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Cysteine cathepsins: their role in tumor progression and recent trends in the development of imaging probes.

Löser R, Pietzsch J - Front Chem (2015)

Bottom Line: The considerable progress in this field over the last two decades has also raised interest in the visualization of these enzymes in their native context, especially with regard to tumor imaging.After a short introduction to structure and general functions of human cysteine cathepsins, we highlight their importance for drug discovery and development and provide a critical update on the current state of knowledge toward their involvement in tumor progression, with a special emphasis on their role in therapy response.In accordance with a radiopharmaceutical point of view, the main focus of this review article will be the discussion of recently developed fluorescence and radiotracer-based imaging agents together with related molecular probes.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiopharmaceutical and Chemical Biology, Institute of Radiopharmaceutical Cancer Research, Helmholtz-Zentrum Dresden-Rossendorf Dresden, Germany ; Department of Chemistry and Food Chemistry, Technische Universität Dresden Dresden, Germany.

ABSTRACT
Papain-like cysteine proteases bear an enormous potential as drug discovery targets for both infectious and systemic human diseases. The considerable progress in this field over the last two decades has also raised interest in the visualization of these enzymes in their native context, especially with regard to tumor imaging. After a short introduction to structure and general functions of human cysteine cathepsins, we highlight their importance for drug discovery and development and provide a critical update on the current state of knowledge toward their involvement in tumor progression, with a special emphasis on their role in therapy response. In accordance with a radiopharmaceutical point of view, the main focus of this review article will be the discussion of recently developed fluorescence and radiotracer-based imaging agents together with related molecular probes.

No MeSH data available.


Related in: MedlinePlus

Fluorogenic substrate-based probes for targeting of cysteine cathepsins containing only one luminophor. Emitting fluorophoric moieties are shown in red, moieties that act as spacer are highlighted in green and additional targeting moieties are shown in blue.
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Figure 8: Fluorogenic substrate-based probes for targeting of cysteine cathepsins containing only one luminophor. Emitting fluorophoric moieties are shown in red, moieties that act as spacer are highlighted in green and additional targeting moieties are shown in blue.

