Ternary complex dissociation kinetics contribute to mutant-selective EGFR degradation

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• Chimeric heterobifunctional degraders are a novel drug discovery approach.
• Understanding protein degradation mechanisms is crucial for degrader development.
• Current understanding of degrader criteria is limited, with target engagement not always correlating with degradation.

Purpose of the Study:

• To biochemically characterize an epidermal growth factor receptor (EGFR) degrader.
• To investigate the mechanistic basis for mutant-selective EGFR degradation.
• To elucidate the relationship between ternary complex dynamics and protein degradation efficacy.

Main Methods:

• Biochemical characterization of an EGFR degrader.
• Ternary complex half-life determination.
• Ubiquitination assays with purified components.
• Cell-based degradation assays.
• Cryoelectron microscopy (cryo-EM) and hydrogen-deuterium exchange mass spectroscopy (HDX-MS).

Main Results:

• The EGFR degrader potently binds both wild-type and mutant EGFR.
• Degradation was observed specifically for mutant EGFR variants.
• Ternary complex half-life correlated with processive ubiquitination and mutant-selective degradation.
• Cryo-EM and HDX-MS data showed degradation is achievable without stable compound-induced protein-protein interactions.

Conclusions:

• Target conformation is a critical factor in degrader development.
• Heterobifunctional ligand binding kinetics influence target degradation robustness.
• Ternary complex stability, rather than just binding affinity, dictates degradation efficiency.
• This work provides insights into achieving selective protein degradation for therapeutic applications.