title: Oxygenated metabolites of anandamide and 2-arachidonoylglycerol: conformational analysis and interaction with cannabinoid receptors, membrane transporter, and fatty acid amide hydrolase.
abstract: This study was aimed at finding structural requirements for the interaction of the acyl chain of endocannabinoids with cannabinoid receptors, membrane transporter protein, and fatty acid amide hydrolase (FAAH). To this end, the flexibility of the acyl chain was restricted by introduction of an 1-hydroxy-2Z,4E-pentadiene system in anandamide (N-arachidonoylethanolamine, AEA) and 2-arachidonoylglycerol (2-AG) at various positions using different lipoxygenases. This brought about selectivity and attenuated the binding potency of AEA and 2-AG. Although the displacement constants were modest, 15(S)-hydroxy-eicosa-5Z,8Z,11Z,13E-tetraenoyl-N-(2-hydroxyethyl)amine was found to bind selectively to the CB(1) receptor, whereas its 1-arachidonoyl-sn-glycerol analogue and 13(S)-hydroxy-octadeca-9Z,11E-dienoyl-N-(2-hydroxyethyl)amine could selectively bind to the CB(2) receptor. 11(S)-Hydroxy-eicosa-5Z,8Z,12E,14Z-tetraenoyl-N-(2-hydroxyethyl)amine did not bind to either receptor, whereas 12(S)-hydroxy-eicosa-5Z,8Z,10E,14Z-tetraenoyl-N-(2-hydroxyethyl)amine did bind to both CB receptors with an affinity similar to that of AEA. All oxygenated anandamide derivatives were good inhibitors of FAAH (low micromolar K(i)) but were ineffective on the AEA transporter. 2-AG rapidly isomerizes into 1(3)-arachidonoyl-sn-glycerol. Both 1- and 3-arachidonoyl-sn-glycerol did not bind to either CB receptor and did not interfere with AEA transport. Thus, after it is isomerized, 2-AG is inactivated, thereby decreasing effective concentrations of 2-AG. Analysis of (1)H NMR spectra revealed that chloroform did not induce notably different conformations in the acyl chain of 15(S)-hydroxy-eicosa-5Z,8Z,11Z,13E-tetraenoic acid as compared with water. Molecular dynamics (MD) simulations of AEA and its analogues in the presence of explicit water molecules revealed that a tightly folded conformation of the acyl chain is not the only requirement for CB(1) binding. Structural details of the C(2)-C(15) loop, such as an sp(2) carbon at position 11, are necessary for receptor binding. The MD simulations may suggest that the average orientations of the pentyl tail of AEA and 12(S)-hydroxy-eicosa-5Z,8Z,10E,14Z-tetraenoyl-N-(2-hydroxyethyl)amine are different from that of the low-affinity, inactive ligands.
authors.affiliation: Department of Bio-organic Chemistry, Bijvoet Center for Biomolecular Research, Utrecht University, The Netherlands.
authors.forename: Mario
authors.initials: M
authors.lastname: van der Stelt
authors.forename: J Albert
authors.initials: JA
authors.lastname: van Kuik
authors.forename: Monica
authors.initials: M
authors.lastname: Bari
authors.forename: Guus
authors.initials: G
authors.lastname: van Zadelhoff
authors.forename: Bas R
authors.initials: BR
authors.lastname: Leeflang
authors.forename: Gerrit A
authors.initials: GA
authors.lastname: Veldink
authors.forename: Alessandro
authors.initials: A
authors.lastname: Finazzi-Agrò
authors.forename: Johannes F G
authors.initials: JF
authors.lastname: Vliegenthart
authors.forename: Mauro
authors.initials: M
authors.lastname: Maccarrone
mesh_terms: D000581:Amidohydrolases / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001095:Arachidonic Acids / Q000737:chemistry / Q000378:metabolism*; D001667:Binding, Competitive; D001692:Biological Transport; D001921:Brain / Q000378:metabolism; D063385:Cannabinoid Receptor Modulators; D002186:Cannabinoids / Q000737:chemistry / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002725:Chloroform / Q000737:chemistry; D003511:Cyclohexanols / Q000737:chemistry; D063388:Endocannabinoids; D005989:Glycerides / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006893:Hydroxyeicosatetraenoic Acids / Q000737:chemistry; D066298:In Vitro Techniques; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D008958:Models, Molecular; D008968:Molecular Conformation; D053284:Polyunsaturated Alkamides; D051381:Rats; D017208:Rats, Wistar; D043885:Receptor, Cannabinoid, CB2; D043882:Receptors, Cannabinoid; D011955:Receptors, Drug / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014867:Water / Q000737:chemistry; D000099252:Fatty Acid Amide Hydrolases
chemical_list: D000581:Amidohydrolases; D001095:Arachidonic Acids; D063385:Cannabinoid Receptor Modulators; D002186:Cannabinoids; D002352:Carrier Proteins; D002725:Chloroform; D003511:Cyclohexanols; D063388:Endocannabinoids; D005989:Glycerides; D006893:Hydroxyeicosatetraenoic Acids; D053284:Polyunsaturated Alkamides; D043885:Receptor, Cannabinoid, CB2; D043882:Receptors, Cannabinoid; D011955:Receptors, Drug; D014867:Water; D000099252:Fatty Acid Amide Hydrolases; C469526:Cnr2 protein, rat; C025984:15-hydroxy-5,8,11,13-eicosatetraenoic acid; C054649:3-(2-hydroxy-4-(1,1-dimethylheptyl)phenyl)-4-(3-hydroxypropyl)cyclohexanol; C094503:glyceryl 2-arachidonate; C078814:anandamide