Identification of novel covalent ligands that disrupt Acetyl-Lysine binding to the SETDB1 tudor domain.

SETDB1 is a histone methylation reader that recognizes the H3K9me2/K14ac marks via two pockets (Kme and Kac) within its triple Tudor domain. Overexpression of SETDB1 is frequently associated with cancer and neurodegenerative diseases, highlighting its potential as a therapeutic target. However, ligands targeting the Kac pocket of the triple Tudor domain remain poorly explored. In this study, differential scanning fluorimetry (DSF) coupled with iodoacetamide-rhodamine (IA-rhodamine, IAR) screening was used to identify two covalent ligands, XY047 and XY087, which primarily target Cys329 and occupy the Kac pocket of the SETDB1 Tudor domain. Cell lysate-based assay further confirmed their binding activity. These findings provide valuable tools for investigating the biological functions of SETDB1 in disease and advancing the development of targeted therapeutics.