Novel diastereomeric opioid tetrapeptides exhibit differing pharmacological activity profiles

A novel opioid peptide antagonist analogue, [H-3]Dmt-Tic-(2S,3R)beta MePhe-Phe, derived from the potent, delta-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 beta-methyl substituted phenylalanine (beta MePhe(3)) at the third position. As the configuration of beta MePhe(3) side-chain might be different due to diastereomerism, and accordingly can alter the biological activity, both unlabeled threo (2S,3R and 2R,3S) diastercomeric analogues were also prepared and included in this study. The affinity and selectivity (delta-opioid versus mu-opioid receptor) were evaluated by radioreceptor binding assays. Agonist or antagonist potencies were determined in [S-35]GTP gamma S binding experiments using Chinese Hamster Ovary (CHO) cells selectively expressing delta- or I-L-opioid receptors. The equilibrium binding of the radiolabeled peptide derivative [H-3]Dmt-Tic-(2S,3R)beta MePhe-Phe to rat brain membranes was saturable and the Scatchard analysis indicated a single binding receptor-type specific opioid ligands showed the rank order of competitor's potency delta > mu > kappa, suggesting selective labeling of opioid delta-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 4 membranes transfected with different receptors. Both isomers were quite weak partial agonists at the delta-receptor and reasonable partial agonists at the mu-receptor, with a prevalence of (2S,3R) over (2R,3S) for the R-receptor. Consistent with these observations both stereomers competitively inhibited the stimulation of [S-35]GTP gamma S binding induced by the prototype delta-agonist peptide (pClPhe(4))-DPDPE in delta mCHO cell membranes, and still the (2S,3R) compound exerted more potent delta-antagonist effect. [H-3]Dmt-Tic-(2S,3R)beta MePhe-Phe represents a high affinity new radioligand and also constitute further example of the influence of P-methyl substitution on the potency and selectivity of TIPP analogues, thus becoming a valuable biochemical and pharmacological tool in opioid research. (c) 2007 Elsevier Inc. All rights reserved.