title: Synthesis of calcineurin-resistant derivatives of FK506 and selection of compensatory receptors.
abstract: We used olefin metathesis to synthesize C40 derivatives of FK506 and measured their ability, when complexed to FKBP12, to inhibit calcineurin's phosphatase activity. We identified modular dimerization domains (CABs) containing segments of the calcineurin A and B polypeptides. These CABs respond to FK506 both when overexpressed in mammalian cells and in yeast or mammalian three-hybrid assays. Using chemical genetic selection, we identified compensatory mutant CABs that respond to a calcineurin-resistant FK506 derivative at concentrations well below the response threshold for CABs containing only wild-type calcineurin sequence. These reagents provide a small molecule-protein combination orthogonal to existing dimerizer systems and may be used with existing systems to increase the complexity of induced-proximity experiments. This new use of the "bump-hole" strategy protects target cells from complications arising from the inhibition of endogenous calcineurin.
authors.affiliation: Howard Hughes Medical Institute at Harvard University, Cambridge, MA 02138, USA.
authors.forename: Paul A
authors.initials: PA
authors.lastname: Clemons
authors.forename: Brian G
authors.initials: BG
authors.lastname: Gladstone
authors.forename: Abhinav
authors.initials: A
authors.lastname: Seth
authors.forename: Elizabeth D
authors.initials: ED
authors.lastname: Chao
authors.forename: Michael A
authors.initials: MA
authors.lastname: Foley
authors.forename: Stuart L
authors.initials: SL
authors.lastname: Schreiber
mesh_terms: D019703:Calcineurin / Q000737:chemistry*; D065095:Calcineurin Inhibitors; D019281:Dimerization; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D006801:Humans; D007166:Immunosuppressive Agents / Q000737:chemistry* / Q000494:pharmacology; D019169:Jurkat Cells; D008958:Models, Molecular; D009154:Mutation; D011993:Recombinant Fusion Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics; D016559:Tacrolimus / Q000737:chemistry* / Q000494:pharmacology; D022061:Tacrolimus Binding Protein 1A / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology
chemical_list: D065095:Calcineurin Inhibitors; D004791:Enzyme Inhibitors; D007166:Immunosuppressive Agents; D011993:Recombinant Fusion Proteins; D019703:Calcineurin; D022061:Tacrolimus Binding Protein 1A; D016559:Tacrolimus