BRETTSA: A BRET-based assay for ultra-sensitive measurement of target engagement through protein denaturation in live cells

Understanding ligand-target interactions within the native cellular environment is a persistent challenge in early drug discovery. Conventional thermal shift assays (TSAs) enable evaluation of target engagement in cells with minimal prior knowledge of the target, but their reliance on protein aggregation limits both sensitivity and scalability for high-throughput screening (HTS). We introduce a novel bioluminescence resonance energy transfer-based thermal shift assay (BRETTSA) that detects ligand-protein interactions in intact cells by monitoring protein denaturation events. In this approach, cell-permeable, denaturation-sensitive dyes report on the unfolded state of a NanoLuc-tagged target protein following a controlled thermal challenge. Ligand binding is quantified as a shift in the protein's thermal stability profile, observed as a dose-dependent decrease in BRET signal within live cells. BRETTSA is broadly applicable and has been successfully deployed to assess ligand binding for more than 100 targets spanning 20 protein families and six subcellular compartments, including transcription factors and integral membrane proteins. The method exhibits a wide dynamic range, enabling detection of ligand interactions across at least five orders of magnitude-surpassing the performance of aggregation-based TSAs. Beyond classical small-molecule binding, BRETTSA can also characterize molecular glues, bifunctional degraders, and cooperative ternary complexes. Together, these results establish BRETTSA as a robust, sensitive, and scalable platform for quantifying ligand engagement directly in living cells, offering a powerful tool for hit discovery and target validation in drug development.