Objective

Methods are needed to study the localization of drugs or drug candidates at cellular and subcellular resolution in relevant clinical specimen.This is necessary to evaluate early drug candidates and to investigate the effects of mutations potentially causing resistance to targeted therapy. We describe a target engagement-mediated amplification (TEMA) technique, wheretarget binding by oligonucleotide-linked drug molecules is visualized and measured via rolling-circle amplification of circularized padlock probes. We established TEMA using kinase inhibitor precursor compounds, and we applied the assay to investigate targets interaction in pathology tissue sections and in blood samples from patients, as well as in commercial arrays of 9000 human proteins. The TEMA method proved useful toreveal the localization of drug binding in fixated cells and tissues, and it allowed us to comprehensively characterize drug-target interaction by clinically approved kinase inhibitors among a large collection of kinases in arrayed proteins. We conclude that TEMA is a promising, physiological relevant technology to evaluate target engagement of candidate drugs in ex-vivo pre-clinical models in drug discovery and as a basis for therapy selection.