Small Neutralizing Molecules to Inhibit Actions of theChemokine CXCL12*□S
Received for publication, May 22, 2008, and in revised form, June 13, 2008 Published, JBC Papers in Press, June 13, 2008, DOI 10.1074/jbc.M803947200
Muriel Hachet-Haas‡1, Karl Balabanian§2, Francois Rohmer‡, Francoise Pons¶, Christel Franchet‡3, Sandra Lecat‡,Ken Y. C. Chow§, Rania Dagher‡, Patrick Gizzi‡, Bruno Didier‡, Bernard Lagane§, Esther Kellenberger‡,Dominique Bonnet‡, Francoise Baleux§, Jacques Haiech‡, Marc Parmentier�, Nelly Frossard¶,Fernando Arenzana-Seisdedos§, Marcel Hibert‡, and Jean-luc Galzi‡4
From the ‡Institut Gilbert Laustriat “Biomolecules, Biotechnologie, Innovation therapeutique” LC1/Unite Mixte de Recherche (UMR)7175, Institut Federatif de Recherche (IFR) 85, Universite Louis Pasteur, 67401 Illkirch, France, §Unite pathogenie Virale Moleculaire,Institut Pasteur, 75015 Paris, France, ¶Inflammation et environnement dans l’asthme EA 3771, IFR 85, Faculte de Pharmacie, 74route du Rhin, BP 60024, 67401 Illkirch Cedex, France, �Institut de Recherche Interdisciplinaire en Biologie humaine et moleculaire(IRIBHM), Universite Libre de Bruxelles, 1070 Brussels, Belgium
The chemokineCXCL12and the receptorCXCR4playpivotalroles in normal vascular and neuronal development, in inflam-matory responses, and in infectious diseases and cancer. Forinstance, CXCL12 has been shown to mediate human immuno-deficiency virus-induced neurotoxicity, proliferative retinopa-thy and chronic inflammation, whereas its receptor CXCR4 isinvolved in human immunodeficiency virus infection, cancermetastasis and in the rare disease known as the warts, hypoga-mmaglobulinemia, immunodeficiency, and myelokathexis(WHIM) syndrome. As we screened chemical libraries to findinhibitors of the interaction between CXCL12 and the receptorCXCR4, we identified synthetic compounds from the family ofchalcones that reduce binding of CXCL12 to CXCR4, inhibitcalcium responses mediated by the receptor, and prevent CXCR4internalization in response to CXCL12.We found that the chemi-cal compounds display an original mechanism of action as theybind to thechemokinebutnot toCXCR4.Thehighest affinitymol-ecule blocked chemotaxis of humanperipheral blood lymphocytesex vivo. It was also active in vivo in a mousemodel of allergic eosi-nophilic airway inflammation in which we detected inhibition ofthe inflammatory infiltrate. The compound showed selectivity forCXCL12 and not for CCL5 and CXCL8 chemokines and blockedCXCL12 binding to its second receptor, CXCR7. By analogy to theeffect of neutralizing antibodies, this molecule behaves as a smallorganic neutralizing compound that may prove to have valuablepharmacological and therapeutic potential.
Chemokines are small (8–10-kDa) secreted proteins thatplay roles in the normal physiology of the immune system aswell as in orchestrating leukocyte recruitment and activation inthe context of inflammatory and infectious diseases (1).Most ofthem belong to one of two major subfamilies: the � or CC che-mokines in which two conserved cysteines from the amino ter-minus are adjacent to each other and the� or CXC chemokinesin which these two cysteines are separated by one residue. Che-mokine receptors aremembers 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),5
now named CXCL12, binds to and activates the chemokinereceptor CXCR4 as well as themore recently identified CXCR7receptor (19). CXCL12 stimulates a rapid receptor-mediatedintracellular calciummobilization and signaling through a Per-tussis toxin-sensitive Gi protein. The response to CXCL12 andexpression of the CXCR4 receptor occur at a very early stage ofembryonic development and appear to be widely used when-ever cell migration is required (2). Indeed mice lacking eitherCXCL12 or CXCR4 die prenatally and exhibit defects in vascu-lar development, neuronal development, hematopoiesis, andcardiogenesis (3–6).Besides the regulation of homeostatic processes, the CXCR4
receptor is implicated in tumormetastasis (7) aswell as in infec-tious and inflammatory diseases. Indeed in different mousemodels of allergic eosinophilic airway inflammation, it wasshown that either competitive antagonists (8) or antibodiesagainst the CXCR4 receptor as well as antibodies neutralizingthe CXCL12 chemokine (9) significantly lower eosinophilrecruitment in lung and reduce airway hyperreactivity.Also inherited heterozygous autosomal dominant mutations
of the CXCR4 gene, which result in the truncation of the car-boxyl terminus (C-tail) of the receptor, are associated with therare disease known as the warts, hypogammaglobulinemia,immunodeficiency, andmyelokathexis (WHIM) syndrome (10,
* This work was supported by the Agence Nationale de Recherches sur leSyndrome d’Immunodeficience Acquise (ANRS), Sidaction, the CNRS, theRegion Alsace, the Association Francaise contre les Myopathies, TheReseau National des Genopoles, and the Agence Nationale de la Recher-che. The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked“advertisement” in accordance with 18 U.S.C. Section 1734 solely to indi-cate this fact.
□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Fig. 1.
1 A fellow of the ANRS.2 Supported by a young investigator fellowship from INSERM. Present
address: INSERM U764, Universite Paris-Sud 11, Faculte de Medecine ParisSud, IFR 13, 92140 Clamart, France.
3 Present address: Faust-Pharmaceutical SA, 67401 Illkirch, France.4 To whom correspondence should be addressed: CNRS UMR 7175 et IFR 85
Gilbert Laustriat Biomolecules et innovations therapeutiques, EcoleSuperieure de Biotechnologie, Bld. Sebastien Brant BP 10413, F-67412Illkirch, France. E-mail: [email protected].
5 The abbreviations used are: SDF1, stromal cell-derived factor-1; HIV, humanimmunodeficiency virus; EGFP, enhanced green fluorescent protein; biot,biotin; HPLC, high performance liquid chromatography; HEK, humanembryonic kidney.
11). Finally the chemokine receptor CXCR4 also serves as acoreceptor to HIV type 1 to infect T cells (12).Considering both qualitative and quantitative aspects of the
involvement of the CXCR4/CXCL12 pair in the above men-tioned physiological and pathological functions on the onehand and the limited number of pharmacological tools to inves-tigate their function or to correct for defects in their function-ing, we set up a screening program to identify new moleculesinterferingwith the binding ofCXCL12 to the receptorCXCR4.Here we describe the discovery of a new class of pharmacolog-ically active molecules that bind to the chemokine itself andneutralize its biological activity in a way similar to that of neu-tralizing antibodies.
