Abstract: Methods and materials for specific imaging of active enzyme in a live or intact cell are disclosed. The enzyme of interest tagged to reporter protein (donor) is exogenously expressed in a cell. The conversion of proenzyme to active enzyme (containing reporter protein) is achieved upon applying an appropriate stimulus to the target cells. The activated enzyme is labelled with an activity-based probe carrying a fluorophore (acceptor). The covalent labelling of active enzyme by the activity-based probe creates a FRET pair which provides the opportunity to exquisitely image the function of an “active enzyme”. This method is used for specific imaging of the function of active caspase-3,-7,-8,-9 and cathepsin-B and also for profiling of inhibitors of caspases and cathepsin B.

Abstract: The present disclosure relates to an assay for determining drug-target engagement in real-time. Specifically, the present disclosure provides a method for determining drug candidate-target interaction in real-time in living systems using Activity-based Reporter Gene Technology-Bioluminescence Resonance Energy Transfer (AbRGT-BRET) based assay. The said assay is useful for High Throughput Screening of compounds to a specific target.

Abstract: Methods and materials for specific imaging of active enzyme in a live or intact cell are disclosed. The enzyme of interest tagged to reporter protein (donor) is exogenously expressed in a cell. The conversion of proenzyme to active enzyme (containing reporter protein) is achieved upon applying an appropriate stimulus to the target cells. The activated enzyme is labelled with an activity-based probe carrying a fluorophore (acceptor). The covalent labelling of active enzyme by the activity-based probe creates a FRET pair which provides the opportunity to exquisitely image the function of an “active enzyme”. This method is used for specific imaging of the function of active caspase-3,-7,-8,-9 and cathepsin-B and also for profiling of inhibitors of caspases and cathepsin B.