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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

Quenched substrate-based probes for optical imaging of cysteine cathepsins. Emitting fluorophoric moieties are shown in red, chromophoric moieties that act as quencher are highlighted in green and additional targeting moieties are shown in blue.
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Figure 7: Quenched substrate-based probes for optical imaging of cysteine cathepsins. Emitting fluorophoric moieties are shown in red, chromophoric moieties that act as quencher are highlighted in green and additional targeting moieties are shown in blue.

Mentions: However, the latter-type of imaging agents offer the potential advantage of enzyme-mediated signal amplification. A recently reported substrate-based probe is compound 17, which has been developed for the imaging of tumor-associated cathepsin S (Figure 7) (Hu et al., 2014a). The compound has been designed on the basis of a potent cathepsin S inhibitor bearing cyclohexylalanine in P2 and a cyclic ketone in the P1 position. The ketone moiety gives rise to a reversible covalent interaction with the active-site cysteine by thiohemiketal formation. The N-terminal amino group of this dipeptide derivative is connected to a morpholine moiety via a urea linkage. A morpholino group in this position will be engaged in favorable interactions with the side-chain of Lys64 in the S3 region of cathepsin S via its oxygen atom (Pauly et al., 2003). Furthermore, cyclohexylalanine has been identified to be optimal to address the S2 pocket of cathepsin S (Ward et al., 2002). The combination of these two favorable moieties makes this inhibitor highly selective for cathepsin S (Link and Zipfel, 2006). In order to convert the inhibitor into a substrate and to allow the attachment of fluorophores and further targeting elements, the cyclic ketone in the P1 position was replaced by diaminobutyric acid. Such a strategy to design selective protease substrates starting from covalent inhibitors is termed reverse design. This concept seems to be promising because small peptide-derived inhibitors very often were structurally optimized using non-proteinogenic amino acids to achieve optimal targeting of protease subsites. It has been successfully applied for the design of optical imaging probes for cathepsin K and cathepsin S (Watzke et al., 2008). By employing lysine with orthogonally protected amino groups a Cy5.5 as NIR fluorophore and a palmitoyl group was attached to the diaminobutyric acid in the P1 position. Palmitoylation of the probe was done with the intention to facilitate its localisation to the cell surface, because the secreted cathepsin S will remain in proximity to the cellular membrane. Via an ethylenediamine linker the chromophore of black hole quencher 3 (BHQ-3) was attached as dark quencher to silence the Cy5.5 reporter. The kinetic characterisation of the substrate-based probe 17 toward hydrolysis catalyzed by the cathepsins B, K, L, S, and V has revealed that it is selectively cleaved by cathepsin S with a specificity constant kcat/Km of 2700 M−1s−1. Interestingly, the cathepsin S-catalyzed hydrolysis of 17 was significantly enhanced in the presence of liposomes as membrane model. Microscopic evaluation of compound 17 together with its non-lipidated counterpart containing a maleimidylhexanoyl group instead of palmitoyl was done toward human THP-1 cell-derived macrophages. The studies indicated that both probes were cleaved rapidly by the cells and associated with them. Preincubation with cell-impermeable E64 (6) resulted in blocking of the cleavage of 17 only, whereas that of the non-lipidated probe was not influenced. In contrast, treatment with E64d (8b), a cell-permeable analog of E64 prevented the hydrolysis of both probes, which indicates that they are not recognized as substrates by other proteases. These results have been interpreted to indicate that the palmitoylated probe undergoes hydrolysis catalyzed by surface-located cathepsin S, whereas the non-lipidated probe is subjected to internalization before it is cleaved. Because compound 8b inhibited the activation of both probes, they are probably not recognized as substrates by other proteases, and thus, cleaved by cathepsin S in a highly specific manner. Performing the experiment at 4°C did not lead to activation of the non-lipidated probe, whereas the signal from activation of 17 was restricted to the cell membrane due to attenuated endocytosis. In vivo evaluation in 4T1 tumor-carrying mice revealed that for both probes a significant fluorescence signal was observable in the tumor 30 min p.i. that did not vanish up to 24 h p.i. At his time the signal caused by 17 was as twice as high compared to its non-lipidated counterpart. The tumor-muscle ratio of 17 reached a maximum of 18 after 5 d p.i. The non-lipidated probe was also detectable in the kidneys, whereas the uptake in other organs was very low. Compound 17 is taken up by the kidneys to a similar extent as its non-lipidated counterpart, whereas lipidation resulted in a significantly higher uptake in all other investigated organs (lung, liver, spleen), but the levels were greatest for the tumor. The significantly increased tumor uptake of 17 compared to the non-lipidated probe was confirmed in ex vivo investigations of tumor sections by fluorescence microscopy (Hu et al., 2014a).


