Authors
Hammed A Badmos, Colin Steele, Ross Cagan
Published in
Bio-protocol. Volume 16. Issue 14. Pages e5752. Jul 20, 2026. Epub Jul 20, 2026.
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.
PMID:
42516565
Bibliographic data and abstract were imported from PubMed on 28 Jul 2026.
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