Design And Application Of Cysteine Cathepsin Activity-Based Melecular Probes

Objective:The World Health Organization?WHO?statistic data shows that the top five diseases cause death worldwide are ischaemic heart disease,stroke,respiratory infections,chronic obstructive pulmonary disease and cancers.These diseases have remained the top killers during the past decade.Therefore,early diagnosis,effective treatment and accurate evaluation of these diseases are of great significance and have become an important direction in the current biomedical researches.We found that cysteine Cathepsin?Cat?plays an important role in the pathological process of various diseases.Thus,selective tools that can discriminate between members of this highly related Cat will be critical to further delineate the unique biological functions of individual cathepsins.However,the current challenge is to identify their endogenous substrates,in order to gain an insight into the mechanisms of substrate degradation and processing.Therefore,targeting the cysteine cathepsin as a target,designing specifically targeted“smart”probes and exploring its application in cardiovascular diseases,respiratory diseases and cancers is the main purpose of this study.Method:Five probes,GB123,BMV109,BMV101,and BMX2 with combinations of the AOMK electrophiles were synthesized using an optimized,solution-chemistry-based procedure.In the pulmonary fibrosis study,female C57BL/6J mice?20–25 g?were treated on day 0 with1.5 unit/kg body weight of bleomycin by endotracheal instillation and control animals received sterile saline.At the indicated times?day 7,14 and day 21?,the probe was injected to mice through tail vein and were imaged non-invasively and quantified using the IVIS 100system and microPET/CT at different time point post injection.Animals were sacrificed under isoflurane anesthesia and BAL fluid extracted as previously describe.Lungs were then removed,imaged ex vivo using an FMT system.Five patients with IPF or IPF-related condition were enrolled in this imaging study after obtaining written informed consent.Controls without lung disease were recruited from patients undergoing PET/CT scans for non-pulmonary indications.For atherosclerosis,the carotid lesions were induced in FVB mice fed on a high-fat diet by streptozotocin injection followed by ligation of the left common carotid artery.Mice with carotid atherosclerotic plaques were injected with the optical or dual-modality probes BMV109 and BMV101,respectively,via the tail vein and noninvasively imaged by optical and small-animal PET/CT at different time points.After noninvasive imaging,the murine carotid arteries were imaged in situ and ex vivo,followed by immuno-fluorescence staining to confirm target labeling.Additionally,human carotid plaques were topically labeled with the probe and analyzed by both sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunofluorescence staining to confirm the primary targets of the probe.For cancer imaging study,U87MG,MDA-MB-231,4T1,and PC3 cells were cultured and then were lysed to test the amounts of Cat expression by western blot.Cells were also treated with BMV109 probe and co-stained with cathepsine antibody and then were imaged with confocal.Subcutaneously 4T1 tumor bearing mice were injected with 100?Ci ⁶⁸Ga-BMV101 via the tail vein and were set to optical imaging and PET imaging studies.After imaging,all tumors and normal organs were resected and weighed,and radioactivity was measured by gamma-counter.The radioactivity uptake in the tumor and normal tissues was expressed as a percentage of the injected radioactive dose per gram of tissue?%ID/g?.Tumors were then homogenized and total protein was labeled with GB123?1 mM final concentration?for 1 h at 37?and were resolved on SDS-PAGE?15%?.Labeled proteases were visualized by scanning the gel and labeling intensities were quantified.Statistics were performed using the data analysis package within GraphPad Prism 6.0.For the human studies,SUVmax values from subjects and controls were compared using the Wilcoxon Mann Whitney U test to account for non-normally distributed data.Statistical significance was defined as a p value<0.05.Unless otherwise stated,tests comparing two means are a Student's t-test,with equal variance assumed.Error bars indicate standard error of the mean?SEM?unless otherwise stated.Result:Cat targeting ABP probes were synthesized successfully and purified for later use?purity of all probes over 99.5%?.With direct administration of bleomycin into the lungs,the animals were being highly effective in eliciting fibrosis and showed extent of probe accumulation in the lungs,focusing on a time course between 7 and 14 days,a time period in which immune cell in filtration and initiation of fibrosis takes place.Lung tissue at day 7 and at day 14 showed histological evidence of pulmonary fibrosis.At Day 7,bleomycin-treated mice showed significantly higher fluorescence signal in the lungs when compared to the saline controls.Signal intensity further increased at Day 14.The levels of signal observed using non-invasive imaging in the lungs directly correlated with the progression of the disease.Subsequent analysis of lung tissues by western blot revealed increased levels of active cathepsins in the bleomycin-induced lung samples and confirmed that the primary targets of the probe were cathepsins X,B,S and L,and that levels of labelling of these targets correlated with the extent of disease burden and intensity of signals observed by non-invasive imaging.In the atherosclerosis study,quantitative analysis of the signal intensity from both optical and PET/CT imaging showed significantly higher levels of accumulation of BMV109 and BMV101 in the ligated left carotid arteries than the right carotid or healthy arteries?P<0.005 and P<0.05,respectively?.Immunofluorescence staining for macrophages in cross-sectional slices of the murine artery demonstrated substantial infiltration of macrophages in the neointima and adventitia of the ligated left carotid arteries compared with the right.Analysis of the human plaque tissues by sodium dodecyl sulfate polyacrylamide gel electrophoresis confirmed that the primary targets of the probe were cathepsins X,B,S,and L.Immunofluorescence labeling of the human tissue with the probe demonstrated co-localization of the probe with CD68,elastin,and cathepsin S,similar to that observed in the experimental carotid inflammation murine model.In cancer imaging study,we found that the MDA-MB-231,4T1 and PC3 cell lines have high Cat-B expression except for U87MG.However,all cell lines have a certain degree of Cat-S and Cat-L expression.At the cellular level,after co-incubation with 4T1 breast cancer tumor cells with BMX2 probes and Cat antibody S-12,the confocal microscopy imaging showed BMX2 cell uptake and co-localized with Cat expression.In PET/CT,Cerenkov and optical imaging study,4T1 breast cancer bearing mice injected with ⁶⁸Ga-BMX2 showed that the tumor had a higher uptake of ⁶⁸Ga-BMX2 and increased with time,while blocking with Cat inhibitor significantly decreased the uptake of ⁶⁸Ga-BMX2 in the tumor,confirming the specificity of the probe in the tumor,which consistent with biodistribution results.Conclusion:In this project we presented the design and synthesis of the activity-based probes,BMV109,GB123,BMV101,and BMX2,that selectively targets cathepsins which is highly expressed in immune cells and cancer cells.Importantly,this high degree of selectivity is retained both in vitro and in vivo of IPF,atherosclerosis,and cancers.The probes could also be used to topically label human carotid plaques demonstrating similar specific labeling of activated macrophage populations.Therefore,ABPs targeting the cysteine cathepsins are potentially valuable new reagents for rapid and noninvasive imaging the progression of disease both in pre-clinic and clinic.