NSD2 catalyzes the mono- and di-methylation of the e-amine of lysine 36 from histone H3, utilizing the methyl donor S-adenosyl-L-methionine. Increased catalytic activity of NSD2, either by overexpression or point mutations, is associated with multiple human cancers. To date, potent, selective and cell-active NSD2 inhibitors have not been described. A platform was established for the discovery of selective NSD2 inhibitors from quantitative high-throughput screening in a 1536-well format using a nucleosome substrate. Active compounds from the primary screen were triaged by a panel of assays and tested against two clinically relevant NSD2 mutants, E1099K and T1150A. Five confirmed inhibitors were further evaluated by a radiolabeled validation assay, surface plasmon resonance studies, methyltransferase profiling, and histone methylation in U-2 OS cells. Several inhibitors bound the NSD2 catalytic SET domain and demonstrated activity in cells, which validates the use of this workflow for the discovery and development of selective NSD2 inhibitors.
 

Learning Objectives: 

1. The importance of orthogonal and counter assays for hit selection
2. The value of assay miniaturization for expensive/limited reagents