Assays for Measuring the Cell Permeability of Proteolysis-Targeting Chimeras (PROTACs): Performance, Correlations,
Christina Brenner1,2, Yunhai Cui3, Denis Schmidt4
1Drug Discovery Sciences, Boehringer Ingelheim RCV GmbH & Co KG, Dr. Boehringer Gasse 5-11, Vienna A-1121, Austria.
Molecular Pharmaceutics
|May 27, 2026
Summary
This study evaluates Proteolysis-Targeting Chimeras (PROTACs) cell permeability assays, finding strong correlations between NanoBRET and Caco-2 assays. Guidelines are provided for optimizing PROTACs with beyond-Rule-of-Five properties.
Area of Science:
- Biochemistry
- Medicinal Chemistry
- Pharmacology
Background:
- Proteolysis-Targeting Chimeras (PROTACs) are often beyond-Rule-of-Five, posing challenges for cell membrane permeability.
- Optimizing PROTAC cell permeability is crucial for therapeutic development.
Purpose of the Study:
- To systematically investigate the performance and assay correlations of PROTAC cell permeability assays.
- To define the chemical space where these assays yield reliable results for PROTAC optimization.
Main Methods:
- Utilized a comprehensive dataset of 3271 PROTACs.
- Employed NanoBRET E3 ligase target engagement, Caco-2, and P-glycoprotein (P-gp) efflux assays.
- Analyzed correlations between assay readouts and physicochemical properties (Rule-of-Five).
Main Results:
- Strong correlation observed between NanoBRET E3 ligase target engagement and Caco-2 assay readouts.
- P-gp efflux liability showed limited translation to Caco-2 efflux ratio.
- Physicochemical properties influence assay applicability.
Conclusions:
- Developed data-driven guidelines for selecting and using cell permeability assays in PROTAC optimization.
- Findings offer insights into optimizing beyond-Rule-of-Five drug modalities.
- Highlights the utility of integrated assay approaches for PROTAC development.
