In vitro and in vivo characterization of opioid activities of endomorphins analogs with novel constrained C-terminus: Evidence for the important role of proper spatial disposition of the third aromatic ring

In the present study, the C-terminus of endomorphin (Tyr1-Pro2-Trp/Phe3-Phe4-NH2, EMs) analogs [Xaa4-R]EMs, modified by substitution of a non-aromatic residue for Phe4 and ending up with –NH-benzyl, were designed to generate an atypical conformationally constrained peptide set. We investigated the effects of these analogs on the opioid receptors affinity, guinea pig ileum (GPI) and mouse vas deferens (MVD) activity, system arterial pressure (SAP), heart rate (HR), antinociception and colonic motility. Analogs 5 ([D-V4-Bzl]EM1) and 10 ([D-V4-Bzl]EM2), which exhibit appropriate spatial orientations of the third aromatic ring, were about 3–4 times more potent than their parents both in vivo and in vitro. However, a drastic loss of activity was found in analogs 2 ([A4-Bzl]EM1) and 7 ([A4-Bzl]EM2), which possess improper spatial orientations of the third aromatic ring. Interestingly, analog 7 or 3 ([G4-Bzl]EM1), when injected intravenously (i.v.), produced significantly different changes in SAP from their parents. Surprisingly, analog 4 displayed relatively higher vasodepressor activity but significantly less potent colonic contractile activity than analog 5. This may be elicited by the differences in the spatial disposition of the third aromatic ring, which were verified by molecular modeling. Our results indicate that the proper spatial disposition of the third aromatic ring plays an important role in the regulation of pharmacological activities of EMs.