NOVEL PROBES FOR IMMUNOPHILIN MEDIATED CELL CONTROL

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Marine invertebrates produce a diverse array of natural products some of which target specific cellular receptors. We have found that bastadin-5, a macrocycle from the marine sponge Ianthella basta is a potent agonist of the ryanodine-sensitive Ca2+ channel of the sarcoplasmic reticulum, SR (ER502muM). FKBP12 is a protein physically associated with the ryanodine receptor that affects Ca2+ transport across SR/ER. Bastadin-5 binds to FKBP12/ryanodine receptor complex and stimulates release of Ca2+. Direct modulation of Ca2+ release from intracellular stores by FKBP12 and binding of FKBP12 to calcineurin and the TGF-beta1 receptor are involved in a cascade of signal transduction events that lead to among other responses, cell proliferation and differentiation. These are important targets for intervention in tumorigenesis. The locus of binding and binding motif of bastadin-5 to the FKBP12/ryanodine receptor complex are unknown. The plan of this proposal is, 1. Isolation and characterization of new Ca2+ channel modulators of the FKBP12/ryanodine receptor complex from extracts of marine invertebrates. We will concentrate on the structures of additional bastadin-like compounds in Ianthella basta and six unidentified marine invertebrate samples which have been screened and shown to contain potent Ca2+ modulatory activity. 2. Determine the quantitative structure-activity relationships (QSAR) for all bastadins for their ability to activate the FKBP/ryanodine receptor complex and Ca2+ efflux from SR/ER preparations. This will provide data for later development of a functional model for bastadin-5 interaction with the FKBP12/ryanodine complex. 3. Total synthesis of bastadin-5 and bastadin-5 photoaffinity-labelled analogs for use in localization of the bastadin-5 effector site on FKBP12/ryanodine complex. 4. Test the hypothesis that bastadins interact directly with FKBP12 or the FKBP12 effector site on the FKBP12/ryanodine receptor complex to affect a significant (unprecedented) change in the function of ryanodine- sensitive Ca2+ channels. 5. To understand how modulation of the FKBP12/ryanodine receptor complex by bastadins influences cellular growth and differentiation using the BC3H1 cell line as model.