Functional imaging of proteases: recent advances in the design and application of substrate-based and activity-based probes

https://doi.org/10.1016/j.cbpa.2011.10.012Get rights and content

Proteases are enzymes that cleave peptide bonds in protein substrates. This process can be important for regulated turnover of a target protein but it can also produce protein fragments that then perform other functions. Because the last few decades of protease research have confirmed that proteolysis is an essential regulatory process in both normal physiology and in multiple disease-associated conditions, there has been an increasing interest in developing methods to image protease activity. Proteases are also considered to be one of the few ‘druggable’ classes of proteins and therefore a large number of small molecule based inhibitors of proteases have been reported. These compounds serve as a starting point for the design of probes that can be used to target active proteases for imaging applications. Currently, several classes of fluorescent probes have been developed to visualize protease activity in live cells and even whole organisms. The two primary classes of protease probes make use of either peptide/protein substrates or covalent inhibitors that produce a fluorescent signal when bound to an active protease target. This review outlines some of the most recent advances in the design of imaging probes for proteases. In particular, it highlights the strengths and weaknesses of both substrate-based and activity-based probes and their applications for imaging cysteine proteases that are important biomarkers for multiple human diseases.

Highlights

► Explanation of the need for tools to image protease activity. ► Examples of recently developed substrate-based imaging probes for cysteine proteases. ► Examples of recently developed activity-based imaging probes for cysteine proteases. ► Discussion of the pros and cons of substrate and activity based probes.

Introduction

The protease family contains approximately 560 members, comprising nearly 2% of the human genome. The primary function of this diverse family of enzymes is to cleave specific peptide bonds of substrates. While this activity is important for normal cellular processes, it is also a critical regulatory mechanism for many pathologies including cancer, arthritis, atherosclerosis, and neurodegenerative disorders such as Alzheimer's and Huntington's Disease, among others. Proteases are classified into seven subfamilies, according to their mechanism of catalysis. Cysteine, serine, and threonine proteases use a nucleophilic amino acid side chain to catalyze the hydrolysis of the peptide substrate (Figure 1). Metallo and aspartic proteases, on the contrary, use active site residues to deprotonate a water molecule for substrate attack.

Because unchecked proteolysis would be highly detrimental to the cell, proteases are subject to tight regulatory mechanisms. They are synthesized as inactive zymogens that can be activated by a number of mechanisms. Once activated, proteases are often negatively regulated by endogenous protein-based inhibitors. Therefore, to obtain a clear understanding of both the normal and pathological function of proteases, direct assessment of the regulation of their enzymatic activities is required. Traditional tools, such as antibodies or proteomic methods survey total protein levels and therefore do not provide information on the dynamic regulation of protease activity. For this reason, new biochemical tools to study protease activity have been in high demand. This review will primarily discuss two major classes of probes, substrate-based and activity-based probes, and how these reagents have been applied to study the biological function of cysteine proteases biochemically and using optical imaging methods. We aim to provide a crucial interpretation of the pros and cons of each type of probe and to provide insight regarding the future of this technology.

Section snippets

Substrate-based probes

Although proteases were originally thought to completely degrade proteins in order to maintain homeostasis of protein levels in the cell, it is now clear that they perform limited proteolysis of substrates at defined cleavage sites. This allows proteases to regulate structure, function, and localization of substrates. Although the ability to cleave a specific site on a protein substrate can be controlled by a number of factors including tertiary structure and localization of target and

Activity-based probes

As an alternative to substrate-based probes, it is also possible to monitor protease activity using activity-based probes (ABPs). ABPs are compounds that have been engineered to covalently modify enzyme targets in an activity dependent manner. ABPs typically contain a reactive functional group (often referred to as a warhead) linked to a targeting sequence and a tag for visualization or affinity purification (Figure 3A). ABPs have been developed for a number of enzyme families, including

