Structure-Based Design of Potent Pan-YTHDF Ligands

Abstract The reader proteins YTHDF1-3 recognize m6A-RNA in the cytosol. Here, we present a structure-based medicinal chemistry campaign that culminated in the pan-YTHDF inhibitor 34 (Kd = 0.14 μM for YTHDF2 by calorimetry), which is selective against YTHDC1 and YTHDC2. The high-resolution crystal structures of the complex of YTHDF2 with six members of the series validate the structure-based design. Using the structural information, we designed compound 31 (IC50 = 0.22 μM for YTHDF2) and a negative control, i.e., its methylated analogue 31b. The cellular thermal shift assay provides evidence of YTHDF2 target engagement in cells for inhibitors 34 and 31, while the negative control 31b is inactive. Compounds 34 and 31 inhibit the proliferation of the AML cell lines MOLM-13 and THP-1. The inhibition of the YTHDF readers by 31 increases the level and stability of the transcript of TNFRSF1B. This renders THP-1 cells more sensitive to TNF-α-induced toxicity.