Discovery of YTHDF2 ligands by fragment-based design

N6-Adenosine methylation is the most abundant modification of mRNA. The three members of the YTH domain family proteins (YTHDF1-3) recognize in the cytoplasm the m6A-RNA modification. We carried out a screening campaign by fragment-based high-throughput docking into YTHDF2 that resulted in the identification of six ligands with a hit rate of 13%. The acquisition of 28 analogues of the docking hits provided an additional set of 10 active compounds (IC50 < 100 µM). Further optimization of a ligand-efficient fragment by the synthesis of 32 derivatives culminated in a series of YTHDF2 ligands which show low-micromolar affinity measured by a fluorescence-polarization (FP) assay and a homo-geneous time-resolved fluorescence-based (HTRF) assay. The series is characterized by very favorable ligand efficiency (of about 0.3 – 0.4 kcal/mol per non-hydrogen atom). Compound 23 binds to YTHDF2 according to the FP and HTRF assays with IC50 values of 2 µM and 10 µM, respectively, and it is selective against all the other YTH reader proteins. Sev-eral compounds of the series bind to the three YTHDF proteins with similar low-micromolar affinity, while they are less potent for YTHDC1 and YTHDC2. In contrast, compounds 17 and 30 bind also to YTHDC2, with affinity of 9 µM and 7 µM, respectively. We also disclose six crystal structures of YTHDF2 in the complex with the fragments identified by docking.