Three families of cyclic depsipeptides, all recently isolated from different shallow water sponges in the Pacific, display cytotoxicity against a broad spectrum of tumor cell lines (mean 1C50= 75 ng/mL), most notably including multidrug-resistant cell lines. All three families also protect T-cells against infection by HIV-1 (EC50= 3.6 ng/mL). Additionally, preliminary studies in our laboratory have indicated that at least one of these molecules may induce apoptosis in tumor cells. The origins of these effects are unknown at present. These families share many common structural features, including a number of novel, non-proteinogenic amino acids, but have not been fully structurally characterized. The first goal of this proposal is to complete the structural elucidation of these natural products through solid phase synthesis, correlation studies, NMR spectroscopy and molecular modeling. The second goal of this project is to investigate the cellular modes of action of these molecules through the synthesis and use of fluorescently labeled derivatives and affinity labeling probes. The fluorescent probes will be used to study cellular localization and investigate whether such molecules can cross plasma membranes. The affinity probes will be used to label high affinity binding sites for these molecules and to identify potential receptor proteins. A third goal of this project is to dissect the roles played by many of the novel residues in these molecules. This will be accomplished by mutating selected residues and examining the effects on structure, cytotoxicity and/or interaction with a cellular receptor. The combination of structural studies and cytotoxicity assays will be used to create a pharmacophore model that accounts for the similar activities of all three families. Development of a pharmacophore model will permit the design of non-peptidic analogues that mimic the activities of the natural products. The final goal of this project will be to fmd an endogenous protein ligand to the receptor for the cyclic depsipeptides. Monoclonal antibodies raised against a cyclic depsipeptide hapten will be used to search for such a protein ligand.
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