SET Domain-Containing Protein 8 (SETD8) is the sole enzyme that catalyzes mono-methylation of histone 4 lysine 20. SETD8 plays important roles in gene transcription, genome integrity, cell cycle progression and DNA damage and repair. Overexpression of SETD8 has been reported in many cancers, including lung cancer and bladder cancer. SETD8 also contributes to epithelial–mesenchymal transition (EMT) in breast cancer. Therefore, SETD8 is a potential therapeutic target and inhibitors of SETD8 would be valuable tools to study its biological roles. After screening our quinazoline-focused compound library, we identified UNC0379 as the first selective small molecule inhibitor of SETD8, which was characterized in scintillation proximity assay (SPA) and microfluidic capillary electrophoresis assay (MCE). Its binding affinity to SETD8 has also been confirmed in a battery of biophysical assays. Importantly, UNC0379 is selective for SETD8 over 15 other histone methyltransferases (HMTs). Subsequent studies have demonstrated UNC0379 is active in multiple malignant diseases, both ex vivo and in vivo (in press in Cancer Cell and unpublished results). In addition, our structure-activity relationship (SAR) studies resulted in the discovery of compound MS2177 as a more potent SETD8 inhibitor (IC50 = 1.9 ± 1.1 µM). We successfully obtained the first co-crystal structure of SETD8 with this inhibitor, which revealed a reactive cysteine residue (C311) nearby the inhibitor binding site. Taking advantage of C311, we developed the first targeted covalent inhibitor (TCI) for SETD8, MS453, displaying improved potency (IC50 = 804 nM). Furthermore, mutagenesis studies confirmed MS453 selectively forms covalent bond with C311 but not with C343 which is also on the surface of SETD8. MS453 is selective for SETD8 over 28 other HMTs (in press in J. Med. Chem.). As ongoing effort to develop chemical probe for SETD8, we have developed the next generation SETD8 inhibitor with IC50 of 285 nM. This new inhibitor has displayed robust activity in reducing H4K20me1 level in multiple cell lines at low micromolar concentrations (unpublished results).

Discovery of selective SETD8 inhibitors via structure-based approach

253rd National Meeting (2017)

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