High throughput E3 ligase degron binding assays for novel PROTAC ligand discovery

Area of Science:

• Biochemistry
• Chemical Biology
• Drug Discovery

Background:

• Targeted protein degradation using PROTACs and molecular glues offers a novel therapeutic strategy.
• Identifying small molecule ligands for E3 ligases is crucial for developing these modalities.
• Undruggable proteins remain a significant challenge in medicine.

Purpose of the Study:

• To establish an integrated screening platform for rapid identification and optimization of E3 ligase ligands.
• To present a method for evaluating E3 ligase ligands using AlphaScreen technology.
• To adapt AlphaScreen for interrogating E3 ligase-degron interactions.

Main Methods:

• Utilized AlphaScreen, a bead-based proximity technology, for evaluating molecular interactions.
• Developed AlphaScreen-based E3 ligase ligand competition assays.
• Applied the assay to screen for novel small molecule ligands targeting E3 ligases.

Main Results:

• Demonstrated the successful development of AlphaScreen for E3 ligase ligand discovery.
• Showcased the adaptability of AlphaScreen for various affinities and interactions.
• Provided a protocol for interrogating E3 ligase-degron interactions.

Conclusions:

• The developed AlphaScreen method enables rapid identification and optimization of E3 ligase ligands.
• This platform is vital for advancing targeted protein degradation strategies.
• Diversifying E3 ligase ligands and ligase types will maximize the potential of these modalities.