenhanced green fluorescent proteinbiotbiotinHPLChigh performance liquid chromatographyHEKhuman embryonic kidney. now named CXCL12, binds to and activates the chemokine receptor CXCR4 as well as the more recently identified CXCR7 receptor (19Balabanian K. Lagane B. Infantino S. Chow K.Y. Harriague J. Moepps B. Arenzana-Seisdedos F. Thelen M. Bachelerie F. J. Biol. Chem. 2005; 280: 35760-35766Abstract Full Text Full Text PDF PubMed Scopus (840) Google Scholar). CXCL12 stimulates a rapid receptor-mediated intracellular calcium mobilization and signaling through a Pertussis toxin-sensitive Gi protein. The response to CXCL12 and expression of the CXCR4 receptor occur at a very early stage of embryonic development and appear to be widely used whenever cell migration is required (2McGrath K.E. Koniski A.D. Maltby K.M. McGann J.K. Palis J. Dev. Biol. 1999; 213: 442-456Crossref PubMed Scopus (399) Google Scholar). Indeed mice lacking either CXCL12 or CXCR4 die prenatally and exhibit defects in vascular development, neuronal development, hematopoiesis, and cardiogenesis (3Ma Q. Jones D. Borghesani P.R. Segal R.A. Nagasawa T. Kishimoto T. Bronson R.T. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9448-9453Crossref PubMed Scopus (1428) Google Scholar, 4Nagasawa T. Nakajima T. Tachibana K. Iizasa H. Bleul C.C. Yoshie O. Matsushima K. Yoshida N. Springer T.A. Kishimoto T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14726-14729Crossref PubMed Scopus (268) Google Scholar, 5Tachibana K. Hirota S. Iizasa H. Yoshida H. Kawabata K. Kataoka Y. Kitamura Y. Matsushima K. Yoshida N. Nishikawa S. Kishimoto T. Nagasawa T. Nature. 1998; 393: 591-594Crossref PubMed Scopus (1323) Google Scholar, 6Zou Y.R. Kottmann A.H. Kuroda M. Taniuchi I. Littman D.R. Nature. 1998; 393: 595-599Crossref PubMed Scopus (2127) Google Scholar). stromal cell-derived factor-1 human immunodeficiency virus enhanced green fluorescent protein biotin high performance liquid chromatography human embryonic kidney. Besides the regulation of homeostatic processes, the CXCR4 receptor is implicated in tumor metastasis (7Muller A. Homey B. Soto H. Ge N. Catron D. Buchanan M.E. McClanahan T. Murphy E. Yuan W. Wagner S.N. Barrera J.L. Mohar A. Verastegui E. Zlotnik A. Nature. 2001; 410: 50-56Crossref PubMed Scopus (4473) Google Scholar) as well as in infectious and inflammatory diseases. Indeed in different mouse models of allergic eosinophilic airway inflammation, it was shown that either competitive antagonists (8Lukacs N.W. Berlin A. Schols D. Skerlj R.T. Bridger G.J. Am. J. Pathol. 2002; 160: 1353-1360Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar) or antibodies against the CXCR4 receptor as well as antibodies neutralizing the CXCL12 chemokine (9Gonzalo J.A. Lloyd C.M. Peled A. Delaney T. Coyle A.J. Gutierrez-Ramos J.C. J. Immunol. 2000; 165: 499-508Crossref PubMed Scopus (177) Google Scholar) significantly lower eosinophil recruitment in lung and reduce airway hyperreactivity. Also inherited heterozygous autosomal dominant mutations of the CXCR4 gene, which result in the truncation of the carboxyl terminus (C-tail) of the receptor, are associated with the rare disease known as the warts, hypogammaglobulinemia, immunodeficiency, and myelokathexis (WHIM) syndrome (10Hernandez P.A. Gorlin R.J. Lukens J.N. Taniuchi S. Bohinjec J. Francois F. Klotman M.E. Diaz G.A. Nat. Genet. 2003; 34: 70-74Crossref PubMed Scopus (539) Google Scholar, 11Balabanian K. Lagane B. Pablos J.L. Laurent L. Planchenault T. Verola O. Lebbe C. Kerob D. Dupuy A. Hermine O. Nicolas J.F. Latger-Cannard V. Bensoussan D. Bordigoni P. Baleux F. Le Deist F. Virelizier J.L. Arenzana-Seisdedos F. Bachelerie F. Blood. 