Molecular Pharmacology, Vol 20, 484-491, Copyright © 1981 by the American Society for Pharmacology and Experimental Therapeutics

Structural Requirements for Specific Recognition of µ or Opiate Receptors

¹ Département de Chimie Organique ERA 613 du Centre National de la Recherche Scientifique et SCN 21 de l’Institut National de la Santé et de la Recherche Médicale, Faculté de Pharmacie, 75006 Paris, et Laboratoire de Biophysique Moléculaire ERA 828 du Centre National de la Recherche Scientifique, Université de Nancy I, Centre de 1er Cycle, C.O. 140, 54037 Nancy Cedex, France

A comparison of the inhibitory potency of rationally selected new analogues of enkephalins on the evoked contractions of guinea pig ileum (µ receptors) and mouse vas deferens ( receptors) allowed the definition of the structural components required for specific recognition of µ or receptors. A high specificity for µ binding sites is obtained (a) by shortening the enkephalin sequence and removing the terminal carboxyl group; (b) by replacing the aromatic Phe⁴ residue with a lipophilic alkyl chain; and (c) by introducing as a second residue a hydrophobic amino acid strictly of the D-configuration whose side-chain probably interacts with a specific µ-receptor subsite. Compared with methionine-enkephalin, such modifications lead to a 100-times loss of potency on mouse vas deferens but a 2-fold enhanced activity on guinea pig ileum. These short peptides display a high folding tendency as shown by ¹H-NMR experiments and conformational calculations. The low-energy conformer of the highest µ-specific peptide exhibits a T-shaped structure similar to that of morphine, evidencing the close correspondence of several biologically critical components in both compounds. All of the proposed µ-specific requirements account for the reported variations in the biological activity of various modified enkephalms. The µ-agonist potency of endogenous enkephalins could be related to a fitting of the side-chain of their fifth amino acid in the µ hydrophobic subsite. This feature is precluded in Tyr-Gly-Gly-Phe and could explain its very low potency. The requirements for receptor specificity are even more strict and involve (a) an aromatic moiety in the fourth position; (b) a conformational key role of the amino acid(s) following the Phe⁴, improving the fitting of the Phe⁴ side-chain in a specific -receptor subsite; and (c) a lengthening of the enkephalin sequence and the introduction of a hydrophilic side-chain in position 2 which decrease the µ specificity. These modifications lead to peptides almost three orders of magnitude more active on mouse vas deferens than on guinea pig ileum. On the whole, µ receptors bind preferentially highly hydrophobic compounds with compact structures, whereas receptors exhibit a stronger affinity for larger peptides with hydrophilic components. These findings obtained at the periphery are corroborated at the level of rat brain receptors by preliminary results from binding experiments. Thus ³H-labeled Tyr-D-Ser-Gly-Phe-Leu-Thr, the most selective agonist, interacts exclusively with -binding sites at concentrations up to 20 nM. In contrast, Tyr-D-Ala-Gly-NH-CH(CH₃)-CH₂-CH(CH₃)₂ exhibits a specificity almost 50 times greater for µ receptors than for receptors as shown by competition experiments. This extensive structure-activity study could permit an exploration of the pharmacological significance of µ and receptors through a rational design of highly specific ligands.

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ACKNOWLEDGMENTS We thank Monique David and Dr. Jean-Claude Meunier for binding studies, Dr Yves Audigier for pharmacological assays on two compounds, Annick Bouju for typing the manuscript and Anne Kornowski for stylistic revision.