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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.

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Figure 1: Pathways for tethering a ligand conjugate to a protein, and the structures of compounds used in the study.
Figure 2: iBOLT of MEK1(XXX-1) variants by 3.
Figure 3: iBOLT is sensitive to inhibitor potency and linker length in inhibitor tetrazine conjugates.
Figure 4: Reversible optical toggling of protein function in live mammalian cells via photo-BOLT.
Figure 5: iBOLT of MEK2(XXX-1) variants.
Figure 6: Inhibition of MEK1(XXX-1) and MEK2(XXX-1) variants with sunitinib tetrazine conjugate 15.
Figure 7: iBOLT of LCK(250-16)-GFP using conjugate 15 inhibits ZAP70-mCherry phosphorylation and TCR phosphorylation and elicits rapid ZAP70-mCherry dissociation from the membrane.

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Acknowledgements

This work was supported by the UK Medical Research Council (grants nos. U105181009 and UD99999908) and the European Research Council. Y-H.T. was supported by an EMBO Long-Term Fellowship and S.E. by a DFG Research Fellowship. J.R.J. is a Sir Henry Dale fellow of The Wellcome Trust and The Royal Society. The authors thank K. Wang, L. Davis and M. Mohan for discussions, S. Elsaesser for plasmids and T. Elliot for compound 16.

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J.W.C. conceived the project. Y-H.T., S.E. and J.R.J. conceived, designed and performed the experiments. S.E. designed and performed the photo-BOLT experiments. K.L. contributed materials. Y-H.T., S.E., J.W.C. and J.R.J. analysed the data. Y-H.T., S.E. and J.W.C. wrote the paper.

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Correspondence to Jason W. Chin.

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Tsai, YH., Essig, S., James, J. et al. Selective, rapid and optically switchable regulation of protein function in live mammalian cells. Nature Chem 7, 554–561 (2015). https://doi.org/10.1038/nchem.2253

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