15-hydroxy-5-8-11-13-eicosatetraenoic-acid and glyceryl-2-arachidonate

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.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Binding, Competitive; Biological Transport; Brain; Cannabinoid Receptor Modulators; Cannabinoids; Carrier Proteins; Chloroform; Cyclohexanols; Endocannabinoids; Glycerides; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Magnetic Resonance Spectroscopy; Male; Models, Molecular; Molecular Conformation; Polyunsaturated Alkamides; Rats; Rats, Wistar; Receptor, Cannabinoid, CB2; Receptors, Cannabinoid; Receptors, Drug; Tumor Cells, Cultured; Water

The endocannabinoid, 2-arachidonylglycerol (2-AG), is an endogenous ligand for the central (CB1) and peripheral (CB2) cannabinoid receptors and has been shown to be efficiently and selectively oxygenated by cyclooxygenase (COX)-2. We have investigated 2-AG/COX-2 interactions through site-directed mutagenesis. An evaluation of more than 20 site-directed mutants of murine COX-2 has allowed for the development of a model of 2-AG binding within the COX-2 active site. Most strikingly, these studies have identified Arg-513 as a critical determinant in the ability of COX-2 to efficiently generate prostaglandin H(2) glycerol ester, explaining, in part, the observed isoform selectivity for this substrate. Mutational analysis of Leu-531, an amino acid located directly across from Arg-513 in the COX-2 active site, suggests that 2-AG is shifted in the active site away from this hydrophobic residue and toward Arg-513 relative to arachidonic acid. Despite this difference, aspirin-treated COX-2 oxygenates 2-AG to afford 15-hydroxyeicosatetraenoic acid glycerol ester in a reaction analogous to the C-15 oxygenation of arachidonic acid observed with acetylated COX-2. Finally, the differences in substrate binding do not alter the stereospecificity of the cyclooxygenase reaction; 2-AG-derived and arachidonic acid-derived products share identical stereochemistry.

Topics: Amino Acid Sequence; Amino Acids; Animals; Arachidonic Acid; Arachidonic Acids; Arginine; Binding Sites; Cannabinoid Receptor Modulators; Cannabinoids; Cyclooxygenase 1; Cyclooxygenase 2; DNA Mutational Analysis; Endocannabinoids; Esters; Glycerides; Glycerol; Hydroxyeicosatetraenoic Acids; Isoenzymes; Leucine; Mass Spectrometry; Membrane Proteins; Mice; Models, Chemical; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Oxygen; Prostaglandin H2; Prostaglandin-Endoperoxide Synthases; Prostaglandins H; Protein Binding; Protein Isoforms; Time Factors