Fragment Screening Yields a Small-Molecule Stabilizer of 14-3-3 Dimers That Modulates Client Protein Interactions

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• Protein-protein interaction (PPI) inhibitors are established drug development tools.
• Identifying PPI stabilizers presents significant challenges in therapeutic research.

Purpose of the Study:

• To develop a fragment-based screening approach for identifying PPI stabilizers.
• To utilize the regulatory hub protein 14-3-3 as a platform for this discovery.

Main Methods:

• Employed a homogenous time-resolved FRET assay to monitor 14-3-3/peptide binding stabilization.
• Screened an in-house fragment library against the 14-3-3/estrogen receptor alpha interaction.
• Utilized fragment 2 (VUF15640) as a putative PPI stabilizer.

Main Results:

• Fragment 2 demonstrated cooperative stabilization of 14-3-3 PPIs with Fusicoccin-A.
• Mechanistically, fragment 2 enhances 14-3-3 dimerization, increasing client-protein binding.
• Functionally, fragment 2 boosted 14-3-3 potency, inhibiting nitrate reductase activity in a cell-free system.

Conclusions:

• A general PPI stabilizer targeting 14-3-3 was identified.
• This compound, fragment 2, can serve as a valuable tool for studying 14-3-3 client protein interactions.