Discovery of a novel selective and cell-active N6-methyladenosine RNA demethylase ALKBH5 inhibitor
N6-methyladenosine (m6A), the most abundant methylation on mRNA, plays pivotal roles in regulating mRNA biological functions, resulting in affecting cell functions. ALKBH5, an m6A demethylase, has been found to be an oncogene in several cancer types, including triple negative breast cancer (TNBC). Here we reported a novel ALKBH5 inhibitor. Firstly, we utilized virtual screening to obtain ALKBH5 inhibitor candidates. Biochemical tests demonstrated that compound AG-690/34437009 could inhibit ALKBH5 with an IC50 value of 11.76 µM. After structure optimization, we acquired a more potent ALKBH5-specific inhibitor, compound W23-1006. Its IC50 value against ALKBH5 came down to 3.848 µM, representing a tripled increase in its inhibitory effect on ALKBH5 as well as roughly 30-fold and 8-fold stronger inhibitory activity than that against FTO and ALKBH3, respectively. Furthermore, we proved that W23-1006 formed a covalent bond with ALKBH5 C200. Cellular experiments demonstrated that W23-1006 could efficiently enhance the m6A level on fibronectin 1 (FN1) mRNA, leading to the strong suppression of TNBC cell migration. Moreover, it could significantly suppress TNBC tumor growth and metastasis in vivo. Collectively, our study developed the novel, selective and cell-active ALKBH5 inhibitor, W23-1006, which could be a potential therapeutic option for cancer such as TNBC treatment. Overall design: RNA-seq in Human MDA-MB-231 WT treated with DMSO (n=2) and MDA-MB-231 WT treated with ALKBH5 inhibitor W23-1006 (n=2)