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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

Radiometal-based cysteine-cathepsin targeting radiotracers.
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Figure 12: Radiometal-based cysteine-cathepsin targeting radiotracers.

Mentions: Most of the known radionuclides are of metallic character, which is simply due the fact that the majority of the chemical elements are metals. Many among them are valuable for SPECT and PET imaging (Wadas et al., 2010). A well-studied metallic PET nuclide is copper-64 (Table 1). Compared to fluorine-18, it offers the advantage of extended half-life and coordinative bond formation under rather mild conditions. However, the latter advantage implicates the drawback of modification with spatially demanding chelators for radiotracer design, which potentially can compromise target interaction, especially in the case of small molecules. The 64Cu-labeled acyloxymethyl ketones shown in Figure 12 have been the first reported PET tracers which target cysteine cathepsins (Ren et al., 2011). The design of compound [64Cu]40 was guided by the structure of GB123, a fluorescent AOMK capable of cathepsin B and L optical imaging7. Its Cy5 fluorophore has been replaced by the macrocyclic chelator DOTA which has been connected via an amide bond with one of its carboxymethyl groups to the Nε of the lysine in P1 to result in the labeling precursor of [64Cu]40. To enable equipment with a copper-64 and fluorescent label, the Z-group was replaced by phenylalanine and the chelator relocated from the lysine side chain to the N-terminus, which provided the vacancy to attach a Cy5 label (precursor for [64Cu]41). To be able to compare the activity-based probe [64Cu]41 with a substrate analog as control, its corresponding primary amide [64Cu]42 was prepared. Labeling with copper-64 was done by incubating the chelator-functionalized precursors with [64Cu]CuCl2 in aqueous solution at pH 5.5 and 50°C to obtain the corresponding radiotracers in high radiochemical purity and specific activities ranging between 1.1 and 17.8 GBq/μmol. The whole-body biodistribution of the three 64Cu-labeled probes was studied in athymic nude mice bearing subcutaneously grafted tumors derived from the human breast cancer cell line MDA-MB-435 and the oncogenically transformed murine C2C12/Ras myeloblastoma line. The tumor uptake of [64Cu]40 at 24 h p.i. was significantly higher in the C2C12/Ras tumors, which correlates with a higher cysteine cathepsin activity in this cell line compared to MDA-MB-435. The difference in the absolute uptake is also reflected in differing tumor/muscle ratios. While the radiotracer exhibited a low uptake in most non-tumor tissues, activity accumulation in the liver was high. This observation has been interpreted to result from de- or transchelation of the 64Cu-copper complex, which is in accordance with results that render DOTA suboptimal for stable complexation of Cu2+ ions (Maheshwari et al., 2012; Cai and Anderson, 2014; Price and Orvig, 2014; Zarschler et al., 2014). Interestingly, the uptake of the bimodal probe [64Cu]41 in the C2C12/Ras tumors was more than 10 times higher than that of [64Cu]40 with a tumor/muscle ratio of 13 at 24 h p.i. This phenomenon might be due to the amphiphilic character conferred by the Cy5 moiety. Also the uptake of the corresponding substrate [64Cu]42 was higher compared to [64Cu]40, but considerably lower than that of its AOMK analog [64Cu]41. Notably, the tumor/muscle ratio at 2 h p.i. for the substrate [64Cu]42 was in the same range as that for the irreversible inhibitor [64Cu]40. μPET investigations mainly confirmed the results of the biodistribution study. PET imaging included also the investigation of the bimodal AOMK-based probe [64Cu]41 in mice bearing murine 4T1 tumors with high cysteine cathepsin expression. Subsequent to the end point of imaging at 24 p.i., the cathepsin activity was investigated in tumor homogenates for all three cell lines by SDS-PAGE. This was performed both by quantifying the fluorescence originating from the [64Cu]41-cathepsin complexes as well as by analyzing the residual cysteine cathepsin activity upon addition of the fluorescence-only probe GB123 to the tumor homogenates. For both methods the measured in-gel fluorescence signals correlated well with the tumor/muscle ratios determined by PET imaging. Furthermore, the tumor uptake of [64Cu]41 was reduced by more than factor 2 upon pretreatment with large amounts of 4, a vinyl sulfone-based broad spectrum inhibitor of cysteine cathepsins, in mice carrying tumors derived from the human breast cancer cell line MDA-MB-231MFP. In conclusion, the work of Ren et al. established very well that the intratumoral imaging signals of [64Cu]41 are associated with the cysteine cathepsin activity. Moreover, the superiority of [64Cu]41 over [64Cu]40 demonstrates that the conjugation of radiotracers with fluorophores may improve their pharmacokinetic properties in addition to providing the opportunity of bimodal imaging.


