Binding to opioid receptors of enkephalin derivatives taking α-helical conformation and its dimer

The opioid receptor binding of [Leu]enkephalin derivatives with extended address segment to the C-terminal was studied. The extension peptide is designed to take an amphiphilic helical structure in order to evaluate effects of helical conformation and membrane affinity of enkephalin moiety of the derivatives on receptor binding. In the σ-receptor-selective binding assay, Tyr-Gly-Gly-Phe-Leu-Lys-Aib-Leu-Aib-OH (1) showed the same affinity as enkephalinamide, whereas in the μ-receptor-selective binding assay, a 7-fold reduction in affinity was observed. On the other hand, Tyr-GIy-Gly-Phe-Leu-(Lys-Aib-Leu-Aib)₂-OH (2) showed 51-and 96-fold decreases in affinities for δ- and μ-receptors, respectively, compared with enkephalinamide. The low receptor affinity of derivative 2 is considered due to α-helical conformation, which might not be compatible with topological requirements of δ- and μ-receptors. A dimer, Tyr-Gly-GIy-Leu-Phe-(Lys-Aib-Leu-Aib)₂-Lys(X)-Aib-OCH₃ (X = Tyr-Gly-Gly-Phe-Leu-, (4)), showed 2.5- and 3.0-fold increases in affinities respectively for δ- and μ-receptors compared with the monovalent derivative 2, possibly due to cross-linking of neighboring receptors. The Hill plot of the binding of the dimer to bovine brain membranes was composed of two phases, although such a heterogeneity of receptors was not observed in the presence of naloxone or in the binding to NG108-15 cell membranes. These findings indicate the presence of the bivalent-ligand-induced interactions between δ- and μ-receptors in bovine brain membranes.