FUNCTIONAL ASSESSMENT OF A JP342 FLUORESCENT PROBE FOR CATHEPSIN G DETECTION IN TUMOR CELLS

Abstract

Cathepsin G (CatG) is a serine protease secreted by activated neutrophils that performs essential functions related to immune defense, inflammation, and cancer. Accurately detecting its enzymatic activity is crucial for understanding the role of CatG in health and disease. This project assesses an innovative boron-dipyrromethene (BODIPY) labeled activity-based probe JP342 for the detection of CatG, while comparing its efficacy with the established MARS116-FAM probe across different experimental conditions. Of these, SDS-PAGE, flow cytometry, and confocal microscopy were applied to both JP342 and MARS116-FAM. JP342 and MARS116-FAM exhibited a concentration-dependent affinity for labeling purified CatG, with MARS116-FAM showing greater sensitivity at all tested concentrations in gel-based assays and confocal microscopy. In contrast, the flow cytometry analysis determined that JP342 exhibited a markedly elevated median fluorescence intensity (MFI), irrespective of the presence of a CatG-specific inhibitor, which suggests a higher affinity of JP342 towards CatG in flow cytometry. Overall, the findings indicate that JP342 serves as a compelling activity-based probe for detecting CatG activity in flow cytometry applications, while MARS116-FAM continues to demonstrate optimal performance in gel-based assays and cell imaging. These results emphasize the importance of matching the activity-based probe to the type of experiment.