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