EXPERIMENTAL PROCEDURES
Antibodies and Reagents—All antibodies were purchasedfrom BD Biosciences. Chalcone and baicalin stock solutionswere prepared in sterile DMSO and then stored at �20 °Cbefore use. The human chemokines CXCL12 and CXCL12-Texas Red were synthesized as described previously (13, 14).The strategy used for the introduction of the Texas Red mol-ecule was the same as for the biotin molecule. After 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl pro-tection, Texas Red was introduced using Texas Redsuccinimidylester, mixed isomers (Invitrogen). The humanchemokines CCL5 and CXCL8 were purchased from BDBiosciences.Chemical Library Screening—The collection of 3,200
screenedmolecules was taken from the Chemical Library of theSchool of Pharmacy of Strasbourg (Institut Federatif de Recher-che 85). Human embryonic kidney 293 cells expressing thefusion receptor EGFP-hCXCR4 (14) were harvested in phos-phate-buffered saline (137 mM NaCl, 2.7 mM KCl, 4.3 mMNa2PO4�7H2O, 1.4 mM KH2PO4, pH 7.4) supplemented with 5mM 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, 0.4mMNaH2PO4, 1% bovine serum albumin (w/v), pH 7.4) supple-mented with protease inhibitors (40 �M/ml bestatin and baci-tracin, 20 �M/ml phosphoramidon, 50 �M/ml chymostatin, 1mg/ml leupeptin). Cells were distributed (75 000 cells/70�l/well) into 96-half-well polystyrene plates (Cliniplates,Thermo Labsystems) previously filled (2 �l/well) with fluores-cent CXCL12 (100 nM final concentration) and amolecule fromthe chemical library (20 �M final concentration). After 15 minat room temperature, fluorescence of cells was recorded at 510nm (excitation at 465 nm) using a multilabel counter (Victor 2,BD Biosciences). Hit compounds were confirmed by repeatingthe experiment.Real Time Fluorescence Monitoring of Ligand-Receptor
Interactions—Experiments were performed on cells stablyexpressing the EGFP-CXCR4 receptor suspended inHepes-bo-vine serum albumin buffer (typically at 106 cells/ml). Time-based recordings of the fluorescence emitted at 510 nm (exci-tation at 470 nm) were performed at 21 °C using aspectrofluorometer and sampled every 0.3 s. Fluorescencebinding measurements were initiated by adding at 30 s 100 nMCXCL12-Texas Red to the 1-ml cell suspension. For competi-
tion experiments, EGFP-CXCR4-expressing cells were prein-cubated for 5min in the absence or presence of various concen-trations of unlabeled drugs. Then CXCL12-Texas Red (100 nM)was added, and fluorescence was recorded until equilibriumwas reached (300 s). Data were analyzed using Kaleidagraph3.08 software (Synergy Software, Reading, PA).Intracellular Ca2� Release Measurement—Intracellular
Ca2� release measurement was carried out as described previ-ously (15, 16) using indo-1 acetoxymethyl ester as the calciumprobe. Cellular responses were recorded at 37 °C in a stirred1-ml cuvette with excitation set at 355 nm and emission set at405 and 475 nm using a spectrofluorometer.Internalization of EGFP-CXCR4 Receptors—Internalization
of EGFP-CXCR4 receptors was recorded as described previ-ously (17) using cell surface labeling of EGFP with monoclonalmouse 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 stainingwas quantified by flow cytometric analysis (10,000 cells/sam-ple) on a cytometer (FACSCalibur, BD Biosciences). The meanof CXCR4 fluorescence intensity was calculated usingCellQuest (BD Biosciences) software.Chemotaxis Assays—CD4� T lymphocytes were isolated
from fresh blood samples of healthy volunteers as describedpreviously (18) and cultured overnight in RPMI 1640 mediumsupplemented with 10% heat-inactivated fetal calf serum, 10mM Hepes, 100 units/ml penicillin, and 100 �g/ml streptomy-cin. Chemotaxis of CD4� T cells was evaluated using the Tran-swell system as described previously (11, 18). The fraction oftransmigrated T cells was calculated as follows: ((number of Tcells migrating to the lower chamber)/(number of T cells addedto the upper chamber at the start of the assay)) � 100.Binding Experiments on CXCR7 Receptor—The binding
experimentswere performed using the same experimental con-ditions as reported previously (19) with the exception thatCXCL12-biotin (CXCL12-biot) concentration was used here at1 nM. The incubation of increasing concentrations of chalcone4 with CXCL12-biot was made in the binding buffer during 1 hat room temperature before addition to cell suspension.Untagged CXCL12 was used at 1 �M as a control in the compe-tition experiments.Tryptophan Fluorescence Assay—Binding of chalcone 4 and
chalcone 1 to CXCL12was examined bymonitoring changes inthe emission intensity of intrinsic Trp fluorescence of the che-mokine. Increasing amounts of molecule were added toCXCL12 protein (2 �M in Hepes buffer without bovine serumalbumin in a 1-ml quartz microcuvette. Fluorescence measure-ments were carried out in triplicate using a Fluorolog 3 spec-trofluorometer (Jobin-Yvon/Spex). The excitation wavelengthwas set to 295 nm, and emission was collected from 310 to 400nm. All solutions were thermostated at 20 °C and continuouslystirred using a small magnetic bar. All fluorescence emissionspectra were corrected for the Raman peak by subtracting theemission scan of the buffer alone.Solubility Measurements—Solubility measurements of chal-
cones 1 and 4 were done by dissolving the compounds up tosaturation in solutions of CXCL12 prepared in the following
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buffer: Tris 50 mM (pH � 8), NaCl 200 mM, CaCl2 1 mM, imid-azole 10 mM. For each chalcone, the maximal solubility wasmeasured in four solutions containing 0, 312, 625, and 1000 �MCXCL12. Samples were shaken for 24 h at 20–22 °C, and foreach solution, the saturation was confirmed by the presence ofundissolved chalcone in excess. After ultracentrifugation (Sor-vall Discovery M120 SE ultracentrifuge with S45-A rotor cen-trifuged at 40,000 rpm), the concentration in the supernatantsolution was determined using high performance liquid chro-matography (HPLC).The measurements were done using a Gilson HPLC chain
