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

Nature Chemical Biology volume 3, pages 668–677 (2007)Cite this article

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.

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Figure 1: Structures and serum sensitivity of the NIRF-ABPs.
Figure 2: Optical imaging of tumors in live mice using nonquenched NIRF-ABPs.
Figure 3: Biochemical characterization of in vivo–labeled proteases.
Figure 4: Direct comparison of the nonquenched and quenched NIRF-ABPs GB123 and GB137.
Figure 5: Imaging of small-molecule inhibitor efficacy.

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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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Authors and Affiliations

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

    Galia Blum & Matthew Bogyo

  2. Baxter Laboratory in Genetic Pharmacology, Stanford University School of Medicine, 300 Pasteur Dr., Stanford, 94305, California, 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, 94305, California, USA

    Matthew Bogyo

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Correspondence to Matthew Bogyo.

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Blum, G., von Degenfeld, G., Merchant, M. et al. Noninvasive optical imaging of cysteine protease activity using fluorescently quenched activity-based probes. Nat Chem Biol 3, 668–677 (2007). https://doi.org/10.1038/nchembio.2007.26

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