We propose to synthesize potent and stable opioid peptide analogs designed to be i) highly selective delta antagonists, ii) mixed mu agonist/delta antagonists, and iii) structurally rigid mu-selective agonists. The compounds to be developed might also be useful for the further demonstration of putative mu-, delta- and kappa-opioid receptor subtypes and as selective ligands for such subtypes. Highly receptor-selective opioid peptide agonists, antagonists or mixed agonist/antagonists are needed in studies aimed at correlating specific opioid effects with a distinct receptor class or subclass as well as for the development of potential therapeutic agents showing minimal side effects. To reach these goals, we use an interdisciplinary approach incorporating peptide synthesis, conformational investigations and extensive pharmacological characterization. The peptide analog design will be based on various principles, including substitutions of natural and artificial amino acids, peptide bond replacements and introduction of conformational constraints.
Specific aims i nclude: a) synthesis of highly potent and selective TIPP(H-Tyr-Tic-Phe-Phe-OH) and TIPP[psi](H-Tyr-Tic-psi[CH2-NH]Phe-Phe-OH) analogs with delta antagonist or inverse delta agonist properties that contain additional conformational constraints or lipophilic structural elements to promote crossing of the blood-brain barrier (BBB); b) development of mixed mu agonist/delta antagonists based on structural modification of H-Dmt-Tic(psi)[CH2-NH]Phe-Phe-NH2 (DIPP-NH2[psi]), H-Tyr-c[-D-Om2-Nal-D-Pro-Gly-] and H-Dmt-Tic-(CH2)3-C6H5, with the goal of optimizing the mu agonist/delta agonist potency ratio and BBB penetration; c) synthesis of highly potent and delta-selective analogs of the novel delta agonist H-Tyr-Tic-CH2-CH(C6H5)2 recently discovered in our laboratory; d) further development of novel cyclic mu agonist peptides with possible mu receptor subtype selectivity; e) conformational studies by molecular mechanics and molecular dynamics techniques and by NMR spectroscopy; f) determination of receptor affinities and selectivities of the new compounds in binding assays based on displacement of mu-, delta-, and kappa- selective radioligands from rat or guinea pig membrane binding sites; g) evaluation of agonist and antagonist properties of the analogs in bioassays based on inhibition of electrically evoked contractions of the guinea pig ileum and of the vasa deferentia of the mouse, rat and rabbit, and in GTPase and adenylate cyclase assays using NG108-15 or SH-SY5Y cells; h) tests of the analgesic profiles of the analogs using the mouse writhing and hot plate assays and the rat tail flick test.
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