2005; 105: 2449-2457Crossref PubMed Scopus (243) Google Scholar). Finally the chemokine receptor CXCR4 also serves as a coreceptor to HIV type 1 to infect T cells (12Granelli-Piperno A. Moser B. Pope M. Chen D. Wei Y. Isdell F. O'Doherty U. Paxton W. Koup R. Mojsov S. Bhardwaj N. Clark-Lewis I. Baggiolini M. Steinman R.M. J. Exp. Med. 1996; 184: 2433-2438Crossref PubMed Scopus (219) Google Scholar). Considering both qualitative and quantitative aspects of the involvement of the CXCR4/CXCL12 pair in the above mentioned physiological and pathological functions on the one hand and the limited number of pharmacological tools to investigate their function or to correct for defects in their functioning, we set up a screening program to identify new molecules interfering with the binding of CXCL12 to the receptor CXCR4. Here we describe the discovery of a new class of pharmacologically active molecules that bind to the chemokine itself and neutralize its biological activity in a way similar to that of neutralizing antibodies. Antibodies and Reagents—All antibodies were purchased from BD Biosciences. Chalcone and baicalin stock solutions were prepared in sterile DMSO and then stored at -20 °C before use. The human chemokines CXCL12 and CXCL12-Texas Red were synthesized as described previously (13Amara A. Lorthioir O. Valenzuela A. Magerus A. Thelen M. Montes M. Virelizier J.L. Delepierre M. Baleux F. Lortat-Jacob H. Arenzana-Seisdedos F. J. Biol. Chem. 1999; 274: 23916-23925Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar, 14Valenzuela-Fernandez A. Palanche T. Amara A. Magerus A. Altmeyer R. Delaunay T. Virelizier J.L. Baleux F. Galzi J.L. Arenzana-Seisdedos F. J. Biol. Chem. 2001; 276: 26550-26558Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). The strategy used for the introduction of the Texas Red molecule was the same as for the biotin molecule. After 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl protection, Texas Red was introduced using Texas Red succinimidylester, mixed isomers (Invitrogen). The human chemokines CCL5 and CXCL8 were purchased from BD Biosciences. Chemical Library Screening—The collection of 3,200 screened molecules was taken from the Chemical Library of the School of Pharmacy of Strasbourg (Institut Fédératif de Recherche 85). Human embryonic kidney 293 cells expressing the fusion receptor EGFP-hCXCR4 (14Valenzuela-Fernandez A. Palanche T. Amara A. Magerus A. Altmeyer R. Delaunay T. Virelizier J.L. Baleux F. Galzi J.L. Arenzana-Seisdedos F. J. Biol. Chem. 2001; 276: 26550-26558Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) were harvested in phosphate-buffered saline (137 mm NaCl, 2.7 mm KCl, 4.3 mm Na2PO4·7H2O, 1.4 mm KH2PO4, pH 7.4) supplemented with 5 mm EDTA, pH 7.4; centrifuged; and resuspended in Hepes-bovine serum albumin buffer (10 mm Hepes, 137.5 mm NaCl, 1.25 mm MgCl2, 1.25 mm CaCl2, 6 mm KCl, 10 mm glucose, 0.4 mm NaH2PO4, 1% bovine serum albumin (w/v), pH 7.4) supplemented with protease inhibitors (40 μM/ml bestatin and bacitracin, 20 μM/ml phosphoramidon, 50 μM/ml chymostatin, 1 mg/ml leupeptin). Cells were distributed (75 000 cells/70 μl/well) into 96-half-well polystyrene plates (Cliniplates, Thermo Labsystems) previously filled (2 μl/well) with fluorescent CXCL12 (100 nm final concentration) and a molecule from the chemical library (20 μm final concentration). After 15 min at room temperature, fluorescence of cells was recorded at 510 nm (excitation at 465 nm) using a multilabel counter (Victor 2, BD Biosciences). Hit compounds were confirmed by repeating the experiment. Real Time Fluorescence Monitoring of Ligand-Receptor Interactions—Experiments were performed on cells stably expressing the EGFP-CXCR4 receptor suspended in Hepes-bovine serum albumin buffer (typically at 106 cells/ml). Time-based recordings of the fluorescence emitted at 510 nm (excitation at 470 nm) were performed at 21 °C using a spectrofluorometer and sampled every 0.3 s. Fluorescence binding measurements were initiated by adding at 30 s 100 nm CXCL12-Texas Red to the 1-ml cell suspension. For competition experiments, EGFP-CXCR4-expressing cells were preincubated for 5 min in the absence or presence