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

Cancer Biology Program, Stanford University School of Medicine, 300 Pasteur Dr., Stanford, CA 94305-5324, USA.
Current opinion in chemical biology (Impact Factor: 6.81). 11/2011; 15(6):798-805. DOI: 10.1016/j.cbpa.2011.10.012
Source: PubMed


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.

Download full-text


Available from: Laura E Edgington-Mitchell, Mar 02, 2014
  • Source
    • "Under acidic conditions cathepsins are far more efficient proteases than at neutral pH, partially due to the instability of their substrates, and partially due to their higher stability [13], thereby suggesting that extracellular cathepsins may even have a leading role in the degradation of ECM [83]. A further confirmation of such cathepsin activity was obtained by in vivo imaging approaches using activitybased probes against different cathepsins [84] [85] [86] "
    [Show abstract] [Hide abstract]
    ABSTRACT: Cysteine cathepsins are normally found in the lysosomes where they are involved in intracellular protein turnover. Their ability to degrade the components of the extracellular matrix in vitro was first reported more than 25 years ago. However, cathepsins were for a long time not considered to be among the major players in ECM degradation in vivo. During the last decade it has, however, become evident that abundant secretion of cysteine cathepsins into extracellular milieu is accompanying numerous physiological and disease conditions, enabling the cathepsins to degrade extracellular proteins. In this review we will focus on cysteine cathepsins and their extracellular functions linked with ECM degradation, including regulation of their activity, which is often enhanced by acidification of the extracellular microenvironment, such as found in the bone resorption lacunae or tumor microenvironment. We will further discuss the ECM substrates of cathepsins with a focus on collagen and elastin, including the importance of that for pathologies. Finally, we will overview the current status of cathepsin inhibitors in clinical development for treatment of ECM-linked diseases, in particular osteoporosis. Due to their major role in ECM remodelling cysteine cathepsins have emerged as an important group of therapeutic targets for a number of ECM-related diseases, including, osteoporosis, cancer and cardiovascular diseases. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties..
    Biochimica et Biophysica Acta 03/2014; 1840(8). DOI:10.1016/j.bbagen.2014.03.017 · 4.66 Impact Factor
  • Source
    • "In this study, it is evident that targeting of NE on NETs could minimize adverse effects of unregulated proteolysis associated with tissue damage and inflammation. The selective cleavage sequences of the neutrophil serine proteases identified in this study will be valuable for designing substrates, inhibitors, activity based imaging agents [36] and protease-activated prodrugs [37]. In addition, the substrate signature of NETs-associated protease activity can be monitored as a biomarker for inflammatory diseases driven by extracellular neutrophil proteases. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Neutrophil extracellular traps (NETs) consist of antimicrobial molecules embedded in a web of extracellular DNA. Formation of NETs is considered to be a defense mechanism utilized by neutrophils to ensnare and kill invading pathogens, and has been recently termed NETosis. Neutrophils can be stimulated to undergo NETosis ex vivo, and are predicted to contain high levels of serine proteases, such as neutrophil elastase (NE), cathepsin G (CG) and proteinase 3 (PR3). Serine proteases are important effectors of neutrophil-mediated immunity, which function directly by degrading pathogenic virulent factors and indirectly via proteolytic activation or deactivation of cytokines, chemokines and receptors. In this study, we utilized a diverse and unbiased peptide library to detect and profile protease activity associated with NETs induced by phorbol-12-myristate-13-acetate (PMA). We obtained a "proteolytic signature" from NETs derived from healthy donor neutrophils and used proteomics to assist in the identification of the source of this proteolytic activity. In addition, we profiled each neutrophil serine protease and included the newly identified enzyme, neutrophil serine protease 4 (NSP4). Each enzyme had overlapping yet distinct endopeptidase activities and often cleaved at unique sites within the same peptide substrate. The dominant proteolytic activity in NETs was attributed to NE; however, cleavage sites corresponding to CG and PR3 activity were evident. When NE was immunodepleted, the remaining activity was attributed to CG and to a lesser extent PR3 and NSP4. Our results suggest that blocking NE activity would abrogate the major protease activity associated with NETs. In addition, the newly identified substrate specificity signatures will guide the design of more specific probes and inhibitors that target NET-associated proteases.
    PLoS ONE 09/2013; 8(9):e75141. DOI:10.1371/journal.pone.0075141 · 3.23 Impact Factor
  • Source
    • "One class of tools that has been developed to meet this need is activity-based probes (ABPs). ABPs are small molecules that bind specifically and irreversibly to active proteases (Edgington et al., 2011; Deu et al., 2012; Serim et al., 2012). Fluorescent ABPs contain a dye that allows for detection and are thus amenable to a wide variety of applications . "
    [Show abstract] [Hide abstract]
    ABSTRACT: Activity-based probes (ABPs) are reactive small molecules that covalently bind to active enzymes. When tagged with a fluorophore, ABPs serve as powerful tools to investigate enzymatic activity across a wide variety of applications. In this article, detailed protocols are provided for using fluorescent ABPs to biochemically characterize the activity of proteases in vitro. Furthermore, descriptions are provided of how these probes can be applied to image protease activity in live animals and tissues along with subsequent analysis by histology, flow cytometry, and SDS-PAGE. Curr. Protoc. Chem. Biol. 5:25-44 © 2013 by John Wiley & Sons, Inc.
    03/2013; 5(1):25-44. DOI:10.1002/9780470559277.ch120235
Show more