Advances in BRET probes for intracellular target engagement studies

Assessing drug-target engagement in living cells is essential for verifying the activity of pharmacological agents. Traditional binding assays often overlook key factors such as permeability, intracellular distribution and complex formation that influence target occupancy in cells. Bioluminescence resonance energy transfer (BRET)-based probes enable direct, quantitative assessment of small-molecule binding to proteins in live, intact cells. BRET provides sensitive detection of target engagement across a wide range of target classes, including less-tractable proteins in membrane compartments. Compared to other existing methods, BRET offers quantification of drug occupancy at steady state and open system regimens. Recent innovations in this platform have expanded its utility beyond occupancy confirmation to include applications in polypharmacology and mechanism-of-action studies. Here, we provide an updated perspective on BRET target engagement assays as versatile tools for chemical biology and early-stage drug discovery.