Discovery and Design of YTHDF2-Selective and Pan-YTHDF Inhibitors for m⁶A Binding by the YTH Aromatic Cage

Recognition of N6-methyl-adenosine (m⁶A) in the RNA sequences by the YT521-B homology domain-containing protein family paralogs 1-3 (YTHDF1-3) is a dominant layer of myriad cellular regulation. Dysregulated YTHDF paralog expression and activities have been reported in cancer, including glioblastoma multiforme (GBM) in which YTHDF2 has been identified as a druggable dependency. Guided by structure-based in silico screening, we identified structurally related small-molecule compound families of promising YTHDF inhibitor compounds. Following in vitro screening, two hits, DT11 and DT15, emerged and were further examined. We identified DT11 as a YTHDF2-selective inhibitor of the YTH domain using proximity-based bead assays, microscale thermophoresis, molecular dynamics, and nuclear magnetic resonance spectroscopy. DT11 demonstrated 10-100-fold YTHDF2-selectivity (IC₅₀: YTHDF2 16.6 μM; YTHDF1 185 μM; YTHDF3 1300 μM) in inhibiting m⁶A-RNA binding, whereas the pan-inhibitor DT15 effectively inhibited all the paralogs (IC₅₀: YTHDF2 12.6 μM; YTHDF1 18.8 μM; YTHDF3 31 μM). In LN229 glioblastoma cells, DT11 reduced cell viability and showed relevant additive efficacy with the gold standard Temozolomide. DT15 blocked GBM cell invasion and scratch wound closure. Similar effects were observed after inhibition of the m⁶A writer METTL3 activity by STM2457. No such effects were observed with DT11. A minutely modified DT11 derivative, DT11-1, showed 10-fold increases in YTHDF2-selective m⁶A binding and LN229 cell viability inhibitory potencies. All the compounds produced high GBM preference over primary neurons and HEK293 cells in their effects. Our results further define pharmacological molecular comprehensions for paralog-dependent engagement of m⁶A in RNA by the aromatic cage for the selective modulation of YTHDF functions.