Vol. 295, Issue 3, 960-966, December 2000

Tetrapeptide Derivatives of [D-Pen²,D-Pen⁵]-Enkephalin (DPDPE) Lacking an N-Terminal Tyrosine Residue Are Agonists at the µ-Opioid Receptor¹

Iain J. McFadyen² , Katarzyna Sobczyk-Kojiro² , Michael J. Schaefer, Jeffrey C. Ho, John R. Omnaas, Henry I. Mosberg and John R. Traynor

Department of Pharmacology (I.J.M., M.J.S., J.R.T.) and College of Pharmacy (J.C.H., J.R.O., K.S.-K., H.I.M.), The University of Michigan, Ann Arbor, Michigan

The Phe¹ cyclic tetrapeptide Phe-c[D-Cys-Phe-D-Pen]NH₂ (Et) (JH-54) has been shown previously to exhibit high affinity and selectivity for the µ-opioid receptor. To examine the role of the Phe¹ residue in the unexpected high affinity of this peptide, 11 analogs of JH-54 have been synthesized and evaluated for opioid ligand binding and for efficacy using the [³⁵S]GTPS assay. Alteration of the bridging groups between the D-Cys² and D-Pen⁴ residues of JH-54 from dithioether to disulfide revealed the importance of the relative position of the aromatic rings of the first and third residues in determining µ- and -affinities. The one carbon distance between the  carbon and phenyl ring in the N-terminal residue was critical. Additional steric bulk in the N-terminal Phe¹ residue was accommodated without large reductions in affinity in two naphthyl analogs, but not with 3,3-(diphenyl)alanine. Conformational restriction of the C-C and/or C-C bonds had little effect on affinities in two peptides with 2-amino-2-carboxytetralin in position 1, but it abolished activity in an isoquinoline analog and differentially altered activity in four phenylproline¹-containing peptides. Most surprisingly, replacement of the Phe¹ aromatic ring with cyclohexyl resulted in a peptide of moderate affinity (K_i = 32.5 nM) and potency (EC₅₀ = 58.8 nM). Thus, the tyrosyl para-hydroxyl substituent and even aromaticity in the N-terminal amino acid of these tetrapeptides are shown to be important, but not critical, features for µ-opioid receptor affinity, agonist potency, and efficacy.