Quantifying the thermodynamics of target engagement remains a major challenge in proximity-driven pharmacology. Existing in vitro methods define ligand affinity and cooperativity but fail to capture how the intracellular environment governs complex assembly. We developed BRETSA (BRET Shift Assay), a biophysical platform for measuring drug target engagement (TE) in intact cells via protein denaturation. Cell-permeable, denaturation-sensitive probes generate energy transfer with a luciferase-tagged target upon thermal challenge; ligand binding is detected as dose-dependent protection from the probe, reflected in a shifted thermal stability profile. BRETSA is broadly applicable across 100+ targets — including transcription factors and intrinsically disordered proteins — and is highly scalable for isothermal, hit-finding workflows. Applied to E3 ligases, isothermal BRETSA revealed potent engagement at DCAF1, expanding accessible chemical space.

Beyond binary interactions, BRETSA resolves cooperative binding and induced-proximity mechanisms directly in live cells. By quantifying intracellular cooperativity (α values) at defined protein-protein interfaces, BRETSA enables mechanistic analysis of molecular glues and tricomplex inhibitors — including those targeting GTP-bound KRAS. This platform offers a uniquely sensitive and scalable approach for dissecting ternary complex pharmacology in the native cellular context.