| A general technique has been developed to control the proximity of proteins in cells with non-toxic small molecules. Rapamycin, through its ability to bind to FKBP and the FRB domain of FRAP simultaneously, has been used to activate cell surface receptors, cytosolic signaling molecules, and transcription factors in cells, mice, and primates. One limitation of using rapamycin in this context, however, is its ability to act as an immunosuppressant.;C16-(R) alkoxy and C20-β-allyl substituted rapamycins can be prepared in a single step from rapamycin. These molecules, which harbor large, bulky substituents on the FRB-binding surface, are rendered non-toxic as they are impaired in their ability to bind to FKBP and the FRB domain simultaneously, do not block p70 S6 kinase activity at low concentrations, and have diminished antiproliferative effects towards rapamycin-sensitive cell lines including CTLL-2 cells and yeast.;To use these non-toxic rapamycin variants to control protein associations in vivo, an FRB-based receptor for them must be discovered. Using a mammalian three-hybrid screen, a triple mutant of FRB (T2098L, W2101F, and K2095P) which restores binding to the non-toxic molecule, C20-methallyl rapamycin, was identified. C20-methallyl rapamycin (rap*) has been used to induce protein expression and protein translocation with an EC50 of 5–10 nM, and should serve as a general tool for regulating protein associations in cells and animals.;Attempts to use rapamycin to control gene expression in the developing central nervous system of mouse embryos encountered several limitations which should be addressed in future experiments. An enhancer which governs expression of the rapamycin transcription factor I to high levels in a blood-accessible tissue or cell type, such as in hematopoietic tissue or muscle cells, would be excellent for rapamycin-inducible gene expression experiments in animals.;In the course of performing rapamycin tissue distribution experiments, organic-soluble components of mouse embryos which induce apoptosis in TAg Jurkat T cells were identified, purified, and characterized as members of a family of polyunsaturated fatty acids, including docosatetraenoic, docosahexaenoic, and arachidonic acids. The apoptosis-inducing properties of these compounds can be blocked by anti-oxidants, consistent with a lipid peroxidation mechanism known to occur during programmed cell death. |
