Biology subjects

Barthels, F.

Publications and source records attributed to Barthels, F..

2 recordsLinked to original sources

bioRxiv2025

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

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

molecular biology↗

bioRxiv2024

SARS-CoV-2 methyltransferase nsp10-16 in complex with natural and drug-like purine analogs for guiding structure-based drug discovery

Non-structural protein 10 (nsp10) and non-structural protein 16 (nsp16) are part of the RNA synthesis complex, which is crucial for the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Nsp16 exhibits 2-O-methyltransferase activity during viral messenger RNA capping and is active in a heterodimeric complex with enzymatically inactive nsp10. It has been shown that inactivation of the nsp10-16 protein complex interferes severely with viral replication, making it a highly promising drug target. As information on ligands binding to the nsp10-16 complex (nsp10-16) is still scarce, we screened the active site for potential binding of drug-like and fragment-like compounds using X-ray crystallography. The screened set of 234 compounds consists of derivatives of the natural substrate S-adenosyl methionine (SAM) and adenine derivatives, of which some have been described previously as methyltransferase inhibitors and nsp16 binders. A docking study guided the selection of many of these compounds. Here we report structures of binders to the SAM site of nsp10-16 and for two of them, toyocamycin and sangivamycin, we present additional crystal structures in the presence of a second substrate, Cap0-analog/Cap0-RNA. The identified hits were tested for binding to nsp10-16 in solution and antiviral activity in cell culture. Our data provide important structural information on various molecules that bind to the SAM substrate site which can be used as novel starting points for selective methyltransferase inhibitor designs.

biochemistry↗