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Alkylamides from Echinacea Are a New Class of Cannabinomimetics

2006; Elsevier BV; Volume: 281; Issue: 20 Linguagem: Inglês

10.1074/jbc.m601074200

1083-351X

Stefan Raduner, Adriana Majewska, Jian‐Zhong Chen, Xiang‐Qun Xie, Jacques Hamon, Bernard Faller, Karl‐Heinz Altmann, Jürg Gertsch,

Cannabis and Cannabinoid Research

Alkylamides (alkamides) from Echinacea modulate tumor necrosis factor α mRNA expression in human monocytes/macrophages via the cannabinoid type 2 (CB2) receptor (Gertsch, J., Schoop, R., Kuenzle, U., and Suter, A. (2004) FEBS Lett. 577, 563–569). Here we show that the alkylamides dodeca-2E,4E,8Z,10Z-tetraenoic acid isobutylamide (A1) and dodeca-2E,4E-dienoic acid isobutylamide (A2) bind to the CB2 receptor more strongly than the endogenous cannabinoids. The Ki values of A1 and A2 (CB2 ∼60 nm;CB1 >1500 nm) were determined by displacement of the synthetic high affinity cannabinoid ligand [3H]CP-55,940. Molecular modeling suggests that alkylamides bind in the solvent-accessible cavity in CB2, directed by H-bonding and π -π interactions. In a screen with 49 other pharmacologically relevant receptors, it could be shown that A1 and A2 specifically bind to CB2 and CB1. A1 and A2 elevated total intracellular Ca2+ in CB2-positive but not in CB2-negative promyelocytic HL60 cells, an effect that was inhibited by the CB2 antagonist SR144528. At 50 nm, A1, A2, and the endogenous cannabinoid anandamide (CB2 Ki >200 nm) up-regulated constitutive interleukin (IL)-6 expression in human whole blood in a seemingly CB2-dependent manner. A1, A2, anandamide, the CB2 antagonist SR144528 (Ki 1500 nm) were determined by displacement of the synthetic high affinity cannabinoid ligand [3H]CP-55,940. Molecular modeling suggests that alkylamides bind in the solvent-accessible cavity in CB2, directed by H-bonding and π -π interactions. In a screen with 49 other pharmacologically relevant receptors, it could be shown that A1 and A2 specifically bind to CB2 and CB1. A1 and A2 elevated total intracellular Ca2+ in CB2-positive but not in CB2-negative promyelocytic HL60 cells, an effect that was inhibited by the CB2 antagonist SR144528. At 50 nm, A1, A2, and the endogenous cannabinoid anandamide (CB2 Ki >200 nm) up-regulated constitutive interleukin (IL)-6 expression in human whole blood in a seemingly CB2-dependent manner. A1, A2, anandamide, the CB2 antagonist SR144528 (Ki 50% were significantly higher than background interference and represent specific positive interactions with the radioligand-binding sites. CB2 Homology Model and Docking Study—The program HOMOLOGY/InsightII (MSI-Biosym InsightII/Homology version 98, MSI Inc., San Diego) was used to generate the initial three-dimensional structural model of the CB2 receptor based on the x-ray crystal structure of bovine rhodopsin (30Palczewski K. Kumasaka T. Hori T. Behnke C.A. Motoshima H. Fox B.A. Trong I. Le Teller D.C. Okada T. Stenkamp R.E. Yamamoto M. Miyano M. Science. 2000; 289: 739-745Crossref PubMed Scopus (5056) Google Scholar). Multiple sequence alignment among 10 selective GPCRs, including the CB2 receptor and bovine rhodopsin from the rhodopsin GPCR family, was first performed to distinguish the seven transmembrane domains and extra- and intra-loop regions of the receptors, and the results were refined and evaluated by mutation scores, pairwise hydrophobicity profiles, and Kyte-Doolittle plots. The CB2 three-dimensional structural model was then constructed by mapping the CB2 sequence on the homologous residues of the rhodopsin x-ray structure in 7TM regions and searching for homologous C-α backbone sequences in published structures from the Protein Data Bank in loop regions. The energy minimization and molecular dynamics (MD/MM) simulation was finally carried out to optimize the CB2 three-dimensional structural model (25Xie X.Q. Chen J.Z. Billings E.M. Proteins. 