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• W2016128034 abstract "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 <10 nm), and also the non-CB2-binding alkylamide undeca-2E-ene,8,10-diynoic acid isobutylamide all significantly inhibited lipopolysaccharide-induced tumor necrosis factor α, IL-1β, and IL-12p70 expression (5–500 nm) in a CB2-independent manner. Alkylamides and anandamide also showed weak differential effects on anti-CD3-versus anti-CD28-stimulated cytokine expression in human whole blood. Overall, alkylamides, anandamide, and SR144528 potently inhibited lipopolysaccharide-induced inflammation in human whole blood and exerted modulatory effects on cytokine expression, but these effects are not exclusively related to CB2 binding. 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 <10 nm), and also the non-CB2-binding alkylamide undeca-2E-ene,8,10-diynoic acid isobutylamide all significantly inhibited lipopolysaccharide-induced tumor necrosis factor α, IL-1β, and IL-12p70 expression (5–500 nm) in a CB2-independent manner. Alkylamides and anandamide also showed weak differential effects on anti-CD3-versus anti-CD28-stimulated cytokine expression in human whole blood. Overall, alkylamides, anandamide, and SR144528 potently inhibited lipopolysaccharide-induced inflammation in human whole blood and exerted modulatory effects on cytokine expression, but these effects are not exclusively related to CB2 binding. Purple coneflower (Echinacea purpurea and Echinacea angustifolia) preparations are widely used herbal medicines for the treatment of the common cold and upper respiratory infections (1Goel V. Lovlin R. Barton R. Lyon M.R. Bauer R. Lee T.D. Basu T.K. J. Clin. Pharmacol. Ther. 2004; 29: 75-83Crossref PubMed Scopus (93) Google Scholar, 2Barnes J. Anderson L.A. Gibbons S. Phillipson J.D. J. Pharm. Pharmacol. 2005; 57: 929-954Crossref PubMed Scopus (319) Google Scholar). It is generally believed that Echinacea affords its benefits through interactions with the immune system (3Randolph R.K. Gellenbeck K. Stonebrook K. Brovelli E. Qian Y. Bankaitis-Davis D. Cheronis J. Exp. Biol. Med. 2003; 228: 1051-1056Crossref PubMed Scopus (57) Google Scholar), but data on the clinical efficacy of Echinacea in the treatment of the common cold and upper respiratory infections are contradictory (4Turner R.B. Bauer R. Woelkart K. Hulsey T.C. Gangemi J.D. N. Engl. J. Med. 2005; 353: 341-348Crossref PubMed Scopus (196) Google Scholar, 5Brinkeborn R.M. Shah D.V. Degenring F.H. Phytomedicine. 1999; 6: 1-6Abstract Full Text PDF PubMed Scopus (110) Google Scholar). In contrast to the significant investments into the clinical assessment of the efficacy of Echinacea (6Percival S. Biochem. Pharmacol. 2000; 60: 155-158Crossref PubMed Scopus (157) Google Scholar), the molecular mechanism of action of this medicinal plant has remained elusive, and comprehensive studies on the immunomodulatory actions of Echinacea constituents are scarce. We have reported previously that unsaturated fatty acid N-alkylamides (alkylamides) from Echinacea preparations can modulate the expression of TNF-α 2The abbreviations used are: TNF-α, tumor necrosis factor α; 2-AG, 2-arachidonoyl-glycerol; CB,cannabinoidreceptor;FACS,fluorescence-activatedcellsorting;GM-CSF,granulocyte colony-stimulating factor; GPCR, G-protein-coupled receptor; LPS, lipopolysaccharide; NF-κB, nuclear factor κB; IL, interleukin; Mϕs, macrophages; PLC, phospholipase C; PMA, phorbol ester (12-tetradecanoylphorbol-13 acetate); BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; A1, dodeca-2E,4E,8Z,10Z-tetraenoic acid isobutylamide; A2, dodeca-2E,4E-dienoic acid isobutylamide; A3, undeca-2E-ene,8,10-diynoic