Exploring the Relationship between Cell Permeability, Intracellular Exposure, and Target Engagement for PROTACs

Abstract Proteolysis-targeting chimeras (PROTACs) occupy beyond-rule-of-5 chemical space, where cellular activity depends on molecular recognition, membrane permeation and unbound exposure at the target site. Here, we examined how these properties shape apparent E3 ligase target engagement for 20 PROTACs spanning different E3 ligase ligands, linkers, and target-binding ligands. Intracellular NanoBRET assays showed a wide range of IC50 values for competition with cereblon or VHL tracer binding when referenced to externally applied concentrations. Passive Caco-2 permeability related only weakly to these values. Recalculating IC50 values using measured intracellular unbound exposure narrowed the spread from approximately 4000-fold to 150-fold, in quantitative agreement with IC50 values measured directly in permeabilized cells. Thus, much of the apparent variation in target engagement reflects differential intracellular exposure rather than intrinsic E3 ligase recognition, and incorporating unbound exposure provides a more mechanistic basis for interpreting cellular target engagement data and distinguishing exposure-driven effects from genuine structure−activity relationships.