Application of activity-based probes to the study of enzymes involved in cancer progression

https://doi.org/10.1016/j.gde.2007.12.001Get rights and content

Many tumor cells have elevated levels of hydrolytic and proteolytic enzymes, presumably to aid in key processes such as angiogenesis, cancer cell invasion, and metastasis. Functional roles of enzymes in cancer progression are difficult to study using traditional genomic and proteomic methods because the activities of these enzymes are often regulated by post-translational mechanisms. Thus, methods that allow for the direct monitoring of enzyme activity in a physiologically relevant environment are required to better understand the roles of specific players in the complex process of tumorigenesis. This review highlights advances in the field of activity-based proteomics, which uses small molecules known as activity-based probes (ABPs) that covalently bind to the catalytic site of target enzymes. We discuss the application of ABPs to cancer biology, especially to the discovery of tumor biomarkers, the screening of enzyme inhibitors, and the imaging of enzymes implicated in cancer.

Introduction

During the development of many types of cancer, a complex series of events occurs, including the formation of new tumor blood vessels (angiogenesis), escape of tumor cells from the primary tumor, cell migration and invasion of adjacent tissues and blood vessels, and the establishment of new tumor colonies at distant sites (metastasis) [1]. A crucial step in all of these processes is the degradation and remodeling of the extracellular matrix (ECM). For cancer cells to invade and metastasize to a new organ, the cells must produce hydrolytic enzymes that break down the proteins of the ECM to permit the passage of tumor cells to the blood and lymphatic vessels [2, 3, 4]. Hydrolytic enzymes are also produced when new tumor blood vessels remodel and migrate through the ECM [1, 2, 3]. Extracellular and cell-surface enzymes from the matrix metalloproteinase and the serine hydrolase families, as well as intracellular enzymes from the cysteine cathepsin protease family, are associated with the degradation of the ECM [2, 3, 4, 5, 6]. Many of these hydrolytic and proteolytic enzymes show great promise as tumor biomarkers for the diagnosis and prognosis of human cancers [3].

Numerous proteomic methods have been developed in the past 20 years to aid us in our understanding of enzyme function in biological processes and human disease states, including cancer [7, 8, 9]. However, many of these techniques, such as two-dimensional gel electrophoresis [8] or isotope-coded affinity-tagging [7], only focus on measuring changes in protein abundance. Protein abundance does not necessarily correlate with activity because most enzymes are expressed as inactive zymogens or reside in complex with their endogenous inhibitors. Other methods, such as protein microarrays [9], can provide information about an enzyme's activity state but generally require recombinantly expressed proteins that are monitored in isolation. Therefore, these technologies do not provide us with a functional understanding of native proteins in the physiologically relevant environment of a cell or whole organism.

To address the limitations in classical proteomics methods, a new research field termed activity-based proteomics or chemical proteomics has been established. Activity-based proteomics uses small molecules known as activity-based probes (ABPs) that covalently bind to the catalytic site of specific target enzymes in complex proteomes derived from cells, tissues, and in some cases, whole organisms. ABPs can be designed to react only with the functionally active form of target enzymes because many of the regulatory mechanisms for enzyme activity alter a protein's active site (i.e. endogenous inhibitors sterically block the active site; zymogens have misaligned catalytic residues). Modification of protein targets by these probes thus provides an indirect measure of enzyme activity and also allows for their purification and identification by mass spectrometry (MS). Several recent reviews have outlined the design of ABPs and their biological applications [10, 11••, 12, 13, 14, 15, 16]. In this review, we will focus on the use of small molecule probes in cancer biology. Particular attention will be given to the application of ABPs to the discovery of novel cancer biomarkers, the screening of potential enzyme inhibitors, and the imaging of enzymes involved in tumorigenesis.