acid isobutylamide; CBA, cytometric bead array. in human monocytes and macrophages (Mϕs) in vitro (7Gertsch J. Schoop R. Kuenzle U. Suter A. FEBS Lett. 2004; 577: 563-569Crossref PubMed Scopus (171) Google Scholar). We ascribed these effects to interactions of alkylamides with the cannabinoid type 2 receptor (CB2) on monocytes, resulting in the activation of c-Jun N-terminal kinase, mitogen-activated protein kinase, and of the nuclear factor κB(NF-κB), which ultimately leads to TNF-α mRNA expression (7Gertsch J. Schoop R. Kuenzle U. Suter A. FEBS Lett. 2004; 577: 563-569Crossref PubMed Scopus (171) Google Scholar). In the same study it was demonstrated that alkylamides inhibit LPS-stimulated TNF-α protein expression from isolated monocytes/Mϕs. These findings were independently confirmed in a more recent study, which demonstrated binding of alkylamides from E. angustifolia to rodent cannabinoid receptors and inhibition of fatty acid amide hydrolase, the enzyme that controls the half-life of the endogenous cannabinoid ligand anandamide (8Woelkart K. Xu W. Pei Y. Makriyannis A. Picone R.P. Bauer R. Planta Med. 2005; 71: 701-705Crossref PubMed Scopus (85) Google Scholar). Cannabinoid receptors belong to the G-protein-coupled receptor (GPCR) family and are ubiquitously found in the central nervous system and in the periphery. So far, two types of receptors have been characterized, which are referred to as type 1 (CB1) and type 2 (CB2) receptors. The CB1 receptor is predominantly but not exclusively found in the nervous system, whereas the CB2 receptor is mainly expressed on immune cells and in the spleen (9De Petrocellis L. Cascio M.G. Di Marzo V. Br. J. Pharmacol. 2004; 141: 765-774Crossref PubMed Scopus (416) Google Scholar, 10Munro S. Thomas K.L. Abu Shaar M. Nature. 1993; 365: 61-65Crossref PubMed Scopus (4180) Google Scholar). Because of its role in the cellular immune system, the CB2 receptor is of particular interest for our ongoing studies on the immunomodulatory activity of alkylamides from Echinacea. In fact, the CB2 receptor is abundantly expressed in different types of inflammatory and immune-competent cells (11Galiegue S. Mary S. Marchand J. Dussossoy D. Carriere D. Carayon P. Bouaboula M. Shire D. Fur G. Le Casellas P. Eur. J. Biochem. 1995; 232: 54-61Crossref PubMed Scopus (1389) Google Scholar, 12Matias I. Pochard P. Orlando P. Salzet M. Pestel J. Di Marzo V. Eur. J. Biochem. 2002; 269: 3771-3778Crossref PubMed Scopus (157) Google Scholar), and there is accumulating evidence that the CB2 receptor plays a role in inflammatory reactions and the immune response (13Oka S. Yanagimoto S. Ikeda S. Gokoh M. Kishimoto S. Waku K. Ishima Y. Sugiura T. J. Biol. Chem. 2005; 280: 18488-18497Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 14Quartilho A. Mata H.P. Ibrahim M.M. Vanderah T.W. Porreca F. Makriyannis A. Malan Jr., T.P. Anesthesiology. 2003; 99: 955-960Crossref PubMed Scopus (199) Google Scholar, 15Carlisle S.J. Marciano-Cabral F. Staab A. Ludwick C. Cabral G.A. Int. Immunopharmacol. 2002; 2: 69-82Crossref PubMed Scopus (275) Google Scholar), as well as related pathophysiological conditions (16Parolaro D. Massi P. Rubino T. Monti E. Chem. Phys. Lipids. 2002; 108: 169-190Google Scholar, 17Klein T.W. Nat. Rev. Immunol. 2005; 5: 400-411Crossref PubMed Scopus (589) Google Scholar). It is also well established that cannabinoids mediate both inhibitory and stimulatory effects on the immune system by modulating cytokine expression (18Klein T.W. Newton C. Larsen K. Lu L. Perkins I. Nong L. Friedman H. J. Leukocyte Biol. 2003; 74: 486-496Crossref PubMed Scopus (428) Google Scholar, 19Croxford J.L. Yamamura T. J. Neuroimmunol. 