with a UV detector set at 280 nm and a Rheodyne injector witha 50-�l loop. Data acquisition and processing were performedwith Unipoint software version 1.71. The reverse phase meas-urements were carried out at room temperature on a 5-�mLuna C18(2) Phenomenex column (150 � 4.6 mm). The aque-ous mobile phase contained 0.1% trifluoroacetic acid (solventA). 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 thefollowing programwas applied for the elution: 0–2.5min, 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 inDMSO. To establish external calibration curves, four differentconcentrations in the range of 10–400 �Mwere prepared fromstandard stock solutions. The chromatograms were recordedby injecting 50 �l of each standard solution, and the calibrationcurves were plotted using peak areas and concentrations. Theretention times for chalcones 1 and 4 were 17.3 and 16.5 min,respectively. 50 �l were injected for the eight saturated solu-tions. The solutions with 625 and 1000 �M CXCL12 had to bediluted before HPLC analysis because their chalcone concen-trations were beyond the calibration ranges.Isothermal TitrationMicrocalorimetry—Isothermal titration
calorimetry measurements were carried out at 25.0 °C using aVP-ITC (MicroCal) titration calorimeter. All solutions werethoroughly degassed before use by stirring under vacuum. Thesample cell was loaded with 1.4 ml of 1 �M CXCL12 in 50 mMHepes, 100 mM KCl buffer, pH 7.5, and the reference cell con-tained distilled water. Titration was carried out using a 300-�lsyringe filledwith 0.2mMchalcone at 10%DMSO inHepes/KClbuffer with stirring at 300 rpm. Injections were started afterbase-line stability had been achieved. A titration experimentconsisted of 15 consecutive injections of 2-�l volume and 6.8-sduration for each with a 4-min interval between injections. Theheat of dilution was measured by injecting chalcone into buffersolution without protein. The enthalpy change for each injec-tion was calculated by integrating the area under the peaks ofrecorded time course of power change and then subtractingthat from the control titration. Data were analyzed usingMicroCalOrigin software with equations corresponding to setsof identical sites and to sets of independent sites.Mouse Model of Allergic Eosinophilic Airway Inflammation—
The protocol used BALB/cmice (9 weeks; Charles River, Saint-Germain-sur-l’Arbresle, France) according Ref. 20. Brieflymicewere sensitized on days 1 and 7 by intraperitoneal injections of
50 �g of ovalbumin � 2 mg of Al(OH)3 in saline (phosphate-buffered saline) and challenged on days 18–21 by ovalbumin(10 �g intranasally, 12.5 �l/nostril). Chalcone 4 (350 �mol/kgintraperitoneally) or vehicle (1% carboxymethylcellulose) wasadministered 2 h before each ovalbumin challenge. On day 22,the lungswere lavaged (10� 0.5ml of saline-EDTA). The bron-choalveolar lavage fluid was centrifuged to pellet cells, anderythrocytes were lysed by hypotonic shock. Cells were resus-pended in 500 �l of ice-cold saline-EDTA. Total and differen-tial cell counts were determined after cytocentrifugation of50,000 cells/slide andHemacolor (Merck) staining. At least 400cells were counted and identified as macrophages, eosinophils,lymphocytes, or neutrophils expressed as an absolute numberfrom the total cell count.Modeling of SDF1-Chalcone 4 Complex Three-dimensional
Structure—The 5.26 release of the Cambridge Structural Data-base (21) was searched to retrieve the crystal structures ofchemically similar compounds. The naked chalcone scaffold ofchalcone 4 was used as Conquest query. The 2006 release of thescreening Protein Data Bank (22) was searched to retrieve thecrystal structure of chalcone 4 chemical analogs bound to pro-tein. The three-dimensional structure of 2�,4,4�-trihy-droxychalcone in complex with the chalcone o-methyltrans-ferase (Protein Data Bank code 1FP1) was edited in Sybyl(Tripos, Inc., St. Louis,MO) to generate chalcone 4 coordinatesstored in the MOL2 file. Hydrogens were added according tothe Jchem (ChemAxon Kft., Budapest, Hungary) preferred tau-tomer at physiological pH.A few rotameric states were modified in the monomeric
structure of CXCL12 (Protein Data Bank code 1VMC) toenlarge the existing cleft at the dimer interface. The largestchanges concerned Leu-26 (�1 moved from gauche� togauche�, and �2 moved from trans to gauche�), Ile-58 (�1moved from gauche� to gauche�), Tyr-61 (�2 moved fromgauche� to gauche�), and Leu-62 (�1 moved from gauche� totrans, and �2moved from trans to gauche�). The protein struc-ture was energy-minimized using Sybyl (default settings) andserved as the target for chalcone 4 docking.Docking experiments were carried out using Gold (Cam-
bridge Crystallographic Data Centre, Cambridge, UK). Genericalgorithm default parameters were set, and the Goldscore scor-ing function was chosen. The protein site was defined with aradius of 10 Å around a point in the center of the cavity. Twodistance restraints of 1.5–4.5 Åwith a spring constant of 5wereset between the halogen atom of the chalcone 4 chlorophenylmoiety and Ile-51 and Trp-57 side chains. The chalcone 4 bestpose was manually edited to solvent expose the ligand carbonylgroup (which was buried in the hydrophobic region of the pro-tein) and to fix unrealistic torsion angles around the vinylgroup. The optimized complex between SDF1 and chalcone 4was further energy-minimized using Sybyl (default settings).
