High-throughput screening with nucleosome substrate identifies small-molecule inhibitors of the human histone lysine methyltransferase NSD2

Abstract

The histone lysine methyltransferase nuclear receptor-binding SET domain protein 2 (NSD2, also known as WHSC1/MMSET) is an epigenetic modifier and is thought to play a driving role in oncogenesis. Both NSD2 overexpression and point mutations that increase its catalytic activity are associated with several human cancers. Although NSD2 is an attractive therapeutic target, no potent, selective, and bioactive small molecule inhibitors of NSD2 have been reported to date, possibly due to the challenges of developing high-throughput assays for NSD2. Here, to establish a platform for the discovery and development of selective NSD2 inhibitors, we optimized and implemented multiple assays. We performed quantitative high-throughput screening with full-length WT NSD2 and a nucleosome substrate against a diverse collection of bioactive small molecules comprising 16,251 compounds. We further interrogated 174 inhibitory compounds identified in the primary screen with orthogonal and counter assays and with activity assays based on the clinically relevant NSD2 variants E1099K and T1150A. We selected five confirmed inhibitors for follow-up, which included a radiolabeled validation assay, surface plasmon resonance studies, methyltransferase profiling, and histone methylation in cells. We found that all five NSD2 inhibitors bind the catalytic SET domain and one exhibited apparent activity in cells, validating the workflow and providing a template for identifying selective NSD2 inhibitors. In summary, we have established a robust discovery pipeline for identifying potent NSD2 inhibitors from small-molecule libraries.

Histone Tail Analysis Reveals H3K36me2 And H4K16ac As Epigenetic Signatures of Diffuse Intrinsic Pontine Glioma

“…Further, we detected high H4K16ac abundance in DIPG cells and tissue specimens. A recently published report characterizing high-throughput screening with nucleosome substrate methods demonstrates small-molecule inhibitors of the human histone lysine methyltransferase NSD2 [36], the writer of mono-and di-methylation of H3K36 [37]; another report identi ed DC_M01_7 as a novel inhibitor of MOF, the H4K16ac-speci c acetyltransferase [38,39].…”

Milestones in transcription and chromatin published in the Journal of Biological Chemistry

“…Similar to studies with inhibitors of acetyl-lysine readers (91,92), both the histone lysine methyltransferases (KMT) and methylated histone readers have been the subject of anti-cancer drug development efforts (101,102). In one recent study, Coussens et al (101) used nucleosome substrates to screen compound libraries for inhibitors of the KMT NSD2, which is overexpressed or mutated in a variety of human cancers. Active molecules were found to bind the KMT nuclear receptor-binding SET domain.…”

Screening for histone codebreakers

“…Thus, NSD2 is a very good target for drug discovery. Unfortunately, ongoing efforts to identify specific NSD2 inhibitors have largely been unsuccessful, with lead compounds either being weak (LEM-06) or nonspecific (suramin, chaetocin, sinefungin, and BIX-01294) (8). Progress in this field has been slow in large part because NSD2 requires a nucleosomal substrate and will not efficiently catalyze the methylation of peptides or recombinant histones, which has made the development of HTS 2 challenging (9).…”

“…In a recently published study, Coussens et al (8) will help to address this gap with their development of a battery of orthogonal HTS, using nucleosomes as a substrate, which will likely facilitate the identification and detailed characterization of NSD2 inhibitors. A histone octamer assembles when a tetramer, containing two copies of both H3 and H4, complexes with two H2A/H2B dimers.…”

“…The efforts by Coussens et al (8) to develop screens to identify inhibitors of the enzymatic activity of NSD2 will certainly inspire new efforts in molecular discovery. However, NSD2 also harbors a number of domains that can potentially "read" the histone code, including four PHD fingers and two PWWP domains that could also serve as targets.…”