DNA-encoded library screening uncovers potent DNMT2 inhibitors targeting a cryptic allosteric binding site

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• DNMT2 (TRDMT1) is a human RNA methyltransferase linked to various diseases.
• Existing small-molecule inhibitors of DNMT2 suffer from poor selectivity and low cellular uptake.
• Targeting DNMT2 is crucial for developing novel therapeutics.

Purpose of the Study:

• To identify novel, selective small-molecule inhibitors of DNMT2.
• To explore new allosteric binding sites on DNMT2.
• To develop potential therapeutic agents for DNMT2-related diseases.

Main Methods:

• DNA-encoded library (DEL) screening to identify non-SAH-like chemotypes.
• Orthogonal assays for target engagement validation.
• X-ray crystallography to elucidate binding modes and allosteric pockets.
• Lead compound optimization and in vitro/cellular assays.

Main Results:

• Identification of five non-SAH-like chemotypes, including peptidomimetics, that selectively bind DNMT2.
• Discovery of a novel allosteric binding pocket through structural analysis.
• Optimization of a lead compound with a K_D of 3.04 μM.
• Demonstration of reduced m⁵C levels in tRNA and synergistic effects with doxorubicin in cancer cells.
• Achieved unprecedented selectivity over other methyltransferases.

Conclusions:

• Novel non-SAH-like inhibitors provide a promising scaffold for DNMT2-targeting therapeutics.
• The study advances understanding of DNMT2 allosteric modulation and structural plasticity.
• These findings open new possibilities for treating diseases associated with aberrant DNMT2 function.