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

Nature Chemical Biology volume 13, pages 168–173 (2017)Cite this article

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Abstract

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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Figure 1: Measuring single-cell drug binding through anisotropy competition imaging.
Figure 2: Ibrutinib target engagement.
Figure 3: Intracelullar PARP inhibitor target engagement.
Figure 4: In vivo olaparib target engagement.

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Acknowledgements

We thank T. Mitchison for his thoughts on experimental approaches and comments on the manuscript. This work was supported by US National Institutes of Health grants T32CA079443 (J.M.D., M.C. and R.W.), K99CA198857 (J.M.D.), R01CA164448, P50CA086355 and R01HL122208 (R.W.), and Department of Defense grant BCRP #BC134081 (R.J.G.).

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  1. Eunha Kim

    Present address: Present address: Department of Molecular Science and Technology, Ajou University, Suwon, Korea.,

Authors and Affiliations

  1. Center for Systems Biology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA

    J Matthew Dubach, Eunha Kim, Katherine Yang, Michael Cuccarese, Randy J Giedt, Labros G Meimetis, Claudio Vinegoni & Ralph Weissleder

  2. Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA

    Ralph Weissleder

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Contributions

J.M.D., C.V. and R.W. designed the experiments. E.K. and L.G.M. synthesized CIPs. E.K. performed in-gel experiments. J.M.D., K.Y. and R.J.G. performed cell experiments. J.M.D. performed in vivo experiments. J.M.D. and M.C. analyzed data. J.M.D. and C.V. performed imaging experiments. J.M.D. and R.W. wrote the paper, and all of the authors reviewed and approved the final manuscript.

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Correspondence to Claudio Vinegoni or Ralph Weissleder.

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The authors declare no competing financial interests.

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Supplementary Results, Supplementary Figures 1–12 and Supplementary Table 1. (PDF 15117 kb)

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Synthetic Procedures. (PDF 321 kb)

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Dubach, J., Kim, E., Yang, K. et al. Quantitating drug-target engagement in single cells in vitro and in vivo. Nat Chem Biol 13, 168–173 (2017). https://doi.org/10.1038/nchembio.2248

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