The expansion of the target landscape of covalent inhibitors requires the engagement of nucleophiles beyond cysteine. Although the conserved catalytic lysine in protein kinases is an attractive candidate for a covalent approach, selectivity remains an obvious challenge. Moreover, few covalent inhibitors have been shown to engage the kinase catalytic lysine in animals. We hypothesized that reversible, lysine-targeted inhibitors could provide sustained kinase engagement in vivo, with selectivity driven in part by differences in residence time. By strategically linking benzaldehydes to a promiscuous kinase binding scaffold, we developed chemoproteomic probes that reversibly and covalently engage >200 protein kinases in cells and mice. Probe–kinase residence time was dramatically enhanced by a hydroxyl group ortho to the aldehyde. Remarkably, only a few kinases, including Aurora A, showed sustained, quasi-irreversible occupancy in vivo, the structural basis for which was revealed by X-ray crystallography. We anticipate broad application of salicylaldehyde-based probes to proteins that lack a druggable cysteine.
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All mass spectrometry raw files have been deposited into the MassIVE database (http://massive.ucsd.edu) and can be downloaded by the identifier MSV000088924, as well as in ProteomeXchange (http://www.proteomexchange.org) with accession number PXD031899. Source data are provided in a source data file or in the Supplementary Datasets. Coordinates and structure factors have been deposited in the PDB under accession code 7FIC. Source data are provided with this paper.
The script used for LFQ quantification is available upon request.
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Funding for this study was provided by the National Cancer Institute (NCI) (NIH NCI F31CA214028, A.C.), Ono Pharma Foundation (J.T.) and Pfizer. Mass spectrometry was supported in part by the University of California, San Francisco (UCSF) Program for Breakthrough Biomedical Research and the Adelson Medical Research Foundation (A.L.B.).
T.Y., B.H., P.K., J.R.M., J.C.K., J.D.L., S.N. and J.D.C. are current or former employees of Pfizer. J.T. is a founder of Global Blood Therapeutics, Principia Biopharma, Kezar Life Sciences, Cedilla Therapeutics and Terremoto Biosciences, and is a scientific advisor to Entos.
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(a) Chemical structures of 1 and 2. (b) Jurkat cells were treated with 1 or 2 (2 μM, 30 min), followed by compound washout for the indicated times. Cells were lysed in the presence of 25 mM sodium cyanoborohydride, except as indicated (#). After copper-promoted click conjugation with TAMRA-azide, samples were analyzed by in-gel fluorescence and Coomassie blue staining. Data are representative of two independent experiments.
(a) COS-7 cells were transfected with WT or K295Q (#) Flag-Src, or not transfected (*), and then treated with the indicated concentrations of probe 3 (30 min). After lysis in the presence of sodium borohydride and TAMRA-azide conjugation, samples were analyzed by in-gel fluorescence and western blotting. Data are representative of two independent experiments. (b) Concentration-dependent labeling of Flag-Src (n = 2, mean values from two independently performed experiments were plotted).
(a) COS-7 cells were transfected with Flag-Src, or not transfected (*), and treated with probe 3 (2 μM, 30 min), followed by washout for the indicated times. After lysis in the presence of sodium borohydride and TAMRA-azide conjugation, samples were analyzed by in-gel fluorescence and western blotting. (b) Normalized fluorescence intensity of ~65 kDa band corresponding to Flag-Src (mean ± SD, n = 3).
AURKA or Src (5 μM) was treated with probe 3 (5.1 μM) in 50 mM HEPES, pH 8.0 at RT for 5 min, followed by 20-fold dilution into buffer containing 10 μM XO44. The percentage of XO44-modified and unmodified kinase was quantified by LC-MS at the indicated time points, and % unmodified kinase (corresponding to probe 3-bound kinase) was plotted vs. time (n = 2, mean values from two independently performed experiments were plotted).
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Yang, T., Cuesta, A., Wan, X. et al. Reversible lysine-targeted probes reveal residence time-based kinase selectivity. Nat Chem Biol 18, 934–941 (2022). https://doi.org/10.1038/s41589-022-01019-1
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