of various concentrations of unlabeled drugs. Then CXCL12-Texas Red (100 nm) was added, and fluorescence was recorded until equilibrium was reached (300 s). Data were analyzed using Kaleidagraph 3.08 software (Synergy Software, Reading, PA). Intracellular Ca2+ Release Measurement—Intracellular Ca2+ release measurement was carried out as described previously (15Palanche T. Ilien B. Zoffmann S. Reck M.P. Bucher B. Edelstein S.J. Galzi J.L. J. Biol. Chem. 2001; 276: 34853-34861Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 16Vollmer J.Y. Alix P. Chollet A. Takeda K. Galzi J.L. J. Biol. Chem. 1999; 274: 37915-37922Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) using indo-1 acetoxymethyl ester as the calcium probe. Cellular responses were recorded at 37 °C in a stirred 1-ml cuvette with excitation set at 355 nm and emission set at 405 and 475 nm using a spectrofluorometer. Internalization of EGFP-CXCR4 Receptors—Internalization of EGFP-CXCR4 receptors was recorded as described previously (17Gicquiaux H. Lecat S. Gaire M. Dieterlen A. Mely Y. Takeda K. Bucher B. Galzi J.L. J. Biol. Chem. 2002; 277: 6645-6655Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar) using cell surface labeling of EGFP with monoclonal mouse anti-green fluorescent protein (Roche Applied Science; 1:100 dilution) as primary antibody and a R-phycoerythrin-conjugated AffiniPure F(ab′)2 fragment goat anti-mouse IgG (Immunotech; 1:100) as secondary antibody. CXCR4 staining was quantified by flow cytometric analysis (10,000 cells/sample) on a cytometer (FACSCalibur, BD Biosciences). The mean of CXCR4 fluorescence intensity was calculated using CellQuest (BD Biosciences) software. Chemotaxis Assays—CD4+ T lymphocytes were isolated from fresh blood samples of healthy volunteers as described previously (18Balabanian K. Harriague J. Decrion C. Lagane B. Shorte S. Baleux F. Virelizier J.L. Arenzana-Seisdedos F. Chakrabarti L.A. J. Immunol. 2004; 173: 7150-7160Crossref PubMed Scopus (69) Google Scholar) and cultured overnight in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, 10 mm Hepes, 100 units/ml penicillin, and 100 μg/ml streptomycin. Chemotaxis of CD4+ T cells was evaluated using the Transwell system as described previously (11Balabanian K. Lagane B. Pablos J.L. Laurent L. Planchenault T. Verola O. Lebbe C. Kerob D. Dupuy A. Hermine O. Nicolas J.F. Latger-Cannard V. Bensoussan D. Bordigoni P. Baleux F. Le Deist F. Virelizier J.L. Arenzana-Seisdedos F. Bachelerie F. Blood. 2005; 105: 2449-2457Crossref PubMed Scopus (243) Google Scholar, 18Balabanian K. Harriague J. Decrion C. Lagane B. Shorte S. Baleux F. Virelizier J.L. Arenzana-Seisdedos F. Chakrabarti L.A. J. Immunol. 2004; 173: 7150-7160Crossref PubMed Scopus (69) Google Scholar). The fraction of transmigrated T cells was calculated as follows: ((number of T cells migrating to the lower chamber)/(number of T cells added to the upper chamber at the start of the assay)) × 100. Binding Experiments on CXCR7 Receptor—The binding experiments were performed using the same experimental conditions as reported previously (19Balabanian K. Lagane B. Infantino S. Chow K.Y. Harriague J. Moepps B. Arenzana-Seisdedos F. Thelen M. Bachelerie F. J. Biol. Chem. 2005; 280: 35760-35766Abstract Full Text Full Text PDF PubMed Scopus (840) Google Scholar) with the exception that CXCL12-biotin (CXCL12-biot) concentration was used here at 1 nm. The incubation of increasing concentrations of chalcone 4 with CXCL12-biot was made in the binding buffer during 1 h at room temperature before addition to cell suspension. Untagged CXCL12 was used at 1 μm as a control in the competition experiments. Tryptophan Fluorescence Assay—Binding of chalcone 4 and chalcone 1 to CXCL12 was examined by monitoring changes in the emission intensity of intrinsic Trp fluorescence of the chemokine. Increasing amounts of molecule were added to CXCL12 protein (2 μm in Hepes buffer without bovine