RESULTS
Searching for Small Compounds That Could Inhibit the Inter-action of the Chemokine CXCL12 with Its CXCR4 Receptor—We screened 3,200molecules from the collection of themedic-inal chemistry laboratories from Strasbourg University in afluorescent binding assay on whole living cells described previ-
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ously (14). Briefly the CXCR4 receptor was stably transfected inhuman embryonic kidney cells as a fusion protein with EGFPfused to the extracellular amino-terminal part of the receptor(EGFP-CXCR4), and the chemokine CXCL12 was covalentlylabeled with the fluorophore Texas Red. Association with fluo-rescent CXCL12 was detected as a decrease of EGFP fluores-cence emission that results from energy transfer to the TexasRed group of CXCL12 (Fig. 1A). CXCL12 binding saturationwas reached at concentrations beyond 300 nM, and the dissoci-ation constant of fluorescent CXCL12 for the CXCR4 receptoris 55 � 15 nM (Ref. 14 and this work). Unlabeled moleculescompeting with fluorescent CXCL12 prevented the decrease ofEGFP emission as a function of receptor sites occupancy as isillustrated in Fig. 1A. The detected variation of fluorescenceintensity can be quantified (15, 16, 23) to derive binding con-stants of hit molecules.The collection of small organicmolecules from the academic
medicinal chemistry laboratories of Strasbourg University is ofrelatively small size but exhibits important chemical diversity(24). Screening of this collection to find inhibitors of the inter-
action of CXCL12 with the CXCR4receptor led to the identification ofmolecules with a fairly high rate ofhit identification (2.5%) and confir-mation (10% of hits).Chalcone 4 Inhibits Binding of
CXCL12 to CXCR4—About 80 hitcompounds were identified in thechemical library as capable, at 10�M, of inhibiting more than 30% offluorescent CXCL12 binding toCXCR4. Of these, seven moleculeswere potent inhibitors of CXCL12binding because they were stillactive at a concentration of 1 �M.The most potent compound
emerging from confirmed hit mole-cules belongs to the family of chal-cones (Table 1); the remainder of hitcompounds exhibited IC50 valuesbeyond 20 �M and belong either tothe chalcone group or to anotherchemical class, the triazines (to bedescribed elsewhere). In the groupof chalcones, three molecules,namely chalcone 2, chalcone 3, andchalcone 4, are analogs of the lowaffinity chemical platform chalcone1 that is devoid of side chains(IC50 � 500 �M). As the two aro-matic rings progressively becomemore substituted (chalcone 2, chal-cone 3, and chalcone 4), the dissoci-ation constants incrementallydecreased to reach a submicromolarvalue (IC50 � 150 � 50 nM for chal-cone 4; see Fig. 1A for an example ofthe inhibition of the association of
CXCL12 to its receptor by chalcone 4). The structure-activityrelationship that we observed points to the importance of sub-stitution of ring A by the chloride atom at position 4� and to thesimultaneous substitutions at positions 3 and 4 of ring B (datanot shown). The affinity of chalcone 4 is only 1 order of magni-tude lower than that of the reference competitive antagonistpeptide T134 (25) (Fig. 1B and Table 1). The unsubstitutedplatform chalcone 1 is also present in the collection. However,because of its veryweak affinity, themoleculewas not identifiedas a hit compound.Chalcone 4 Inhibits CXCL12-evoked Calcium Cellular
Responses—The next step toward pharmacological character-ization of the most potent compound, chalcone 4, consisted indetermining its effects on CXCR4-mediated cellular responses.Chalcone 4 by itself did not induce any calcium response (datanot shown).Fig. 1C shows that chalcone 4 inhibited CXCL12-evoked cal-
cium responses in a dose-dependent manner and with anapparent inhibitory constant (210 � 50 nM) that is in goodagreement with its potency for inhibition of CXCL12 binding
[Log Chalcone 4 M]
% o
f cal
cium
Res
pons
e
-9 -8 -7 -5-6
100
90
80
70
60
50
40
30
A B
C D
% o
f sur
face
exp
ress
ion
Time (min)
100
90
80
70
60
50
400 10 20 30
EGFP
-CXC
R4
fluor
esce
nce
(510
nm
)
Time (sec)
1.1
1.0
0.9
0.8
0.7
0.6
0.50 50 100 200150 Concentration [Log M]
CXC
L12-
TR b
ound
1.0
0.8
0.6
0.4
0.2
0.0
-9 -8 -7 -5-6 -4 -3-10
FIGURE 1. Chalcones inhibit CXCL12 binding to CXCR4 and alter associated responses. A, binding kineticsof Texas Red-labeled CXCL12 (100 nM) to EGFP-tagged CXCR4. Normalized EGFP emission intensity at 510 nmwas followed as a function of time on a whole living cell suspension. CXCL12 was added at 30 s. Interactionbetween CXCL12 and CXCR4 resulted in a rapid decrease of EGFP fluorescence due to fluorescence resonanceenergy transfer between EGFP and Texas Red that reached a plateau at equilibrium (dashed squares). The solidline shows receptor fluorescence detected when cells were preincubated with an excess of peptide antagonistT134 (20 �M) to prevent CXCL12 binding. Preincubation with chalcone 4 at 100 nM (filled triangles) or 300 nM
(open triangles) dose-dependently inhibited binding of CXCL12 to its receptor. B, inhibition of CXCL12 bindingto CXCR4 as a function of increasing concentration of peptide T134 (squares), chalcone 4 (triangles), chalcone 2(diamonds), and baicalin (circles) was monitored using fluorescence resonance energy transfer intensity varia-tion as shown in A. IC50 values derived from the competition curves are given in Table 1. C, inhibition ofCXCL12-evoked calcium responses in HEK 293 cells. The graph reports the maximal amplitude of the calciumresponse peak to 5 nM CXCL12 determined in the absence (triangles) or presence (squares) of increasingconcentrations of chalcone 4 at 37 °C. Each data point represents the mean � S.D. of three independentexperiments performed in duplicates. D, inhibition of CXCL12-induced CXCR4 internalization in HEK 293 cells.Cell surface EGFP-tagged CXCR4 receptors were measured by antibody labeling and flow cytometry analysisand are represented as a function of time of incubation with 200 nM CXCL12 in the presence (squares) orabsence (triangles) of chalcone 4 (1 �M). Control cells were either exposed to no ligand (circles) or to chalcone4 alone (diamonds). Each data point represents the mean � S.D. of three experiments.
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(Fig. 1B). Yet in contrast to the known competitive peptideT134 that fully blocks calcium signaling at high concentration(20 �M; data not shown), chalcone 4 did not block more than60–70% of the response to 5 nM CXCL12. Maximal inhibi-tion by chalcone 4was not improvedwhen preincubation dura-tion with cells was increased from typically 30 s to 30 min,supporting our view that the mechanism of inhibition by chal-cone 4 differs from that of peptide T134.Chalcone 4 Inhibits CXCL12-evoked CXCR4 Internalization—
As an antagonist of CXCR4 responses, chalcone 4 also alteredchemokine-induced receptor internalization (Fig. 1D). Recep-tor endocytosis was monitored on HEK cells expressing EGFP-CXCR4andquantifiedbyflowcytometry.Endocytosiswastime-dependent and reached 55 � 4% in 30 min when cells wereexposed to 200 nMCXCL12. Chalcone 4 (1�M) did not alter thelevel of surface receptor on its ownbut significantly reduced theCXCL12 effect because only 20� 8% of the receptor moleculeswere internalized in 30 min.
