1. Metkephamid (Tyr-D-ala-Gly-Phe-N(Me)Met-NH2), a potent opioid peptide: receptor binding and analgesic properties
C Burkhardt, R C Frederickson, G W Pasternak Peptides. 1982 Sep-Oct;3(5):869-71. doi: 10.1016/0196-9781(82)90029-8.
The interaction of metkephamid (Tyr-D-Ala-Gly-Phe-N(Me)Met-NH2) with 3H-dihydromorphine and 3H-D-Ala2-D-Leu5-enkephalin binding has been examined in rat brain homogenates. Displacements of both 3H-ligands by metkephamid indicate that metkephamid interacts competitively with greatest potency to the high affinity binding component for both ligands (mu1 site). Unlike most enkephalins and opiates, metkephamid binds equipotently to both morphine-selective (mu2) and enkephalin-selective (delta) binding sites. Metkephamid is differentiated from morphine by its better than 12-fold higher affinity for the delta receptor. Blockade of the high affinity (mu1) binding in vivo with high doses of naloxazone dramatically reduces metkephamid's analgesic potency.
2. Design of opioid peptides for a potential delta-receptor affinity label function: comparison with the mu-specific Tyr-D-Ala-Gly-(Me)Phe-chloromethyl ketone
A Z Rónai, J Hepp, A Magyar, A Borsodi, K Medzihradszky Pharmacology. 1994 Aug;49(2):121-31. doi: 10.1159/000139224.
To find a delta-opioid receptor preferring peptide structure containing an Asp residue in a potentially interacting position, Tyr-Pro-Phe-Asp, Tyr-D-Ala-Phe-Asp, Tyr-D-Ala-Gly-Phe-Asp, Tyr-D-Ala-Gly-Phe-Asp alpha- and beta-methyl ester and Tyr-Gly-Gly-Phe-Asp peptides were synthesized and their biological activities were analyzed in vitro in mouse vas deferens and longitudinal muscle strip of guinea pig ileum. Changing the beta-methyl ester for an alkylating chloromethyl ketone moiety in the delta-receptor-selective agonist Tyr-D-Ala-Gly-Phe-Asp-beta-methyl ester enhanced further the delta-receptor preference. The delta-receptor selective chloromethyl ketone but not the beta-methyl ester gave a very slow washout after prolonged incubation in the mouse vas deferens bioassay; however, it was still readily displaceable by naloxone. The washout pattern of mu-specific Tyr-D-Ala-Gly-(Me)Phe chloromethyl ketone did not differ in the bioassays from that of the corresponding Gly5-ol derivative. Both chloromethyl ketones gave irreversible characteristics in the receptor binding assay.
3. Tyr-D-Ala-Gly-(Me)Phe-chloromethyl ketone: a mu specific affinity label for the opioid receptor
S Benyhe, J Hepp, J Simon, A Borsodi, K Medzihradszky, M Wollemann Neuropeptides. 1987 Apr;9(3):225-35. doi: 10.1016/0143-4179(87)90043-6.
An alkylating tetrapeptide enkephalin derivative, Tyr-D-Ala-Gly-(Me)Phe-chloromethyl ketone (DAMK) was synthesized, and its binding characteristics on rat brain membranes were evaluated. In competition experiments, the product shows high affinity for the mu opioid binding site of the rat brain membranes, whereas its binding to the delta and kappa subtypes is weak. Micromolar concentrations of this ligand produce a dose-dependent, apparently irreversible inhibition of /3H/-naloxone binding, with apparent IC50 value of 1-5 uM. Neither reversibly binding opioids nor tosyl-amino acid chloromethyl ketones show these effects. Saturation binding analysis with /3H/-naloxone of membranes preincubated with Tyr-D-Ala-Gly-(Me)Phe-CH2Cl reveal a selective and irreversible inhibition of the high affinity /3H/-naloxone binding site. Irreversible blockade of mu-selective /3H/-ligand binding by Tyr-D-Ala-Gly-(Me)Phe-CH2Cl is much more effective than that of the binding of /3H/-enkephalin or /3H/-ethylketocyclazocine. The mu-selective binding properties of this new irreversible enkephalin analogue suggest that it could serve as an affinity label for the mu opioid receptor subtype.
