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Protocol for Measuring Drug-Target Engagement in Mouse Colorectal Cancer Organoids Using NanoBRET Assay
Hammed A Badmos1, Colin Steele1, Ross Cagan1
1School of Cancer Sciences, Wolfson Wohl Cancer Research Centre, University of Glasgow, Glasgow, UK.
Bio-Protocol
|July 28, 2026
Summary
Researchers developed a novel organoid-compatible Nanoluciferase (Nluc) vector for advanced drug discovery. This tool enables real-time measurement of drug-target engagement in 3D organoid models, improving drug development accuracy.
Area of Science:
- Biotechnology
- Drug Discovery
- Molecular Biology
Background:
- Organoids are emerging as powerful 3D models for drug discovery, offering more physiological relevance than traditional 2D cell lines.
- NanoBRET (bioluminescence resonance energy transfer) is a sensitive assay for measuring molecular interactions, but its application in 3D organoid systems is limited.
- Understanding drug-target engagement in 3D models is crucial due to observed differences in drug response compared to 2D systems.
Purpose of the Study:
- To develop an organoid-compatible Nanoluciferase (Nluc) vector for NanoBRET assays in 3D model systems.
- To establish a protocol for utilizing NanoBRET to measure drug-target engagement in mouse colorectal cancer organoids.
- To provide a method for quantifying drug binding and the impact of drug combinations in living 3D organoids.
Main Methods:
- Development and characterization of an organoid-compatible Nluc vector.
- Lentiviral delivery of the Nluc vector and antibiotic selection for generating MEK1-Nluc engineered organoids.
- Detailed protocol for performing NanoBRET assays in 3D organoid cultures, including seeding, transfection, and replating.
Main Results:
- Successful development of the first organoid-compatible Nluc vector for 3D NanoBRET assays.
- Demonstration of real-time measurement of drug-target engagement in living 3D organoids.
- Quantitative assessment of competitive drug binding and the effects of drug combinations on target engagement within organoids.
Conclusions:
- The developed organoid-compatible Nluc vector and NanoBRET assay protocol enable robust measurement of drug-target engagement in 3D organoid models.
- This platform facilitates the study of drug response differences between 2D and 3D systems at the molecular level.
- The protocol serves as a template for advancing drug discovery and development using patient-derived organoids and other 3D models.