2005; 166: 3-18Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). These differential effects are also dependent on ligand concentration (19Croxford J.L. Yamamura T. J. Neuroimmunol. 2005; 166: 3-18Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar), but the underlying mechanisms of the concentration effects are not yet understood. Recent studies also highlight the potential of CB2 receptor ligands in the treatment of cancer (20Sarfaraz S. Afaq F. Adhami V.M. Mukhtar H. Cancer Res. 2005; 65: 1635-1641Crossref PubMed Scopus (203) Google Scholar, 21Blazquez C. Casanova M.L. Planas A. Pulgar T.G. Del Villanueva C. Fernandez-Acenero M.J. Aragones J. Huffman J.W. Jorcano J.L. Guzman M. FASEB J. 2003; 17: 529-531Crossref PubMed Scopus (251) Google Scholar) and atherosclerosis (22Steffens S. Veillard N.R. Arnaud C. Pelli G. Burger F. Staub C. Karsak M. Zimmer A. Frossard J.L. Mach F. Nature. 2005; 434: 782-786Crossref PubMed Scopus (369) Google Scholar). Therefore, the general aim of this study was to characterize the interaction of alkylamides from the medicinal plant Echinacea with the human CB2 receptor with respect to binding affinity, ligand specificity, and functional consequences at physiological drug concentrations in cellular systems in vitro. Binding studies were based on radioligand displacement assays with the bicyclic cannabinoid ligand [3H]CP-55,940, which has played an important role in the discovery of the cannabinoid receptors (23Devane W.A. Dysarz F.A. II I Johnson M.R. Melvin L.S. Howlett A.C. Mol. Pharmacol. 1988; 34: 605-613PubMed Google Scholar, 24Kaminski N.E. Abood M.E. Kessler F.K. Martin B.R. Schatz A.R. Mol. Pharmacol. 1992; 42: 736-742PubMed Google Scholar), and which strongly binds to the cannabinoid-binding site in CB2 (Ki = 0.7 nm). Despite the fact that more than one plausible binding site in CB2 has been postulated (25Xie X.Q. Chen J.Z. Billings E.M. Proteins. 2003; 53: 307-319Crossref PubMed Scopus (103) Google Scholar), the CP-55,940-binding site is shared by all cannabinoids reported so far. The experimental investigations were complemented by molecular modeling studies based on a previously established homology model (25Xie X.Q. Chen J.Z. Billings E.M. Proteins. 2003; 53: 307-319Crossref PubMed Scopus (103) Google Scholar). No experimental information is currently available on the structure of the receptor. We have shown previously that alkylamides inhibit forskolin-induced cAMP production (7Gertsch J. Schoop R. Kuenzle U. Suter A. FEBS Lett. 2004; 577: 563-569Crossref PubMed Scopus (171) Google Scholar) via the CB2 receptor. To further characterize the direct functional effects of alkylamide binding to the CB2 receptor, the total intracellular free Ca2+ concentration ([Ca2+]i) in both CB2-expressing (CB2-positive) and CB2-nonexpressing (CB2-negative) HL60 cells was measured. In addition to defined cellular systems, we have also investigated the effects of nanomolar concentrations of alkylamides on cytokine expression in human peripheral whole blood cultures, both under nonstimulating and stimulating conditions. Immunomodulatory actions of endogenous and exogenous cannabinoids have been investigated in numerous studies (18Klein T.W. Newton C. Larsen K. Lu L. Perkins I. Nong L. Friedman H. J. Leukocyte Biol. 2003; 74: 486-496Crossref PubMed Scopus (428) Google Scholar), mostly performed with isolated cells or transformed cell lines, but only sparse data exist for ex vivo studies or studies with whole blood. Recent reports have shown that plasma levels of alkylamides in the lower nanomolar range can be achieved in humans after oral administration of commercial alkylamide-containing Echinacea preparations (26Woelkart K. Koidl C. Grisold A. Gangemi J.D. Turner R.B. Marth E. Bauer R. J. Clin. Pharmacol. 