Coupling cellular drug-target engagement to downstream pharmacology with CeTEAM

Abstract

Cellular target engagement technologies are reforming drug discovery by enabling quantification of intracellular drug binding; however, concomitant assessment of drug-associated phenotypes has proven challenging. We have developed cellular target engagement by accumulation of mutant (CeTEAM) as a platform that can seamlessly evaluate drug-target interactions and phenotypic responses in a single multiparametric experiment. In the presence of binding ligand, accumulation of an initially unstable target protein acts as a biosensor that permits holistic assessment of drug pharmacology under physiological conditions. We demonstrate this proof-of-concept by uncoupling target binding from divergent cellular activities of MTH1 inhibitors, repurposing the R139C variant to dissect complex NUDT15-thiopurine interactions, and profiling the live-cell dynamics of DNA trapping by PARP inhibitors. Further, PARP1-derived drug biosensors facilitated multimodal ex vivo analysis of drug-target engagement and non-invasive tracking of drug binding in live animals. CeTEAM empowers real-time, comprehensive characterization of target engagement by bridging drug binding events and their biological consequences.