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

ARTICLE

PUBLISHED ONLINE: 5 DECEMBER 2016 | http://dx.doi.org/10.1038/nchembio.2248

Web End =DOI: 10.1038/NCHEMBIO.2248

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

J Matthew Dubach1, Eunha Kim1,3, Katherine Yang1, Michael Cuccarese1, Randy J Giedt1, Labros G Meimetis1, Claudio Vinegoni1 & Ralph Weissleder1,2

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.

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To achieve the desired biological response, drugs must first reach the intended organ or tissue, enter the cell (for intracellular proteins), and engage the target1. The duration, com

pleteness and cellular heterogeneity of drugtarget engagement dictate success. Most pharmacokinetic studies rely on bulk sampling of plasma or tissue providing modest information on drug activity at the cellular target. Furthermore, new mechanistic targets are not easily invalidated in failed treatments if target engagement was not confirmed, which, for example, was the case in 43% of phase II failures in a recent study2. Given the complexity of in vivo drug action3, and recent clinical failures of drugs that were not properly characterized4, methods to determine cellular drug binding could, in theory, reduce the considerable clinical failure rates and associated high costs.

Direct chemical modification of drugs can attach small labels such as biotin or fluorophores, enabling tissue distribution and target engagement measurements by pulldown assays or imaging58.

However, the addition of a label changes the physiochemical properties of a small molecule, and the results may not be directly relevant to the parent drug candidate. Conversely, labeling target proteins with genetic fluorescent labels, such as GFP, may alter protein activity or trafficking9. Out of several creative labelfree approaches to measure target engagement1012, PET imaging is...