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

Löser R, Pietzsch J - Front Chem (2015)

Radiometal-based cysteine-cathepsin targeting radiotracers.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 12: Radiometal-based cysteine-cathepsin targeting radiotracers.
Mentions: Most of the known radionuclides are of metallic character, which is simply due the fact that the majority of the chemical elements are metals. Many among them are valuable for SPECT and PET imaging (Wadas et al., 2010). A well-studied metallic PET nuclide is copper-64 (Table 1). Compared to fluorine-18, it offers the advantage of extended half-life and coordinative bond formation under rather mild conditions. However, the latter advantage implicates the drawback of modification with spatially demanding chelators for radiotracer design, which potentially can compromise target interaction, especially in the case of small molecules. The 64Cu-labeled acyloxymethyl ketones shown in Figure 12 have been the first reported PET tracers which target cysteine cathepsins (Ren et al., 2011). The design of compound [64Cu]40 was guided by the structure of GB123, a fluorescent AOMK capable of cathepsin B and L optical imaging7. Its Cy5 fluorophore has been replaced by the macrocyclic chelator DOTA which has been connected via an amide bond with one of its carboxymethyl groups to the Nε of the lysine in P1 to result in the labeling precursor of [64Cu]40. To enable equipment with a copper-64 and fluorescent label, the Z-group was replaced by phenylalanine and the chelator relocated from the lysine side chain to the N-terminus, which provided the vacancy to attach a Cy5 label (precursor for [64Cu]41). To be able to compare the activity-based probe [64Cu]41 with a substrate analog as control, its corresponding primary amide [64Cu]42 was prepared. Labeling with copper-64 was done by incubating the chelator-functionalized precursors with [64Cu]CuCl2 in aqueous solution at pH 5.5 and 50°C to obtain the corresponding radiotracers in high radiochemical purity and specific activities ranging between 1.1 and 17.8 GBq/μmol. The whole-body biodistribution of the three 64Cu-labeled probes was studied in athymic nude mice bearing subcutaneously grafted tumors derived from the human breast cancer cell line MDA-MB-435 and the oncogenically transformed murine C2C12/Ras myeloblastoma line. The tumor uptake of [64Cu]40 at 24 h p.i. was significantly higher in the C2C12/Ras tumors, which correlates with a higher cysteine cathepsin activity in this cell line compared to MDA-MB-435. The difference in the absolute uptake is also reflected in differing tumor/muscle ratios. While the radiotracer exhibited a low uptake in most non-tumor tissues, activity accumulation in the liver was high. This observation has been interpreted to result from de- or transchelation of the 64Cu-copper complex, which is in accordance with results that render DOTA suboptimal for stable complexation of Cu2+ ions (Maheshwari et al., 2012; Cai and Anderson, 2014; Price and Orvig, 2014; Zarschler et al., 2014). Interestingly, the uptake of the bimodal probe [64Cu]41 in the C2C12/Ras tumors was more than 10 times higher than that of [64Cu]40 with a tumor/muscle ratio of 13 at 24 h p.i. This phenomenon might be due to the amphiphilic character conferred by the Cy5 moiety. Also the uptake of the corresponding substrate [64Cu]42 was higher compared to [64Cu]40, but considerably lower than that of its AOMK analog [64Cu]41. Notably, the tumor/muscle ratio at 2 h p.i. for the substrate [64Cu]42 was in the same range as that for the irreversible inhibitor [64Cu]40. μPET investigations mainly confirmed the results of the biodistribution study. PET imaging included also the investigation of the bimodal AOMK-based probe [64Cu]41 in mice bearing murine 4T1 tumors with high cysteine cathepsin expression. Subsequent to the end point of imaging at 24 p.i., the cathepsin activity was investigated in tumor homogenates for all three cell lines by SDS-PAGE. This was performed both by quantifying the fluorescence originating from the [64Cu]41-cathepsin complexes as well as by analyzing the residual cysteine cathepsin activity upon addition of the fluorescence-only probe GB123 to the tumor homogenates. For both methods the measured in-gel fluorescence signals correlated well with the tumor/muscle ratios determined by PET imaging. Furthermore, the tumor uptake of [64Cu]41 was reduced by more than factor 2 upon pretreatment with large amounts of 4, a vinyl sulfone-based broad spectrum inhibitor of cysteine cathepsins, in mice carrying tumors derived from the human breast cancer cell line MDA-MB-231MFP. In conclusion, the work of Ren et al. established very well that the intratumoral imaging signals of [64Cu]41 are associated with the cysteine cathepsin activity. Moreover, the superiority of [64Cu]41 over [64Cu]40 demonstrates that the conjugation of radiotracers with fluorophores may improve their pharmacokinetic properties in addition to providing the opportunity of bimodal imaging.

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