serum albumin in a 1-ml quartz microcuvette. Fluorescence measurements were carried out in triplicate using a Fluorolog 3 spectrofluorometer (Jobin-Yvon/Spex). The excitation wavelength was set to 295 nm, and emission was collected from 310 to 400 nm. All solutions were thermostated at 20 °C and continuously stirred using a small magnetic bar. All fluorescence emission spectra were corrected for the Raman peak by subtracting the emission scan of the buffer alone. Solubility Measurements—Solubility measurements of chalcones 1 and 4 were done by dissolving the compounds up to saturation in solutions of CXCL12 prepared in the following buffer: Tris 50 mm (pH = 8), NaCl 200 mm, CaCl2 1 mm, imidazole 10 mm. For each chalcone, the maximal solubility was measured in four solutions containing 0, 312, 625, and 1000 μm CXCL12. Samples were shaken for 24 h at 20–22 °C, and for each solution, the saturation was confirmed by the presence of undissolved chalcone in excess. After ultracentrifugation (Sorvall Discovery M120 SE ultracentrifuge with S45-A rotor centrifuged at 40,000 rpm), the concentration in the supernatant solution was determined using high performance liquid chromatography (HPLC). The measurements were done using a Gilson HPLC chain with a UV detector set at 280 nm and a Rheodyne injector with a 50-μl loop. Data acquisition and processing were performed with Unipoint software version 1.71. The reverse phase measurements were carried out at room temperature on a 5-μm Luna C18(2) Phenomenex column (150 × 4.6 mm). The aqueous mobile phase contained 0.1% trifluoroacetic acid (solvent A). The organic phase was HPLC grade acetonitrile (Sigma-Aldrich CHROMASOLV) containing 0.1% trifluoroacetic acid (solvent B). The mobile phase flow rate was 1 ml/min, and the following program was applied for the elution: 0–2.5 min, 0% B; 2.5–17 min, 0–100% B; 17–21 min, 100% B; 21–24.50 min, 100–0% B; and 24.50–30 min, 0% B. Standard stock solutions of chalcones 1 and 4 at a 1 mm concentration were prepared by dissolving molecules in DMSO. To establish external calibration curves, four different concentrations in the range of 10–400 μm were prepared from standard stock solutions. The chromatograms were recorded by injecting 50 μl of each standard solution, and the calibration curves were plotted using peak areas and concentrations. The retention times for chalcones 1 and 4 were 17.3 and 16.5 min, respectively. 50 μl were injected for the eight saturated solutions. The solutions with 625 and 1000 μm CXCL12 had to be diluted before HPLC analysis because their chalcone concentrations were beyond the calibration ranges. Isothermal Titration Microcalorimetry—Isothermal titration calorimetry measurements were carried out at 25.0 °C using a VP-ITC (MicroCal) titration calorimeter. All solutions were thoroughly degassed before use by stirring under vacuum. The sample cell was loaded with 1.4 ml of 1 μm CXCL12 in 50 mm Hepes, 100 mm KCl buffer, pH 7.5, and the reference cell contained distilled water. Titration was carried out using a 300-μl syringe filled with 0.2 mm chalcone at 10% DMSO in Hepes/KCl buffer with stirring at 300 rpm. Injections were started after base-line stability had been achieved. A titration experiment consisted of 15 consecutive injections of 2-μl volume and 6.8-s duration for each with a 4-min interval between injections. The heat of dilution was measured by injecting chalcone into buffer solution without protein. The enthalpy change for each injection was calculated by integrating the area under the peaks of recorded time course of power change and then subtracting that from the control titration. Data were analyzed using MicroCal Origin software with equations corresponding to sets of identical sites and to sets of independent sites. Mouse Model of Allergic Eosinophilic Airway Inflammation—The protocol used BALB/c mice (9 weeks; Charles River, Saint-Germain-sur-l'Arbresle, France) according Ref. 20Delayre-Orthez C. Becker J. de Blay F. Frossard N. Pons F. Int. Arch. Allergy Immunol. 