Article | Published:

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

Nature Chemical Biology volume 13, pages 168173 (2017) | Download Citation

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.

  • Compound

    (S)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one

  • Compound

    4-(3-(4-(cyclopropanecarbonyl)piperazine-1-carbonyl)-4-fluorobenzyl)phthalazin-1(2H)-one

  • Compound

    (S,E)-N-(4-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-4-oxobut-2-en-1-yl)-3-(5,5-difluoro-7,9-dimethyl-5H-5l4,6l4-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinin-3-yl)propanamide

  • Compound

    4-(3-(4-(3-(5,5-difluoro-1,3-dimethyl-5,10a-dihydro-1H-5l4,6l4-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinin-7-yl)propanoyl)piperazine-1-carbonyl)-4-fluorobenzyl)phthalazin-1(2H)-one

  • Compound

    methyl (3aR,3a1R,4R,5S,5aR,10bR)-4-acetoxy-3a-ethyl-9-((5S,7R,9S)-5-ethyl-5-hydroxy-9-(methoxycarbonyl)-1,4,5,6,7,8,9,10-octahydro-2H-3,7-methano[1]azacycloundecino[5,4-b]indol-9-yl)-5-hydroxy-8-methoxy-6-methyl-3a,3a1,4,5,5a,6,11,12-octahydro-1H-indolizino[8,1-cd]carbazole-5-carboxylate

  • Compound

    methyl (5S,7R,9S)-9-((3aR,3a1R,4R,5S,5aR,10bR)-5-((2-(3-(5,5-difluoro-7,9-dimethyl-5H-5l4,6l4-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinin-3-yl)propanamido)ethyl)carbamoyl)-3a-ethyl-4,5-dihydroxy-8-methoxy-6-methyl-3a,3a1,4,5,5a,6,11,12-octahydro-1H-indolizino[8,1-cd]carbazol-9-yl)-5-ethyl-5-hydroxy-1,4,5,6,7,8,9,10-octahydro-2H-3,7-methano[1]azacycloundecino[5,4-b]indole-9-carboxylate

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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.).

Author information

Author notes

    • Eunha Kim

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

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.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Claudio Vinegoni or Ralph Weissleder.

Supplementary information

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    Supplementary Text and Figures

    Supplementary Results, Supplementary Figures 1–12 and Supplementary Table 1.

  2. 2.

    Supplementary Note

    Synthetic Procedures.

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DOI

https://doi.org/10.1038/nchembio.2248

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