The long-term objectives of this research are to better understand the interactions of the opioid peptides, specifically dynorphin, with opioid receptors and to study at a molecular level how interactions of antagonists with opioid receptors may differ from those of agonists. Another objective is ultimately to identify selective and potent antagonists for kappa receptors which can be used as pharmacological tools in further studies. Since kappa opioid receptors are present in the human brain and spinal cord in high concentrations, a better understanding of how these receptors function at a molecular level and the availability of specific antagonists for these receptors could be very important in the development of non-additive analgesics.
The specific aims of this project are to explore the structural, conformational and topographical requirements for kappa receptor antagonists derived from dynorphin and to compare the effects of structural modifications on the agonist vs antagonist activity of dynorphin analogues. Modifications chosen to potentially impart antagonist activity will involve the N-terminal tyrosine residue and Phe4. Topographical restrictions of the N-terminal tyrosine will also be investigated to evaluate whether the topography of the side chain and/or orientation of the N-terminal nitrogen is important for distinguishing agonist vs antagonist activity of dynorphin analogues. Other structural modifications which may affect the relative orientations of the important residues Tyr1, Phe4 and Arg7 will be examined to determine how they affect antagonist as compared to agonist activity. These will include cyclizations involving Tyr1 and other residues. Topographically constrained phenylalanine derivatives will also be incorporated into the peptides. Conformational constraints affecting Arg7 will also be investigated. The peptides will be prepared by solid phase peptide synthesis. The conformations of the constrained peptides will be examined using NMR and CD spectroscopy and by computer-assisted molecular modeling with the INSIGHT and DISCOVER programs. The analogues will be evaluated for opioid activity, both agonism and antagonism, in the guinea pig ileum and other isolated smooth muscle assays. Receptor affinity will be determined in radioligand binding assays in guinea pig cerebellum and rat brain. In vivo analgesia and antagonist activity will be evaluated in the mouse phenylquinone writhing assay.
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