Discovery of a new inhibitor for YTH domain-containing m6A RNA readers†

N ⁶-methyladenosine (m⁶A) is an abundant modification in mammalian mRNAs and plays important regulatory roles in gene expression, primarily mediated through specific recognition by “reader” proteins. YTH family proteins are one major family of known m⁶A readers, which specifically recognize m⁶A-modified transcripts via the YTH domains. Despite the significant relevance of YTH-m⁶A recognition in biology and diseases, few small molecule inhibitors are available for specifically perturbing this interaction. Here we report the discovery of a new inhibitor (“N-7”) for YTH-m⁶A RNA recognition, from the screening of a nucleoside analogue library against the YTH domain of the YTHDF1 protein. N-7 is characterized to be a pan-inhibitor in vitro against five YTH domains from human YTHDF1, YTHDF2, YTHDF3, YTHDC1, and YTHDC2 proteins, with IC₅₀ values in the range of 30–48 μM measured using a fluorescence polarization competition assay. We demonstrated that N-7 directly interacts with the YTH domain proteins via a thermal shift assay. N-7 expands the chemical structure landscape of the m⁶A YTH domain-containing reader inhibitors and potentiates future inhibitor development for reader functional studies and therapeutic efforts in targeting the epitranscriptome.