Stereochemical requirements for receptor recognition of the μ-opioid peptide endomorphin-1

A series of diastereoisomers of endomorphin-1 (EM1, Tyr¹-Pro²-Trp³- Phe⁴-NH₂) have been synthesized and their potency measured using the guinea pig ileum assay. [D-Phe⁴]EM1 possessed 1/10 the potency of EM1, while potencies of [D-Tyr¹]EM1 and [D-Trp³]EM1 were 50- and 100-fold lower, respectively. Drastic loss of activity occurred in the [D-Pro²]EM1 peptide. The structural determinants for the inactivity and reduced potency of the diastereoisomers were investigated using NMR spectroscopy and conformational analysis. Simulations of trans-[D-Pro²]EM1 using NOE-derived distance constraints afforded well-defined structures in which Tyr and Trp side chains stack against the proline ring. The inactivity of [D- Pro²]EM1 was explained by structural comparison with EM1 (Podlogar et al., 1998, FEBS Lett. 439:13- 20). The two peptides showed an opposite orientation of the Trp³ residue with respect to Tyr¹, thus suggesting a role of Pro² as a stereochemical spacer in orienting Trp³ and Phe⁴ toward regions suitable for μ-receptor interaction. The agonist activity of [D-Tyr¹]EM1 and [D-Trp³]EM1 was attributed to their ability to adopt low-energy conformations that mimic those of EM1. The requirements for μ-receptor activation were examined further by comparing EM1 with the μ-peptide [D-Ala², MePhe⁴, Gly-ol]- enkephalin (DAMGO). Conformations of DAMGO with a Tyr -MePhe⁴ phenyl ring separation of ~12 Å, were found to mimic Tyr¹-Phe⁴ of EM1, thus suggesting overlapping binding modes between these two peptides.