Fragment-Based Screening of NSD2-PWWP1 Identifies Novel Covalent Allosteric Ligands That Diminish Methyllysine and DNA Binding Abilities of NSD2

The PWWP1 domain of NSD2 recognizes both H3K36me2/3 and DNA, a function critical for its subcellular localization and oncogenic activity, making it a promising therapeutic target. In this study, through fragment library screening and structure–activity relationship studies, we identified compounds that covalently bind to the C294 residue of NSD2-PWWP1. Structural and biochemical analyses demonstrated that compounds 13 and 16 competitively block NSD2-PWWP1′s recognition of both H3K36me2 and DNA, thereby impairing its nucleosome-binding ability. This study uncovers a novel allosteric regulatory mechanism and provides a structural framework for the development of more effective cancer therapeutics targeting NSD2-PWWP1.