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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 ABPs targeting cysteine cathepsins. Emitting fluorophoric moieties are shown in red, chromophoric moieties that act as quencher are highlighted in green.
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Figure 6: Fluorogenic ABPs targeting cysteine cathepsins. Emitting fluorophoric moieties are shown in red, chromophoric moieties that act as quencher are highlighted in green.

Mentions: More than for in vivo imaging, ABPs have been used to detect active proteases in cell lysates by incubating the crude protein mixtures with the labeled probes and subsequent electrophoretic separation followed by label-specific detection. This approach is called activity-based or chemical proteomics and offers the advantage of coupling the detection signal to enzyme activity and therefore provides more accurate information about the functions of the enzyme of interest in the biological process to be studied (Fonovič and Bogyo, 2008; Deu et al., 2012). A recently developed ABP to target cathepsin K is compound 14 (Figure 6) (Frizler et al., 2013). This probe is based on the vinyl sulfone warhead and equipped with an innovative tricyclic luminescent group which represents a conformationally restricted analog of the 4-(4-hydroxybenzylidene)-1H-imidazol-5(4H)-one fluorophore in green fluorescent protein (Baranov et al., 2012). Compound 14 proved to be an irreversible inactivator of the cathepsins S, K, L, and B with a slight selectivity toward S and K over L and B, while it showed the strongest potency against cathepsin S. Its suitability to visualize cathepsin K ex vivo by in-gel fluorescence imaging after electrophoretic separation of the cathepsin K-14 complex has been demonstrated. Labeling of cathepsin K by 14 could be prevented upon preincubation with a reversible azadipeptide nitrile inhibitor that is highly selective for this cathepsin. The probe enabled the detection of external cathepsin K among the proteins present in HEK cell lysate. A linear correlation between the fluorescence intensity of the electrophoretic spots and the amount of enzyme was observed over a range of 17–280 ng of cathepsin K and the detection limit was found to be superior to Western blot. The ABP 15 is of very similar design as 14 (Mertens et al., 2014). In contrast, it is equipped with an alternative fluorophore on the basis of a novel coumarin-tetrahydroquinoline hybrid. This fluorophore can be considered as a conformationally locked 7-N,N-diethylaminocoumarin and shows improved photophysical properties over its non-rigidified counterpart such as bathochromically shifted absorption and emission maxima (Frizler et al., 2012; Mertens et al., 2014). Among the cathepsins S, K, L, and B, inactivation by 15 was strongest for cathepsin S with approximately threefold increased kinact/KI-values for this cathepsin compared to compound 14. Probe 15 is more than 50-fold less potent for cathepsin K than for S. This result has been explained on the basis of docking studies, which revealed that the coumarin-tetrahydroquinoline moiety of 15 partially occupies the S3 pocket of cathepsin S and its position is probably stabilized by hydrogen bond interactions between its two carbonyl groups and the side chain of Arg141 and the backbone NH of Val162. This aptly illustrates that also the fluorophore may interact with the enzyme and thus can contribute to the selectivity of the probe. Compound 15 was evaluated for in-gel detection of cathepsin S in a similar fashion as 14 for cathepsin K. The fluorescent cathepsin S-15 complex enabled to visualize enzyme amounts as low as 0.5 ng. Preincubation of cathepsin S with the broad-spectrum cysteine protease inhibitor E64 (6) abolished its labeling by 15, which shows that inactivation is dependent on the catalytic activity and labeling does not occur due to indiscriminate reaction with nucleophiles on the protein surface. In addition, ABP 15 has been shown to be capable of detecting cathepsin S specifically in a protein extract derived from human placental tissue.


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

Löser R, Pietzsch J - Front Chem (2015)

Fluorogenic ABPs targeting cysteine cathepsins. Emitting fluorophoric moieties are shown in red, chromophoric moieties that act as quencher are highlighted in green.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 6: Fluorogenic ABPs targeting cysteine cathepsins. Emitting fluorophoric moieties are shown in red, chromophoric moieties that act as quencher are highlighted in green.
Mentions: More than for in vivo imaging, ABPs have been used to detect active proteases in cell lysates by incubating the crude protein mixtures with the labeled probes and subsequent electrophoretic separation followed by label-specific detection. This approach is called activity-based or chemical proteomics and offers the advantage of coupling the detection signal to enzyme activity and therefore provides more accurate information about the functions of the enzyme of interest in the biological process to be studied (Fonovič and Bogyo, 2008; Deu et al., 2012). A recently developed ABP to target cathepsin K is compound 14 (Figure 6) (Frizler et al., 2013). This probe is based on the vinyl sulfone warhead and equipped with an innovative tricyclic luminescent group which represents a conformationally restricted analog of the 4-(4-hydroxybenzylidene)-1H-imidazol-5(4H)-one fluorophore in green fluorescent protein (Baranov et al., 2012). Compound 14 proved to be an irreversible inactivator of the cathepsins S, K, L, and B with a slight selectivity toward S and K over L and B, while it showed the strongest potency against cathepsin S. Its suitability to visualize cathepsin K ex vivo by in-gel fluorescence imaging after electrophoretic separation of the cathepsin K-14 complex has been demonstrated. Labeling of cathepsin K by 14 could be prevented upon preincubation with a reversible azadipeptide nitrile inhibitor that is highly selective for this cathepsin. The probe enabled the detection of external cathepsin K among the proteins present in HEK cell lysate. A linear correlation between the fluorescence intensity of the electrophoretic spots and the amount of enzyme was observed over a range of 17–280 ng of cathepsin K and the detection limit was found to be superior to Western blot. The ABP 15 is of very similar design as 14 (Mertens et al., 2014). In contrast, it is equipped with an alternative fluorophore on the basis of a novel coumarin-tetrahydroquinoline hybrid. This fluorophore can be considered as a conformationally locked 7-N,N-diethylaminocoumarin and shows improved photophysical properties over its non-rigidified counterpart such as bathochromically shifted absorption and emission maxima (Frizler et al., 2012; Mertens et al., 2014). Among the cathepsins S, K, L, and B, inactivation by 15 was strongest for cathepsin S with approximately threefold increased kinact/KI-values for this cathepsin compared to compound 14. Probe 15 is more than 50-fold less potent for cathepsin K than for S. This result has been explained on the basis of docking studies, which revealed that the coumarin-tetrahydroquinoline moiety of 15 partially occupies the S3 pocket of cathepsin S and its position is probably stabilized by hydrogen bond interactions between its two carbonyl groups and the side chain of Arg141 and the backbone NH of Val162. This aptly illustrates that also the fluorophore may interact with the enzyme and thus can contribute to the selectivity of the probe. Compound 15 was evaluated for in-gel detection of cathepsin S in a similar fashion as 14 for cathepsin K. The fluorescent cathepsin S-15 complex enabled to visualize enzyme amounts as low as 0.5 ng. Preincubation of cathepsin S with the broad-spectrum cysteine protease inhibitor E64 (6) abolished its labeling by 15, which shows that inactivation is dependent on the catalytic activity and labeling does not occur due to indiscriminate reaction with nucleophiles on the protein surface. In addition, ABP 15 has been shown to be capable of detecting cathepsin S specifically in a protein extract derived from human placental tissue.

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