Quantitating drug-target engagement in single cells in vitro and in vivo

The use of fluorescence-polarized microscopy, combined with competitive binding with matched fluorescence companion imaging probes, enable target engagement measurements of covalent and reversible small molecule inhibitors in a single cell. 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 .