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

Abstract

Protease activity is tightly regulated in both normal and disease conditions. However, it is often difficult to monitor the dynamic nature of this regulation in the context of a live cell or whole organism. To address this limitation, we developed a series of quenched activity-based probes (qABPs) that become fluorescent upon activity-dependent covalent modification of a protease target. These reagents freely penetrate cells and allow direct imaging of protease activity in living cells. Targeted proteases are directly identified and monitored biochemically by virtue of the resulting covalent tag, thereby allowing unambiguous assignment of protease activities observed in imaging studies. We report here the design and synthesis of a selective, cell-permeable qABP for the study of papain-family cysteine proteases. This probe is used to monitor real-time protease activity in live human cells with fluorescence microscopy techniques as well as standard biochemical methods.

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

“…In contrast, ABPs covalently bind to active enzymes, thus permitting assignment of imaging signals to specific enzymes [13,14]. In fact, a number of ABPs that target cysteine proteases have been used to image enzyme activity in tumor cells both in vitro and in vivo [43,48,49].…”

“…In a recent advance, a cathepsin-specific ABP that becomes fluorescent only upon binding to its enzyme target has been developed [48]. Since tagged ABPs used in imaging are constitutively fluorescent, they generate a high nonspecific fluorescent background when used in living cells.…”

“…Covalent modification of cysteine cathepsins by this probe liberates the quencher moiety and the probe becomes fluorescent. This cell-permeable, quenched probe has been used to image cathepsin activity levels in both the murine fibroblast cell line NIH-3T3 and the human MCF-10A breast cancer cell line [48]. Cells treated with the qABP showed distinct punctuate fluorescent staining of lysosomal compartments, whereas the unquenched control probe produced bright, nonspecific intracellular fluorescence that required extensive washing to reveal specific target labeling.…”

Specificity of aza-peptide electrophile activity-based probes of caspases

“…Bovine cathepsin B (Sigma) inhibition kinetics were determined as previously described. 18 Recombinant human caspase-3 was expressed in E. coli and purified as described. 19 Inhibition rates were determined as previously described.…”

Molecular tools for cell and systems biology

“…This concept was successfully applied to measure matrix metalloproteases, caspases, cathepsins, as is summarized in several reviews (Baruch et al, 2004;Fonovic and Bogyo, 2007). Although many proteases act in extracellular space, it was in some cases possible to generate a membrane-penetrating sensor, for instance for caspase-3 and some cysteine proteases (Blum et al, 2005;Komoriya et al, 2000). For the future, it would be desirable to use amino acid derivatives carrying bioactivatable protecting groups (see the following) to generally allow peptides and peptide-based sensors to enter cells.…”