2005; 45: 683-689Crossref PubMed Scopus (61) Google Scholar, 27Matthias A. Addison R.S. Penman K.G. Dickinson R.G. Bone K.M. Lehmann R.P. Life Sci. 2005; 77: 2018-2029Crossref PubMed Scopus (66) Google Scholar). So far, however, it is not known whether alkylamides can exert immunomodulatory effects via the CB2 receptor at such low concentrations. Whole blood rather than isolated leukocytes were chosen for these studies in order to simulate physiological conditions as closely as possible. Whole blood studies were performed with three major alkylamides from Echinacea, the endogenous cannabinoid arachidonoylethanolamide (anandamide), and the CB2 antagonist SR144528 (28Calignano A. La Rana G. Giuffrida A. Piomelli D. Nature. 1998; 394: 277-281Crossref PubMed Scopus (956) Google Scholar). Cell Culture—Human promyelocytic leukemia non-CB2-expressing (negative) HL60 cells (obtained from Prof. Dr. Verena Dirsch, Vienna, Austria) were grown in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal bovine serum, 1 g/ml fungizone (amphotericin B), 100 units/ml penicillin, 100 g/ml streptomycin, and 2 mm l-glutamine (all from Invitrogen). Human promyelocytic leukemia CB2-expressing (positive) HL60 cells (obtained from the ATCC, CCL-240) were grown in Iscove's modified Dulbecco's medium with 4 mm l-glutamine and 1.5 g/liter sodium bicarbonate (ATCC, Manassas, VA) supplemented with 20% fetal bovine serum, 1 g/ml fungizone (amphotericin B), 100 units/ml penicillin, and 100 g/ml streptomycin. The human CB2-expressing CHO-K1 cells were grown in the same medium as the CB2-negative HL60 cells but supplemented with 400 μg/ml G418 (10131-027; Invitrogen). All cells were grown in a humidified incubator at 37 °C and 5% CO2. Human Peripheral Whole Blood Cultures—10 ml of peripheral whole blood was obtained from healthy volunteers in the early afternoon by a medical doctor. The blood was collected into heparinized tubes (BD Vacutainer Systems) and gently shaken for 1 min. 200-μl portions were then immediately aliquoted into a 96-well plate under sterile conditions. Each experiment was carried out in triplicate. Test compounds and vehicle controls were added. After 45 min of incubation in a humidified incubator at 37 °C and 5% CO2, stimulation of cells was initiated by addition of either 313 ng/ml LPS, 1 μg/ml αCD3 (combined with 1.5 μg/ml PMA), or 0.5 μg/ml αCD28 (combined with 1.5 μg/mlPMA) to the blood culture under gentle stirring. Volumes of stimulatory mixtures were set to 2 μl. PMA stimulation alone did not markedly induce cytokines in whole blood. Again, vehicle controls (ethanol or H2O) of the same dilutions were included. The plate was then incubated at 37 °C and 5% CO2 for 18 h. After incubation the plates were centrifuged at room temperature for 5 min at 450 rpm in an MSE Mistral 3000i centrifuge to facilitate plasma collection. For each assay at least three experiments were performed in triplicate with blood from at least three different donors (total of at least nine measurements). FACS Analysis of CB2 Expression—HL60 or CB2-transfected CHO-K1 cells (106) were washed in phosphate-buffered saline (Invitrogen) supplemented with 0.1% NaN3 and 2% fetal bovine serum and incubated (1:100) with the rabbit polyclonal CB2-specific antibody (3561) for 45 min on ice in the dark. After two washing steps, the cells were incubated (1:32) with a monoclonal anti-rabbit fluorescein isothiocyanate-labeled antibody for 45 min on ice in the dark. The cells were washed twice and resuspended in 500 μl of phosphate-buffered saline with 0.1% NaN3 and 1% p-formaldehyde prior to analysis on a FACScan cytometer (BD