Small-Molecule Ebselen Binds to YTHDF Proteins Interfering with the Recognition of N ⁶-Methyladenosine-Modified RNAs

Area of Science:

• Molecular Biology
• Epigenetics
• Drug Discovery

Background:

• YTHDF proteins bind N6-methyladenosine (m6A)-modified mRNAs, regulating gene expression.
• Dysregulation of YTHDF proteins is implicated in various physiological and pathological conditions.
• YTHDF proteins possess a specific pocket that binds m6A within the RRACH sequence on mRNA.

Purpose of the Study:

• To identify small molecules that can competitively bind to the m6A pocket of YTHDF proteins.
• To characterize the interaction of identified ligands with YTHDF proteins.
• To explore the therapeutic potential of targeting the YTHDF m6A-binding domain.

Main Methods:

• High-throughput screening using an m6A-competitive assay.
• Orthogonal biochemical and biophysical assays (X-ray crystallography, mass spectrometry, NMR) to validate ligand binding.
• Cell-based assays to assess the in-cell engagement and function of the identified inhibitor.
• Structure-activity relationship studies by synthesizing ebselen analogs.

Main Results:

• Ebselen was identified as the first-in-class inhibitor of the YTHDF m6A-binding domain.
• Ebselen binds to YTHDF proteins, disrupting their interaction with m6A-modified mRNAs.
• Structural studies revealed ebselen binds near the m6A pocket, with covalent or reversible interaction depending on the environment.
• Ebselen analogs also demonstrated interaction with the YTHDF m6A domain.

Conclusions:

• Targeting the YTH domain of YTHDF proteins with small molecules is feasible.
• Ebselen is a validated chemical probe for studying YTHDF protein function.
• The development of ebselen analogs offers a promising strategy for creating novel disruptors of m6A recognition.