Reversible lysine-targeted probes reveal residence time-based kinase selectivity

Abstract

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.

Extended Data Fig. 1. Reversible protein labeling by benzaldehyde probe 2.

(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.

Extended Data Fig. 2. Probe 3 modification of overexpressed SRC.

(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).

Extended Data Fig. 3. Rapid dissociation of probe 3 from overexpressed SRC.

(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).

Extended Data Fig. 4. Dissociation of probe 3 from recombinant AURKA and SRC.

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).

Extended Data Fig. 5. Related to Figure 5.

Volcano plot showing log₂ fold-change and significance (−log₁₀ p-value; two tailed t-test assuming unequal variance) of proteins enriched by probe 3 (t=3 h post-dose) vs. vehicle. Red, kinases; gray, non-kinases.

Figure 1.

Benzaldehyde probe 1 reversibly labels cellular proteins. (a) Chemical structures of XO44 and probe 1. (b) Jurkat cells were treated with XO44 or 1 (2 μM, 30 min), followed by compound washout for the indicated times. Cells treated with 1 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 (Supplementary Figure 1). Data are representative of two independent experiments.

Figure 2.

Salicylaldehyde probes exhibit prolonged kinase residence time in cells. (a) Salicylaldehyde probes 3 and 4. (b,c) Jurkat cells were treated with 3 (b) or 4 (c) (2 μM, 30 min), followed by compound washout for the indicated times and lysis in the presence of sodium borohydride. After click conjugation with TAMRA-azide, samples were analyzed by in-gel fluorescence and Coomassie blue staining (Supplementary Figure 2). Data are representative of two independent experiments. (d-f) Jurkat cells were treated with DMSO or probes 1, 3, and 4 (2 μM, 30 min), followed by washout as described in the main text. After click conjugation with biotin-azide, modified proteins were enriched with Neutravidin-agarose and digested on-bead with trypsin. Enriched peptides were analyzed by LC-MS/MS and label-free quantitation (LFQ). (d) Shared and unique kinases enriched by each probe. (e) Median intensity values (LFQ) for identified kinases and non-kinases (mean ± SD, n=3). (f) Heat map showing estimated half-lives (t_1/2) of kinases enriched from cells treated with probes 3 and 4, followed by washout for 0, 1, 3, and 6 hr. Inset depicts kinases with prolonged engagement post-washout. Grey bars (N.D.): kinase not detected or did not meet criteria for t_1/2 determination (see main text).

Figure 3.

Quasi-irreversible engagement of AURKA in cells. (a) COS-7 cells were transfected with WT or K162Q (#) Flag-AURKA and treated with 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. (b) Concentration-dependent labeling of Flag-AURKA (~50 kDa TAMRA band, mean ± SD, n=3). (c,d) COS-7 cells were transfected with Flag-AURKA and treated with probe 3 (2 μM, 30 min), followed by washout for the indicated times. Samples were analyzed as described above (mean ± SD, n=3). * and NT indicate non-transfected cells.

Figure 4.

Co-crystal structure of AURKA bound to salicylaldehyde 3. (a) Salicylaldehyde 3 forms an imine bond with the catalytic lysine of AURKA. Electron density map (2F_o-F_c) is shown at a contour level of 1σ. (b) Surface rendering of AURKA bound to probe 3, highlighting the Gly-rich loop (red, top right) and the activation segment (red, bottom right), both of which shield the salicylaldimine from bulk water (imine carbon in green).

Figure 5.

Quantifying kinase occupancy by salicylaldehyde 3 in mice. (a) Mice were dosed with vehicle or probe 3 (25 or 50 mg/kg) by subcutaneous injection. At 1, 3, and 7 h post-dose, plasma and spleens were collected for quantification of probe 3 concentrations and kinase occupancy, respectively. Spleens from each time point were selected for chemoproteomic analysis (biotin-azide conjugation, streptavidin pulldown, 10-plex TMT quantitation) based on the probe 3 plasma levels quantified from the same mice (see main text and Supplementary Table 2). Spleens from vehicle-treated mice were collected at t=7 h post-injection. (b-c) Plasma concentrations of probe 3 and relative occupancy of the indicated kinases (normalized to t=3 h) are plotted for each time point (t=1 and 7 h, n=2; t=3 h, n=3). (d) Scatter plot comparing relative kinase occupancy (log2 fold-change vs. kinase occupancy at t=3 h) at t=7 h vs. t=1 h. Each circle represents one kinase, with AURKA, AURKB, and SGK3 showing the highest occupancy at t=7 h, despite decreased plasma levels of probe 3.

Figure 6.

Salicylaldehyde probe 3 reveals PF-06873600 target engagement in vivo. (a) Mice were dosed orally with vehicle (n=5) or PF-06873600 (50 mg/kg, n=5), and all mice were co-dosed with probe 3 (10 mg/kg) by subcutaneous injection. After 1 h, spleens were collected for chemoproteomic analysis. (b) Chemical structure of PF-06873600. (c) Volcano plot showing log₂ fold-change and significance (−log₁₀ p-value) between proteins enriched by probe 3 from mice treated with PF-06873600 vs. vehicle. Red, kinases; gray, non-kinases.