Mentions: Despite quenched substrates offer the advantage of enzyme-catalyzed signal amplification, substrate-based imaging is also possible with probes that contain a fluorogenic leaving group in the absence of a quencher. This principle is based on the deactivation of substituents that exert a +M effect on the aromatic core of fluorophoric entities by the attachment of acyl groups to these substituents. Cleavage of those acyl groups by the action of enzymes will result in unblocked electron donation to the fluorophore and thus increased fluorescence. This principle has been used extensively for the activity determination of proteases and other enzymes that catalyze acyl transfer reactions (Grimm et al., 2013) while reports concerning their application for imaging purposes are scarce. Probes of this type that have been recently developed for cellular imaging are compounds 19 and 20 (Figure 8). Development of compound 19 started from the established dipeptidic fluorogenic substrate Z-Arg-Arg-AMC, which is sensitive and selective for the fluorimetric detection of cathepsin B activity. For the sake of better synthetic accessibility, its two arginine residues were replaced by lysines. This structural change had almost no influence on the kcat/Km-values (148 and 167 mM−1s−1 for Z-Lys-Lys-AMC and Z-Arg-Arg-AMC, respectively). To increase the stability toward degradation by other proteases and the affinity toward cathepsin B a para-aminobenzyloxycarbonyl (PABA) linker was placed between the peptidic and AMC moiety, as this will enable a more effective targeting of the primed binding sites (presumably S1′ and S2′) of cathepsin B (Chowdhury et al., 2014). PABA is a self-destructive linker, as release of its amino group will result in spontaneous fragmentation due to imino-quinone methide formation, which has been successfully used for prodrug design (Carl et al., 1981). Indeed, 19 showed a slightly increased kcat/Km-value (231 mM−1s−1) than Z-Lys-Lys-AMC, which seems to be due to a decrease in Km. Noteworthy, the introduction of the PABA linker was not tolerated by cathepsin L as the analog of 19 containing Phe instead of Lys in P2 (Z-Phe-Lys-PABA-AMC) was not converted by this enzyme. Therefore, the combination of the two Lys residues with the self-destructive PABA linker makes 19 to a substrate which is cleaved by cathepsin B with excellent selectivity over cathepsins L and S. Probe 19 was also effectively cleaved by lysates of HeLa cervical tumor cells. Pretreatment of HeLa cells by the cell-permeable pro-inhibitors E64d (8b) or CA074Me (9b), which inhibit several cysteine cathepsins or selectively cathepsin B, respectively, after intracellular activation by esterases, resulted in a complete blocking of the conversion of 19. Fluorescence microscopic observation of live HeLa cells in the presence of Z-Arg-Arg-AMC or Z-Phe-Arg-AMC did not result in the detection of any cellular fluorescence. Contrary to this, the three-component probe 19 containing the PABA linker led to blue fluorescent cells, while no intracellular fluorescence could be visualized in the presence of CA074Me (9b) or E64d (8b). Similar results were obtained when the HeLa cells were replaced by the Her2-positive breast cancer cell line MDA-MB-231-H2N. These results clearly indicate that 19 undergoes cathepsin B-dependent activation inside the cells and that the PABA linker obviously facilitates membrane permeation of the peptidic probe. When Z-Phe-Lys-PABA-AMC was investigated in the same manner, it was revealed that its cellular activation was only completely blocked by treatment with E64d, while the presence of CA074Me resulted in considerable residual cellular fluorescence. These observations suggest that the latter probe is activated by cathepsin B and other cysteine cathepsins, which is in accordance to results from kinetic investigations and cell lysate experiments. Furthermore, Z-Phe-Lys-PABA-AMC compromises cell viability in both cell lines as assessed by the MTT assay, while 19 and the ordinary two-component probe Z-Arg-Arg-AMC are well-tolerated (Chowdhury et al., 2014).


Cysteine cathepsins: their role in tumor progression and recent trends in the development of imaging probes.

Löser R, Pietzsch J - Front Chem (2015)

Fluorogenic substrate-based probes for targeting of cysteine cathepsins containing only one luminophor. Emitting fluorophoric moieties are shown in red, moieties that act as spacer are highlighted in green and additional targeting moieties are shown in blue.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4477214&req=5

