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Carbobenzoxy-capped Phe-Lys(BODIPY TMR-X-acyloxymethyl ketone(QSY7).


Shan L1.


Molecular Imaging and Contrast Agent Database (MICAD) [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2004-2013.
2010 May 19 [updated 2010 May 25].

Author information

National Center for Biotechnology Information, NLM, NIH


The human cysteine cathepsins have 11 members (cathepsins B, C, H, F, K, L, O, S, V, W, and X/Z) and share a conserved active site that is formed by cysteine, histidine, and asparagine residues (1-5). Cathepsins B, L, H, F, O, X/Z, and C are expressed ubiquitously, whereas the expression of cathepsins S, K, W, and V are relatively organ-limited. Cysteine cathepsins interact with other proteases (aspartic, metallo, serine, and threonine) in a cascade-like manner, involving various physiological processes, including protein degradation, precursor protein activation, MHC-II–mediated antigen presentation, bone remodeling, keratinocytes differentiation, hair follicle cycle, reproduction, and apoptosis (6, 7). Increased expression and activity, and relocalization to the plasma membrane of cysteine cathepsins are associated with the pathogenesis of a number of human diseases such as cancer, atherosclerosis, and neurodegenerative diseases, and changes related to cysteine cathepsins have been shown to be of diagnostic and prognostic value for the diseases (2, 8-11). Significant efforts have been made in developing optical molecular probes of protease activity (4, 7, 12, 13). These probes are either substrate-based or activity-based, providing a readout of the enzyme activity rather than simple protein abundance. Substrate-based probes (SBPs) are designed using fluorescent peptide sequences tethered to a large polymer or dendramer backbone, and these SBPs are internally quenched by the high density of fluorophores loaded onto the backbone structure. Similar to conventional enzymatic tests, SBPs appear to be less reliable because of overlapping substrate specificities, enzymatic activity instability, and interactions with endogenous inhibitors. Activity-based probes (ABPs) label target proteases through the formation of a covalent bond with the active site cysteine. The selectivity of an ABP is controlled by both its peptide selectivity sequence and reactive functional group. The fluorescent reporter allows probe-labeled cathepsins to be directly visualized. Because ABPs tend to be small molecules, the in vivo half-lives of ABPs are relatively short, which results in the production of high-contrast images. A drawback of using a covalent probe is the lack of signal amplification because the target proteases are inactivated upon binding the probe. However, sufficient levels of the active proteases have been found to exist in tumor tissues, which allows the generation of reasonable contrast images using noninvasive methods (4, 7, 12). Blum et al. synthesized a group of near-infrared fluorescent activity-based probes (NIRF-ABPs) for noninvasive optical imaging of cysteine protease activity (4, 7, 12, 14). These probes can be subgrouped into quenched ABPs (qABPs) (e.g., GB117, GB119, GB135, and GB137) and nonquenched ABPs (e.g., GB111, GB123, and GB138). In short, the probes consist of a reactive group of peptide acyloxymethyl ketone (AOMK) that targets diverse members of cysteine cathepsins. The ketone in AOMK reacts with the cysteine in the enzyme active site and produces a stable thiomethyl ketone adduct. The covalent binding involves the loss of the acyloxy group of AOMK. Thus, a probe carrying a fluorescent reporter group on its peptide scaffold and a highly efficient quenching molecule attached to the acyloxy-losing group should result in a quenched probe that only becomes fluorescent upon covalent binding with an enzyme. In addition, a spacer is designed to reduce the steric congestion between the reporter and the fluorescence quencher. Blum et al. have shown that the NIRF-ABPs are nontoxic to cells, reasonably water soluble, potentially valuable as imaging agents for disease diagnosis, and powerful tools for in vivo preclinical and clinical testing of small-molecule therapeutic agents. Although NIRF-ABPs present different features regarding stability, specificity, and kinetics, the quenched probes can be used to image specific protease activity at considerably earlier time points than can be used for substrate-based methods or nonquenched ABPs. However, high levels of signal in large organs with high cathepsin activity such as liver, kidney, and spleen make activity-based imaging of specific locations within the central body cavity difficult. In this chapter, the synthesis and analytic results of quenched GB117 were introduced, which were compared with that of GB111. The quenched GB117 and its corresponding nonquenched control GB111 are the first generation of this group of NIRF-ABPs.

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