A study of the conformational and pharmacophoric requirements of ligands of the cannabinergic system using NMR and computer modeling

The interaction of different ligands with various components of the cannabinergic system was studied to determine the role of the ligands\u27 conformations. Such ligands can modulate many physiological and psychological responses controlled by that system. Therefore knowledge of their conformations may aid in developing potent cannabinergic drugs. Three objectives were achieved. ^ First, NOESY and molecular dynamics were performed, and a method based upon relaxation rate calculations was developed, to determine the arachidonyl backbone conformations of six anandamide analogs in isotropic bicelles ( q=1.0, 3% w/v, and 10:1 [DMPC]:[ligand]) and DMSO. No long range NOEs were observed, suggesting extended conformations for the backbones. In bicelles, anandamide preferred an inverse-L shape while R-methyl anandamide preferred either a fully extended or compact-C shape. Additionally, analysis of measured coupling constants revealed a possible intramolecular hydrogen bond between the carbonyl oxygen and the hydroxy proton of those ligands in DMSO. NOESY spectra analysis, however, suggests this bond occurs infrequently in bicelles. Finally, experiments with chemical shift reagents showed that the analogs orient within the bicelle with their head groups at the aqueous interface and their alkyl tails within the interior. ^ Second, quantum mechanical calculations were employed to explain differences in kinetic parameters (Vmax, Km) among six anandamide analogs with different head group structures when hydrolyzed by fatty acid amide hydrolase. For all analogs, head group conformations with a dihedral angle (C1-N-C1\u27-C2\u27) of -140° yielded transition state energies that correlated with the Vmax values. The binding energies, however, did not correlate with the Km values. Since the active site is buried within the enzyme, solvation energies, strain energies, and head group surface areas were needed to explain the mechanism of ligand binding. ^ Finally, cannabinoid receptor docking was used to explain differences in binding affinity among various classical cannabinoids. Calculated receptor volume maps revealed the ideal sizes needed by different ligand moieties for optimal binding. Surprisingly, docking showed that although large ligand moieties may be accommodated by the receptors instead of clashing with nearby residues, unfavorable interactions may result between the binding pocket and other areas of the ligand normally well accommodated by the receptor.