Biosciences). Measurements were carried out with the CellQuest™ software, and relative expressions were compared with secondary antibody controls. Radioligand Displacement Assays on CB1 and CB2 Receptors—For the CB1 receptor, binding experiments were performed in the presence of 0.39 nm of the radioligand [3H]CP-55,940 at 30 °C in siliconized glass vials together with 7.16 μg of membrane recombinantly overexpressing CB1 (RBHCB1M; PerkinElmer Life Sciences), which was resuspended in 0.2 ml (final volume) of binding buffer (50 mm Tris-HCl, 2.5 mm EGTA, 5mm MgCl2, 0.5 mg/ml fatty acid free bovine serum albumin, pH 7.4). Test compounds were present at varying concentrations, and the nonspecific binding of the radioligand was determined in the presence of 10 μm CP-55,940. After 90 min of incubation, the suspension was rapidly filtered through 0.05% polyethyleneimine pre-soaked GF/C glass fiber filters on a 96-well cell harvester and washed nine times with 0.5 ml of ice-cold washing buffer (50 mm Tris-HCl, 2.5 mm EGTA, 5 mm MgCl2, 2% bovine serum albumin, pH 7.4). Radioactivity on filters was measured with a Beckman LS 6500 scintillation counter in 3 ml of Ultima Gold scintillation liquid. Data collected from three independent experiments performed in triplicate were normalized between 100 and 0% specific binding for [3H]CP-55,940. These data were graphically linearized by projecting Hill plots, which allowed the calculation of IC50 values. Derived from the dissociation constant (KD) of [3 H]CP-55,940 and the concentration-dependent displacement (IC50 value), inhibition constants (Ki) of competitor compounds were calculated using the Cheng-Prusoff equation (Ki = IC50/(1 + L/KD)) (29Cheng Y.C. Prusoff W.H. Biochem. Pharmacol. 1973; 22: 3099-3108Crossref PubMed Scopus (12330) Google Scholar). For CB2 receptor binding studies, 3.8 μg of membrane recombinantly overexpressing CB2 (RBXCB2M; PerkinElmer Life Sciences) was resuspended in 0.6 ml of binding buffer (see above) together with 0.11 nm of the radioligand [3H]CP-55,940. The CB2-binding assay was conducted in the same manner as for CB1. Western Blotting—HL60 cells and CB2-transfected CHO-K1 cells were resuspended and homogenized in ice-cold buffer A (15 mm Tris-HCl, 2 mm MgCl2, 0.3 mm EDTA, 1 mm EGTA, pH 7.5) followed by centrifugation at 40,000 × g for 25 min at 4 °C. The pellet was washed with buffer A and centrifuged again at 40,000 × g for 25 min at 4 °C. The membrane was resuspended in buffer B (75 mm Tris-HCl, 12.5 mm MgCl2, 0.3 mm EDTA, 1 mm EGTA, 250 mm sucrose, pH 7.5) and stored at –80 °C until used. All membrane preparation steps were performed in the presence of protease inhibitor mixture (P8340; Sigma). Membrane proteins were separated on 4–12% Nupage™ Novex BisTris pre-cast gels (Invitrogen) under denaturing and nonreducing conditions and subsequently transferred to nitrocellulose membranes. Blocking of membrane, incubation of the primary and secondary antibodies, and detection by chemiluminescence following ECL Plus Western blotting Detection Reagents (Amersham Biosciences) were performed according to the manufacturer's instructions. Receptor Screen—The receptor screen was carried out at the Novartis Institute for Biomedical Research in Basel, Switzerland. 10 μm of test compound was subjected to cell membrane preparations from cell lines overexpressing specific receptors in order to test for competitive binding with the corresponding radioligands. Inhibitions of >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 a" @default.
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• W2016128034 doi "https://doi.org/10.1074/jbc.m601074200" @default.
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