Novel diastereomeric opioid tetrapeptides exhibit differing pharmacological activity profiles.
A novel opioid peptide antagonist analogue, [3H]Dmt-Tic-(2S,3R)betaMePhe-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, Dmt1) replacing tyrosine (Tyr1) at the N-terminus, and beta-methyl substituted phenylalanine (betaMePhe3) at the third position. As the configuration of betaMePhe3 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 (delta-opioid versus mu-opioid receptor) were evaluated by radioreceptor binding assays. Agonist or antagonist potencies were determined in [35S]GTPgammaS binding experiments using Chinese Hamster Ovary (CHO) cells selectively expressing delta- or mu-opioid receptors. The equilibrium binding of the radiolabeled peptide derivative [3H]Dmt-Tic-(2S,3R)betaMePhe-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)betaMePhe-Phe binding displacement by various 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 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 mu-receptor. Consistent with these observations both stereomers competitively inhibited the stimulation of [35S]GTPgammaS binding induced by the prototype delta-agonist peptide (pClPhe4)-DPDPE in delta(m) CHO cell membranes, and still the (2S,3R) compound exerted more potent delta-antagonist effect. [3H]Dmt-Tic-(2S,3R)betaMePhe-Phe represents a high affinity new radioligand and also constitute further example of the influence of beta-methyl substitution on the potency and selectivity of TIPP analogues, thus becoming a valuable biochemical and pharmacological tool in opioid research.
Institute of Biochemistry, Biological Research Center, Hungarian Academy of Sciences, Temesvari krt. 62, H-6726 Szeged, Hungary.
Eniko
E
Ioja
Dirk
D
Tourwé
István
I
Kertész
Géza
G
Tóth
Anna
A
Borsodi
Sándor
S
Benyhe
D000701:Analgesics, Opioid / Q000494:pharmacology; D000818:Animals; D001921:Brain / Q000166:cytology / Q000187:drug effects; D016466:CHO Cells; D002462:Cell Membrane / Q000187:drug effects; D006224:Cricetinae; D003412:Cricetulus; D004305:Dose-Response Relationship, Drug; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)- / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000493:pharmacokinetics; D009292:Narcotic Antagonists / Q000494:pharmacology; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D011869:Radioligand Assay; D051381:Rats; D017208:Rats, Wistar; D011957:Receptors, Opioid / Q000187:drug effects / Q000378:metabolism*; D013237:Stereoisomerism; D013329:Structure-Activity Relationship; D044005:Tetrahydroisoquinolines / Q000494:pharmacology; D014316:Tritium; D014443:Tyrosine / Q000378:metabolism
D000701:Analgesics, Opioid; D009292:Narcotic Antagonists; D009842:Oligopeptides; D011957:Receptors, Opioid; D044005:Tetrahydroisoquinolines; D014316:Tritium; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; C082263:tyrosyl-1,2,3,4-tetrahydro-3-isoquinolinecarbonyl-phenylalanyl-phenylalanine; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D014443:Tyrosine