Figure 2.

Osimertinib inactivates GAPDH via covalent modification of cysteine residues. (A) HUVECs were treated with the compounds from the FDA-approved inhibitors at a concentration of 1 μmol/L for 12 h, followed by the assessment of GAPDH enzyme activity. Dots in gray represent inhibitors. Osimertinib is indicated in fuchsine. Data represent the mean of three independent experiments. n = 3. (B) Recombinant GAPDH was treated with the FDA-approved inhibitors (final concentration 100 μmol/L) or DMSO for 2 h, followed by enzyme activity assay. Data represent the mean of three independent experiments. n = 3. (C) Dose-dependent inactivation of GAPDH enzyme activity in HUVECs were treated with the indicated osimertinib concentrations for 12 h n = 5. (D) Immunoblot confirmed the stabilization of GAPDH by osimertinib (100 μmol/L) treatment for 2 h in HUVEC cell lysates in the DARTS assay. (E) Tandem mass spectrometry spectrum of the peptide in recombinant human GAPDH containing the osimertinib-adducted at residue Cys247. (F) Schematic diagram of GAPDH thiol group that was covalently modified by osimertinib. (G) Concentration of osimertinib in mouse plasma, whole tumor tissues, CD31⁺ tumor endothelial cells and CD31^– non-endothelial cells at 4 h after osimertinib treatment (25 mg/kg) by oral gavage. n = 5. (H) Mice were treated with osimertinib (25 mg/kg) by oral gavage; after 4 h, the mice were sacrificed and lysates of CD45^–CD31⁺ endothelial cells from mouse transplanted CT26 tumors were used for GAPDH enzyme activity assay. n = 5. (I) Immunoblot confirmed the stabilization of GAPDH by osimertinib (25 mg/kg) or vehicle for 4 h in CD31⁺ endothelial cell lysates from mouse transplanted CT26 tumors in the DARTS assay. (J) Alignment of the GAPDH motif in the indicated species. (K) DARTS confirmed that GAPDH could not bind with osimertinib in zebrafish embryos lysates after osimertinib (100 μmol/L) treatment for 2 h. Statistical analysis by unpaired Student's t-test or one-way ANOVA. Data are presented as mean ± SEM.