STRUCTURE-BASED VIRTUAL SCREENING, <EM>IN VITRO</EM> AND <EM>IN SILICO </EM>ANALYSIS IDENTIFIED NOVEL POTENT M6A DEMETHYLASE FTO INHIBITORS AS PROMISING NEUROTHERAPEUTIC AGENTS

Dysregulation of the N6-methyladenosine (m6A) RNA demethylase FTO has been linked to cancer, metabolic disorders, and neurodegenerative diseases like Alzheimer’s disease (AD). Although several FTO inhibitors have been created, key challenges remain, such as poor blood–brain barrier (BBB) penetration, limited selectivity over the related RNA demethylase ALKBH5, and suboptimal pharmacokinetic properties. In this study, we discovered new potent FTO inhibitors through structure-based virtual screening of a CNS-optimized chemical library with 496,644 compounds. Using hierarchical docking at the Fe(II)/2-oxoglutarate catalytic site, combined with MM-GBSA binding energy calculations and CNS multiparameter optimization scoring, we shortlisted 12 virtual hits. An in vitro membrane permeation test confirmed excellent brain permeability for compounds I, V, VI, VII, VIII, and X, which performed better than reference FTO inhibitors like FB23. Enzymatic assays showed IC₅₀ values in the low micromolar range, with maintained selectivity over ALKBH5. Molecular dynamics simulations over 500 ns demonstrated stable interactions between FTO and the inhibitors involving key residues such as His231, Asp233, His307, Arg316, Ser318, and Tyr295. Cytotoxicity tests on SH-SY5Y cells challenged with β-amyloid (Aβ) (1–42) indicated these inhibitors have a favorable therapeutic window and provide significant neuroprotection. In vivo pharmacokinetic studies in mice showed that only compound VI effectively crossed the BBB and reached the brain tissue after oral administration. Overall, the data reveals new FTO inhibitor chemotypes with improved brain permeability and pharmacological profiles, highlighting compound VI as a highly promising candidate for CNS-targeted FTO modulation in neurodegenerative diseases.