2005; 138: 298-304Crossref PubMed Scopus (33) Google Scholar. Briefly mice were sensitized on days 1 and 7 by intraperitoneal injections of 50 μg of ovalbumin + 2 mg of Al(OH)3 in saline (phosphate-buffered saline) and challenged on days 18–21 by ovalbumin (10 μg intranasally, 12.5 μl/nostril). Chalcone 4 (350 μmol/kg intraperitoneally) or vehicle (1% carboxymethylcellulose) was administered 2 h before each ovalbumin challenge. On day 22, the lungs were lavaged (10 × 0.5 ml of saline-EDTA). The bronchoalveolar lavage fluid was centrifuged to pellet cells, and erythrocytes were lysed by hypotonic shock. Cells were resuspended in 500 μl of ice-cold saline-EDTA. Total and differential cell counts were determined after cytocentrifugation of 50,000 cells/slide and Hemacolor (Merck) staining. At least 400 cells were counted and identified as macrophages, eosinophils, lymphocytes, or neutrophils expressed as an absolute number from the total cell count. Modeling of SDF1-Chalcone 4 Complex Three-dimensional Structure—The 5.26 release of the Cambridge Structural Database (21Taylor R. Acta Crystallogr. Sect. D Biol. Crystallogr. 2002; 58: 879-888Crossref PubMed Scopus (88) Google Scholar) was searched to retrieve the crystal structures of chemically similar compounds. The naked chalcone scaffold of chalcone 4 was used as Conquest query. The 2006 release of the screening Protein Data Bank (22Kellenberger E. Muller P. Schalon C. Bret G. Foata N. Rognan D. J. Chem. Inf. Model. 2006; 46: 717-727Crossref PubMed Scopus (169) Google Scholar) was searched to retrieve the crystal structure of chalcone 4 chemical analogs bound to protein. The three-dimensional structure of 2′,4,4′-trihydroxychalcone in complex with the chalcone o-methyltransferase (Protein Data Bank code 1FP1) was edited in Sybyl (Tripos, Inc., St. Louis, MO) to generate chalcone 4 coordinates stored in the MOL2 file. Hydrogens were added according to the Jchem (ChemAxon Kft., Budapest, Hungary) preferred tautomer at physiological pH. A few rotameric states were modified in the monomeric structure of CXCL12 (Protein Data Bank code 1VMC) to enlarge the existing cleft at the dimer interface. The largest changes concerned Leu-26 (χ1 moved from gauche- to gauche+, and χ2 moved from trans to gauche+), Ile-58 (χ1 moved from gauche- to gauche+), Tyr-61 (χ2 moved from gauche+ to gauche-), and Leu-62 (χ1 moved from gauche- to trans, and χ2 moved from trans to gauche+). The protein structure was energy-minimized using Sybyl (default settings) and served as the target for chalcone 4 docking. Docking experiments were carried out using Gold (Cambridge Crystallographic Data Centre, Cambridge, UK). Generic algorithm default parameters were set, and the Goldscore scoring function was chosen. The protein site was defined with a radius of 10 Å around a point in the center of the cavity. Two distance restraints of 1.5–4.5 Å with a spring constant of 5 were set between the halogen atom of the chalcone 4 chlorophenyl moiety and Ile-51 and Trp-57 side chains. The chalcone 4 best pose was manually edited to solvent expose the ligand carbonyl group (which was buried in the hydrophobic region of the protein) and to fix unrealistic torsion angles around the vinyl group. The optimized complex between SDF1 and chalcone 4 was further energy-minimized using Sybyl (default settings). Searching for Small Compounds That Could Inhibit the Interaction of the Chemokine CXCL12 with Its CXCR4 Receptor—We screened 3,200 molecules from the collection of the medicinal chemistry laboratories from Strasbourg University in a fluorescent binding assay on whole living cells described previously (14Valenzuela-Fernandez A. Palanche T. Amara A. Magerus A. Altmeyer R. Delaunay T. Virelizier J.L. Baleux F. Galzi J.L. Arenzana-Seisdedos F. J. Biol. Chem. 2001; 276: 