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

Abstract

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.

Figure 1

General and specific structures of activity-based probes (ABPs) (A) General structure of an ABP. (B) DCG-04 is an ABP that targets cysteine proteases and contains a biotin tag (red), a dipeptide-containing linker (blue), and an epoxide as a warhead (green). FP-rhodamine is an ABP that targets the serine hydrolase superfamily of enzymes and contains a rhodamine fluorophore (red), a poly(ethylene glycol) linker (blue), and a fluorophosphonate as a warhead (green).

Figure 2

Tumor biomarker discovery using ABPs. Enzymes from cancer cells and normal cells are reacted with a biotin-containing ABP and are then separated and analyzed by gel electrophoresis. Probe-labeled enzymes are visualized, and enzymes with altered activities in normal and cancerous cells are identified. These potential tumor biomarkers can then be identified by MS analysis.

Figure 3

Enzyme inhibitor discovery using an ABP-based assay. Cell lysates or whole cells are treated with a range of concentrations of an inhibitor. These samples are then reacted with an ABP and subjected to gel electrophoresis to separate active enzymes. A decrease in residual activity corresponds to more potent inhibition by the inhibitor. Additionally, the selectivity of the inhibitor for one or multiple enzymes can be determined using this assay.

Figure 4

Quenched ABPs for the non-invasive imaging of tumors in vivo. (A) Covalent labeling of a cysteine protease target by a quenched ABP (GB137). Activity-based labeling of the target enzyme results in the loss of the quenching group and subsequent generation of a fluorescently-labeled enzyme. (B) Optical imaging of MDA-MB-231 breast cancer xenograft tumors in nude mice using a quenched cysteine cathepsin-specific ABP. The quenched probe was injected intravenously and fluorescent images of the mice were taken at various time points after injection. Images taken from reference .