Fig 2: Pharmacological inhibition of BTK with ibrutinib after the induction of stress in mice attenuates hyper-anxious behavior. Mice were injected daily with ibrutinib (3 mg/kg, i.p.) or vehicle two days after restraint stress and underwater trauma. One week after the induction of stress mice were assessed for the hyper-anxious behavior. A Open field test: physically stressed mice dosed with ibrutinib after the induction of stress exhibited significantly decreased anxiety levels when compared to the vehicle controls, as illustrated by increased exploration time in the central area of the OFT. B Light–dark test: physically stressed mice dosed with ibrutinib after the induction of stress displayed significant rescue from hyper-anxiety as explained by the significant increase in their time spent in the light chamber of the LDT. C Elevated plus maze test: physically stress mice injected with ibrutinib after the induction of stress exhibited significantly reduced anxiety, as evidenced by the increased duration of time spent in the open arms of the EPM. All values are presented as mean with 95% CI (n = 20/group); ***p < 0.001, ns (not significant); 2X2X2 factorial ANOVA, followed by Bonferroni post hoc test

Fig 3: Selectivity of ibrutinib derivatives. (A) isoTOP ABPP using desthiobiotin–valine–azidolysine light or heavy peptides, schematic description, and result summary. Mino cells treated with 1 μM compound for 2 h (n = 4). Proteins in the box have a heavy to light (H/L) ratio ≥ 3. (B) Pull-down proteomics schematic description and result summary. Mino cells treated with 1 μM compound for 1 h and 10 μM ibrutinib-alkyne for an additional 1 h (n = 4). Proteins in the box show significant change (fold change > 2; p < 0.05). (C) In vitro kinase activity assays with selected kinases; see additional plots for BTK, BMX, and ITK in Figure S15.

Fig 4: Site-selective labeling of BTK using CoLDR chemistry. (A) Chemical structures of ibrutinib-directed methacrylamides with various functional tags. (B) Typical example of the reaction of BTK (2 μM) with 1i (2 μM) in a 20 mM Tris buffer at pH 8, 25 °C. (C) Deconvoluted LC/MS spectrum shows the labeling of a BODIPY probe and demonstrates Ibr-H leaving. (D) Percent of labeling of BTK (2 μM) with the probes (1a–1m; 2 μM) at 10, 30, and 120 min in 20 mM Tris buffer at pH 8, 25 °C. (E) Kinetics of the increase in fluorescence intensity measured at Ex/Em = 550/620 nm (n = 4) upon addition of BTK (2 μM) to 1h (2 μM) in 20 mM Tris buffer at pH 8, 37 °C (blue). Control experiments without BTK (red), preincubation of ibrutinib (4 μM) and Ibr-H (4 μM) prior to adding 1h (green and orange, respectively), and incubation of K-RasG12C (pink) with 1h show no fluorescence. (F) Deconvoluted LC/MS spectra for BTK incubated with 1h at the end of the fluorescence measurement (shown in E). The adduct mass corresponds to a labeling event in which the Ibr-H moiety was released, validating the proposed mechanism.

Fig 5: Labeling BTK with CoLDR probes does not inhibit its activity in cells. (A) Cellular labeling profile of 1b, 1f, and 1i after 2 h of incubation with Mino cells and 1g in Mino cell lysate. 1b and 1f samples were further reacted with TAMRA-azide in lysate before imaging. An arrow indicates BTK’s MW. (B) Time-dependent labeling profile of 1f with BTK after incubation of Mino cells with 100 nM probe followed by a click reaction with TAMRA-azide in lysate prior to imaging. (C) Competition experiment of 1b, 1d, 1f, and 1i with ibrutinib. The cells were preincubated for 30 min with either 0.1% DMSO or 1 μM ibrutinib, followed by 2 h of incubation with 200 nM 1b or 1f or 100 nM 1d or 1i. (D) Mino cells were incubated with 0.1% DMSO or 1b (100 nM). Samples were further reacted with biotin-azide in lysate, followed by enrichment, trypsin digestion, and peptide identification by LC/MS/MS. The log(fold-ratio) of proteins enriched by 1b over DMSO is plotted as a function of statistical significance. BTK is clearly identified as the most enriched target; additional prominent targets that correspond to bands identified by in-gel fluorescence (panel C) are indicated. (E) BTK activity assay in Mino cells as measured by autophosphorylation of BTK. The cells were incubated for 1 h with either 0.1% DMSO, 1 μM ibrutinib, 1 μM Ibr-H, or 100 nM 1b, 1f, 1h, or 1i. The cells were either washed or not before induction of BTK activity by anti-IgM. (F) BTK activity assay: Mino cells were incubated for 2 h with either DMSO or 1 μM 1b, 1f, 1i, and 1h, washed, and then incubated for 45 min with ibrutinib (100 nM). The cells were washed again before induction of BTK activity by anti-IgM. The CoLDR probes were able to rescue BTK activity from inhibition by ibrutinib. (G) Primary B cell activation induced by anti-IgM after 24 h of treatment with increasing doses of either ibrutinib, 1b, or 1f, showing no inhibition of the CoLDR probes.