Identification of a Selective YTHDF1 Inhibitor Targeting the m⁶A Recognition Domain for Breast Cancer

Abstract As a key N 6 ‐methyladenosine (m 6 A) reader, YTH domain‐containing family protein 1 (YTHDF1) promotes protein synthesis by recognizing m 6 A‐modified mRNA, and its abnormal expression is closely related to breast cancer (BC) progression. To date, the scarce reported YTHDF1 inhibitors suffer from poor selectivity and limited potency, primarily due to the high homology of the YTH domain within the YTHDF family, which poses significant challenges for the discovery of subtype‐selective inhibitors. Here, we report SKLB‐Y13 , the first small‐molecule inhibitor achieving exclusive targeting of the YTHDF1 m 6 A‐binding pocket (IC 50 = 0.76 µM), via structural optimization of a novel 4,5,6,7‐tetrahydrothieno[2,3‐ c ]pyridine scaffold. Uniquely, SKLB‐Y13 interacts with YTHDF1‐specific residues Tyr397 and Trp470, as confirmed by site‐directed mutagenesis, and demonstrates improved selectivity for YTHDF1 over YTH family proteins. Cellular and in vivo studies reveal that SKLB‐Y13 disrupts YTHDF1‐ PRPF6 mRNA interaction in an m 6 A‐dependent manner, thereby impairing the translation of PRPF6 and inhibiting BC proliferation while promoting apoptosis. Chemical proteomics profiling confirms its good target specificity, while pharmacokinetic analysis shows favorable in vivo properties. This study introduces the first selective YTHDF1 inhibitor, serving as a novel chemical probe to elucidate m 6 A‐dependent oncogenesis and a promising starting point for developing precision therapies against YTHDF1‐overexpressing BC.