BIOCHEMICAL STUDIES AND CLONING OF DELTA SUBTYPES

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This proposal is part of a program project that seeks to produce and characterize non-peptidic compounds acting at delta opioid receptors. A basic hypothesis of this program project is that drugs acting on delta opioid receptors will be potent analgesics, but will lack many of the undesirable side effects of the opiate drugs now in use. Furthermore, it is hypothesized that there are multiple delta opioid receptor subtypes and that an additional increase in therapeutic specificity can be achieved by drugs selective for particular delta receptor subtypes. Two recent developments, the discovery of the non-peptidic compound BW373U86 and the cloning of mouse delta opioid receptors, provide a foundation for the testing of these hypotheses and the work proposed here. BW373U86 is the only known selective delta receptor agonist that is not a peptide. The racemic mixture of BW373U86 was resolved into its two (+) and (-) isomers by Dr. Kenner Rice who proposes to have each form labeled with tritium for studies by radioligand binding. We propose to do this characterization by tissue homogenate binding and receptor autoradiography studies using mouse and rat neural tissue. Parallel studies with radiolabeled forms of the established ligands [4'-Cl- Phe4]DPDPE and naltrindole in addition to binding inhibition studies designed to characterize the site(s) labeled by BW373U86 will be used to define its properties at CNS receptors. This information will assist the design of new analogs of BW373U86 and help to explain some of the unusual pharmacological properties of this novel compound. Some of these properties, including high potency at delta receptors in the mouse vas deferens but low analgesic potency suggest that BW373U86 may be selective for delta receptor subtypes. We also intend to explore the hypothesis of delta opioid receptor subtypes by cloning mouse and human cDNAs encoding the proposed subtypes. Since at least one subtype of the mouse delta opioid receptor has been cloned, we will use polymerase chain reaction methods to product oligonucleotide probes selective for delta opioid receptors. These probes will be used to initially screen mouse and subsequently human brain cDNA libraries for the presence of multiple delta opioid receptors. The identification of cDNAs for delta receptor subtypes is important since selective radioligands are not available which impedes the development of selective drugs for these subtypes. Furthermore, the only way by which any human delta opioid receptors can be studied is through the use of recombinant cells expressing these receptors. cDNAs for delta receptors will be expressed in mammalian cells to produce cell lines having defined delta receptor subtypes. The cell lines expressing different human delta opioid receptor subtypes will be used for the development of specific radioligand binding and functional assays for the screening and characterization of the new compounds proposed elsewhere by this program project proposal.