Quantifying target engagement and cooperativity in proximity-driven pharmacology remains challenging, as in vitro methods fail to capture intracellular influences on complex assembly. To this end, we developed BRETSA, a BRET-based thermal shift assay using denaturation-sensitive probes to detect binding to NanoLuc-tagged proteins in live cells. BRETSA is scalable and broadly applicable across 100+ targets. Beyond binary interactions, BRETSA quantifies intracellular cooperativity and induced-proximity mechanisms, enabling analysis of RIPTAC and molecular glue pharmacology in cells.
