The objectives of the project are to develop a through understanding of the functional conformations of the opioid peptides Beta-endorphin and dynorphin A(1-17), and the relationship of these conformations to the selectivities of these peptides for mu-delta-and kappa-opioid receptors and to the activation of these receptors to produce agonist activities. This knowledge will then be used to design opioid peptides and peptide mimetics with novel receptor selectivities, enhanced potencies and/or specificities and varied agonist-antagonist character. These compounds will be useful as biochemical and pharmacological probes to characterize the structure and function of the opioid receptors, and will provide new directions for pharmaceutical design. A model for the functional conformations of Beta-endorphin and dynorphin has been developed, based on the expectation that the interface-induced, amphiphilic structures in these peptides will bind to the hydrophilic/lipophilic interface of the cell surface. This controls receptor selectivity by limiting the availability of the enkephalin segments of these peptides at their amino termini for binding to their recognition sites on the opioid receptors. In addition, these amphiphilic structures appear to interact directly with certain receptors to enhance receptor activation. This model will be tested and refined through the chemical synthesis of analogues of these opioid peptides that incorporate rigid or constricted models of their amphiphilic structures. The amphiphilic alpha-helix in beta-endorphin residues 13-29 will be constrained by introducing multiple side-chain to side-chain crosslinks on its surface, using a new synthetic method. The amphiphilic Beta-strand in dynorphin A residues 7-15 will be replaced by a non-natural peptide mimetic consisting of several units of a rigid indole-based structure that substitutes for a dipeptide segment of the Beta-strand structure. In parallel studies, more detailed structure-activity information, pertaining particularly to mu-opioid receptor activation, will be obtained through the study of single-residue substituted analogues of a Beta-endorphin model peptide. These peptides will be prepared through site-directed mutagenesis and expression in E. coli cells, using a newly developed expression system. All of the above peptides will be tested in binding, adenylyl cyclase inhibition, smooth muscle and analgesic assays, as appropriate, with an emphasis on determining agonist-antagonist character. The combined information obtained from these two approaches will be used to design more novel opioid peptides, including delta- receptor specific beta-endorphin analogues, receptor-specific photoaffinity ligands, and morphine/beta-endorphin hybrid structures with enhanced efficacy on mu-opioid receptors.
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