Phenylpyrazoles as Inhibitors of the m6A RNA-Binding Protein YTHDF2.

The N6-methyladenosine (m6A) modification, which is the most common RNA modification in eukaryotes, is regulated by the "writer" methyltransferases, the "reader" m6A binding proteins, and the "eraser" demethylases. m6A plays a multifunctional role in physiological and pathological processes, regulating all aspects of RNA metabolism and function, including RNA splicing, translation, transportation, and degradation. Accumulating evidence suggests that the YT521-B homology domain family 2 (YTHDF2), one of the m6A "readers," is associated with various biological processes in cancers and noncancerous disorders, impacting migration, invasion, metastasis, proliferation, apoptosis, and cell cycle. Here, we describe our work in the identification of a series of functionalized pyrazoles, such as CK-75, as new YTHDF2 inhibitors, which potentially bind to a small hydrophobic pocket on the YTH domain. Cellular evaluations revealed that the small-molecule YTHDF2 inhibitors induced cell cycle arrest, induced apoptosis, and significantly inhibited the cell viability of cancer cells. Furthermore, we evaluated the transcriptome-wide change in the global RNA-binding protein and RNA-binding patterns of CK-75 via an enhanced cross-linking and immunoprecipitation assay. Our work demonstrated the feasibility of targeting the YTH domain of YTHDF2 with small molecules. The phenylpyrazoles studied in this work provided a lead structure for the further development of small molecules targeting YTHDF2 for both biological and therapeutic applications.