2003; 53: 307-319Crossref PubMed Scopus (103) Google Scholar). To explore the possible binding pocket or domain(s) inside the CB2 receptor, molecular surface and physicochemical property maps, i.e. electrostatic and hydrophobicity (lipophilicity) potentials, were generated on the Connolly solvent-accessible surface by using the MOLCAD program (SYBYL7.0) (molecular modeling software packages, version 7.0, Tripos Associates, Inc., St. Louis). MOLCAD's rendering techniques allow the rapid calculation and display of property-coded surfaces for the molecular recognition. The generated surface property maps were further examined for the complementary biological data. The alkylamide docking and CB2 protein-ligand complex studies were performed on the basis of the following docking protocol by using Tripos molecular modeling packages Sybyl7.0 on an SGI octane computer. First, a three-dimensional structure of the alkylamide molecule was built by the Sketch module in Sybyl7.0 and optimized by using the Tripos force field. The initial docking position of alkylamide molecules was established inside the hypothetic binding pocket that was defined on the basis of the MOLCAD-generated solvent-accessible cavity model of the CB2 receptor. Then the receptor-ligand binding geometry was optimized by flexible docking using the FlexiDock module in Sybyl7.0. During flexible docking simulation, the single bonds of the alkylamide and all side chains within the defined binding region, or 3 Å around the target ligand, of the CB2 receptor were defined as rotatable or flexible bonds, and the ligand was allowed to move flexibly within the tentative binding site/pocket. The atomic charges were recalculated by using Kollman all-atom for the protein and Gasteiger-Hückel for the ligand. The interaction energy was calculated using van der Waals, electrostatic, and torsional energy terms of the Tripos force field. The iterations were set at 20,000 generations for genetic algorithms. Subsequently, further optimization was carried out on the FlexiDock-generated CB2 receptor-ligand complexes by using energy minimization and molecular dynamics. In this study, the AMBER force field along with a 15-Å cut-off distance for nonbonded interactions was applied to optimizetheintermediateligand-boundCB2receptormodel.Adistance-dependent (ϵ = 5r) dielectric function was used. Before the optimization, a binding pocket was defined to include the ligand and the residues within 7.5 Å around the ligand in the complex. The molecular dynamics protocol consisted of the following. (i) Initial minimization for 500 iterations of steepest descents, followed by conjugate gradients minimization, until the root mean square deviation became less than 0.1 kcal ·mol–1 ·Å–1. (ii) MD simulations were then performed at a constant temperature of 1000 K and a time step of 1 fs for a total of 50 ps. Initially, a constraint was applied to keep the backbone atoms in the seven transmembrane domains inside the binding pocket and all of other atoms outside the binding pocket of the CB2 receptor. Fifty representative snapshots of the ligand receptor complex from the molecular dynamics run were retrieved, minimized with 500 iterations of steepest descent, and followed by conjugate gradient minimization until the maximum derivative was less than 0.1 kcal ·mol–1 ·Å–1. The minimization and molecular dynamics simulation of the ligand receptor complex were further analyzed and evaluated as described later. Measurement of [Ca2+]i—HL60 CB2-positive cells were washed once, and cells (107 cells/ml) were incubated at 37 °C for 20 min in Hanks' balanced salt solution containing fluo3/AM in a final concentration of 4 μm and 0.15 mg/ml Pluronic F-127. The cells were then diluted 1:5 in Hanks' balanced salt solution containing 1% fetal bovine