Synthesis and pharmacological characterization of a novel, highly potent, peptidomimetic δ-opioid radioantagonist, [3H]Tyr-Tic-(2S, 3R)-β-MePhe-Phe-OH

[3H]Tyr-Tic-(2S, 3R)-β-MePhe-Phe-OH (where Tic: 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) with a specific radioactivity of 53.7 Ci/mmol was synthesized and characterized in receptor binding assays at 25 °C in rat brain membranes. The specific binding was saturable and displayed high affinity, with a KD of 0.16 ± 0.005 nM and Bmax of 85.9 ± 6.3 fmol/mg protein. NaCl increased its affinity by about 4-fold in membranes of rat brain and Chinese Hamster Ovary Cells stably transfected with the human δ-opioid receptors (hDOR-CHO) showing that the new ligand is an antagonist. The prototypic δ-opioid ligands were much more potent than μ- or κ-specific ligands in competition assays. The autoradiographic distribution of the binding sites of the new ligand agreed with the known locations of the δ-opioid receptors in rat brain. The unlabeled new ligand was about 7-fold more potent than the parent peptide in competing for the binding sites of [3H]Tyr-Tic-(2S, 3R)-β-MePhe-Phe-OH in rat brain membranes. Likewise, the threo-β-methyl analog was 3.8-fold more potent than the parent compound in antagonizing the effect of DPDPE in the [35S]GTPγS functional assay in hDOR-CHO membranes. The new, highly potent, conformationally constrained antagonist may be a valuable pharmacological tool in understanding the structural and topographical requirements of peptide ligand binding to the δ-opioid receptors.