Cysteine cathepsins are proteolytic enzymes that belong to the papain subfamily of cysteine proteases (1). The human family of cysteine cathepsins consists of 11 members: cysteine cathpsin B, C, F, H, K, L, O, S, V, W and X (2). The active site in these enzymes contains a cysteine residue that acts as a nucleophile for attacking the peptide bond of substrates, and a His residue that acts as a general base for shuttling protons (3). As a key component in cell division, cell death and antigen presentation, cysteine cathepsins are found to be associated with various diseases, including cancer, arthritis, osteoporosis, cardiovascular diseases and neurodegenerative disorders (4). Increased expression, activity and mislocation of several cysteine cathepsins (B and L) are significantly correlated with malignant progressions in tumors (2). Like the other four catalytic types of mammalian protease (aspartic, metallo, serine and threonine), the cysteine cathepsins are synthesized as inactive zymogens and their activity is post-translationally regulated (5). Thus, the abundance of these enzymes may not directly correlate to their proteolytic activity. Activity-based proteomics is a novel technology that has been developed recently to characterize the activity of proteolytic enzymes (6, 7). In this method, small molecules known as activity-based probes (ABPs) covalently bind to the catalytic site of specific target enzymes in complex proteomes. ABPs comprise three basic elements: a reactive functional group known as a warhead for labeling enzymes via covalent binding, a linker for preventing steric congestions, and a reporter tag for identification, purification and visualization of the probe-labeled enzymes (8). For instance, acyloxymethyl ketone (AOMK) is a warhead specific for cysteine cathepsins (B, L and S). The ketone in AOMKs reacts with the cysteine in the enzyme active via a sulfhydryl nucleophile to produce a stable thiomethyl ketone adduct, leading to blockade of cysteine cathepsins (8). With an optical labeling as the tag, the ABPs can be used to image the activity of proteolytic enzymes in vivo (1).
CBz-Phe-Lys(Cy5)-methyl ketone-2,6,dimethylterephthalic amide-hexyl-QSY 21(GB137) is an optical agent used for imaging cysteine cathepsins in vivo (1). GB137 consists of four components: an AOMK type of warhead to bind specifically to cysteine cathepsin B and cysteine cathepsin L, a hexyl and a Lys as spacers to reduce steric congestion, the infrared fluorescent probe Cy5 (646/664 nm excitation/emission), and the commercial fluorescence quencher QSY 21 (absorption ~661 nm). As a quenched ABP (qABP), Cy5 and QSY 21 are separated by a Lys-methyl ketone in the molecule. In the absence of cysteine cathepsin, the fluorescence emitted by the Cy5 is quenched by the QSY 21, and no fluorescence is observed. Upon binding to the enzyme, GB137 interacts with the enzyme active site to release the quenching group QSY 21 as a side product, and produces a Cy5-labeled cysteine cathepsin that is detectable with infrared fluorescent imaging.