Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Reversible targeting of noncatalytic cysteines with chemically tuned electrophiles


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.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

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.


  1. Copeland, R.A., Pompliano, D.L. & Meek, T.D. Opinion — Drug-target residence time and its implications for lead optimization. Nat. Rev. Drug Discov. 5, 730–739 (2006).

    Article  CAS  Google Scholar 

  2. Potashman, M.H. & Duggan, M.E. Covalent modifiers: an orthogonal approach to drug design. J. Med. Chem. 52, 1231–1246 (2009).

    Article  CAS  Google Scholar 

  3. Smith, A.J.T., Zhang, X.Y., Leach, A.G. & Houk, K.N. Beyond picomolar affinities: quantitative aspects of noncovalent and covalent binding of drugs to proteins. J. Med. Chem. 52, 225–233 (2009).

    Article  CAS  Google Scholar 

  4. Singh, J., Petter, R.C., Baillie, T.A. & Whitty, A. The resurgence of covalent drugs. Nat. Rev. Drug Discov. 10, 307–317 (2011).

    Article  CAS  Google Scholar 

  5. Fry, D.W. et al. Specific, irreversible inactivation of the epidermal growth factor receptor and erbB2, by a new class of tyrosine kinase inhibitor. Proc. Natl. Acad. Sci. USA 95, 12022–12027 (1998).

    Article  CAS  Google Scholar 

  6. Cohen, M.S., Zhang, C., Shokat, K.M. & Taunton, J. Structural bioinformatics-based design of selective, irreversible kinase inhibitors. Science 308, 1318–1321 (2005).

    Article  CAS  Google Scholar 

  7. Honigberg, L.A. et al. The Bruton tyrosine kinase inhibitor PCI-32765 blocks B-cell activation and is efficacious in models of autoimmune disease and B-cell malignancy. Proc. Natl. Acad. Sci. USA 107, 13075–13080 (2010).

    Article  CAS  Google Scholar 

  8. Zhou, W. et al. Novel mutant-selective EGFR kinase inhibitors against EGFR T790M. Nature 462, 1070–1074 (2009).

    Article  CAS  Google Scholar 

  9. Zhou, W. et al. A structure-guided approach to creating covalent FGFR inhibitors. Chem. Biol. 17, 285–295 (2010).

    Article  CAS  Google Scholar 

  10. Hagel, M. et al. Selective irreversible inhibition of a protease by targeting a noncatalytic cysteine. Nat. Chem. Biol. 7, 22–24 (2011).

    Article  CAS  Google Scholar 

  11. Leproult, E., Barluenga, S., Moras, D., Wurtz, J.M. & Winssinger, N. Cysteine mapping in conformationally distinct kinase nucleotide binding sites: application to the design of selective covalent inhibitors. J. Med. Chem. 54, 1347–1355 (2011).

    Article  CAS  Google Scholar 

  12. Zhang, J., Yang, P.L. & Gray, N.S. Targeting cancer with small molecule kinase inhibitors. Nat. Rev. Cancer 9, 28–39 (2009).

    Article  Google Scholar 

  13. Wissner, A. et al. Synthesis and structure-activity relationships of 6,7-disubstituted 4-anilinoquinoline-3-carbonitriles. The design of an orally active, irreversible inhibitor of the tyrosine kinase activity of the epidermal growth factor receptor (EGFR) and the human epidermal growth factor receptor-2 (HER-2). J. Med. Chem. 46, 49–63 (2003).

    Article  CAS  Google Scholar 

  14. Weerapana, E. et al. Quantitative reactivity profiling predicts functional cysteines in proteomes. Nature 468, 790–795 (2010).

    Article  CAS  Google Scholar 

  15. Uetrecht, J. Idiosyncratic drug reactions: past, present, and future. Chem. Res. Toxicol. 21, 84–92 (2008).

    Article  CAS  Google Scholar 

  16. Evans, D.C., Watt, A.P., Nicoll-Griffith, D.A. & Baillie, T.A. Drug-protein adducts: an industry perspective on minimizing the potential for drug bioactivation in drug discovery and development. Chem. Res. Toxicol. 17, 3–16 (2004).

    Article  CAS  Google Scholar 

  17. Park, B.K. et al. Managing the challenge of chemically reactive metabolites in drug development. Nat. Rev. Drug Discov. 10, 292–306 (2011).

    Article  CAS  Google Scholar 

  18. Lee, G. et al. Novel inhibitors of hepatitis C virus RNA-dependent RNA polymerases. J. Mol. Biol. 357, 1051–1057 (2006).

    Article  CAS  Google Scholar 

  19. Patch, R.J. et al. Identification of diaryl ether-based ligands for estrogen-related receptor α potential antidiabetic agents. J. Med. Chem. 54, 788–808 (2011).

    Article  CAS  Google Scholar 

  20. Pritchard, R.B., Lough, C.E., Currie, D.J. & Holmes, H.L. Equilibrium reactions of N-butanethiol with some conjugated heteroenoid compounds. Can. J. Chem. 46, 775–781 (1968).

    Article  CAS  Google Scholar 

  21. Pearson, R.G. & Dillon, R.L. Rates of ionization of pseudo acids. IV. Relation between rates and equilibria. J. Am. Chem. Soc. 75, 2439–2443 (1953).

    Article  CAS  Google Scholar 

  22. Cohen, M.S., Hadjivassiliou, H. & Taunton, J. A clickable inhibitor reveals context-dependent autoactivation of p90 RSK. Nat. Chem. Biol. 3, 156–160 (2007).

    Article  CAS  Google Scholar 

  23. Fabian, M.A. et al. A small molecule–kinase interaction map for clinical kinase inhibitors. Nat. Biotechnol. 23, 329–336 (2005).

    Article  CAS  Google Scholar 

  24. Frödin, M. & Gammeltoft, S. Role and regulation of 90 kDa ribosomal S6 kinase (RSK) in signal transduction. Mol. Cell. Endocrinol. 151, 65–77 (1999).

    Article  Google Scholar 

  25. Doehn, U. et al. RSK Is a principal effector of the RAS-ERK pathway for eliciting a coordinate promotile/invasive gene program and phenotype in epithelial cells. Mol. Cell 35, 511–522 (2009).

    Article  CAS  Google Scholar 

  26. Kang, S. et al. p90 ribosomal S6 kinase 2 promotes invasion and metastasis of human head and neck squamous cell carcinoma cells. J. Clin. Invest. 120, 1165–1177 (2010).

    Article  CAS  Google Scholar 

  27. Smolen, G.A. et al. A genome-wide RNAi screen identifies multiple RSK-dependent regulators of cell migration. Genes Dev. 24, 2654–2665 (2010).

    Article  CAS  Google Scholar 

  28. Malakhova, M. et al. Structural basis for activation of the autoinhibitory C-terminal kinase domain of p90 RSK2. Nat. Struct. Mol. Biol. 15, 112–113 (2008).

    Article  CAS  Google Scholar 

Download references


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).

Author information

Authors and Affiliations



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.

Corresponding author

Correspondence to Jack Taunton.

Ethics declarations

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.

Supplementary information

Supplementary Text and Figures

Supplementary Methods and Supplementary Results (PDF 1685 kb)

Supplementary Table 1

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

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Serafimova, I., Pufall, M., Krishnan, S. et al. Reversible targeting of noncatalytic cysteines with chemically tuned electrophiles. Nat Chem Biol 8, 471–476 (2012).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing