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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.
Abstract:
Organoids as a drug discovery platform represent an emerging field that continues to refine its tools. NanoBRET (bioluminescence resonance energy transfer) has emerged as a proximity-based and highly sensitive assay to measure protein-protein and protein-ligand interactions. NanoBRET assays were developed and are currently used for 2D cell line experiments. Here, we present the development of the first organoid-compatible Nanoluciferase (Nluc) for 3D model systems. We utilise the Nluc for NanoBRET assays to test drug-target engagement. We describe steps for seeding, transfecting, and replating of mouse colorectal cancer organoids. In addition, we provide detailed procedures for the NanoBRET assay. Various lines of evidence have shown significant difference in drug response between 2D human cell lines and 3D model systems, including patient-derived organoids. Our protocol provides a template for measuring this difference in the context of drug-target engagement. Key features • Development of organoid-compatible Nluc vector. • Lentiviral delivery and antibiotic selection of MEK1-Nluc organoids. • NanoBRET assay for the real-time measurement of drug-target engagement in organoids. • Quantitative measurement of competitive drug binding in living 3D organoids. • Impact of drug combinations on drug-target engagement in organoids.
Insights
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.