Chalcone 4 Selectivity among Chemokine Receptors—To gaininsight into compound selectivity, we next characterized theeffect of chalcone 4 on calcium responses of various chemo-kines/receptor pairs (Fig. 2). Consistent with data from Fig. 1C,chalcone 4 inhibited 50% of CXCL12-evoked calciumresponses in HEK EGFP-CXCR4 cells (Fig. 2, left panel). Incontrast, it had no effect on CCL5-evoked calcium responses inHEKCCR5 cells (Fig. 2,middle panel) and inhibited only 15%ofthe maximal CXCL8-evoked responses in HEK EGFP-CXCR1cells (Fig. 2, right panel). These results support the idea thatchalcone 4 shows selectivity for the CXCL12/CXCR4 pair.Chalcone 4 Inhibits Chemotactic Responses to CXCL12—In
contrast to other known antagonists of CXCR4 receptors, suchas T134, P2G-CXCL12, or AMD3100 (26–28), we found thatchalcone 4 does not inhibit infection of humanCD4� CXCR4�
T lymphocytes by HIV in an assay carried out as describedpreviously (29) (data not shown). In such an assay, there is noimplication of the chemokine CXCL12. The data could be
TABLE 1Binding inhibition constants of chalconesIC50 values of chalcones and reference peptide T134 (14-mer, DK � D-Lys; Ci � L-citrulline) (30) were determined as values leading to 50% inhibition of CXCL12-TRbinding to EGFP-CXCR4 receptor. Each value is a mean � S.D. of three independent determinations carried out in duplicate.
Compound Structure IC50
Chalcone 1 or (E)-1,3-
diphenylprop-2-en-1-one
O
BA
2
3
4
56
11'
2'
3'
4'
5'
6'IC50 > 500 µM
Chalcone 2 or (E)-3-(4-
hydroxyphenyl)-1-
phenylprop-2-en-1-one)
IC50 = 15 ± 3 µM
Chalcone 3 or (E)-1-(6'-
hydroxyphenyl)-3-(4-
hydroxyphenyl)prop-2-en-1-
one)
IC50 = 24.2 ± 5 µM
Chalcone 4 or ((E)-1-(4'-
chlorophenyl)-3-(4-hydroxy-
3-metoxyphenyl)prop-2-en-1-
one)
IC50 = 150 ± 50 nM
T134 RRWCYRKDKPYRCiCR-COOH IC50 = 6.9 ± 0.5 nM
O
OH
O
OHOH
O
OH
O
Cl
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interpreted if one proposes that chalcone 4 binds to the chemo-kine CXCL12 instead of directly binding to the CXCR4 recep-tor. There is precedent because the natural flavone isolatedfrom Scutellaria baicalensis, baicalin, has been shown to bind,although with low affinity, to various chemokines (30), andinterestingly, chalcones are precursors of flavones and antho-cyanins.Wedid confirm that baicalin is an inhibitor of CXCL12binding to CXCR4 (Fig. 1B) although weaker than chalcone 4.We then decided to further explore the mode of action of chal-cone 4 using complementary experimental approaches.We first investigated the effect of chalcone 4 on the chemo-
tactic activity of CXCL12. The assay, carried out with CD4�-enriched T cells isolated from human blood, revealed a drasticdifference depending on the protocol. Indeed when chalcone 4(0.5, 2, or 10 �M) was preincubated with the cells, no inhibitionof CD4� T cell chemotaxis was detected (Fig. 3A), whereas thespecific CXCR4 antagonist, AMD3100 (1 �M), inhibited 90% ofCD4� T cell chemotaxis (18). In contrast, when chalcone 4 waspreincubated with CXCL12 (Fig. 3A), we observed a dose-de-pendent inhibition of chemotaxis with an apparent affinityclose to 1 �M suggesting that chalcone 4 may bind to the che-mokine rather than to the receptor. The absence of effects ofchalcone 4 alone on cells showed in addition that the moleculeis non-toxic to human T lymphocytes and does not trigger che-motaxis on its own.Chalcone 4 Inhibits CXCL12 Binding to CXCR7—If chalcone
4 acts as a ligand of the CXCL12 chemokine, one straightfor-ward prediction related to this mechanism of action is thatbinding of CXCL12 to its second natural receptor, CXCR7 (19,31), should be blocked by chalcone 4 as well. We tested thishypothesis in a binding assaymaking use of fluorescence detec-tion of bound CXCL12-biot by flow cytometry (19). A0.01 cellsthat do not expressCXCL12 binding siteswere transfectedwithCXCR7. They displayed significant CXCL12-biot binding (Fig.3B) of which nearly 80%was displaced by unlabeled CXCL12 inexcess. If chalconewas used to prevent CXCL12-biot binding, a
clear dose-dependent inhibition was detected (Fig. 3B). Theefficacy of chalcone 4, however, was strongly enhanced if thechemokine was preincubated with the chalcone for 1 h. Atthe concentration that blocks 50% of the binding of CXCL12 toCXCR4, namely 200 nM, chalcone 4 also displaced 50% of spe-cific CXCL12 binding to CXCR7. This effect was clearlydetected if the chalcone was preincubated with the chemokine
FIGURE 2. Selectivity of chalcone effects. Shown is inhibition by 10 �M chal-cone 4 of calcium responses triggered by either 10 nM CXCL12 on HEK EGFP-CXCR4 -expressing cells, by 10 nM CCL5 on HEK CCR5-expressing cells, or by50 nM CXCL8 on HEK EGFP-CXCR1-expressing cells. Typical kinetics of thecalcium response recorded during 80 s are shown. Results are expressed asthe percentage of the maximal peak amplitude of the calcium response in theabsence (black bars) or in the presence of 10 �M chalcone 4 (gray bars) andrepresent the mean � S.D. of three independent experiments. Statistical anal-ysis consisted of unpaired two-tailed Student’s t tests and was conductedwith Prism software (GraphPad). *, p � 0.05; **, p � 0.005.
