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

N-6-methyladenosine (m(6)A) 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 m(6)A readers, which specifically recognize m(6)A-modified transcripts via the YTH domains. Despite the significant relevance of YTH-m(6)A 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-m(6)A 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 mu M 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 m(6)A YTH domain-containing reader inhibitors and potentiates future inhibitor development for reader functional studies and therapeutic efforts in targeting the epitranscriptome.