Structure-Based Design of a Highly Potent Dual-Competitive FTO Inhibitor for Targeted m ⁶ A Demethylase Inhibition in AML

Fat mass and obesity-associated protein (FTO), an Fe²⁺/2-oxoglutarate (<b>2-OG</b>)-dependent RNA demethylase, removes <i>N</i>⁶-methyladenosine (m⁶A) modification. FTO is overexpressed in AML, promoting pathogenesis through c-Myc upregulation. Using fragment linking of meclofenamic acid (<b>MA</b>) and <b>2-OG</b> mimetics, we developed <b>8a</b>, a substrate/<b>2-OG</b> dual-competitive FTO inhibitor. <b>8a</b> substantially inhibits FTO demethylation, exceeding its constituent fragments' activity, with high selectivity over ALKBH3 and ALKBH5. <b>2-OG</b> competition assay and docking confirm simultaneous occupation of substrate and <b>2-OG</b> pockets, although the cocrystal structure revealed a different binding site. To circumvent the limitation of poor cellular permeability of <b>8a</b>, we synthesized the prodrug ester <b>8a-1</b>, 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. <b>8a</b> represents a highly potent FTO inhibitor with therapeutic potential, providing a framework for future development.