title: Inducible gene expression and protein translocation using nontoxic ligands identified by a mammalian three-hybrid screen.
abstract: The natural product rapamycin has been used to provide temporal and quantitative control of gene expression in animals through its ability to interact with two proteins simultaneously. A shortcoming of this approach is that rapamycin is an inhibitor of cell proliferation, the result of binding to FKBP12-rapamycin-associated protein (FRAP). To overcome this limitation, nontoxic derivatives of rapamycin bearing bulky substituents at its C16-position were synthesized, each in a single step. The isosteric isopropoxy and methallyl substituents with the nonnatural C16-configuration abolish both binding to FRAP and inhibition of T cell proliferation. Binding proteins for these derivatives were identified from libraries of cDNAs encoding mutants of the FKBP12-rapamycin-binding (FRB) domain of FRAP by using a mammalian three-hybrid transcription assay. Targeting of the mutations was guided by the structure of the FKBP12-rapamycin-FRB ternary complex. Three compensatory mutations in the FRB domain, all along one face of an alpha-helix in a rapamycin-binding pocket, were identified that together restore binding of the rapamycin derivatives. Using this mutant FRB domain, one of the nontoxic rapamycin derivatives induced targeted gene expression in Jurkat T cells with an EC50 below 10 nM. Another derivative was used to recruit a cytosolic protein to the plasma membrane, mimicking a process involved in many signaling pathways.
authors.affiliation: Howard Hughes Medical Institute, Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, MA 02138, USA.
authors.forename: S D
authors.initials: SD
authors.lastname: Liberles
authors.forename: S T
authors.initials: ST
authors.lastname: Diver
authors.forename: D J
authors.initials: DJ
authors.lastname: Austin
authors.forename: S L
authors.initials: SL
authors.lastname: Schreiber
mesh_terms: D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics; D008024:Ligands; D015394:Molecular Structure; D011090:Polyenes / Q000494:pharmacology; D011506:Proteins / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D020123:Sirolimus; D022021:Tacrolimus Binding Proteins
chemical_list: D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D006360:Heat-Shock Proteins; D008024:Ligands; D011090:Polyenes; D011506:Proteins; D011993:Recombinant Fusion Proteins; D022021:Tacrolimus Binding Proteins; D020123:Sirolimus