26550-26558Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Briefly the CXCR4 receptor was stably transfected in human embryonic kidney cells as a fusion protein with EGFP fused to the extracellular amino-terminal part of the receptor (EGFP-CXCR4), and the chemokine CXCL12 was covalently labeled with the fluorophore Texas Red. Association with fluorescent CXCL12 was detected as a decrease of EGFP fluorescence emission that results from energy transfer to the Texas Red group of CXCL12 (Fig. 1A). CXCL12 binding saturation was reached at concentrations beyond 300 nm, and the dissociation constant of fluorescent CXCL12 for the CXCR4 receptor is 55 ± 15 nm (Ref. 14Valenzuela-Fernandez A. Palanche T. Amara A. Magerus A. Altmeyer R. Delaunay T. Virelizier J.L. Baleux F. Galzi J.L. Arenzana-Seisdedos F. J. Biol. Chem. 2001; 276: 26550-26558Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar and this work). Unlabeled molecules competing with fluorescent CXCL12 prevented the decrease of EGFP emission as a function of receptor sites occupancy as is illustrated in Fig. 1A. The detected variation of fluorescence intensity can be quantified (15Palanche T. Ilien B. Zoffmann S. Reck M.P. Bucher B. Edelstein S.J. Galzi J.L. J. Biol. Chem. 2001; 276: 34853-34861Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 16Vollmer J.Y. Alix P. Chollet A. Takeda K. Galzi J.L. J. Biol. Chem. 1999; 274: 37915-37922Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 23Ilien B. Franchet C. Bernard P. Morisset S. Weill C.O. Bourguignon J.J. Hibert M. Galzi J.L. J. Neurochem. 2003; 85: 768-778Crossref PubMed Scopus (62) Google Scholar) to derive binding constants of hit molecules. The collection of small organic molecules from the academic medicinal chemistry laboratories of Strasbourg University is of relatively small size but exhibits important chemical diversity (24Krier M. Bret G. Rognan D. J. Chem. Inf. Model. 2006; 46: 512-524Crossref PubMed Scopus (109) Google Scholar). Screening of this collection to find inhibitors of the interaction of CXCL12 with the CXCR4 receptor led to the identification of molecules with a fairly high rate of hit identification (2.5%) and confirmation (10% of hits). Chalcone 4 Inhibits Binding of CXCL12 to CXCR4—About 80 hit compounds were identified in the chemical library as capable, at 10 μm, of inhibiting more than 30% of fluorescent CXCL12 binding to CXCR4. Of these, seven molecules were potent inhibitors of CXCL12 binding because they were still active at a concentration of 1 μm. The most potent compound emerging from confirmed hit molecules belongs to the family of chalcones (Table 1); the remainder of hit compounds exhibited IC50 values beyond 20 μm and belong either to the chalcone group or to another chemical class, the triazines (to be described elsewhere). In the group of chalcones, three molecules, namely chalcone 2, chalcone 3, and chalcone 4, are analogs of the low affinity chemical platform chalcone 1 that is devoid of side chains (IC50 > 500 μm). As the two aromatic rings progressively become more substituted (chalcone 2, chalcone 3, and chalcone 4), the dissociation constants incrementally decreased to reach a submicromolar value (IC50 = 150 ± 50 nm for chalcone 4; see Fig. 1A for an example of the inhibition of the association of CXCL12 to its receptor by chalcone 4). The structure-activity relationship that we observed points to the importance of substitution of ring A by the chloride atom at position 4′ and to the simultaneous substitutions at positions 3 and 4 of ring B (data not shown). The affinity of chalcone 4 is only 1 order of magnitude lower than that of the reference competitive antagonist peptide T134 (25Tamamura H. Omagari A. Oishi S. Kanamoto T. Yamamoto N. Peiper S.C. Nakashima H. Otaka A. Fujii N. Bioorg. Med. Chem. Lett. 2000; 10: 2633-2637Crossref PubMed Scopus (116) Google Scholar) (Fig. 1B and Table 1). The unsubstituted platform chalcone 1 is also present in the collection. However, because of its very weak affinity, the molecule was not identified as a hit compound.TABLE 1Binding inhibition constants of chalcones IC50 values of chalcones and reference peptide T134 (14-mer, DK = d-Lys; Ci = l-citrulline) (30Li B.Q. Fu T. Gong W.H. Dunlop N. Kung H. Yan Y. Kang J. Wang J.M. Immunopharmacology. 