Section snippets

The structure of an activity-based probe

A typical ABP consists of three basic elements: (1) a reactive functional group (also termed a warhead) that covalently reacts with the enzyme's active site, (2) a linker region that confers specificity, directs binding to the target, and prevents steric congestion, and (3) a reporter tag used for the identification, purification, or direct visualization of the probe-labeled proteins (Figure 1). The warhead is the most crucial component of an ABP — it must be reactive enough to covalently bind

Profiling and discovery of enzymes involved in cancer

Several ABPs have been developed to target enzymes implicated in cancer progression and tumorigenesis, including metalloproteases, cysteine cathepsins, and esterases [11••, 14, 16]. These ABPs have been used to profile human tumors and tumor cell lines and identify novel enzyme activities for the diagnosis and treatment of cancer (Table 1). In a typical experiment, normal and disease proteomes are labeled with an ABP and the proteins are separated and analyzed by gel electrophoresis (Figure 2).

Enzyme inhibitor discovery and verification

In traditional drug discovery, libraries of small molecules are screened in vitro against purified, often recombinant, protein targets to identify inhibitors. However, in vitro assays provide only limited information regarding the in vivo potency and selectivity of an inhibitor for a related series of enzymes. ABPs have been used to develop small molecule inhibitor screens that resolve many of the shortcomings that plague standard in vitro inhibitor assays [11••, 14, 33•] because these probes

Imaging enzyme activity in tumors

One of the major challenges in cancer diagnosis is the early detection of small primary tumors [45]. Probes that report on enzymatic activity represent valuable tools for early diagnostic imaging strategies [45, 46, 47] because many enzyme activities are upregulated in tumor cells. Current methods for imaging enzymes mainly rely on antibody labeling or on substrates that become fluorescent after enzyme cleavage [13, 47]. Although antibodies are specific for their enzyme targets, they are not

Conclusion and future directions

Over the past several years, the field of activity-based proteomics has produced a wealth of new technologies for the direct biological study of enzymes. ABPs that target numerous diverse enzyme classes have been synthesized, and these probes have been applied to many biologically and pathologically relevant fields. Additionally, a number of new tools, including gel-free screening systems and quenched ABPs, have been developed that allow rapid identification and visualization of probe-labeled

References and recommended reading

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

  • • of special interest

  • •• of outstanding interest

Acknowledgements

We thank the members of the Bogyo Lab for helpful discussions and manuscript comments. This work was supported by funding from a NIH National Technology Center for Networks and Pathways (NTCNP) grant U54 RR020843.

References (49)

  • G. Blum et al.

    Dynamic imaging of protease activity with fluorescently quenched activity-based probes

    Nat Chem Biol

    (2005)
  • G. Blum et al.

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

    Nat Chem Biol

    (2007)
  • M. Lee et al.

    Extracellular proteases as targets for treatment of cancer metastases

    Chem Soc Rev

    (2004)
  • C.A. Borgono et al.

    The emerging roles of human tissue kallikreins in cancer

    Nat Rev Cancer

    (2004)
  • E.I. Deryugina et al.

    Matrix metalloproteinases and tumor metastasis

    Cancer Metastasis Rev

    (2006)
  • L.C. Patten et al.

    Role of proteases in pancreatic carcinoma

    World J Surg

    (2005)
  • M.M. Mohamed et al.

    Cysteine cathepsins: multifunctional enzymes in cancer

    Nat Rev Cancer

    (2006)
  • S.P. Gygi et al.

    Quantitative analysis of complex protein mixtures using isotope-coded affinity tags

    Nat Biotechnol

    (1999)
  • H. Zhu et al.

    Proteomics

    Annu Rev Biochem

    (2003)
  • G. MacBeath

    Protein microarrays and proteomics

    Nat Genet

    (2002)
  • M.J. Evans et al.

    Mechanism-based profiling of enzyme families

    Chem Rev

    (2006)
  • A.B. Berger et al.

    Activity-based protein profiling: applications to biomarker discovery, in vivo imaging and drug discovery

    Am J Pharmacogen

    (2004)
  • A. Baruch et al.

    Enzyme activity — it's all about image

    Trends Cell Biol

    (2004)
  • C.I. Phillips et al.

    Proteomics meets microbiology: technical advances in the global mapping of protein expression and function

    Cell Microbiol

    (2005)
  • Cited by (0)

    View full text