Dual‐Mechanism Quenched Fluorogenic Probe Provides Selective and Rapid Detection of Cathepsin L Activity**

Cathepsin L (CTL) is a cysteine protease demonstrating upregulated activity in many disease states. Overlapping substrate specificity makes selective detection of CTL activity difficult to parse from that of its close homologue CTV and the ubiquitous CTB. Current probes of CTL activity have limited applications due to either poor contrast or extra assay steps required to achieve selectivity. We have developed a fluorogenic probe, CTLAP, that displays good selectivity for CTL over CTB and CTV while exhibiting low background fluorescence attributed to dual quenching mechanisms. CTLAP achieves optimum CTL selectivity in the first 10 min of incubation, thus suggesting that it is amenable for rapid detection of CTL, even in the presence of competing cathepsins.