Figure 6. Liganding a hyper-reactive phosphotyrosine site of GSTP1 in live cells.

(A) Gel-based chemical proteomic analysis of GSTP1-HEK293T soluble proteomes pretreated with vehicle or fragment electrophiles (50 μM, 30 min, 37 °C) followed by labeling with HHS-482 under the same treatment conditions. GSTP1 Y8F mutant shows >90% reductions in probe labeling compared with wild-type protein. JWB152 and JWB198 but not JWB146 or JWB191 block HHS-482 labeling to levels comparable with Y8F mutant. Western blot analyses (α-FLAG) confirm equivalent FLAG-tagged GSTP1 expression across all conditions tested. (B) In vitro potency of JWB152 and JWB198 against recombinant GSTP1 lysates as evaluated by GSH substrate assay (JWB152, IC₅₀ = 23 μM, 95% confidence intervals: 14-39 μM; JWB198, IC₅₀ = 16 μM, 95% confidence intervals: 11-22 μM . The negative control probes JWB146 and JWB191 did not show inhibitory activity even at the highest concentration tested (250 μM). The SuFEx analog (SuFEx-2) showed moderate inhibition of GSTP1 activity at the highest concentration tested (250 μM). Data are shown as mean ± s.e.m,; n=3 biologically independent experiments. (C) GSTP1 Y8 site is liganded (~70% blockade) by JWB198 but not JWB146 in live DM93 cells treated with SuTEx fragments followed by quantitative chemical proteomic analysis. (D) Heat map showing quantified tyrosine sites on GSTP1 and the ability of JWB198 to ligand Y8 with site specificity in live cells. JWB146 was inactive against all GSTP1 tyrosine sites quantified. See Supplementary Figure 11 for details on location of quantified tyrosine sites in the GSTP1 crystal structure. All data shown are representative of n=2 biologically independent experiments.