title: Selective, rapid and optically switchable regulation of protein function in live mammalian cells.
abstract: The rapid and selective regulation of a target protein within living cells that contain closely related family members is an outstanding challenge. Here we introduce genetically directed bioorthogonal ligand tethering (BOLT) and demonstrate selective inhibition (iBOLT) of protein function. In iBOLT, inhibitor-conjugate/target protein pairs are created where the target protein contains a genetically encoded unnatural amino acid with bioorthogonal reactivity and the inhibitor conjugate contains a complementary bioorthogonal group. iBOLT enables the first rapid and specific inhibition of MEK isozymes, and introducing photoisomerizable linkers in the inhibitor conjugate enables reversible, optical regulation of protein activity (photo-BOLT) in live mammalian cells. We demonstrate that a pan kinase inhibitor conjugate allows selective and rapid inhibition of the lymphocyte specific kinase, indicating the modularity and scalability of BOLT. We anticipate that BOLT will enable the rapid and selective regulation of diverse proteins for which no selective small-molecule ligands exist.
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.
authors.forename: Yu-Hsuan
authors.initials: YH
authors.lastname: Tsai
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Essig
authors.affiliation: Department of Medicine, Medical Research Council Laboratory of Molecular Biology, University of Cambridge, Francis Crick Avenue, Cambridge CB2 0QH, UK.
authors.forename: John R
authors.initials: JR
authors.lastname: James
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.
authors.forename: Kathrin
authors.initials: K
authors.lastname: Lang
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.
authors.forename: Jason W
authors.initials: JW
authors.lastname: Chin
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D006801:Humans; D008958:Models, Molecular; D010766:Phosphorylation; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry*
chemical_list: D011506:Proteins