2000; 49: 295-306Crossref PubMed Scopus (208) Google Scholar) were determined as values leading to 50% inhibition of CXCL12-TR binding to EGFP-CXCR4 receptor. Each value is a mean ± S.D. of three independent determinations carried out in duplicate. Open table in a new tab Chalcone 4 Inhibits CXCL12-evoked Calcium Cellular Responses—The next step toward pharmacological characterization of the most potent compound, chalcone 4, consisted in determining its effects on CXCR4-mediated cellular responses. Chalcone 4 by itself did not induce any calcium response (data not shown). Fig. 1C shows that chalcone 4 inhibited CXCL12-evoked calcium responses in a dose-dependent manner and with an apparent inhibitory constant (210 ± 50 nm) that is in good agreement with its potency for inhibition of CXCL12 binding (Fig. 1B). Yet in contrast to the known competitive peptide T134 that fully blocks calcium signaling at high concentration (20 μm; data not shown), chalcone 4 did not block more than 60–70% of the response to 5 nm CXCL12. Maximal inhibition by chalcone 4 was not improved when preincubation duration with cells was increased from typically 30 s to 30 min, supporting our view that the mechanism of inhibition by chalcone 4 differs from that of peptide T134. Chalcone 4 Inhibits CXCL12-evoked CXCR4 Internalization—As an antagonist of CXCR4 responses, chalcone 4 also altered chemokine-induced receptor internalization (Fig. 1D). Receptor endocytosis was monitored on HEK cells expressing EGFP-CXCR4 and quantified by flow cytometry. Endocytosis was time-dependent and reached 55 ± 4% in 30 min when cells were exposed
Additional file 5: Figure S4. (Related to Figure 3). Quantitative histological and cytometric parameters of the muscle one month post NTX injury in the WT and the CXCL12Gagtm/Gagtm mice. (A) Quantification of calcium deposits number by Von Kossa staining in 12 days and one month post NTX injured TA from WT (C57Bl6) and CXCL12Gagtm/Gagtm mice. Three animals (n=3) were used per condition and were repeated independently two times. (B) Representative Von Kossa stained TA section 12 post NTX injury in KI (CXCL12Gagtm/Gagtm) mice. Scale bar represent 100μm. Quantification of (C) fibers number and (D) fibers diameter by Hematoxylin-eosin staining in uninjured and post NTX injured TA from WT (C57Bl6) and CXCL12Gagtm/Gagtm mice. Three animals (n=3) were used per condition and were repeated independently two times. (E) Quantification of vessels number by CD31 immunostaining in the uninjured and the post NTX injured TA from WT (C57Bl6) and CXCL12Gagtm/Gagtm mice. Three animals (n=3) were used per condition and were repeated independently two times. (F) Quantification of GFP-positive cells by FACS analysis per TA of the uninjured and the post NTX injured WT (Flk1GFP/+) vs. KI (CXCL12Gagtm/Gagtm :: Flk1GFP/+) mice. (n=5 mice per condition). Data are given as the mean ± SEM. * p < 0.05; ** p < 0.01, *** p < 0.001.
Abstract Chemokines and their receptors determine the distribution of leukocytes within tissues in health and disease. We have studied the role of the constitutive chemokine receptor CXCR4 and its ligand, stromal-derived factor-1 (SDF-1) in the perivascular accumulation of T cells in rheumatoid arthritis. We show that synovial T cells, which are primed CD45RO+CD45RBdull cells and consequently not expected to express constitutive chemokine receptors, have high levels of the chemokine receptor CXCR4. Sustained expression of CXCR4 was maintained on synovial T cells by specific factors present within the synovial microenvironment. Extensive screening revealed that TGF-β