FIGURE 3. Chalcone 4 is able to bind to CXCL12 chemokine. A, inhibition ofCXCL12-induced chemotaxis of human CD4� T cells by chalcone 4. To test theeffect of chalcone 4 on CXCR4-expressing cells, CD4� T cells (2 � 106 cells/ml)were treated in RPMI 1640 medium containing 20 mM Hepes with variousconcentrations of chalcone 4 at 37 °C for 1 h, washed three times, resus-pended in RPMI 1640 medium supplemented with 20 mM Hepes and 1%human AB serum (chemotaxis buffer), and then tested for chemotaxis inresponse to CXCL12 (middle panel). To assess the effect of chalcone 4 on thechemotactic activity of CXCL12, the chemokine was preincubated in RPMI1640 medium containing 20 mM Hepes with different concentrations of chal-cone 4 at room temperature (RT) for 1 h and then loaded in the lower chamberin the chemotaxis buffer (right panel). Chalcone 4 did not modulate sponta-neous T cell migration (left panel). Results are expressed as the percentage ofinput CD4� T cells that migrated to the lower chamber and represent themean � S.D. of three independent experiments performed in duplicate. Sta-tistical analysis consisted of unpaired two-tailed Student’s t tests and wereconducted with Prism software (GraphPad). *, p � 0.05; **, p � 0.005 as com-pared with CXCL12 alone. B, inhibition of CXCL12 binding to CXCR7 receptorby chalcone 4. Binding of CXCL12-biot to CXCR7-expressing A0.01 cells wasdetected, in the presence or absence of the indicated concentrations of chal-cone 4 (in �M), using phycoerythrin (PE)-labeled streptavidin and flow cytom-etry detection. Left panel, mock-transfected A0.01 cells were exposed or notto CXCL12-biot, and nonspecific binding was determined. The right panelshows the results of binding of CXCL12-biot to CXCR7-expressing A0.01 cells:the successive bars (from left to right) correspond to 1) no treatment, 2) incu-bation with CXCL12-biot, 3) displacement of CXCL12-biot by CXCL12 at 1 �M,4 – 6) displacement of CXCL12-biot by chalcone 4 at the indicated concentra-tions without preincubation of CXCL12-biot with chalcone 4, and 7–9) dis-placement of CXCL12-biot by chalcone 4 at the indicated concentrationsafter preincubation of CXCL12-biot with chalcone 4 for 1 h. MFI, geometricmean fluorescence intensity (expressed in arbitrary units).
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aswas shown to be necessary in the chemotaxis assay. Chalcone4 thus inhibited binding of CXCL12 to both CXCR4 andCXCR7 receptors with similar affinity, supporting the notionthat themolecule interacts with the chemokine and neutralizesits binding capacity.Chalcone 4 Binds to the Chemokine CXCL12 but Not to
CXCR4—The second evidence supporting that chalcone 4binds to CXCL12 was provided by tryptophan fluorescenceanalysis. CXCL12 contains a single tryptophan residue, Trp-57.This amino acid belongs to the carboxyl-terminal helicaldomain of the chemokine, and its indole ring is buried in ahydrophobic pocket (32). When CXCL12 is incubated withincreasing concentrations of chalcone 4, we found that trypto-phan fluorescence intensity at 340 nm declined (Fig. 4A). Theresulting Trp fluorescence inhibition curve was satisfactorilyfitted according to a 1:1 stoichiometry interaction model. The
deduced dissociation constant, KD � 220 � 80 nM, was in thesame range as the affinity estimates derived from CXCL12-Texas Red binding assays. Also consistent with the bindingassays shown in Fig. 1B, the analog chalcone 1 devoid of sub-stituent groups displayed poor potency to alter Trp fluores-cence and weak affinity (KD � 50 �M).Chalcone 4 Binds toMultiple Sites onCXCL12—While deter-
mining physicochemical properties of chalcones, we noticedthat they exhibit poor solubility in physiological buffers (5–15�M maximal solubility) and that solubility was significantlyimproved by soluble proteins like serum albumin. In addition,we noticed that the actions of the chalcones were significantlylarger after preincubation of the molecule with the target pro-tein. We therefore addressed the question as to whether thechemokine CXCL12 was able to solubilize the chalcone mole-cules. Solubility of chalcones 1 and 4 was thus determined after24-h incubation in a physiological buffer containing variousamounts of CXCL12. Fig. 4B shows that CXCL12 wasextremely efficient in solubilizing chalcone molecules becauseexperimental values are in the millimolar range in the presenceof chemokine. Interestingly the stoichiometry of solubilizationapproaches three molecules of chalcone 4 solubilized by onemolecule of CXCL12, whereas only about two molecules ofchalcone 1 are solubilized per CXCL12 molecule. This experi-ment strongly argues in favor of chalconemolecules interactingwith CXCL12 with, in addition, chalcone 4 binding to one sup-plementary site presumably mediating the biological effect andaccounting for tryptophan fluorescence quenching.The fact that chalcone molecule solubilization results from
binding to CXCL12 chemokine was further confirmed by iso-thermal titration microcalorimetric measurements. Fig. 5shows a heat effect generated by addition of chalcones 1 and 4to solutions of CXCL12, reflecting differential behaviors of thetwo molecules. The shapes of the two titration curves are sim-ilar for molar ratios beyond 2, i.e. for modest to low affinityinteraction, and exhibit an additional component in the molarratio smaller than 2 for chalcone 4 only, indicating some high
FIGURE 4. Interaction between chalcone 4 and CXCL12. A, tryptophan flu-orescence measurements. Titration of binding of chalcone 4 (filled squares)and chalcone 1 (filled triangles) to CXCL12 was determined by monitoringchanges at the maximal emission of Trp fluorescence of CXCL12 (measured at340 nm). Increasing amounts of molecule were added to CXCL12 solution (2�M in Hepes buffer). The equation used to fit experimental data is the root ofthe following second order equation: (RL)2 � (RL) � (�Ro � Lo � KD) � Ro �Lo � 0 where (RL) � ((Ro � Lo � KD) � ((�Ro � Lo � KD)2 � 4 � Ro � Lo)1/2)/2and where Ro, the concentration of chemokine, is set to 2 �M; Lo is the initialconcentration of chalcone; KD is the dissociation constant of the chalcone forthe chemokine; and RL is the fractional concentration of receptor and ligandcomplex. B, effect of CXCL12 on chalcone 4 and chalcone 1 solubility. Maximalsolubility of chalcones 1 and 4 was determined using HPLC detection in theabsence and presence of increasing concentrations of CXCL12 in 50 mM Tris(pH � 8), 200 mM NaCl, 1 mM CaCl2, 10 mM imidazole buffer. In the absence ofadded CXCL12, maximal chalcone 1 and 4 solubilities are 12 and 6 �M, respec-tively. In the presence of 1 mM CXCL12, chalcone 1 and 4 solubilities increaseto 1.9 mM and 2.7 mM, respectively, indicating a strong and differential effecton chalcone dissolution in the buffer.
