Kinetic Detection of E3:PROTAC:Target Ternary Complexes Using NanoBRET Technology in Live Cells

Area of Science:

• Biochemistry
• Molecular Biology
• Chemical Biology

Background:

• Proteolysis Targeting Chimeras (PROTACs) are heterobifunctional molecules that induce targeted protein degradation.
• PROTACs function by forming a ternary complex between a target protein and an E3 ligase, initiating ubiquitination and proteasomal degradation.
• Successful ternary complex formation is essential for PROTAC efficacy but presents significant design challenges due to nonnative complex formation.

Purpose of the Study:

• To develop and validate novel cellular assays for studying PROTAC-induced ternary complex formation.
• To assess the utility of NanoBRET technology for real-time and endpoint kinetic analysis of ternary complexes.
• To provide insights into how PROTAC chemical modifications impact ternary complex formation and stability in live cells.

Main Methods:

• Utilized NanoBRET technology to develop cellular assays for monitoring ternary complex formation.
• Tested assays with VHL (von Hippel-Lindau disease tumor suppressor) and CRBN (Cereblon) E3 ligase components.
• Performed assays in both endpoint and real-time kinetic formats, assessing compatibility with high-throughput screening.

Main Results:

• Demonstrated the ability of NanoBRET assays to effectively study ternary complex formation in live cells.
• Showcased the application of these assays with commonly used E3 ligases (VHL and CRBN).
• Validated the assays' suitability for high-throughput workflows and kinetic analysis of PROTAC-target-ligase interactions.

Conclusions:

• NanoBRET-based cellular assays are highly enabling for the study and optimization of PROTACs.
• These assays provide valuable insights into the mechanistic steps of targeted protein degradation.
• The developed assays facilitate rapid triaging of early-stage PROTAC compounds by assessing ternary complex formation and stability.