Real-Time NanoBRET Target Engagement Reveals Permeability-Activity Relationships in BET-Targeting Degraders

Area of Science:

• Biochemistry
• Cell Biology
• Pharmacology

Background:

• Membrane permeability is crucial for proteolysis targeting chimera (PROTAC) efficacy.
• Conventional assays like PAMPA and Caco-2 transwell assays have limitations in measuring cytosolic access.

Purpose of the Study:

• To develop and validate a novel method for directly measuring PROTAC membrane permeability into the cytosol of living cells.
• To overcome the limitations of existing assays in assessing real-time cytosolic entry.

Main Methods:

• Utilized NanoBRET (BRET) live-cell target engagement technology.
• Quantified real-time membrane permeability rates of PROTACs and E3 ligase ligands.
• Compared results with traditional transwell assay data.

Main Results:

• The NanoBRET method accurately quantified permeability rates comparable to transwell systems.
• Identified and quantified permeability rates for PROTACs previously undetectable in transwell assays.
• Revealed differential permeabilities for BET-targeting PROTACs with subtle structural modifications, correlating with degradation efficiency and cytotoxicity.

Conclusions:

• The developed NanoBRET-based method enables direct, quantitative measurement of PROTAC membrane permeability into living cell cytosol.
• This approach overcomes limitations of traditional assays, providing a powerful tool for PROTAC design and optimization.
• Facilitates the study of previously unmeasurable PROTACs, aiding in understanding structure-activity relationships and improving drug development.