Activity Based Probes for Proteases: Applications to Biomarker Discovery,Molecular Imaging and Drug Screening

Stanford University, Palo Alto, California, United States
Current pharmaceutical design (Impact Factor: 3.45). 02/2007; 13(3):253-61. DOI: 10.2174/138161207779313623
Source: PubMed


Recent advances in global genomic and proteomic methods have lead to a greater understanding of how genes and proteins function in complex networks within a cell. One of the major limitations in these methodologies is their inability to provide information on the dynamic, post-translational regulation of enzymatic proteins. In particular proteases are often synthesized as inactive zymogens that need to be activated in order to carry out specific biological processes. Thus, methods that allow direct monitoring of protease activity in the context of a living cell or whole animal will be required to begin to understand the systems-wide functional roles of proteases. In this review, we discuss the development and applications of activity based probes (ABPs) to study proteases and their role in pathological processes. Specifically we focus on application of this technique for biomarker discovery, in vivo imaging and drug screening.

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Available from: Matthew Bogyo
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    • "Proteasome inhibitors, for example, have been used to treat hematological malignancies [1], [2] and serum levels of the protease PSA (prostate specific antigen) have been used as a biomarker for monitoring prostate cancer in various contexts [3]. The invention of activity-based probes (ABP) allows the assessment of protease activity within living cells or in whole organisms [4]. In spite of the success of some drugs and probes, however, targeting proteolytic activity for development of drug and biomarkers has not always been very satisfying. "
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    ABSTRACT: The type 2 transmembrane serine protease matriptase is broadly expressed in human carcinomas and hematological cancers. The proteolytic activity of matriptase is a potential target of drugs and imaging probes. We assessed the fate of active matriptase following the induction of matriptase zymogen activation. Exposing eight human carcinoma cells to pH 6.0 buffer induced robust matriptase zymogen activation followed by rapid inhibition of the nascent active matriptase by hepatocyte growth factor activator inhibitor (HAI)-1. Consequently, no enzymatically active matriptase was detected in these cells. Some active matriptase is, however, rapidly shed to the extracellular milieu by these carcinoma cells. The lack of cell-associated active matriptase and the shedding of active matriptase were also observed in two hematological cancer lines. Matriptase shedding is correlated closely with the induction of matriptase activation, suggesting that matriptase activation and shedding are kinetically coupled. The coupling allows a proportion of active matriptase to survive HAI-1 inhibition by rapid shedding from cell surface. Our study suggests that cellular free, active matriptase is scarce and might not be an effective target for in vivo imaging and drug development.
    Full-text · Article · Mar 2014 · PLoS ONE
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    • "Protease probes based on inhibitors that bind covalently to the active site of a protease are more effective in localizing protease activity. On the other hand, they cannot amplify the signal and therefore are less sensitive in detecting proteases that are not highly expressed or reductions in protease activity (Fonovic and Bogyo, 2007). Whether protease probes are based on a substrate or an inhibitor, proteases are ideal targets for selective, activatable contrast agents. "
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    ABSTRACT: Proteases play causal roles in many aspects of the aggressive phenotype of tumors, yet many of the implicated proteases originate from tumor-associated cells or from responses of tumor cells to interactions with other cells. Therefore, to obtain a comprehensive view of tumor proteases, we need to be able to assess proteolysis in tumors that are interacting with their microenvironment. As this is difficult to do in vivo, we have developed functional live-cell optical imaging assays and 3D and 4D (i.e., 3D over time) coculture models. We present here a description of the probes used to measure proteolysis and protease activities, the methods used for imaging and analysis of proteolysis and the 3D and 4D models used in our laboratory. Of course, all assays have limitations; however, we suggest that the techniques discussed here will, with attention to their limitations, be useful as a screen for drugs to target the invasive phenotype of tumors.
    Full-text · Article · Jan 2012 · Methods in enzymology
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    • "Numerous enzymes implicated in diseases have been highlighted to allow the development of inhibitors as potential therapeutics. Existing ABPs and their biological applications were extensively described in several recent reviews (Schmidinger, Hermetter, & Birner-Gruenberger, 2006; Barglow & Cravatt, 2007; Fonovic & Bogyo, 2007; Sadaghiani, Verhelst, & Bogyo, 2007a; Paulick & Bogyo, 2008). Cysteine proteases are enzymes that degrade polypeptides, and share a common catalytic domain that involves a cysteine. "
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    ABSTRACT: Amino acid-tagging strategies are widespread in proteomics. Because of the central role of mass spectrometry (MS) as a detection technique in protein sciences, the term "mass tagging" was coined to describe the attachment of a label, which serves MS analysis and/or adds analytical value to the measurements. These so-called mass tags can be used for separation, enrichment, detection, and quantitation of peptides and proteins. In this context, cysteine is a frequent target for modifications because the thiol function can react specifically by nucleophilic substitution or addition. Furthermore, cysteines present natural modifications of biological importance and a low occurrence in the proteome that justify the development of strategies to specifically target them in peptides or proteins. In the present review, the mass-tagging methods directed to cysteine residues are comprehensively discussed, and the advantages and drawbacks of these strategies are addressed. Some concrete applications are given to underline the relevance of cysteine-tagging techniques for MS-based proteomics.
    Full-text · Article · May 2011 · Mass Spectrometry Reviews
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