serum and incubated for 40 min at 37 °C. Afterward, the cells were washed three times and resuspended in 500 μlofCa2+-free HEPES-buffered saline, containing 137 mm NaCl, 5 mm KCl, 1 mm Na2HPO4,5mm glucose, 0.5 mm MgCl2, 0.1 mm EGTA, 1 g/liter bovine serum albumin, 10 mm Hepes, pH 7.4. Prior to each measurement, the cells were incubated for 7 min in a 37 °C water bath. In some experiments the cells were pretreated for 4 min with SR144528 (1 μm). The cells were subsequently stimulated with drugs and vehicle controls and analyzed with the FL1 channel on a FACScan flow cytometer equipped with a 488 nm argon laser (BD Biosciences). Because the solvent (ethanol) showed an effect on [Ca2+]i in vehicle controls, this solvent effect was subtracted from each value. Quantification of Cytokines with CBAs—Cytokine production in human peripheral whole blood was analyzed in blood plasma or supernatants of cells cultured for 18 h at 37 °C, 5% CO2 using Cytometric Bead Arrays™ (BD Biosciences). Blood cultures were carried out as described above. IL-12p70, TNF-α, IL-10, IL-6, IL-1β, and IL-8 were detected using the human inflammation CBA kit (551811; BD Biosciences), and for GM-CSF, IL-7, IL-5, IL-4, and IL-3 detection the human allergy CBA kit (558022; BD Biosciences) was used. Tests were performed according to the manufacturer's instructions. Briefly, 50 μl of supernatants were mixed with 50 μl of phycoerythrin-conjugated cytokine capture beads. For each set of experiments a standard curve was generated. Prior to each measurement the red and orange channels were adequately compensated, according to instructions. FL-2 was typically compensated for 40% FL-1. After3hof incubation, samples were rinsed, fixed with 1% paraformaldehyde, and analyzed by flow cytometry (FACScan and FACSCanto) with the CBA Analysis Software; BD Biosciences). The results were expressed as pg/ml and then analyzed for their relative expression (control versus treated sample). The lower limit of detection for each cytokine was determined as 20 pg/ml. Drugs and Antibodies—Dodeca-2E,4E-dienoic acid isobutylamide (A2) was isolated from E. purpurea as published previously (31Bauer R. Reminger P. Wagner H. Phytochemistry. 1988; 27: 2339-2342Crossref Scopus (91) Google Scholar) for E. angustifolia root material. Dodeca-2E,4E,8Z,10Z-tetraenoic acid isobutylamide (A1) and undeca-2E-en-8,10-diynoic acid isobutylamide (A3) were gifts from R. Lehmann (MediHerb, Australia). Compounds were checked for identity and integrity by thin layer chromatography and 1H NMR (500 MHz Bruker) spectroscopy prior to use. Anandamide, 2-AG, AM630, and CP-55,940 were obtained from Tocris Cookson Ltd. (UK). SR144528 was obtained as a gift from Sanofi-Synthélabo Recherche (France). Fluo3/AM, Pluronic F-127, and the monoclonal anti-rabbit fluorescein isothiocyanate antibody were purchased from Sigma. CB2 rabbit polyclonal antibody (3561) was obtained from Abcam (UK) and was tested for differential binding to immune cells. The radioligand [3H]CP-55,940 was obtained from PerkinElmer Life Sciences. Anandamide, LPS (E. coli, serotype 055:B5), and PMA (from Euphorbiaceae) were obtained from Fluka Chemie, Switzerland. Thapsigargin was purchased from Alexis Biochemicals, Switzerland. Monoclonal αCD3 (555336) and αCD28 (348040) were purchased from Pharmingen. Calculations and Statistics—Results are expressed as mean values ± S.D. or ± S.E. for each examined group. Statistical significance of differences between groups was determined by the Student's t test (paired t test) with GraphPad Prism software. Outliners in a series of identical experiments were determined by Grubb's test (ESD method) with α set to 0.05. Statistical differences between treated and vehicle control groups were determined by Student's t test for dependent samples. Differences between the analyzed samples were considered as signi