Mechanistic and Structural Features of PROTAC Ternary Complexes.

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• Advancements in proteolysis-targeting chimeras (PROTACs) necessitate a deeper understanding of their mechanism of action.
• The ternary complex, comprising PROTAC, target protein, and E3 ubiquitin ligase, is central to PROTAC-mediated protein degradation.

Purpose of the Study:

• To elucidate the theoretical framework of ternary complex formation in PROTACs.
• To review and detail biophysical methods for interrogating ternary complex formation and PROTAC activity.
• To provide a holistic understanding of PROTAC mode of action in vitro and in cellular environments.

Main Methods:

• Theoretical modeling of the three-component binding model, cooperativity, and hook effect.
• Application of biophysical techniques including X-ray crystallography, AlphaLISA, FRET, FP, ITC, and SPR.
• Detailed isothermal titration calorimetry (ITC) methods for assessing target engagement and degradation.

Main Results:

• Comprehensive overview of the theoretical underpinnings of PROTAC ternary complexes.
• Detailed description of various biophysical methods for studying ternary complex formation.
• Methodological guidance for assessing target engagement, ubiquitination, and degradation.

Conclusions:

• Understanding ternary complex dynamics is crucial for optimizing PROTAC-based therapeutics.
• A combination of theoretical and biophysical approaches provides a comprehensive view of PROTAC action.
• Assessing cellular target engagement and degradation is essential for validating PROTAC efficacy.