Papain-like cysteine proteases, or cysteine cathepsins, were once thought to degrade proteins nonspecifically in the lysosome. However, their roles in normal cellular processes and disease pathologies have become increasingly apparent. Cysteine cathepsins are implicated in cancer progression, due to their roles in angiogenesis, apoptosis, and tumor cell invasion [20
]. They are also key regulators of inflammation in diseases such as atherosclerosis, rheumatoid arthritis, and asthma. A number of probes have been developed for studying the functional roles of cysteine cathepsins. In addition to being valuable biochemical reagents, many of these probes have also become useful as contrast agents for imaging disease processes.
To this end, Blum et al. developed a series of AOMK-based ABPs that target cathepsin B, L, and S [18
]. Both quenched and non-quenched versions are cell permeable and can be used to biochemically profile active cathepsins in live cells and lysates by fluorescent SDS-PAGE. Furthermore, near infrared versions of the AOMK probes can be used for whole-body non-invasive imaging. GB123 and GB137 (non-quenched and quenched, respectively) both accumulated specifically in xenografted tumors of nude mice upon systemic intravenous delivery [19
]. The quenched probe showed specific signal in tumors in as little as 30 minutes, whereas the non-quenched version required longer times to generate contrast, due to the need to clear the unbound probe. Importantly, fluorescent signal in the tumors could be substantially reduced by pretreatment with a potent cathepsin inhibitor indicating the specific nature of the accumulation. Since its initial publication, GB123 has been applied in a number of additional studies. Most recently it was used to show the localization of Cathepsin B activity to caveolae of endothelial cells during tube formation in vitro
, to investigate the role of cathepsins in VEGF-induced angiogenesis, and to image the effects of expression of neutrophilic granule protein (NGP) in tumors [21
In addition to ABPs, there has been significant interest in the use of quenched substrate probes for non-invasive imaging applications. Polymer-based probes have been developed by VisEn Medical (now Perkin Elmer) and are commercially available. In particular, ProSense-680, a cathepsin targeted probe, has been used to assess the contribution of Cathepsin B activity to many inflammatory processes, including asthma [24
], focal inflammation, angiogenesis, and growth of intestinal polyps [3
], immune cell function following rejection of transplanted mouse hearts [25
], and following myocardial infarction [5
]. Substrate based probes have also been used to image cathepsin activities and assess their contributions in other heart-related conditions including early aortic valve disease [26
] and atherosclerosis [27
A variation of the Prosense cathepsin B probe containing a substrate sequence with selectivity towards Cathepsin K, a matrix-degrading elastase, was used to study atherosclerosis [28
]. Using intravital fluorescent microscopy, Cathepsin K activity was identified in strong focal regions of atherotic lesions. Fluorescence was strongly enhanced in the macrophage population and colocalized with immunoreactive Cathepsin K. In this instance, the population of Cathepsin K was broader than the probe fluorescence, suggesting that only a fraction of the total protein was proteolytically active. The same substrate-based probe was also used to image Cathepsin K activity in osteoclasts in models of accelerated bone loss [4
A new class of substrate probes was recently developed using a “reverse design” method in which potent inhibitors are converted back into cleavable reporter substrates [13
]. Because these inhibitors were optimized using extensive medicinal chemistry efforts, they are highly selective and have optimized pharmacokinetic properties making them likely to be better probes than standard peptide substrates. One substrate probe developed by this approach, AW-091, was used in vivo
to image cathepsin S activity in a mouse model of paw inflammation. Comparison with ProSense680 in the same models indicated that AW-091 gave maximal signal-to-noise ratio after 3 hours, while ProSense680 required 24 hours, reflecting enhanced pharmacodynamic properties.
Caspases are cysteine proteases that mediate a programmed form of cell death called apoptosis [29
]. Apoptosis is critical for normal development and tissue homeostasis, as well as for a number of diseases including cancer. In addition, a sub-family of caspases plays a major role in regulating a pro-inflammatory form of cell death called pyroptosis [30
]. Caspases have a unique reactivity compared to other cysteine proteases, in that they only cleave substrates containing aspartic acid residues in the P1 position. Therefore, all peptide-based probes for caspases have made use of this selectivity requirement.
As with cathepsins, one of the major challenges in developing probes for caspases is selectivity for unique proteases within the family (i.e. caspase-3 over caspase-7); however, an even bigger problem is the tendency for caspase probes to target other enzymes, such as cathepsins or legumain [31
]. Legumain, is a lysosomal cysteine protease that has roles in antigen processing [33
], matrix degradation, and tumorigenesis[34
]. Legumain has a preference for substrates containing asparagine in the P1 position, however, it is also capable of binding to activity-based AOMK probes containing a P1 aspartic acid (Asp) [35
]. This is most likely due to the fact that the Asp side chain is protonated in the acidic environment of the lysosome, allowing it to fit into the S1 binding pocket of the active site.
