Discovery of YTHDF2 Ligands by Fragment-Based Design

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• N6-Adenosine methylation (m6A) is a prevalent mRNA modification.
• YTH domain family proteins (YTHDF1-3) are key cytoplasmic readers of m6A.
• Targeting m6A readers offers therapeutic potential.

Purpose of the Study:

• To identify small molecule inhibitors of YTHDF2.
• To characterize the binding affinity and selectivity of identified compounds.
• To guide the optimization of m6A-binding ligands.

Main Methods:

• Fragment-based drug discovery approach using molecular docking.
• In vitro screening of a large fragment library against YTHDF2.
• Structure-guided optimization and synthesis of analogues.
• Biophysical assays including Fluorescence Polarization (FP) and Homogeneous Time-Resolved Fluorescence (HTRF).
• X-ray crystallography of YTHDF2-ligand complexes.

Main Results:

• Screening identified six initial active fragments from ~500,000 molecules.
• Optimization yielded compounds with low-micromolar affinity for YTHDF2.
• Compound 23 demonstrated high affinity (Kd=1.3 μM) and selectivity for YTHDF2.
• Several compounds showed broad affinity for YTHDF1-3, with some binding YTHDC2.
• Six crystal structures revealed YTHDF2-fragment complexes.

Conclusions:

• Successful identification and optimization of YTHDF2-targeting small molecules.
• Developed ligands with favorable ligand efficiency and low-micromolar affinity.
• Demonstrated selectivity profiles against other YTH reader proteins.
• Provided structural insights into YTHDF2-ligand interactions.