Dimeric tetrapeptide enkephalins display extraordinary selectivity for the δ opiate receptor

Abstract

The μ and δ opiate receptors, postulated on the basis of pharmacological observations, have been well characterized in ligand-binding studies^1–6. The several classes of opiate receptor appear to subserve different functions^7–9. Although highly specific μ ligands are available, there has been a paucity of high-affinity ligands with δ specificity that could be used to investigate the functions, properties and spatial distribution of the δ receptor. Previous structure–activity studies of the enkephalins have suggested that a free carboxyl group at the C-terminus is an important determinant of δ selectivity^9,10: amidation of Leu⁵ or Met⁵ results in a non-selective ligand. Recently, however, we have demonstrated that cross-linking enkephalin amides by a methylene bridge of suitable length produces a dimeric pentapeptide, with greater affinity and selectivity for the δ receptor than is found in the original δ ligand^11,12. Evidence that this dimer may interact simultaneously with two δ, but not two μ receptors¹² is consistent with independent demonstrations that the δ receptors are clustered in the membrane^7,13. When the C-terminal amino acid of enkephalin is removed, the resulting tetrapeptide enkephalin amide H-Tyr-D-Ala-Gly-Phe-NH₂ and its analogues are potent and selective ligands for μ opiate sites^14,15. If the δ receptors were closely clustered, as suggested by our findings with dimeric enkephalin pentapeptides^11,12 and by others^7,13, then it might be possible to form a dimeric analogue of the tetrapeptide enkephalin which could interact with two δ receptors but not with two μ receptors. Theoretically, this would confer δ selectivity on the dimer of a μ-selective ligand. We have thus synthesized a series of dimeric analogues of the μ -selective tetrapeptide [D-Ala², des-Leu⁵]enkephalin amide by cross-linking at the C-terminus with NH₂-(CH₂)_n-NH₂, with n = 2–12. We report here that the dimer with n = 12 has a nearly 1,000-fold increase in δ/μ selectivity ratio, and is thus a δ-selective ligand.