Year: 2026
Type: article
Abstract: Introduction and aim.Drug discovery increasingly relies on targeted protein degradation to tackle previously "undruggable" proteins.Proteolysis-targeting chimeras (PROTACs) are leading this field, but progress is often slowed by limitations in how protein degradation is measured.Western blotting, the current standard, is labour-intensive, variable, and poorly suited for high-throughput screening.The NanoLuc HiBiT system offers a faster, more sensitive, and reproducible alternative for real-time protein quantification in live cells.The aim was to establish NanoLuc HiBiT as a superior platform for evaluating PROTAC-mediated protein degradation, demonstrated through the synthesis and testing of trivalent PROTACs targeting BRD4.Material and methods.The trivalent PROTAC 1,2,5T-EG2-MeVHL and its positional isomers were synthesised following published protocols.BRD4 degradation was quantified in live cells using HiBiT, with MZ1 as a benchmark.Lumines-cence was measured across concentrations and time points, and results were compared with published Western blot data to assess sensitivity, reproducibility, and assay fidelity.Results.HiBiT revealed clear, dose-dependent BRD4 degradation.Both 1,2,5T-EG2-MeVHL and MZ1 showed strong activity, while 1,2,6T was the most potent isomer, highlighting linker-position effects.HiBiT outperformed Western blotting, providing faster, higher-resolution, and more reproducible data.Minor differences were attributed to cell line variability.Conclusion.NanoLuc HiBiT is validated as a next-generation assay for targeted protein degradation, offering a scalable, robust, and high-throughput platform to accelerate degrader research, streamline PROTAC development, and advance drug discovery.
Language: English
Topic: Health and Medical Education
Subfield: General Health Professions
Field: Health Professions
Domain: Health Sciences
Open Access status: diamond
OpenAlex ID: W7218097501