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

Löser R, Pietzsch J - Front Chem (2015)

Quenched substrate-based probes for optical imaging of cysteine cathepsins. Emitting fluorophoric moieties are shown in red, chromophoric moieties that act as quencher 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 7: Quenched substrate-based probes for optical imaging of cysteine cathepsins. Emitting fluorophoric moieties are shown in red, chromophoric moieties that act as quencher are highlighted in green and additional targeting moieties are shown in blue.
Mentions: However, the latter-type of imaging agents offer the potential advantage of enzyme-mediated signal amplification. A recently reported substrate-based probe is compound 17, which has been developed for the imaging of tumor-associated cathepsin S (Figure 7) (Hu et al., 2014a). The compound has been designed on the basis of a potent cathepsin S inhibitor bearing cyclohexylalanine in P2 and a cyclic ketone in the P1 position. The ketone moiety gives rise to a reversible covalent interaction with the active-site cysteine by thiohemiketal formation. The N-terminal amino group of this dipeptide derivative is connected to a morpholine moiety via a urea linkage. A morpholino group in this position will be engaged in favorable interactions with the side-chain of Lys64 in the S3 region of cathepsin S via its oxygen atom (Pauly et al., 2003). Furthermore, cyclohexylalanine has been identified to be optimal to address the S2 pocket of cathepsin S (Ward et al., 2002). The combination of these two favorable moieties makes this inhibitor highly selective for cathepsin S (Link and Zipfel, 2006). In order to convert the inhibitor into a substrate and to allow the attachment of fluorophores and further targeting elements, the cyclic ketone in the P1 position was replaced by diaminobutyric acid. Such a strategy to design selective protease substrates starting from covalent inhibitors is termed reverse design. This concept seems to be promising because small peptide-derived inhibitors very often were structurally optimized using non-proteinogenic amino acids to achieve optimal targeting of protease subsites. It has been successfully applied for the design of optical imaging probes for cathepsin K and cathepsin S (Watzke et al., 2008). By employing lysine with orthogonally protected amino groups a Cy5.5 as NIR fluorophore and a palmitoyl group was attached to the diaminobutyric acid in the P1 position. Palmitoylation of the probe was done with the intention to facilitate its localisation to the cell surface, because the secreted cathepsin S will remain in proximity to the cellular membrane. Via an ethylenediamine linker the chromophore of black hole quencher 3 (BHQ-3) was attached as dark quencher to silence the Cy5.5 reporter. The kinetic characterisation of the substrate-based probe 17 toward hydrolysis catalyzed by the cathepsins B, K, L, S, and V has revealed that it is selectively cleaved by cathepsin S with a specificity constant kcat/Km of 2700 M−1s−1. Interestingly, the cathepsin S-catalyzed hydrolysis of 17 was significantly enhanced in the presence of liposomes as membrane model. Microscopic evaluation of compound 17 together with its non-lipidated counterpart containing a maleimidylhexanoyl group instead of palmitoyl was done toward human THP-1 cell-derived macrophages. The studies indicated that both probes were cleaved rapidly by the cells and associated with them. Preincubation with cell-impermeable E64 (6) resulted in blocking of the cleavage of 17 only, whereas that of the non-lipidated probe was not influenced. In contrast, treatment with E64d (8b), a cell-permeable analog of E64 prevented the hydrolysis of both probes, which indicates that they are not recognized as substrates by other proteases. These results have been interpreted to indicate that the palmitoylated probe undergoes hydrolysis catalyzed by surface-located cathepsin S, whereas the non-lipidated probe is subjected to internalization before it is cleaved. Because compound 8b inhibited the activation of both probes, they are probably not recognized as substrates by other proteases, and thus, cleaved by cathepsin S in a highly specific manner. Performing the experiment at 4°C did not lead to activation of the non-lipidated probe, whereas the signal from activation of 17 was restricted to the cell membrane due to attenuated endocytosis. In vivo evaluation in 4T1 tumor-carrying mice revealed that for both probes a significant fluorescence signal was observable in the tumor 30 min p.i. that did not vanish up to 24 h p.i. At his time the signal caused by 17 was as twice as high compared to its non-lipidated counterpart. The tumor-muscle ratio of 17 reached a maximum of 18 after 5 d p.i. The non-lipidated probe was also detectable in the kidneys, whereas the uptake in other organs was very low. Compound 17 is taken up by the kidneys to a similar extent as its non-lipidated counterpart, whereas lipidation resulted in a significantly higher uptake in all other investigated organs (lung, liver, spleen), but the levels were greatest for the tumor. The significantly increased tumor uptake of 17 compared to the non-lipidated probe was confirmed in ex vivo investigations of tumor sections by fluorescence microscopy (Hu et al., 2014a).

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