Conformational Memories and the Endocannabinoid Binding Site at the Cannabinoid CB1 Receptor
Chemistry & Biochemistry
Endocannabinoid structure-activity relationships (SAR) indicate that the CB1 receptor recognizes ethanolamides whose fatty acid acyl chains have 20 or 22 carbons, with at least three homoallylic double bonds and satn. in at least the last five carbons of the acyl chain. To probe the mol. basis for these acyl chain requirements, the method of conformational memories (CM) was used to study the conformations available to an n-6 series of ethanolamide fatty acid acyl chain congeners: 22:4, n-6 (Ki = 34.4±3.2 nM); 20:4, n-6 (Ki = 39.2±5.7 nM); 20:3, n-6 (Ki = 53.4±5.5 nM); and 20:2, n-6 (Ki > 1500 nM). CM studies indicated that each analog could form both extended and U/J-shaped families of conformers. However, for the low affinity 20:2, n-6 ethanolamide, the higher populated family was the extended conformer family, while for the other analogs in the series, the U/J-shaped family had the higher population. In addn., the 20:2, n-6 ethanolamide U-shaped family was not as tightly curved as were those of the other analogs studied. To quantitate this variation in curvature, the radius of curvature (in the C-3 to C-17 region) of each member of each U/J-shaped family was measured. The av. radii of curvature (with their 95% confidence intervals) were found to be 5.8 Å (5.3-6.2) for 20:2, n-6; 4.4 Å (4.1-4.7) for 20:3, n-6; 4.0 Å (3.7-4.2) for 20:4, n-6; and 4.0 Å (3.6-4.5) for 22:4, n-6. These results suggest that higher CB1 affinity is assocd. with endocannabinoids that can form tightly curved structures. Endocannabinoid SAR also indicate that the CB1 receptor does not tolerate large endocannabinoid headgroups; however, it does recognize both polar and nonpolar moieties in the headgroup region. To identify a headgroup orientation that results in high CB1 affinity, a series of di-Me anandamide analogs (R)-N-(1-methyl-2-hydroxyethyl)-2-(R)-methyl-arachidonamide (Ki = 7.42±0.86 nM), (R)-N-(1-methyl-2-hydroxyethyl)-2-(S)-methyl-arachidonamide (Ki = 185±12 nM), (S)-N-(1-methyl-2-hydroxyethyl)-2-(S)-methyl-arachidonamide (Ki = 389±72 nM), and (S)-N-(1-methyl-2-hydroxyethyl)-2-(R)-methyl-arachidonamide (Ki = 233±69 nM) were then studied using CM and computer receptor docking studies in an active state (R*) model of CB1. These studies suggested that the high CB1 affinity of the R,R stereoisomer is due to the ability of the headgroup to form an intramol. hydrogen bond between the carboxamide oxygen and the headgroup hydroxyl that orients the C2 and C1' Me groups to have hydrophobic interactions with valine 3.32(196), while the carboxamide oxygen forms a hydrogen bond with lysine 3.28(192) at CB1. In this position in the CB1 binding pocket, the acyl chain has hydrophobic and C-H···π interactions with residues in the transmembrane helix (TMH) 2-3-7 region. Taken together, the studies reported here suggest that anandamide and its congeners adopt tightly curved U/J-shaped conformations at CB1 and suggest that the TMH 2-3-7 region is the endocannabinoid binding region at CB1.
Barnett-Norris J, Hurst DP, Lynch DL, Guarnieri F, Makriyannis A, Reggio PH. 2002. Conformational memories and the endocannabinoid binding site at the cannabinoid CB1 receptor. J Med Chem 45(17):3649-59.