The long term objective of this research is to better understand at a molecular level how opioids, specifically opioid peptides, interact with opioid receptors. A second objective is to identify peptide-based affinity labels for opioid receptors. These affinity labels which bind covalently to their targets, can be used as pharmacological tools in a variety of studies of opioid receptor structure and function. A better understanding at a molecular level of how opioids interact with their receptors could provide invaluable information for the design of new analgesics with less of the side effects (addiction liability, etc.) associated with the currently used narcotics. The proposed research involves two phases.
The specific aims of the first phase of this research are to prepare a variety of peptide-based potential affinity labels for opioid receptors and to evaluate their ability to interact with receptors in an irreversible manner. The proposed derivatives include peptides with selectivity for each of the opioid receptor types; reactive functionalities will be incorporated at different positions in these peptides. Depending upon the peptides these analogues will be prepared either in solution or by solid phase peptide synthesis. Receptor affinity will be determined in radioligand binding assays using cloned opioid receptors, and the peptides ability to inhibit radioligand binding in a wash-resistant manner determined in these assays. Compounds exhibiting wash-resistant inhibition of binding to receptors will be chosen for further study in the second phase of the research. They will be examined against chimeric receptors to assess which regions of the receptors may be required for irreversible binding of the peptides. The selected peptides will be prepared in labeled form for further study of affinity labeled receptors and a biotin functionality will be attached to these peptides to facilitate receptor isolation. Following isolation the affinity labeled receptors will be subjected to proteolytic digestion and the resulting fragments analyzed by ion spray mass spectrometry to examine the point of attachment of the affinity label.
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