Pharmacological profiles of selective non-peptidic δ opioid receptor ligands

Several non-peptidic opioids have been synthesized recently as part of a program to develop selective δ receptor agonists. In this study, the affinities of a set of compounds for cloned δ and μ opioid receptors expressed in HEK 293 cell lines were determined by competition analysis of [ 3H]bremazocine binding to membrane preparations. All compounds studied exhibited high affinity and selectivity, with apparent dissociation constants in the range of 0.6–1.7 nM for the δ opioid receptor and 240–1165 nM for the μ opioid receptor. We next sought to determine which domain of the δ receptor was critical for mediating the highly selective binding by analysis of ligand affinities for μ/δ receptor chimeras. Receptor binding profiles suggested that a critical site of receptor/ligand interaction was located between transmembrane domain 5 (TM5) and TM7 of the δ receptor. Substitution of tryptophan 284, located at the extracellular surface of TM6, with lysine, which is found at the equivalent position in the μ opioid receptor, led to a spectrum of effects on affinities, depending on the ligand tested. Affinities of SB 219825 and SB 222941 were particularly sensitive to the substitution, displaying a 50-fold and 70-fold decrease in affinity, respectively. Activities of the δ receptor-selective agonists were tested in two functional assays. Brief exposure of HEK 293 cells expressing δ opioid receptors with selective ligands induced phosphorylation of MAP kinase, although the non-peptidic ligands were less efficacious than the enkephalin derivative DADL (Tyr- d-Ala-Gly-Phe- d-Leu). Similarly, chronic exposure of HEK 293 cells expressing δ opioid receptors with selective, non-peptidic ligands, with the exception of SB 206848, caused receptor down-regulation, however, the SB compounds were less efficacious than DADL.