A Phenotypic Approach for the Identification of New Molecules for Targeted Protein Degradation Applications

Area of Science:

• Chemical Biology
• Drug Discovery
• Molecular Biology

Background:

• Targeted protein degradation is a novel strategy for modulating intracellular protein levels.
• Proteolysis targeting chimeras (PROTACs) leverage the ubiquitin-proteasome system for targeted degradation.
• Limited E3 ligase options currently hinder PROTAC-based drug design.

Purpose of the Study:

• To develop a novel phenotypic screening approach for identifying E3 ligase binders.
• To enable the discovery of new E3 ligases for PROTAC applications.
• To expand the toolkit for targeted protein degradation strategies.

Main Methods:

• High-throughput modification of screening compounds with a chloroalkane moiety to generate HaloPROTACs in situ.
• Evaluation of HaloPROTACs for their ability to degrade a GFP-HaloTag fusion protein in a cellular context.
• Screening of approximately 2000 prospective E3 ligase-recruiting molecules.

Main Results:

• Demonstrated successful generation and detection of functional HaloPROTACs in situ.
• Validated the approach using a Von Hippel-Lindau (VHL) binder to degrade a GFP-HaloTag fusion protein.
• Successfully screened a library of 2000 prospective E3 ligase-recruiting molecules.

Conclusions:

• The developed phenotypic screening approach is effective for identifying E3 ligase binders.
• This method expands the utility of PROTACs by enabling the use of a wider range of E3 ligases.
• The findings contribute to advancing targeted protein degradation strategies in chemical biology and drug discovery.