Discovery and biological evaluation of HS13 as a novel covalent ligand targeting the NSD2-PWWP1 domain.

The PWWP1 domain of NSD2 is an epigenetic reader essential for the subcellular localization of NSD2 and is frequently implicated in oncogenesis, making it an attractive therapeutic target. Our previous work has identified the C294 residue of NSD2-PWWP1 as a viable covalent targeting site. In this study, we screened a library of 968 covalent compounds and identified the lead compound, 92-B7, which exhibited an IC50 of 332 μM. Subsequent structural optimization and affinity-guided evaluations led to the development of HS13, which demonstrated significantly improved potency with a Kd of 8.33 μM. Binding assays confirmed that HS13 selectively formed a covalent bond with C294. In cellular studies, HS13 inhibited U2OS cell proliferation, induced apoptosis, and triggered G2-phase cell cycle arrest. Strikingly, HS13 treatment also prompted the degradation of NSD2, likely through destabilization of the PWWP1 domain. Collectively, these findings demonstrate that HS13 functions as a covalent modulator of NSD2-PWWP1 and highlight covalent targeting of this domain as a promising therapeutic strategy against NSD2-driven cancers.