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Reversible targeting of noncatalytic cysteines with chemically tuned electrophiles

Abstract

Targeting noncatalytic cysteine residues with irreversible acrylamide-based inhibitors is a powerful approach for enhancing pharmacological potency and selectivity. Nevertheless, concerns about off-target modification motivate the development of reversible cysteine-targeting strategies. Here we show that electron-deficient olefins, including acrylamides, can be tuned to react with cysteine thiols in a rapidly reversible manner. Installation of a nitrile group increased the olefins' intrinsic reactivity, but, paradoxically, eliminated the formation of irreversible adducts. Incorporation of these electrophiles into a noncovalent kinase-recognition scaffold produced slowly dissociating, covalent inhibitors of the p90 ribosomal protein S6 kinase RSK2. A cocrystal structure revealed specific noncovalent interactions that stabilize the complex by positioning the electrophilic carbon near the targeted cysteine. Disruption of these interactions by protein unfolding or proteolysis promoted instantaneous cleavage of the covalent bond. Our results establish a chemistry-based framework for engineering sustained covalent inhibition without accumulating permanently modified proteins and peptides.

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Figure 1: Thiol reactivity of electron-deficient olefins.
Figure 2: Sustained, reversible inhibition of RSK2-CTD by doubly activated Michael acceptors.
Figure 3: Sustained inhibition of cellular RSK1 and RSK2 by CN-NHiPr.
Figure 4: Specific noncovalent interactions drive covalent bond formation.

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Acknowledgements

We thank D. King (Howard Hughes Medical Institute Mass Spectrometry Laboratory) for protein mass spectrometry expertise, members of the Taunton laboratory for insight, and the staff of Advanced Light Source (ALS) Beamline 8.3.1 for help with data collection. This work was supported by grants from the US National Institutes of Health (NIH) (GM071434 to J.T., CA020535 and K99CA149088 to M.A.P., F32GM087052 to J.M.M.), the Leukemia and Lymphoma Society (5416-7 to M.A.P.), and the California Tobacco Related Disease Research Program (19FT-0091 to S.K.). We acknowledge the University of California San Francisco (UCSF) Mass Spectrometry Facility (supported by NIH grant P41RR001614).

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J.T. conceived of and directed the study. I.M.S., S.K., M.S.C., R.L.M., J.M.M. and R.M.M. synthesized compounds, designed and executed chemical, biochemical and cellular experiments, and analyzed data. K.D. and M.F. designed and executed the cellular multilayering and invasion experiments. M.A.P. solved and refined the cocrystal structure. J.T., I.M.S. and S.K. wrote the manuscript with contributions from all other authors.

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Correspondence to Jack Taunton.

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Competing interests

J.T., I.M.S., S.K., M.S.C., R.L.M., J.M.M. and R.M.M. are co-inventors on a patent application covering the inhibitors described in this paper.

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Supplementary Methods and Supplementary Results (PDF 1685 kb)

Supplementary Table 1

NCHEMB-A110706812C-Taunton_Sup_table1.xls (XLS 77 kb)

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Serafimova, I., Pufall, M., Krishnan, S. et al. Reversible targeting of noncatalytic cysteines with chemically tuned electrophiles. Nat Chem Biol 8, 471–476 (2012). https://doi.org/10.1038/nchembio.925

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