Structural requirements for opioid activity of analogues of the enkephalins

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Abstract
Structure-activity relations of a series of analogues of the two endo­genous morphine-like peptides, leucine-enkephalin and methionine-enkephalin are examined on the basis of (a) effects on the mouse vas deferens and the guinea pig ileum and (b) affinities for the rat brain opiate receptor. In the mouse vas deferens, metabolism of the peptides by proteolysis is not a major influence on activity. In contrast, however, brain opiate re­ceptor preparations contain an abundance of proteolytic enzymes, the effects of which can be minimized by conducting opiate receptor binding assays at 0 °C and in the presence of bacitracin. The potentiation of biological activity and opiate receptor binding affinity by replacing the Gly2 residue in the natural enkephalins by d-Ala, is discussed both in terms of increased stability of the Tyr-d-Ala bond to aminopeptidases and of the stabilization of the peptide conform­ation as present in the receptor-peptide complex. The substitution of the Leu5- or Met5-residue by the corresponding d-amino acid contributes little to proteolytic stability, which emphasizes that the predominating site at which metabolism occurs is the Tyr1-Gly2bond. Of the analogues described, [d-Ala2, d-Leu5]-enkephalin is the most active peptide in the three assay systems, the mouse vas deferens, the guinea pig ileum and the rat brain opiate receptor preparations. Substitutions by the respective d-amino acids d-Tyr and d-Phe at positions 1 and 4 reduce both the potency and binding affinity and emphasize the importance of stereochemical acceptability at these positions. The promotion of receptor binding by d-amino acids is examined, particularly with respect to implied peptide conformations. The experi­mental data have been analysed for the relative influence of metabolic and conformational factors.