Discovery of Covalent and Cell‐Active ALKBH5 Inhibitors with Potent Antileukemia Effects In Vivo

Abstract The N 6 ‐methyladenosine (m 6 A) demethylase ALKBH5 is the only other identified m 6 A eraser except for FTO, and dysregulated ALKBH5 functions were closely associated with leukemogenesis. However, the development of ALKBH5 inhibitors is slow compared to FTO inhibitors. Inspired by a non‐catalytic C200‐covalent strategy, a series of maleimide derivatives were designed and synthesized as potent and covalent ALKBH5 inhibitors in this work. The analog 18 l exhibited excellent inhibitory effects on ALKBH5 (IC 50 =0.62 μM), and exerted a strong antiproliferative effect on NB4 cells with IC 50 of 0.63 μM. The K d value of 18 l binding to ALKBH5 was 804 nM, while no binding was observed with FTO. This result indicated that 18 l was a highly selective inhibitor of ALKBH5 rather than FTO. Additionally, proteomic experiments showed that 18 l directly targeted ALKBH5 in cells and altered m 6 A levels on mRNA, blocked the related downstream signal pathways, promoted differentiation, and induced apoptosis. Furthermore, 18 l exerted excellent in vivo antitumor activity with TGI TV values of 66.3 % at 1 mg/kg in NB4 tumor xenograft models.