DNA-encoded Library Screening Uncovers Potent DNMT2 Inhibitors Targeting a Cryptic Allosteric Binding Site

Abstract

The human RNA methyltransferase DNMT2 is thought to be involved in various pathophysiological processes, yet, a major challenge in drug targeting DNMT2 is given by the fact that current SAH-derived inhibitors have poor target selectivity and limited cellular permeability. In this study, we have performed a DNA-encoded library (DEL) screening on DNMT2 yielding five non-SAH-like hit structures, three of which feature a peptidomimetic scaffold. All DEL hits could be validated by orthogonal biophysical and biochemical assays for DNMT2 binding. At the same time, the lead structure did not interact with related methyltransferases from the DNMT and NSUN families highlighting an unmatched DNMT2-targeting selectivity profile. Subsequent crystallographic studies revealed the unique ligand binding mode including an active site loop rearrangement and the formation of a cryptic allosteric binding pocket able to modulate the enzymatic activity by non-covalent DNMT2 dimerization. Based on the crystallographic results, we performed a structure-activity relationship study around the inhibitor lead structure resulting in an optimized DNMT2 inhibitor (KD=3.04 {micro}M), which was able to reduce m5C levels in MOLM-13 tRNA. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=64 SRC="FIGDIR/small/632061v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1bf92b3org.highwire.dtl.DTLVardef@38498borg.highwire.dtl.DTLVardef@1645740org.highwire.dtl.DTLVardef@9c61d5_HPS_FORMAT_FIGEXP M_FIG C_FIG