FIGURE 5. Microcalorimetric determination of the interaction betweenchalcones 4 and 1 and CXCL12. Shown are typical isothermal titrationcurves of CXCL12 interaction with chalcone 4 (left) or chalcone 1 (right) at25 °C in 50 mM Hepes, 100 mM KCl buffer, pH 7.5. Each experimental data set(n � 3) consisted of 15 consecutive additions of 2 �l of concentrated chalconeto cover molar ratios up to 4 (chalcone 4) or 7 (chalcone 1). Data were ana-lyzed using MicroCal Origin software and were fitted to obtain thermody-namic parameters of the chalcone interaction with each binding site ofCXCL12: chalcone 4 was fitted with a model comprising two sets of independ-ent sites, one high affinity site with K1 � 1.7 � 0.4 � 10�8
M (H1 � 1.2 � 0.2 �105 kcal/mol, S � 426) and two low affinity sites with K2 � 2.1 � 0.2 � 10�6
M (H2 � �1.2 � 0.6 � 105 kcal/mol, S � �382). Chalcone 1 was fitted witha single set of two identical sites with K � 5.5 � 0.3 � 10�6
M (H � �3.6 �0.3 � 105 kcal/mol, S � �1169).
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affinity interaction. Datawere fitted using the equations for oneor two categories of independent site(s). The best fits wereobtained for each chalcone molecule with the number of sitesestimated from the solubility experiment stoichiometries, indi-cating that chalcone 1 (Fig. 5B) binds to two identical sites withlow affinity (KD � 5.5� 0.3� 10�6 M), whereas chalcone 4 (Fig.5A) binds to one high affinity site (KD� 1.7� 0.3� 10�8M) andtwo low affinity sites (KD � 2.1 � 0.2 � 10�6 M).Chalcone 4 Reduces Inflammation in a Murine Model of
Allergic Eosinophilic Airway Inflammation—Altogether theresults show that chalcone 4 directly interacted with CXCL12,reduced the ability of this chemokine to bind to its CXCR4receptor, and thus partially neutralized the biological functionsof the chemokine/receptor pair in vitro. To extend this findingin an in vivomodel involving CXCR4, we analyzed the effects ofchalcone 4 in a model of allergic eosinophilic airway inflamma-tion (8, 9).CXCL12 and CXCR4 have been suspected to take part in
inflammatory processes in particular because CXCR4 isexpressed in a wide variety of leukocytes such as T and B cells,eosinophils, andmast cells, which are involved in asthma-asso-ciated immune responses.We therefore investigated the poten-tial of chalcone 4 to inhibit airway inflammation in a murinemodel of ovalbumin-induced allergic eosinophilic airwayinflammation. Mice were sensitized to and challenged withovalbumin to develop airway inflammation (20) that specifi-cally led to the recruitment of 1.52 � 0.15 � 106 eosinophils(mean � S.E.), i.e. 60% of the recovered cells in the bronchoal-veolar lavage fluid (Fig. 6). We found that intraperitoneal treat-ment with chalcone 4 (at the dose of 350 �mol/kg) significantlyreduced the total number of cells collected in the bronchoal-veolar lavage fluid in particular by reducing to 8.4 � 0.8 � 105the number of recruited eosinophils (45% reduction compared
with solvent) (Fig. 6, inset). The number of macrophages wasnot modified (9.6 � 1.0 versus 9.6 � 1.1 � 105 in chalcone 4-and vehicle (carboxymethylcellulose)-treated animals, respec-tively). In addition, chalcone 4 inhibited the recruitment of lym-phocytes in bronchoalveolar fluid (from 13.7 � 4.0 � 103 to2.4 � 0.8 � 103 cells). No toxicity of chalcone 4 was detected atdoses up to 700�mol/kg. These data clearly show that chalcone4 was active at reducing the accumulation of eosinophils inairways in response to ovalbumin sensitization.This result is in excellent accordance with the previously
reported effect of AMD3100 in a model of allergic asthma tocockroach allergen (8) or of neutralizing antibodies directedeither against CXCR4 orCXCL12 (9). The good activity of chal-cone 4 on eosinophils infiltration supports reports of a directinvolvement of CXCL12 and CXCR4 in the asthmatic responsein an ovalbumin model of allergic eosinophilic airway inflam-mation (9). Both studies showed reduction of eosinophilicinflammation to the same extent as with chalcone 4, indicatingthat full inhibition presumably involves multiple signalingpathways. The inhibitory effectmay result from blockade of theinteraction of CXCL12 with multiple cell populations, includ-ing Th2 lymphocytes and eosinophils, both of which have beendescribed to express CXCR4 (33).
DISCUSSION
In this work we provide convergent functional and biophys-ical data to show that a lowmolecular weightmolecule is able tobind to the chemokine CXCL12 with high affinity to preventbinding of the chemokine to the receptors CXCR4 and CXCR7and thus to alter the functional consequences of this interactionas demonstrated here by inhibition of ex vivo chemotaxis and invivo anti-inflammatory activity in the airways. Considering thatchemokines are rather small proteins, our observations raisequestions concerning themolecularmechanismof action of theneutralizing molecule chalcone 4 and about the location of asuitable site(s) for small chemicals. Three crystallographic andthree NMR structures of CXCL12 are available in the ProteinData Bank (1QG7 (34), 1A15 (32), 2J7Z and 1SDF (35), 2SDF(35), and 1VMC (36)). In all structures, CXCL12 adopts a typi-cal fold comprising an �-helix tightly packed with a three-strand antiparallel �-sheet.In the monomeric state, the CXCL12 fold has been charac-
terized by NMR. It is almost identical to that reported in thedimeric state. In the most acute monomer NMR structureavailable (Protein Data Bank code 1VMC), the CXCL12 hydro-phobic patch consists of a shallow depression at the surface ofthe protein. Minor conformation changes allow us to form apocket that perfectly accommodates chalcone 4 (the overallroot mean square deviation computed over all residue C�atoms of CXCL12 of the starting and optimized structure isonly 0.24Å). The entire pocket, up to itsmouth, is hydrophobic.In the modeled CXCL12-chalcone complex, the chlorophe-
nyl moiety of the chalcone, which appears to be critical for highaffinity binding, is deeply buried inside the pocket (Fig. 7), andthe 3�-methoxy,4�-hydroxyphenyl moiety slightly protrudesfrom the solvent-accessible face of the cavity, thereby plausiblyoccluding the protein dimerization site.
EosinoTotal cells Macro
vehicleChalcone 4
Cell
num
ber
(x 1
06)
0
0.5
1.0
1.5
2.0
2.5*
*Neutro Lympho
Cell
num
ber
(x 1
04 )
0
0.5
1.0
1.5
2.0*
FIGURE 6. Chalcone 4 reduces inflammation in a mouse model of allergiceosinophilic airway inflammation. Shown are total, eosinophil (Eosino),and macrophage (Macro) as well as neutrophil (Neutro) and lymphocyte (Lym-pho) (inset) counts in bronchoalveolar lavage fluids from ovalbumin-sensi-tized and -challenged mice treated either with 350 �mol/kg chalcone 4 orwith its vehicle alone (carboxymethylcellulose). Data are means � S.E. of n �6 –7 animals. *, p � 0.05.
