Structure-based virtual screening, in vitro and in silico analysis identified novel potent m6A demethylase FTO inhibitors as promising neurotherapeutic agents.

Dysregulation of the m6A RNA demethylase FTO has been implicated in neurodegeneration, but brain-penetrant, selective inhibitors remain scarce. Here, we used structure-based virtual screening of a CNS-oriented library to identify novel FTO inhibitors and characterized their permeability, selectivity, and pharmacological profiles. Among them, compound VI showed low-micromolar inhibition of human FTO, selectivity over ALKBH5, high PAMPA-BBB permeability, and oral exposure with measurable plasma levels, moderate brain penetration, and CSF detectability. Molecular dynamics simulations confirmed stable binding of VI within the FTO catalytic pocket, consistent with its enzymatic potency and selectivity. In differentiated SH-SY5Y cells, VI protected against Aβ1-42-induced toxicity while increasing global m6A levels and dampening pro-inflammatory gene expression. In SAMP8 mice, chronic oral treatment with VI (3 mg/kg) ameliorated anxiety-like behavior and rescued hippocampal-dependent spatial and recognition memory, concomitant with increased brain m6A and normalization of synaptic and neuroinflammatory markers. Overall, our findings identify compound VI as a selective, brain-penetrant FTO inhibitor with favorable pharmacokinetics and disease-modifying efficacy in a sporadic Alzheimer's disease model, supporting its further development as a neurotherapeutic candidate.