Comparative Assessment of Substrates and Activity Based Probes as Tools for Non-Invasive Optical Imaging of Cysteine Protease Activity

Recent advances in the field of non-invasive optical imaging have included the development of contrast agents that report on the activity of enzymatic targets associated with disease pathology. In particular, proteases have proven to be ideal targets for development of optical sensors for cancer. Recently developed contrast agents for protease activity include both small peptides and large polymer-based quenched fluorescent substrates as well as fluorescently labeled activity based probes (ABPs). While substrates produce a fluorescent signal as a result of processing by a protease, ABPs are retained at the site of proteolysis due to formation of a permanent covalent bond with the active site catalytic residue. Both methods have potential advantages and disadvantages yet a careful comparison of substrates and ABPs has not been performed. Here we present the results of a direct comparison of commercially available protease substrates with several recently described fluorescent ABPs in a mouse model of cancer. The results demonstrate that fluorescent ABPs show more rapid and selective uptake into tumors as well as overall brighter signals compared to substrate probes. These data suggest that the lack of signal amplification for an ABP is offset by the increased kinetics of tissue uptake and prolonged retention of the probes once bound to a protease target. Furthermore, fluorescent ABPs can be used as imaging reagents with similar or better results as the commercially available protease substrates.

[1]  G. Blum,et al.  Use of fluorescent imaging to investigate pathological protease activity. , 2008, Current opinion in drug discovery & development.

[2]  V. Turk,et al.  Selective activity-based probes for cysteine cathepsins. , 2008, Angewandte Chemie.

[3]  J. Joyce,et al.  Cysteine cathepsin proteases as pharmacological targets in cancer. , 2008, Trends in pharmacological sciences.

[4]  Georges von Degenfeld,et al.  Noninvasive optical imaging of cysteine protease activity using fluorescently quenched activity-based probes. , 2007, Nature chemical biology.

[5]  M. Bogyo,et al.  Specificity of aza-peptide electrophile activity-based probes of caspases , 2007, Cell Death and Differentiation.

[6]  Dustin J. Maxwell,et al.  Biochemical and in vivo characterization of a small, membrane-permeant, caspase-activatable far-red fluorescent peptide for imaging apoptosis. , 2007, Biochemistry.

[7]  M. Bogyo,et al.  Identification of early intermediates of caspase activation using selective inhibitors and activity-based probes. , 2006, Molecular cell.

[8]  Kinneret Keren,et al.  Dynamic imaging of protease activity with fluorescently quenched activity-based probes , 2005, Nature chemical biology.

[9]  Matthew Bogyo,et al.  Activity-based probes that target diverse cysteine protease families , 2005, Nature chemical biology.

[10]  Roger Y Tsien,et al.  Tumor imaging by means of proteolytic activation of cell-penetrating peptides. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[11]  Shiva Gautam,et al.  Development of a novel fluorogenic proteolytic beacon for in vivo detection and imaging of tumour-associated matrix metalloproteinase-7 activity. , 2004, The Biochemical journal.

[12]  M. Bogyo,et al.  Enzyme activity--it's all about image. , 2004, Trends in cell biology.

[13]  C. López-Otín,et al.  Protease degradomics: A new challenge for proteomics , 2002, Nature Reviews Molecular Cell Biology.

[14]  A. Burlingame,et al.  Chemical Approaches for Functionally Probing the Proteome* , 2002, Molecular & Cellular Proteomics.

[15]  R. Weissleder,et al.  In vivo imaging of tumors with protease-activated near-infrared fluorescent probes , 1999, Nature Biotechnology.