Discovery ofa Novel Selective and Cell-Active N⁶‑MethyladenosineRNA Demethylase ALKBH5 Inhibitor

N⁶-methyladenosine (m⁶A), the most abundant methylation on mRNA, plays pivotal roles in regulating mRNA biological functions, which affect cell functions. ALKBH5, an m⁶A demethylase, was found to be an oncogene in several cancer types, including triple-negative breast cancer (TNBC). Here, we report a novel and selective ALKBH5 covalent inhibitor, <b>W23</b>-<b>1006</b>, through virtual screening and structure optimization. It covalently bonds to the ALKBH5 C200 residue with an IC₅₀ value of 3.848 μM, representing roughly 30- and 8-fold stronger inhibitory activity than that against FTO and ALKBH3, respectively. Cellular experiments demonstrated that <b>W23-1006</b> could efficiently enhance the m⁶A level on fibronectin 1 (FN1) mRNA, leading to strong suppression of TNBC cell proliferation and migration in vitro as well as tumor growth and metastasis in vivo. Collectively, our study developed a novel, selective, and cell-active ALKBH5 covalent inhibitor, <b>W23-1006</b>, which could be a potential therapeutic option for cancer, such as TNBC treatment.