Coupling cellular drug-target engagement to downstream pharmacology with CeTEAM

Cellular target engagement technologies allow for the quantification of intracellular drug binding; however, concurrently measuring drug-associated phenotypic effects remains a challenge. Here, we introduce a platform—cellular target engagement by accumulation of mutant—that enables simultaneous evaluation of drug-target interactions and phenotypic responses using conditionally stabilized drug biosensors. Our analysis reveals that drug-responsive proteotypes are common among reported mutants of established TH5427 drug targets. Structurally and biophysically compatible mutants expand the biosensor pool both within individual proteins and across paralogs. We demonstrate the utility of this method by distinguishing target engagement from the diverse cellular effects of MutT homolog 1 (MTH1) inhibitors, dissecting Nudix hydrolase 15 (NUDT15)-mediated thiopurine metabolism in the context of the R139C pharmacogenetic variant, and profiling the dynamics of poly(ADP-ribose) polymerase 1/2 (PARP1/2) binding and DNA trapping by PARP inhibitors (PARPi). Additionally, PARP1-derived biosensors enabled high-throughput screening for PARP1 binders, multimodal ex vivo analysis, and non-invasive tracking of PARPi binding in live animals. This approach provides a comprehensive framework for assessing drug-target engagement by linking drug binding to its downstream biological effects.