isoforms induce the expression of CXCR4 on CD4 T cells in vitro. Depletion studies using synovial fluid confirmed an important role for TGF-β1 in the induction of CXCR4 expression in vivo. The only known ligand for CXCR4 is SDF-1. We found SDF-1 on synovial endothelial cells and showed that SDF-1 was able to induce strong integrin-mediated adhesion of synovial fluid T cells to fibronectin and ICAM-1, confirming that CXCR4 expressed on synovial T cells was functional. These results suggest that the persistent induction of CXCR4 on synovial T cells by TGF-β1 leads to their active, SDF-1-mediated retention in a perivascular distribution within the rheumatoid synovium.
Human immunodeficiency virus entry into target cells requires sequential interactions of the viral glycoprotein envelope gp120 with CD4 and chemokine receptors CCR5 or CXCR4. CD4 interaction with the chemokine receptor is suggested to play a critical role in this process but to what extent such a mechanism takes place at the surface of target cells remains elusive. To address this issue, we used a confocal microspectrofluorimetric approach to monitor fluorescence resonance energy transfer at the cell plasma membrane between enhanced blue and green fluorescent proteins fused to CD4 and CCR5 receptors. We developed an efficient fluorescence resonance energy transfer analysis from experiments carried out on individual cells, revealing that receptors constitutively interact at the plasma membrane. Binding of R5-tropic HIV gp120 stabilizes these associations thus highlighting that ternary complexes between CD4, gp120, and CCR5 occur before the fusion process starts. Furthermore, the ability of CD4 truncated mutants and CCR5 ligands to prevent association of CD4 with CCR5 reveals that this interaction notably engages extracellular parts of receptors. Finally, we provide evidence that this interaction takes place outside raft domains of the plasma membrane.
ABSTRACT De novo synthesized IκBα accumulates transiently in the nucleus where it inhibits NF-κB-dependent transcription and reduces nuclear NF-κB content. A sequence present in the C-terminal domain of IκBα and homologous to the HIV-1 Rev nuclear export signal (NES) has been recently defined as a functional NES conferring on IκBα the ability to export IκBα/NF-κB complexes. Rev utilises its RNA-binding activity and NES sequence to promote specifically the transport of unspliced and monospliced viral RNAs to the cytoplasm. The object of this work was to determine if nuclear IκBα could interfere with Revdependent transport of viral RNA from the nucleus to the cytoplasm. We report that accumulation of IκBα in the cell nucleus blocks viral replication. This effect could be dissociated from the capacity of IκBα to inhibit NF-κB-DNA-binding activity and required a functional IκBα NES motif. Indeed, mutation of the NES abrogated the capacity of IκBα to inhibit Rev-dependent mechanisms involved in the replication of either wild-type or NF-κB-mutated HIV-1 molecular clones. Nuclear accumulation of a reporter protein tagged with a nuclear localization signal (NLS) and fused to the IκBα NES motif (NLS-PK-NES) was sufficient to inhibit HIV-1 replication at a post-transcriptional level by specifically blocking the expression of a Rev-dependent gene. Furthermore, in cells pulsed with TNF, a treatment which favors nuclear accumulation of newly synthesized IκBα, NLS-PK-NES expression promoted sustained accumulation of nuclear NF-κB lacking DNA-binding activity. This NES-mediated accumulation of inactive nuclear NF-κB is likely the consequence of interference in the IκBα-mediated export of NF-κB. These findings indicate that IκBα and Rev compete for the same nuclear export pathway and suggest that nuclear accumulation of IκBα, which would occur during normal physiological cell activation process, may interfere with the Rev-NES-mediated export pathway of viral RNAs, thus inhibiting HIV-1 replication.
