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Noninvasive optical imaging of cysteine protease activity using fluorescently quenched activity-based probes

Nature Chemical Biology volume 3, pages 668677 (2007) | Download Citation

Abstract

We have generated a series of quenched near-infrared fluorescent activity-based probes (qNIRF-ABPs) that covalently target the papain-family cysteine proteases shown previously to be important in multiple stages of tumorigenesis. These 'smart' probes emit a fluorescent signal only after covalently modifying a specific protease target. After intravenous injection of NIRF-ABPs into mice bearing grafted tumors, noninvasive, whole-body imaging allowed direct monitoring of cathepsin activity. Importantly, the permanent nature of the probes also allowed secondary, ex vivo biochemical profiling to identify specific proteases and to correlate their activity with whole-body images. Finally, we demonstrate that these probes can be used to monitor small-molecule inhibition of protease targets both biochemically and by direct imaging methods. Thus, NIRF-ABPs are (i) potentially valuable new imaging agents for disease diagnosis and (ii) powerful tools for preclinical and clinical testing of small-molecule therapeutic agents in vivo.

  • Compound

    2-((5S,8S)-5-benzyl-8-(2-(2,6-dimethylbenzoyloxy)acetyl)-3,6,14,21-tetraoxo-1-phenyl-2-oxa-4,7,13,20-tetraazatricosan-23-yl)-5,5-difluoro-7-(4-methoxyphenyl)-1,3-dimethyl-5H-dipyrrolo[1,2-c:1',2'-f][1,3,2]diazaborinin-4-ium-5-uide

  • Compound

    2-((5S,8S)-5-benzyl-8-(2-(2-(1-(2-((E)-6-(methyl(phenyl)amino)-3-(methyl(phenyl)iminio)-3H-xanthen-9-yl)phenylsulfonyl)piperidine-4-carboxamido)acetoxy)acetyl)-3,6,14,21-tetraoxo-1-phenyl-2-oxa-4,7,13,20-tetraazatricosan-23-yl)-5,5-difluoro-7-(4-methoxyphenyl)-1,3-dimethyl-5H-dipyrrolo[1,2-c:1',2'-f][1,3,2]diazaborinin-4-ium-5-uide

  • Compound

    2-((1E,3E,5Z)-5-(1-((5S,8S)-5-benzyl-8-(2-(2,6-dimethylbenzoyloxy)acetyl)-3,6,14-trioxo-1-phenyl-2-oxa-4,7,13-triazanonadecan-19-yl)-3,3-dimethyl-5-sulfoindolin-2-ylidene)penta-1,3-dienyl)-1-ethyl-3,3-dimethyl-5-sulfo-3H-indolium

  • Compound

    2-((1E,3E,5Z)-5-(1-((5S,8S)-5-benzyl-8-carbamoyl-3,6,14-trioxo-1-phenyl-2-oxa-4,7,13-triazanonadecan-19-yl)-3,3-dimethyl-5-sulfoindolin-2-ylidene)penta-1,3-dienyl)-1-ethyl-3,3-dimethyl-5-sulfo-3H-indolium

  • Compound

    2-((1E,3E,5Z)-5-(1-((5S,8S)-5-benzyl-8-(2-(2-(1-(2-((E)-6-(indolin-1-yl)-3-(indolinium-1-ylidene)-3H-xanthen-9-yl)phenylsulfonyl)piperidine-4-carboxamido)acetoxy)acetyl)-3,6,14-trioxo-1-phenyl-2-oxa-4,7,13-triazanonadecan-19-yl)-3,3-dimethyl-5-sulfoindolin-2-ylidene)penta-1,3-dienyl)-1-ethyl-3,3-dimethyl-5-sulfo-3H-indolium

  • Compound

    2-((1E,3E,5Z)-5-(1-((5S,8S)-5-benzyl-8-(2-(2-(1-(2-((E)-6-(indolin-1-yl)-3-(indolinium-1-ylidene)-3H-xanthen-9-yl)phenylsulfonyl)piperidine-4-carboxamido)-2-methylpropanoyloxy)acetyl)-3,6,14-trioxo-1-phenyl-2-oxa-4,7,13-triazanonadecan-19-yl)-3,3-dimethyl-5-sulfoindolin-2-ylidene)penta-1,3-dienyl)-1-ethyl-3,3-dimethyl-5-sulfo-3H-indolium

  • Compound

    (S)-3-((S)-2-(benzyloxycarbonylamino)-3-phenylpropanamido)-7-(tert-butoxycarbonylamino)-2-oxoheptyl 4-(6-aminohexylcarbamoyl)-2,6-dimethylbenzoate

  • Compound

    2-((1E,3E,5Z)-5-(1-((5S,8S)-5-benzyl-8-(2-(4-(6-(1-(2-((E)-6-(indolin-1-yl)-3-(indolinium-1-ylidene)-3H-xanthen-9-yl)phenylsulfonyl)piperidine-4-carboxamido)hexylcarbamoyl)-2,6-dimethylbenzoyloxy)acetyl)-3,6,14-trioxo-1-phenyl-2-oxa-4,7,13-triazanonadecan-19-yl)-3,3-dimethyl-5-sulfoindolin-2-ylidene)penta-1,3-dienyl)-1-ethyl-3,3-dimethyl-5-sulfo-3H-indolium

