Discovery of the salicylaldehyde-based compound DDO-02267 as a lysine-targeting covalent inhibitor of ALKBH5.

N-methyladenosine (mA) is a crucial mRNA epigenetic modification in eukaryotes, and its methylation regulation is associated with the proliferation and metastasis of diverse tumor cells. ALKBH5 functions as a demethylase for mA and plays a role in the demethylation process, thus influencing tumor cell growth and migration. However, there are limited reports on selective small molecule inhibitors of ALKBH5. Herein, we designed and synthesized the ALKBH5 covalent inhibitor DDO-02267 by analyzing the protein structure of ALKBH5 and introducing salicylaldehyde warhead into noncovalent small molecule ligand. DDO-02267 specifically targeted Lys132 within ALKBH5, demonstrating significant selectivity for ALKBH5 in vitro. Additionally, DDO-02267 increased mA levels and targeted the ALKBH5-AXL signaling axis in AML cells. The compound DDO-02267 can serve as a probe for investigating the biological function of mRNA demethylase and may inspire the development of future ALKBH5 inhibitors.