Figure 8: Fluorogenic substrate-based probes for targeting of cysteine cathepsins containing only one luminophor. Emitting fluorophoric moieties are shown in red, moieties that act as spacer are highlighted in green and additional targeting moieties are shown in blue.
Mentions: Despite quenched substrates offer the advantage of enzyme-catalyzed signal amplification, substrate-based imaging is also possible with probes that contain a fluorogenic leaving group in the absence of a quencher. This principle is based on the deactivation of substituents that exert a +M effect on the aromatic core of fluorophoric entities by the attachment of acyl groups to these substituents. Cleavage of those acyl groups by the action of enzymes will result in unblocked electron donation to the fluorophore and thus increased fluorescence. This principle has been used extensively for the activity determination of proteases and other enzymes that catalyze acyl transfer reactions (Grimm et al., 2013) while reports concerning their application for imaging purposes are scarce. Probes of this type that have been recently developed for cellular imaging are compounds 19 and 20 (Figure 8). Development of compound 19 started from the established dipeptidic fluorogenic substrate Z-Arg-Arg-AMC, which is sensitive and selective for the fluorimetric detection of cathepsin B activity. For the sake of better synthetic accessibility, its two arginine residues were replaced by lysines. This structural change had almost no influence on the kcat/Km-values (148 and 167 mM−1s−1 for Z-Lys-Lys-AMC and Z-Arg-Arg-AMC, respectively). To increase the stability toward degradation by other proteases and the affinity toward cathepsin B a para-aminobenzyloxycarbonyl (PABA) linker was placed between the peptidic and AMC moiety, as this will enable a more effective targeting of the primed binding sites (presumably S1′ and S2′) of cathepsin B (Chowdhury et al., 2014). PABA is a self-destructive linker, as release of its amino group will result in spontaneous fragmentation due to imino-quinone methide formation, which has been successfully used for prodrug design (Carl et al., 1981). Indeed, 19 showed a slightly increased kcat/Km-value (231 mM−1s−1) than Z-Lys-Lys-AMC, which seems to be due to a decrease in Km. Noteworthy, the introduction of the PABA linker was not tolerated by cathepsin L as the analog of 19 containing Phe instead of Lys in P2 (Z-Phe-Lys-PABA-AMC) was not converted by this enzyme. Therefore, the combination of the two Lys residues with the self-destructive PABA linker makes 19 to a substrate which is cleaved by cathepsin B with excellent selectivity over cathepsins L and S. Probe 19 was also effectively cleaved by lysates of HeLa cervical tumor cells. Pretreatment of HeLa cells by the cell-permeable pro-inhibitors E64d (8b) or CA074Me (9b), which inhibit several cysteine cathepsins or selectively cathepsin B, respectively, after intracellular activation by esterases, resulted in a complete blocking of the conversion of 19. Fluorescence microscopic observation of live HeLa cells in the presence of Z-Arg-Arg-AMC or Z-Phe-Arg-AMC did not result in the detection of any cellular fluorescence. Contrary to this, the three-component probe 19 containing the PABA linker led to blue fluorescent cells, while no intracellular fluorescence could be visualized in the presence of CA074Me (9b) or E64d (8b). Similar results were obtained when the HeLa cells were replaced by the Her2-positive breast cancer cell line MDA-MB-231-H2N. These results clearly indicate that 19 undergoes cathepsin B-dependent activation inside the cells and that the PABA linker obviously facilitates membrane permeation of the peptidic probe. When Z-Phe-Lys-PABA-AMC was investigated in the same manner, it was revealed that its cellular activation was only completely blocked by treatment with E64d, while the presence of CA074Me resulted in considerable residual cellular fluorescence. These observations suggest that the latter probe is activated by cathepsin B and other cysteine cathepsins, which is in accordance to results from kinetic investigations and cell lysate experiments. Furthermore, Z-Phe-Lys-PABA-AMC compromises cell viability in both cell lines as assessed by the MTT assay, while 19 and the ordinary two-component probe Z-Arg-Arg-AMC are well-tolerated (Chowdhury et al., 2014).

Bottom Line: The considerable progress in this field over the last two decades has also raised interest in the visualization of these enzymes in their native context, especially with regard to tumor imaging.After a short introduction to structure and general functions of human cysteine cathepsins, we highlight their importance for drug discovery and development and provide a critical update on the current state of knowledge toward their involvement in tumor progression, with a special emphasis on their role in therapy response.In accordance with a radiopharmaceutical point of view, the main focus of this review article will be the discussion of recently developed fluorescence and radiotracer-based imaging agents together with related molecular probes.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiopharmaceutical and Chemical Biology, Institute of Radiopharmaceutical Cancer Research, Helmholtz-Zentrum Dresden-Rossendorf Dresden, Germany ; Department of Chemistry and Food Chemistry, Technische Universität Dresden Dresden, Germany.

ABSTRACT
Papain-like cysteine proteases bear an enormous potential as drug discovery targets for both infectious and systemic human diseases. The considerable progress in this field over the last two decades has also raised interest in the visualization of these enzymes in their native context, especially with regard to tumor imaging. After a short introduction to structure and general functions of human cysteine cathepsins, we highlight their importance for drug discovery and development and provide a critical update on the current state of knowledge toward their involvement in tumor progression, with a special emphasis on their role in therapy response. In accordance with a radiopharmaceutical point of view, the main focus of this review article will be the discussion of recently developed fluorescence and radiotracer-based imaging agents together with related molecular probes.

No MeSH data available.


Related in: MedlinePlus