  • Compound

    (S)-7-amino-3-((S)-2-(benzyloxycarbonylamino)-3-phenylpropanamido)-2-oxoheptyl 2,6-dimethylbenzoate

  • Compound

    (2S,3S)-ethyl 3-((S)-1-(4-hydroxyphenethylamino)-4-methyl-1-oxopentan-2-ylcarbamoyl)oxirane-2-carboxylate

  • Compound

    1-((5S,8S)-5-benzyl-8-(2-(2,6-dimethylbenzoyloxy)acetyl)-3,6,14-trioxo-1-phenyl-2-oxa-4,7,13-triazanonadecan-19-yl)-2-((E)-2-((E)-3-((E)-2-(3,3-dimethyl-5-sulfo-1-(4-sulfobutyl)indolin-2-ylidene)ethylidene)-2-(4-sulfophenoxy)cyclohex-1-enyl)vinyl)-3,3-dimethyl-5-sulfo-3H-indolium

  • Compound

    4-methyl-N-((S)-1-oxo-3-phenyl-1-((S,E)-5-phenyl-1-(phenylsulfonyl)pent-1-en-3-ylamino)propan-2-yl)piperazine-1-carboxamide hydrochloride

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References

  1. 1.

    , & Activity-based protein profiling: applications to biomarker discovery, in vivo imaging and drug discovery. Am. J. Pharmacogenomics 4, 371–381 (2004).

  2. 2.

    & Mechanism-based profiling of enzyme families. Chem. Rev. 106, 3279–3301 (2006).

  3. 3.

    , & Tagging and detection strategies for activity-based proteomics. Curr. Opin. Chem. Biol. 11, 20–28 (2006).

  4. 4.

    & Assignment of protein function in the postgenomic era. Nat. Chem. Biol. 1, 130–142 (2005).

  5. 5.

    , & Activity-based proteomics: enzymatic activity profiling in complex proteomes. Amino Acids 30, 333–350 (2006).

  6. 6.

    et al. Chemical approaches for functionally probing the proteome. Mol. Cell. Proteomics 1, 60–68 (2002).

  7. 7.

    , , & Direct visualization of serine hydrolase activities in complex proteomes using fluorescent active site-directed probes. Proteomics 1, 1067–1071 (2001).

  8. 8.

    , , , & Activity-based probes for the proteomic profiling of metalloproteases. Proc. Natl. Acad. Sci. USA 101, 10000–10005 (2004).

  9. 9.

    , , & Proteomic profiling of metalloprotease activities with cocktails of active-site probes. Nat. Chem. Biol. 2, 274–281 (2006).

  10. 10.

    , & Human and parasitic papain-like cysteine proteases: their role in physiology and pathology and recent developments in inhibitor design. Chem. Rev. 102, 4459–4488 (2002).

  11. 11.

    et al. Antiamyloidogenic and neuroprotective functions of cathepsin B: implications for Alzheimer's disease. Neuron 51, 703–714 (2006).

  12. 12.

    & Cysteine cathepsins in human cancer. Biol. Chem. 385, 1017–1027 (2004).

  13. 13.

    & Cysteine cathepsins: multifunctional enzymes in cancer. Nat. Rev. Cancer 6, 764–775 (2006).

  14. 14.

    et al. Prognostic significance of cathepsins B and L in primary human breast cancer. J. Clin. Oncol. 16, 1013–1021 (1998).

  15. 15.

    et al. Prognostic impact of proteolytic factors (urokinase-type plasminogen activator, plasminogen activator inhibitor 1, and cathepsins B, D, and L) in primary breast cancer reflects effects of adjuvant systemic therapy. Clin. Cancer Res. 7, 2757–2764 (2001).

  16. 16.

    , , & Prognostic and predictive value of cathepsins D and L in operable breast cancer patients. Neoplasma 52, 1–9 (2005).

  17. 17.

    et al. Cathepsin B, a prognostic indicator in lymph node-negative breast carcinoma patients: comparison with cathepsin D, cathepsin L, and other clinical indicators. Clin. Cancer Res. 6, 578–584 (2000).

  18. 18.

    et al. Distinct roles for cysteine cathepsin genes in multistage tumorigenesis. Genes Dev. 20, 543–556 (2006).

  19. 19.

    et al. Cathepsin cysteine proteases are effectors of invasive growth and angiogenesis during multistage tumorigenesis. Cancer Cell 5, 443–453 (2004).

  20. 20.

    et al. Cathepsin L is required for endothelial progenitor cell-induced neovascularization. Nat. Med. 11, 206–213 (2005).

  21. 21.

    et al. Tumor cell-derived and macrophage-derived cathepsin B promotes progression and lung metastasis of mammary cancer. Cancer Res. 66, 5242–5250 (2006).

