Novel diastereomeric opioid tetrapeptides exhibit differing pharmacological activity profiles

A novel opioid peptide antagonist analogue, [3H]Dmt-Tic-(2S,3R)βMePhe-Phe, derived from the potent, δ-receptor selective TIPP tetrapeptide (Tyr-Tic-Phe-Phe) series was synthesized and radiolabeled by catalytic tritiation of its iodinated precursor peptide. The purified radioprobe exhibited a specific activity of 2.15 TBq/mmol (58 Ci/mmol). The novelty of this compound is that it contains structurally modified tyrosine residue (2′,6′-dimethyltyrosine, Dmt1) replacing tyrosine (Tyr1) at the N-terminus, and β-methyl substituted phenylalanine (βMePhe3) at the third position. As the configuration of βMePhe3 side-chain might be different due to diastereomerism, and accordingly can alter the biological activity, both unlabeled threo (2S,3R and 2R,3S) diastereomeric analogues were also prepared and included in this study. The affinity and selectivity (δ-opioid versus μ-opioid receptor) were evaluated by radioreceptor binding assays. Agonist or antagonist potencies were determined in [35S]GTPγS binding experiments using Chinese Hamster Ovary (CHO) cells selectively expressing δ- or μ-opioid receptors. The equilibrium binding of the radiolabeled peptide derivative [3H]Dmt-Tic-(2S,3R)βMePhe-Phe to rat brain membranes was saturable and the Scatchard analysis indicated a single binding site with a Kd of 0.3 nM and a Bmax of 127 fmol/mg protein. A study of [3H]Dmt-Tic-(2S,3R)βMePhe-Phe binding displacement by various receptor-type specific opioid ligands showed the rank order of competitor's potency δ > μ > κ, suggesting selective labeling of opioid δ-sites. In the functional tests, the (2S,3R) and (2R,3S) peptides exhibited partial agonist behaviour by weakly stimulating regulatory G-proteins in CHO cell membranes transfected with different receptors. Both isomers were quite weak partial agonists at the δ-receptor and reasonable partial agonists at the μ-receptor, with a prevalence of (2S,3R) over (2R,3S) for the μ-receptor. Consistent with these observations both stereomers competitively inhibited the stimulation of [35S]GTPγS binding induced by the prototype δ-agonist peptide (pClPhe4)-DPDPE in δmCHO cell membranes, and still the (2S,3R) compound exerted more potent δ-antagonist effect. [3H]Dmt-Tic-(2S,3R)βMePhe-Phe represents a high affinity new radioligand and also constitute further example of the influence of β-methyl substitution on the potency and selectivity of TIPP analogues, thus becoming a valuable biochemical and pharmacological tool in opioid research.