biostudies-literatureDubach JMNIBIB NIH HHSNHLBI NIH HHSNCI NIH HHS168-173https://www.ebi.ac.uk/biostudies/studies/S-EPMC5630128biostudies-literatureUnknown13(2)Quantitation of drug target engagement in single cells has proven to be difficult, often leaving unanswered questions in the drug development process. We found that intracellular target engagement of unlabeled new therapeutics can be quantitated using polarized microscopy combined with competitive binding of matched fluorescent companion imaging probes. We quantitated the dynamics of target engagement of covalent BTK inhibitors, as well as reversible PARP inhibitors, in populations of single cells using a single companion imaging probe for each target. We then determined average in vivo tumor concentrations and found marked population heterogeneity following systemic delivery, revealing single cells with low target occupancy at high average target engagement in vivo.Nature chemical biologyQuantitating drug-target engagement in single cells in vitro and in vivo.PMC5630128R01 CA241179T32 CA079443R01 HL122208P50 CA086355R01 CA164448K99 CA198857R01 EB010011R00 CA198857Cuccarese MGiedt RJYang KKim EWeissleder RVinegoni CDubach JMMeimetis LGfalseQuantitating drug-target engagement in single cells in vitro and in vivo.Quantitation of drug target engagement in single cells has proven to be difficult, often leaving unanswered questions in the drug development process. We found that intracellular target engagement of unlabeled new therapeutics can be quantitated using polarized microscopy combined with competitive binding of matched fluorescent companion imaging probes. We quantitated the dynamics of target engagement of covalent BTK inhibitors, as well as reversible PARP inhibitors, in populations of single cells using a single companion imaging probe for each target. We then determined average in vivo tumor concentrations and found marked population heterogeneity following systemic delivery, revealing single cells with low target occupancy at high average target engagement in vivo.2017-01-01T00:00:00Z2017 Feb2025-04-03T22:41:19.99Z2019-03-27T02:58:21ZS-EPMC56301282791855810.1038/nchembio.2248