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We thus considered the possibility that chalcone 4 interactswith themonomer of CXCL12. Veldkamp et al. (37) and others(38) investigated the effect of solution composition on the qua-ternary structure of CXCL12, and they showed that CXCL12exists in a monomer-dimer equilibrium, yet only underextreme conditions, and that the dimer dissociation KD ishighly dependent on both the solution pH and the presence ofstabilizing counterions. Specifically for CXCL12 dimerizationto occur, high chemokine concentrations are required (in the�M–mM range); multivalent anions like phosphate, sulfate, cit-rate, or heparin must be present; and the pHmust be above thepresumed pKa ofHis-25, a residue positioned at the interface ofthe dimer (32, 34, 35). In samples containing only Hepes bufferat pH 7.4 the dimer dissociation KD is beyond 10 mM, whereasin 100 mM sodium phosphate at pH 7.4, it is 140–180 mM (37,38). Accordingly in our experimental conditions binding andfunctional responses were measured at 2 orders of magnitudelower concentrations than those at which CXCL12 can dimer-ize. The chemokineCXCL12 is thusmost likely in amonomericstate in our study.The very short distance (around 4 Å) between the Trp indole
and chalcone 4 in the model is also consistent with the ligandbeing able to quench Trp fluorescence. Accordingly the mod-eled site would correspond to the high affinity site at the level ofwhich chalcone 4 blocks chemokine function. On the otherhand, it has been described that smallmoleculesmay be accom-modated into unexpected pockets, arising from adaptative pro-cesses, that could not be predicted on the basis of crystallo-graphic data from protein (39). Further structural studies,including co-crystallization of the NMR solution structure of
the complex,will thus be required torefine structural hypotheses.Chalcone 4 exhibited binding
selectivity for the chemokineCXCL12 as compared with CCL5and CXCL8. Although the overallfold is conserved among the threeproteins (supplemental Fig. 1A),changes are observed in the carbox-yl-terminal helix orientation withrespect to the �-sheet especiallybetween CCL5 and CXCL12. Resi-dues forming the hydrophobic sur-face patch of CXCL12 and CXCL8are very similar, but the putativebinding pocket in CXCL8 is largerthan the one in CXCL12. Thus chal-cone 4 could interact in a differentmanner with these two chemokines.CCL5 has a higher content of aro-matic amino acid residues with ahigher compactness of side chains.As a consequence, CCL5 hydropho-bic incurvation is shallower thanthat in CXCL8 and CXCL12 (sup-plemental Fig. 1B), and it is unlikelyto bind any small molecular weightcompound. The hydrophobic in-
curvation on CXCL8 is larger than the hydrophobic incurva-tion on CXCL12, and the chalcone could interact in a differentmanner.Chalcones constitute a relatively large group endowed with
potential therapeutic biological activities on analgesia, inflam-mation (40–43), infectious diseases (44–46), or cancer (47). Inthemajority of plants, chalcones are precursors of other classesof flavonoids, such as flavanones, dihydroflavonols, and finallyanthocyanins, the major water-soluble pigments in flowers andfruits (for a review, see Ref. 48). The flavone baicalin hasreceived attention because of its potential anti-inflammatoryactions and inhibitory effect on HIV (49–51) reverse tran-scriptase, amechanismbywhich baicalin is thought to be activeat inhibiting HIV infection (49). The action of baicalin as ananti-inflammatory agent may proceed through its interactionwith chemokines as suggested by Li et al. (30). However, it mustbe noticed that the affinity of baicalin for the chemokinesCCL8, CXCL8, CCL4, or CXCL12 is very weak (30), and we didconfirm it in this work with CXCL12.Considering that chalcone 4 exhibits potent anti-inflamma-
tory activity upon binding to CXCL12 and not to CXCR4, itsmechanism of action markedly differs from that of other phar-macological agents acting upon binding to the receptorCXCR4, such as AMD3100 (52), ALX40-4C, or T22/T140 (5,53, 54). Analysis of functional properties of these compoundson constitutively active mutants of CXCR4 receptors (55)reveals that, at high doses, AMD3100 and ALX40-4C are weakpartial agonists and that T22/T140 is an inverse agonist of thereceptor functions.
FIGURE 7. Putative binding model of chalcone 4 to CXCL12. The CXCL12 cavity was obtained by manualediting of Leu-26, Ile-38, Ile-58, Tyr-61, and Leu-62 rotameric states of PDB entry 1VMC using Sybyl7.2 (Tripos,Inc.). The chalcone 4 pose into the CXCL12 cavity was generated by automatic docking using Goldv3.1 (Cam-bridge Crystallographic Data Centre) and then manually refined and energy-minimized using Sybyl7.2 (defaultsettings) (58). A, color-coded Connolly surface of the putative binding site of chalcone 4. The surface is coloredfrom brown to blue according to decreasing hydrophobicity. The chalcone backbone is represented as sticks.B, stereoview of optimal docking of chalcone 4 (white balls and sticks representation) in the CXCL12 monomerdisplayed as a green ribbon. The hydrophobic chlorophenyl moiety interacts with side chains of Val-23, Leu-26,Ile-38, Ala-40, Ile-51, Trp-57, Ile-58, and Tyr-61. The carbonyl group of the ligand faces the solvent, and the3�-methoxy,4�-hydroxyphenyl moiety establishes hydrophobic contacts with the side chains of Leu-28, Leu-36, Leu-62, and Leu-66 as well as one hydrogen bond with Asn-30.
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As a consequence of partial agonism, both AMD3100 andALX40-4C molecules are cautiously considered as antimeta-static agents in the many cancer types involving CXCR4 (56,57). Thus, it is possible that none of the known CXCR4 antag-onists will be found to have a neutral effect on basal CXCR4response levels.Chalcone 4, in contrast to AMD3100, ALX40-4C, and T22/
T140, did not alter the resting/basal level of the CXCR4-asso-ciated responses, as we show here on both calcium and chemo-tactic responses. It behaved as a neutral inhibitor of the ligand.It is thus interesting to consider antiligand molecules, or neu-traligands, as pharmacological tools to investigate the functionsof receptors as well as potential therapeutic agents with mech-anisms of action that differ from traditional competitive ligandsbinding to the receptor.
Acknowledgments—We thankV. Utard (UMR7175), P. Villa (IFR 85,Illkirch, France), F. Daubeuf and A. Degrave (EA 3771, Illkirch,France), and C. Ebel (Institute of Genetics and Molecular and Cellu-lar Biology, Illkirch, France) for technical help.
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