Identification of novel class inhibitors of NSD3 methyltransferase showing a unique, bivalent binding mode in the SET domain

Abstract NSD3/WHSC1L1 lysine methyltransferase promotes the transcription of target genes through di‐ or tri‐methylation at histone H3K36 using SAM as a cofactor. Genetic alterations such as amplification and gain‐of‐function mutation of NSD3 act as oncogenic drivers in several cancers including squamous cell lung cancer and breast cancer. NSD3 is an important therapeutic target for cancers, but the reported NSD3 inhibitors targeting the catalytic SET domain are very rare and show a poor activity. Herein, from a virtual library screening and the subsequent medicinal chemistry optimization, we identified a novel class of NSD3 inhibitors. Our docking analysis and pulldown result suggested that the most potent analogue 13i shows a unique, bivalent binding mode interacting with both SAM‐binding site and BT3‐bindig site within the SET domain. We found 13i inhibits NSD3 activity with IC 50 = 287 μM in vitro and suppresses the proliferation of JIMT1 breast cancer cells with GI 50 = 36.5 μM, which express a high level of NSD3. Also, 13i downregulated the levels of H3K36me2/3 in a dose‐dependent manner. Our study could provide an insight in designing high‐affinity NSD3 inhibitors. Also, as the acrylamide group of 13i was predicted to position near Cys1265 in the BT3‐binding site, further optimization would lead to a discovery of novel irreversible NSD3 inhibitors.