A sensitive Interactome-based approach to characterize drug activity in living human cells.

Abnormal protein-protein interactions (PPIs) involving signaling proteins contribute to numerous human diseases. Targeting such aberrant PPIs with selective inhibitory molecules is a promising therapeutic strategy, yet assessing how many PPIs are affected by a candidate drug, and whether inhibition occurs before or after complex assembly, remains technically challenging. Here, we present a live-cell methodology that captures drug-sensitive PPIs by integrating drug-induced cellular responses with interaction mapping. As a proof of concept, we identify ERK1 PPIs that are selectively altered by a novel chemical molecule exhibiting anti-tumor efficacy. We further apply complementary real-time imaging tools to visualize the dissociation of pre-assembled ERK1/cofactor complexes in living cells. Our approach provides a generalizable framework for decoding drug activity on PPIs for virtually any protein target, enabling more precise and physiologically relevant evaluation of therapeutic mechanisms.