Receptor binding and biological activity of bivalent enkephalins

Two series of dimeric enkephalin analogues were assayed for opioid activity in two isolated smooth muscle preparations: the guinea pig ileum (GPI) and the mouse vas deferens (MVD). Dimers have the general structure: X-(CH 2) n - X, where X is H-Tyr- gd-Ala-Gly-Phe-Leu-NH-( n = 0, 2, 4, 6, 8, 10,12), for the first series of dimeric pentapeptide enkephalins (DPE n ), and H-Tyr- d-Ala-Gly-Phe-NH-( n = 2, 4, 6, 8, 12), for the series of dimeric tetrapeptide enkephalins (DTE n ). Comparison of biological activities with bincling affinities revealed that: (1) the DPE series with n = 2–8 showed increased potency in the MVD assay relative to monomeric [ d-Ala 2, Leu 5]enkephalinamide (DALEA); (2) there was an associated increase affinity for the delta receptor of rat brain or neuroblastoma-glioma hybrid cells. (however, the relative potencies were higher in the MVD assay than predicted on the basis of bincling affinities); (3) the DTE series also showed an increase in delta receptor affinities and MVD potencies relative to DALEA, for n = 2–12; (4) for the DTE series, the increase in MVD activities was less than that expected on the basis of delta bincling affinity; (5) for both the DPE and DTE series, activities in the GPI assay and mu-receptor affinities were highly correlated: as the length of the methylene bridge increased from 2 to 12, there was a progressive loss of activity in both assays, with a similar pattern for DPE and DTE. Two selected dimers and their corresponcling monomers were also assayed for antinociceptive activity in vivo: results were consistent with GPI and μ-bincling but not with MVD and δ-binding. Two alkylamide analogs of penta- and tetrapeptide monomers, representing the monomer with the attached spacer of the most active dimers, were also assayed in biological and bincling assays. Comparison of these compounds with the corresponcling dimers suggest that the changes in activities and selectivities induced by dimerization are not a spurious effect of the presence of an akylamide derivative of the carboxy terminal of enkephalin but rather may represent a specific effect due to the bivalent nature of the ligands.