Discovery of a Potent, Selective, and In Vivo Efficacious Covalent Inhibitor for Lysine Methyltransferase SETD8

Dysregulated signaling of SET domain-containing protein 8 (SETD8) has been implicated in tumorigenesis, yet most SETD8 inhibitors exhibited limited cellular efficacy. Herein, we developed a potent and selective SETD8 covalent inhibitor, MS2928 (<b>3</b>), featuring a propiolamide covalent warhead. Compound <b>3</b> potently and selectively inhibited SETD8 methyltransferase activity. The covalent inhibition mechanism of <b>3</b> was confirmed by mass spectrometry and X-ray crystallography. Moreover, <b>3</b> significantly reduced the histone H4 lysine 20 monomethylation (H4K20me1) levels in cells and robustly inhibited the proliferation of SETD8-overexpressing multiple myeloma (MM) cell lines with no significant antiproliferative effect on SETD8-low expressing MM cells and normal cells. Importantly, <b>3</b> effectively inhibited tumor growth <i>in vivo</i> in two xenograft mouse models of SETD8-overexpressing MM cell lines. Collectively, our results establish <b>3</b> as a valuable chemical tool for exploring the biological functions of SETD8 and pave the way for further development of novel epigenetic therapies for MM.