Cathepsins

Papain-like cysteine proteases, or cysteine cathepsins, were once thought to degrade proteins nonspecifically in the lysosome. However, their roles in normal cellular processes and disease pathologies have become increasingly apparent. Cysteine cathepsins are implicated in cancer progression, owing to their roles in angiogenesis, apoptosis, and tumor cell invasion [20]. They are also key regulators of inflammation in diseases such as atherosclerosis, rheumatoid arthritis, and asthma. A number

Pros and cons of substrate-based and activity-based probes

One of the potential drawbacks of the quenched activity-based probes is that, owing to their covalent nature, there is a one-to-one reaction between probe and enzyme, preventing signal amplification. Because the substrate-based probes do not render the protease inactive, one molecule of enzyme is theoretically able to cleave many probe molecules, potentially leading to enhanced fluorescent signal. In order to assess the potential contribution of signal amplification, Blum et al. performed a

Conclusions

Cysteine proteases have been targeted by a significant number of probes, resulting in a wealth of tools that can be used to monitor their activity. Here we have described key advances in the development of fluorescent probes for imaging cysteine proteases. We have highlighted some of the key differences between substrate-based and activity-based probes, providing insight into the strengths and weaknesses of each. Both classes of probes have utility across a wide range of applications, allowing

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

The authors thank Edgar Deu for helpful discussions of the manuscript. This work was funded by National Institutes of Health grants R01 AI-078947 and R01 EB005011 (both to M.B.).

References (50)

  • P. Pozarowski et al.

    Interactions of fluorochrome-labeled caspase inhibitors with apoptotic cells: a caution in data interpretation

    Cytometry A

    (2003)
  • K.E. Bullok et al.

    Biochemical and in vivo characterization of a small, membrane-permeant, caspase-activatable far-red fluorescent peptide for imaging apoptosis

    Biochemistry

    (2007)
  • E.M. Barnett et al.

    Single-cell imaging of retinal ganglion cell apoptosis with a cell-penetrating, activatable peptide probe in an in vivo glaucoma model

    Proc Natl Acad Sci USA

    (2009)
  • M. Los et al.

    Fluorogenic substrates as detectors of caspase activity during natural killer cell-induced apoptosis

    Methods Mol Biol

    (2000)
  • E. Gounaris et al.

    Live imaging of cysteine-cathepsin activity reveals dynamics of focal inflammation, angiogenesis, and polyp growth

    PLoS ONE

    (2008)
  • K.M. Kozloff et al.

    Non-invasive optical detection of cathepsin K-mediated fluorescence reveals osteoclast activity in vitro and in vivo

    Bone

    (2009)
  • W. Pham et al.

    Developing a peptide-based near-infrared molecular probe for protease sensing

    Bioconjug Chem

    (2004)
  • Y. Choe et al.

    Substrate profiling of cysteine proteases using a combinatorial peptide library identifies functionally unique specificities

    J Biol Chem

    (2006)
  • D.J. Maly et al.

    Combinatorial strategies for targeting protein families: application to the proteases

    Chembiochem

    (2002)
  • D. Cuerrier et al.

    Development of calpain-specific inactivators by screening of positional scanning epoxide libraries

    J Biol Chem

    (2007)
  • A.W. Patterson et al.

    Substrate activity screening (SAS): a general procedure for the preparation and screening of a fragment-based non-peptidic protease substrate library for inhibitor discovery

    Nat Protoc

    (2007)
  • A. Watzke et al.

    Selective activity-based probes for cysteine cathepsins

    Angew Chem Int Ed Engl

    (2008)
  • D. Caglic et al.

    Functional in vivo imaging of cysteine cathepsin activity in murine model of inflammation

    Bioorg Med Chem

    (2011)
  • B.F. Cravatt et al.

    Activity-based protein profiling: from enzyme chemistry to proteomic chemistry

    Annu Rev Biochem

    (2008)
  • D. Greenbaum et al.

    Epoxide electrophiles as activity-dependent cysteine protease profiling and discovery tools

    Chem Biol

    (2000)
  • Cited by (147)

    View all citing articles on Scopus
    View full text