  22. 22.

    , & Current advances in molecular imaging: noninvasive in vivo bioluminescent and fluorescent optical imaging in cancer research. Mol. Imaging 2, 303–312 (2003).

  23. 23.

    & Optical imaging in drug discovery and diagnostic applications. Adv. Drug Deliv. Rev. 57, 1087–1108 (2005).

  24. 24.

    & Molecular optical imaging: applications leading to the development of present day therapeutics. NeuroRx 2, 215–225 (2005).

  25. 25.

    , & Enzyme activity–it's all about image. Trends Cell Biol. 14, 29–35 (2004).

  26. 26.

    et al. Optical imaging of spontaneous breast tumors using protease sensing 'smart' optical probes. Invest. Radiol. 40, 321–327 (2005).

  27. 27.

    et al. Tumor imaging by means of proteolytic activation of cell-penetrating peptides. Proc. Natl. Acad. Sci. USA 101, 17867–17872 (2004).

  28. 28.

    , , & In vivo imaging of proteolytic enzyme activity using a novel molecular reporter. Cancer Res. 60, 4953–4958 (2000).

  29. 29.

    , , & In vivo imaging of tumors with protease-activated near-infrared fluorescent probes. Nat. Biotechnol. 17, 375–378 (1999).

  30. 30.

    et al. Dynamic imaging of protease activity with fluorescently quenched activity-based probes. Nat. Chem. Biol. 1, 203–209 (2005).

  31. 31.

    et al. Cathepsins D, B, and L in malignant human lung tissue. Clin. Cancer Res. 2, 561–568 (1996).

  32. 32.

    et al. Exogenous wt-p53 protein is active in transformed cells but not in their non-transformed counterparts: implications for cancer gene therapy without tumor targeting. J. Gene Med. 2, 11–21 (2000).

  33. 33.

    , , & Breast cancer cell lines grown in vivo: what goes in isn't always the same as what comes out. Cell Cycle 4, 253–255 (2005).

  34. 34.

    , , , & Selective targeting of lysosomal cysteine proteases with radiolabeled electrophilic substrate analogs. Chem. Biol. 7, 27–38 (2000).

  35. 35.

    , , & Vinyl sulfones as mechanism-based cysteine protease inhibitors. J. Med. Chem. 38, 3193–3196 (1995).

  36. 36.

    , , , & Glycosylation of procathepsin L does not account for species molecular-mass differences and is not required for proteolytic activity. Biochem. J. 262, 931–938 (1989).

  37. 37.

    et al. Substrate specificity of prostate-specific antigen (PSA). Chem. Biol. 5, 475–488 (1998).

  38. 38.

    , & Targeting of tumor cells by cell surface urokinase plasminogen activator-dependent anthrax toxin. J. Biol. Chem. 276, 17976–17984 (2001).

  39. 39.

    et al. Activity-based probes that target diverse cysteine protease families. Nat. Chem. Biol. 1, 33–38 (2005).

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Acknowledgements

We thank T. Doyle and S. Keren from the Stanford Small Animal Imaging Facility and T. Troy from Caliper Life Sciences for expert advice and technical assistance. We thank K.B. Sexton and S. Verhelst for valuable discussions and technical assistance. The authors thank C. Gilon for helpful advice on peptide synthesis. We thank P. Jackson (Stanford University) for the mouse fibroblast cells, G.P. Nolan (Stanford University) for the ecotropic ΦNX packaging cell line, S. Gambhir (Stanford University) for the U87MG cells, X. Chen (Stanford University) for the MDA-MB 435 human epithelial adenocarcinoma cells, and B. Cravatt (The Scripps Research Institute) for the MDA-MB 231 MFP human epithelial adenocarcinoma. This work was supported by the US National Institutes of Health National Technology Center for Networks and Pathways (grants U54 RR020843, R01 EB005011 and P01 CA072006), the US Department of Defense Breast Cancer Center of Excellence (grant DAMD-17-02-0693; to M.B.), a Susan G. Komen postdoctoral fellowship (to G.B.) and a grant from the Deutsche Forschungsgemeinschaft (DE 740/1-1; to G.v.D.).

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Affiliations

  1. Department of Pathology, Stanford University School of Medicine, 300 Pasteur Dr., Stanford, California 94305, USA.

    • Galia Blum
    •  & Matthew Bogyo
  2. Baxter Laboratory in Genetic Pharmacology, Stanford University School of Medicine, 300 Pasteur Dr., Stanford, California 94305, USA.

    • Georges von Degenfeld
    • , Milton J Merchant
    •  & Helen M Blau
  3. Department of Microbiology and Immunology, Stanford University School of Medicine, 300 Pasteur Dr., Stanford, California 94305, USA.

    • Matthew Bogyo

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The authors declare no competing financial interests.

Corresponding author

Correspondence to Matthew Bogyo.

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DOI

https://doi.org/10.1038/nchembio.2007.26

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