The NSD (Nuclear receptor SET Domain-containing) family of histone methyltransferases is comprised of three members, NSD1, NSD2 (WHSC1), and NSD3 (WHSC1L1). These NSDs have been reported to target H3K36 and control the levels of methylated H3K36 in vivo [[1], [2], [3]]. NSD proteins, especially NSD2 and NSD3, behave as potent oncoproteins in a wide range of cancer types. High expression of NSD2 is implicated in bladder cancer [4,5], lung cancer [[4], [5], [6], [7], [8]], colon cancer [5,9,10], hepatocellular carcinoma, and myeloma [11,12]. NSD3 is located at 8p11-12 region which displays strong cancer relevance to lung cancer and breast cancer [13,14]. It drives the progression of breast cancer, lung cancer, pancreatic adenocarcinoma, and osteosarcoma [13,[15], [16], [17], [18], [19], [20]]. Collectively, these studies have underlined the critical roles of NSDs in carcinogenesis, and there has been great interest in the discovery of NSD inhibitors.

NSDs contain one catalytic SET (Su(var)3–9, Enhancer of zeste, Trithorax) domain, two PWWP (proline-tryptophan-tryptophan-proline) domain, and multiple PHD (plant homeodomain) fingers. Most current inhibitors of NSDs have been designed to target the SET domain and PWWP domain [21]. In 2015, LEM-06 was discovered by virtual screening and targeted the SET domain of NSD2 with an IC50 of 0.8 mM in vitro [22]. In 2018, Epizyme Inc. USA reported a peptide PTD2 derived from the histone H4 sequence, which exhibited inhibitory potency in vitro towards the NSD2-SET and NSD3-SET with an IC50 of 22 μM and 3.2 μM, respectively [23]. In 2021, Wang et al. reported a 5-aminonaphthalene derivative compound 9c targeting the NSD2-SET domain with an IC50 of 2.7 μM in biochemical assay. Compound 9c effectively suppressed the proliferation of RS4:11 acute lymphoblastic leukaemia (ALL) cells and KMS11 multiple myeloma (MM) cells with micromolar levels [24]. In addition, Coussens et al. also reported several hit compounds targeting the NSD2-SET domain, including DA3003-1 (a Cdc25 phosphatase inhibitor) [25], PF-03882845 (a mineralocorticoid receptor antagonist) [26], TC LPA5 4 (a lysophosphatidic acid receptor 5 antagonist) [27], ABT-199 (a BCL-2 inhibitor) [28], DZNep (a s-adenosyl homocysteine hydrolase inhibitor) [29], and Chaetocin (a nonspecific HMT inhibitor) [30]. These compounds exhibited submicromolar-to-micromolar affinities against the NSD2-SET in SPR [31]. Likewise, Suramin, a nonspecific inhibitor of DNA binding proteins, also displayed the inhibition of NSDs with micromolar in vitro activity [32].

Furthermore, in 2019, Böttcher et al. firstly reported the discovery of BI-9321 targeting the NSD3-PWWP1 domain with submicromolar in vitro activity (e.g., Kd = 166 nM in SPR). BI-9321 inhibited the proliferation of MOLM-13 acute myeloid leukaemia (AML) cells (IC50 = 26.8 μM) and RN2 AML cells (IC50 = 13 μM) [33]. In 2021, a small molecule antagonist 3f was identified to interrupt the interaction of NSD2 with H3K36me2 via the high binding affinity against the NSD2-PWWP1 domain (e.g., Kd = 3.4 μM in SPR) [34]. Later, through optimization, this group reported another chemical probe, UNC6934, which competitively bound to the NSD2-PWWP1 domain with a Kd of 91 nM in SPR [35].

Recently, using BI-9321 as the binder, Xu et al. discovered the first NSD3 proteolysis targeting chimera (PROTAC) degrader, MS9715, which was superior to BI-9321 in repressing NSD3-targeted transcripts in cancer [36]. In 2022, using the same binder BI-9321, Sun et al. reported a NSD3 specific degrader with DC50 of 1.43 μM and 0.94 μM in lung cancer cells [37]. These studies on the inhibitors of NSDs would speed up the understanding of the function of NSDs.

In this study, we report the discovery of small-molecule NSD inhibitors, compound 2 and 3 which bind to the NSD-SET domain with micromolar in vitro activity. These compounds effectively decrease cellular levels of H3K36me2 at sub-micromolar in H460 and H1299 non-small cell lung cancer cells. Treatment of the compound induces S-phase arrest, leads to the increases in the apoptotic cell population, and prohibits cell proliferation. Thus, these results provide further utility of these compounds in a wide range of cancers presented with the aberrant expression of NSDs or NSDs-related signal pathways to explore the roles of NSDs in cancers.