Irreversible labelling of the opioid receptors by a melphalan-substituted [Met(5)]enkephalin-Arg-Phe derivative

[Met(5)]enkephalin-Arg-Phe (Tyr-Gly-Gly-Phe-Met-Arg-Phe) was modified with the methyl esther of melphalan (Mel; 4-bis(2-chloroethyl)amino-L-phenylalan ine) and the resulting compounds were studied for their opioid binding prop erties in guinea pig and rat brain membranes. Three new peptides, with a su bstitution of a single amino acid, were synthesized (Mel-Gly-Gly-Phe-Met-Ar g-Phe, Tyr-Gly-Gly-Mel-Met-Arg-Phe and Tyr-Gly-Gly-Phe-Met-Arg-Mel). In the rat brain, none of these ligands displayed any type specificity, whereas i n guinea pig brain membranes the C-terminally modified peptide, Tyr-Gly-Gly -Phe-Met-Arg-Mel ([Mel(7)]peptide), displayed a kappa-binding profile and w as a weak kappa-opioid-receptor agonist in isolated guinea pig ileum. The e ffect of sodium ions on [Mel(7)]peptide competition against [H-3]naloxone b inding indicated a weak agonist nature of the compound. When guinea pig bra in membranes were preincubated with 1-10 mu M of [Mel(7)]peptide, an appare ntly irreversible inhibition of [H-3]naloxone ligand binding was observed. These results suggest that the heptapeptide containing melphalan at the C-t erminus can be used as a relatively high-affinity irreversible label for th e kappa-opioid receptor. (C) 1999 Elsevier Science B.V. All rights reserved .