Nuclear SET Domain containing (NSD) family member proteins are histone methyltransferases that mono- and di-methylate histone 3 at lysine 36 (H3K36) to alter gene expression in a context-dependent manner. Two members of this family are NSD1 and NSD3. NSD1 is found as a fusion with NUP98 in aggressive cases of pediatric leukemia, and the histone methyltransferase activity (KMTase) of NUP98-NSD1 drives bone marrow progenitor proliferation. Additionally, high levels of catalytic NSD3 mRNA are correlated with lower disease-free survival in breast cancer patients, and expression of enzymatically active NSD3 is sufficient to rescue tumor growth in a xenograft model of triple negative breast cancer. Selective and potent inhibitors of NSD1 and NSD3 KMTase activity are currently missing but are needed to further investigate the therapeutic potential of targeting NSD1 or NSD3 in leukemia and/or solid cancers. Here, we report the development and optimization of small molecule, irreversible inhibitors of the NSD1 and NSD3 catalytic SET domain. We performed NMR-based fragment screening with NSD1 SET domain and identified BT1, which binds to the SET domain of NSD1. We then extensively optimized BT1 to develop a series of potent irreversible ligands. Mass spectrometry and structural studies of these compounds and the SET domain of NSD1/NSD3 reveal covalent engagement with a cysteine near the autoinhibitory loop. Biochemical analysis shows that these compounds strongly block the histone methyltransferase activity of NSD1 and NSD3 and bind to the SET domain in cells. Additionally, our inhibitors exhibit potent anti-proliferative effects against NUP98-NSD1 containing leukemia cells and breast cancer cells. In summary, we present novel irreversible inhibitors of NSD1 and NSD3 histone methyltransferases activity which represent important chemical probes that can be used to investigate the therapeutic potential of targeting NSD family member KMTase activity in cancers.

Development of irreversible inhibitors of NSD family histone methyltransferases

ACS Spring 2022

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