Structure-Based Design of a Highly Potent Dual-Competitive FTO Inhibitor for Targeted m⁶A Demethylase Inhibition in...

Fat mass and obesity-associated protein (FTO), an Fe²⁺/2-oxoglutarate (2-OG)-dependent RNA demethylase, removes N⁶-methyladenosine (m⁶A) modification. FTO is overexpressed in AML, promoting pathogenesis through c-Myc upregulation. Using fragment linking of meclofenamic acid (MA) and 2-OG mimetics, we developed 8a, a substrate/2-OG dual-competitive FTO inhibitor. 8a substantially inhibits FTO demethylation, exceeding its constituent fragments' activity, with high selectivity over ALKBH3 and ALKBH5. 2-OG competition assay and docking confirm simultaneous occupation of substrate and 2-OG pockets, although the cocrystal structure revealed a different binding site. To circumvent the limitation of poor cellular permeability of 8a, we synthesized the prodrug ester 8a-1, which suppressed AML cell viability, reduced m⁶A levels, downregulated c-Myc and CEBPA, and upregulated ASB2 and RARA. It has also shown obvious tumor-inhibiting efficacy at the animal level. 8a represents a highly potent FTO inhibitor with therapeutic potential, providing a framework for future development.