Fig. 2 | Signal Transduction and Targeted Therapy

Fig. 2

From: Discovery of IHMT-337 as a potent irreversible EZH2 inhibitor targeting CDK4 transcription for malignancies

Fig. 2

IHMT-337 covalently binds to EZH2 at Cys663 residue in SET domain. a The CETSA assay: The effect of IHMT-337 on the stability of the EZH2 protein in a temperature-dependent manner was investigated using WSU-DLCL2 cell lysate. b The CETSA assay: The effect of IHMT-337 on the stability of the EZH2 protein in a dose-dependent manner was investigated using WSU-DLCL2 cell lysate. c Washout assay: The effect of washout assay on signal pathway inhibition post-drug washout at different time points after using IHMT-337 and IHMT-338 treatment 72 h on WSU-DLCL2 cell line. d Target-engagement assay: Using Biotin-IHMT-337 and IHMT-337 to investigate the binding of IHMT-337 to EZH2 in Pfeiffer cells. e Predicted mode of binding of IHMT-337 to EZH2 based upon molecular modeling (PDB ID 5IJ7, chain B). f Using the HEK293T EZH2-KO cell line and plasmids with different mutations, investigation of the contribution of three cysteines in the SET domain to the direct binding of EZH2 and IHMT-337, the wt EZH2 was set as control. g The level of H3K27me3 was quantified and graphed. Shown are the representative results of three independent experiments

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