In an attempt to develop selective probes for caspases, Berger et al. used a positional scanning library approach [36
]. Optimal sequences were identified and converted to biotinylated probes, which were evaluated in kinetic studies using cell free extracts and intact cells. In subsequent studies, the most selective caspase ABPs were tagged with NIRF fluorophores for use in non-invasive imaging applications[32
]. Interestingly, the most potent in vitro
probe exhibited substantial cross-reactivity with capthepsin B and legumain. To avoid cathepsin reactivity, a proline was substituted at the P2 position. This new probe, AB50 (Cy5-EPD-AOMK), showed significantly improved in vivo
selectivity properties and was applied in vivo
in two mouse models of apoptosis. These studies show that, in addition to being valuable for non-invasive imaging applications, AB50 can also be used to assess apoptosis by microscopy, flow cytometry, and ex vivo
fluorescence imaging. Importantly, since the probes are covalent labels, caspase modification can be confirmed biochemically using SDS-PAGE. These studies also confirmed that AB50 suffers from cross-reactivity with legumain. Efforts were made to reduce legumain reactivity, but increased selectivity came at the cost of reduced caspase potency.
Addition of the transporter peptide Tat to AB50 (tAB50) significantly enhanced the fluorescent signal in apoptotic cells. Unfortunately, the Tat labeled probes increased labeling of both legumain and Cathepsin B, due to uptake of the Tat-labeled probes by endocytosis. Hence, transporter peptides such as Tat may not be ideal for delivery of probes to cytosolic protease targets, especially when primary off-targets are lysosomal enzymes.
One of the most widely used classes of activity-based probes for caspases contains a fluoromethyl ketone (FMK) electrophile. Carboxyfluorescein and sulphorhodamine-labeled versions are commercially available and marketed under such names as FLICA (fluorochrome-labeled inhibitor of caspases) and CaspaTag. FLICA has been used to assess the kinetics of cell death in response to several stimuli by flow cytometry [37
] and microscopy [38
]. FAM-YVAD-FMK is also a common reagent to visualize caspase-1 activity during inflammasome-dependent cell death [39
]. New versions called FLIVO are currently being marketed for use in in vivo
CaspaTags for caspase 3/7 (SR-DEVD-FMK) and caspase 9 (FAM-LEHD-FMK) were used in a head-to-head comparison with cleaved caspase antibodies for immunofluorescence microscopy of gentamicin-treated chick cochlea [42
]. The overall trend and timing of labeling with both antibodies and the CaspaTag probes were similar; however, at later time points the CaspaTag showed more caspase-3 positive cells than the antibodies. The authors concluded that antibodies showed the activated caspases present at a given time point, whereas the CaspaTag could track cells that had already completed cell death in addition to those currently dying, giving a more complete assessment of apoptotic cells. Alternatively, the enhanced signal from CaspaTag may be due to cross-reactivity with other proteases. Specifically, FMK- and CMK- based inhibitors have been shown to block the activity of cathepsins and legumain [31
]. The increase in fluorescence at late stages of apoptosis in chick cochlea may reflect the involvement of lysosomal proteases in cell death [43
]. This cross-reactivity could also explain the results of studies that show that FLICA signal in flow cytometry could not be decreased by pre-treating cells with concentrations of untagged Z-DEVD-FMK or Z-VAD-FMK sufficient to block caspase activity [44
]. Interestingly, there have been no reported biochemical data regarding the selectivity of the FLICA reagents when used in cells. Inhibitor versions marketed as selective for specific caspases (i.e. z-DEVD-FMK for caspase-3/7, z-LEHD-FMK for caspase-9, z-LETD-FMK, etc.) were demonstrated to have broad reactivity in competition assays [9
]. Given these findings, one should interpret all data obtained using activity-based probes with extreme caution. Data analysis should always be paired with careful biochemical analyses, such that observations of enzyme activity and function can be assigned to the correct enzyme.
Likewise, the same practice should be applied to the use of substrate-based probes, which also suffer from a lack of specificity that is even more difficult to track. The most widely used substrate probes are the fluorogenic substrates, which typically contain a tetrapeptide caspase cleavage site and aminomethylcoumarin (AMC) or aminofluorocoumarin (AFC). These reagents are commercially available and, like FMK probes, are usually marketed as specific for one caspase. The specificity regions have been optimized based on reported data from positional scanning studies [1
]; however it is important to keep in mind that just because an enzyme prefers
one substrate over others does not mean it cannot
cleave the others, especially at higher substrate concentrations.
In 2005, Bullok and Piwnica-Worms first described the synthesis of a novel substrate based probe for imaging caspase activity during apoptosis [46
]. This probe, TcapQ647
, contains the caspase substrate DEVD, flanked by a fluorophore/quencher pair, Alexa Fluor 647/QSY 21. Similar to the tAB50 activity-based probe [32
contains the tat peptide sequence. Initial studies with this probe verified that it was 92 – 99% quenched, and that it could be unquenched in the presence of active caspases-7 and -9. Apoptotic cells could be detected by flow cytometry and fluorescence microscopy. Later, the kinetic properties of this probe towards other caspases were reported, and a non-cleavable control probe was shown to be inactive in apoptotic cells [47
]. These probes were also used in vivo
in a model of parasite-induced apoptosis in human colon xenografts. The degree of fluorescence in tumors was shown to correlate with the rate of apoptosis, as assessed by TUNEL assay.
A second generation probe called KcapQ was later introduced, in which the tat peptide was replaced by the Lys-Arg-rich sequence KKKRKV [48
]. Efficacy of KcapQ was assessed in a mouse model of retinal ganglion cell apoptosis induced by N-methyl-D-aspartate (NMDA), a clinically relevant model of glaucoma. NMDA-treated eyecups showed an increase in fluorescence when compared with PBS-treated controls and localization corresponded with TUNEL-positive cells. TcapQ was also tested in this model [49
]. Pre-treatment with Z-DEVD-FMK reduced the number of probe-positive cells by approximately 60%.