Novel diastereomeric opioid tetrapeptides exhibit differing pharmacological activity profiles

Abstract

A novel opioid peptide antagonist analogue, [ 3H]Dmt-Tic-(2 S,3 R)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, Dmt 1) replacing tyrosine (Tyr 1) at the N-terminus, and β-methyl substituted phenylalanine (βMePhe 3) at the third position. As the configuration of βMePhe 3 side-chain might be different due to diastereomerism, and accordingly can alter the biological activity, both unlabeled threo (2 S,3 R and 2 R,3 S) 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-(2 S,3 R)βMePhe-Phe to rat brain membranes was saturable and the Scatchard analysis indicated a single binding site with a K d of 0.3 nM and a B max of 127 fmol/mg protein. A study of [ 3H]Dmt-Tic-(2 S,3 R)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 (2 S,3 R) and (2 R,3 S) 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 (2 S,3 R) over (2 R,3 S) 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 (pClPhe 4)-DPDPE in δ mCHO cell membranes, and still the (2 S,3 R) compound exerted more potent δ-antagonist effect. [ 3H]Dmt-Tic-(2 S,3 R)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.