Abstract
N(6)-methyladenosine (m(6)A) has emerged as the most abundant mRNA modification that regulates gene expression in many physiological processes. m(6)A modification in RNA controls cellular proliferation and pluripotency and has been implicated in the progression of multiple disease states, including cancer. RNA m(6)A methylation is controlled by a multiprotein "writer" complex including the enzymatic factor methyltransferase-like protein 3 (METTL3) that regulates methylation and two "eraser" proteins, RNA demethylase ALKBH5 (ALKBH5) and fat mass- and obesity-associated protein (FTO), that demethylate m(6)A in transcripts. FTO can also demethylate N(6),2'-O-dimethyladenosine (m(6)A(m)), which is found adjacent to the m(7)G cap structure in mRNA. FTO has recently gained interest as a potential cancer target, and small molecule FTO inhibitors such as meclofenamic acid have been shown to prevent tumor progression in both acute myeloid leukemia and glioblastoma in vivo models. However, current FTO inhibitors are unsuitable for clinical applications due to either poor target selectivity or poor pharmacokinetics. In this work, we describe the structure-based design, synthesis, and biochemical evaluation of a new class of FTO inhibitors. Rational design of 20 small molecules with low micromolar IC(50)'s and specificity toward FTO over ALKBH5 identified two competitive inhibitors FTO-02 and FTO-04. Importantly, FTO-04 prevented neurosphere formation in patient-derived glioblastoma stem cells (GSCs) without inhibiting the growth of healthy neural stem cell-derived neurospheres. Finally, FTO-04 increased m(6)A and m(6)A(m) levels in GSCs consistent with FTO inhibition. These results support FTO-04 as a potential new lead for treatment of glioblastoma.