ALKBH5 Inhibitor, Compound 20m

Compound 20m potently inhibits RNA N⁶-methyladenosine (m⁶A) demethylase ALKBH5, with a fluorescence polarization assay IC₅₀ of 21 nM. Demonstrating high ALKBH5 selectivity, 20m shows minimal activity against FTO, ALKBH1, and ALKBH2 (IC₅₀ > 25 μM in DSF and FP assays). Cellular assays in HepG2 cells confirm 20m′s cell permeability and on-target demethylase activity, evidenced by ALKBH5 stabilization in cellular thermal shift assay and dose-dependent elevation of intracellular m⁶A levels. Molecular docking suggests inhibition occurs via 2-oxoglutarate binding pocket engagement, involving Mn(II) coordination, hydrogen bonds, and hydrophobic interactions.