Ligandability at the Membrane Interface of GPx4 Revealed through a Reverse Micelle Fragment Screening Platform

Area of Science:

• Biochemistry
• Medicinal Chemistry
• Drug Discovery

Background:

• Peripheral membrane proteins (PMPs) are challenging drug targets due to their membrane association.
• The protein-membrane interface is critical for PMP function but underexplored in inhibitor design.
• Current inhibitors for targets like GPx4 are often covalent with limited clinical use.

Purpose of the Study:

• To develop and validate a screening methodology for discovering small-molecule ligands at the protein-membrane interface of PMPs.
• To identify noncovalent ligands for glutathione peroxidase 4 (GPx4), a key enzyme in membrane protection and ferroptosis.

Main Methods:

• Utilized membrane-mimicking reverse micelles (mmRM) for a screening approach.
• Employed NMR-based fragment screening to assess ligandability of GPx4 at the protein-membrane interface.
• Tested fragments under both aqueous and membrane-embedded conditions.

Main Results:

• Discovered 9 noncovalent small-molecule fragments binding to membrane-bound GPx4.
• Fragments showed minimal binding in aqueous conditions, highlighting the importance of membrane association.
• Optimized fragments demonstrated improved affinity (from >200 μM to ~15 μM) with hydrophobic modifications.

Conclusions:

• The mmRM fragment screening methodology advances capabilities for targeting membrane-associated proteins.
• Demonstrated ligandability within the GPx4 protein-membrane interface.
• Provides a foundation for developing novel noncovalent GPx4 inhibitors for cancer therapy.