Figure 2.

Quantitative high-throughput screening and secondary screening for inhibitors of NSD2 WT and variant enzymes. A–D, statistics from the qHTS primary screen against full-length WT NSD2 with the MTase-Glo methyltransferase assay for 43 1,536-well plates. A, overall assay performance of the primary screen was robust with an average Z′-factor value of 0.92. B, average signal-to-background ratio was 3.28. C, average % CV for the DMSO control was 1.68. D, of the 16,251 compounds screened, 536 were identified as active (3.3% hit rate). E, primary and secondary assay triage process. Eight compound libraries (16,251 compounds) were screened in qHTS format at three concentrations, resulting in 536 hits. From the initial hit list, 289 compounds were selected as cherry picks and further evaluated in 11-point dose response. Of the 289 cherry picks, the activities of 48 were confirmed against NSD2 with the primary and orthogonal assays with no activity observed by the counter assays. The majority of these compounds was also active against the NSD2 variants E1099K (45 compounds) and T1150A (44 compounds).