Cathepsin L (CTL) is a cysteine protease that demonstrates upregulated activity and/or altered trafficking in disease states such as cancer. The overlapping substrate specificity of cathepsin family members makes selective detection of activity from a single cathepsin difficult, and CTL activity is particularly difficult to parse from its close homologue CTV and the ubiquitous CTB. To overcome this, screening campaigns have explored the extended chemical space in the cathepsin binding sites and identified unique substrate structures that offer selectivity for one enzyme over the others. In this vein, we present the CTL-activable probe CTLAP, a fluorogenic probe that is rapidly activated by CTL and displays good selectivity over CTB and CTV, the closest competing analytes for CTL activity. We describe the kinetics of this compound against CTL, CTB, and CTV, and demonstrate its use in a rapid test for detection of CTL activity in a mixed-analyte sample. CTLAP demonstrates markedly higher turn-on ratios and improved enzyme selectivity when compared to Z-FR-AMC, a commercially available probe commonly used to detect CTL activity in mixed samples. Notably lower background fluorescence is observed for CTLAP compared to Z-FR-AMC, suggesting a possible internal quenching mechanism between the unique aromatic substrate sequence and the AMC reporter; investigation into the nature of this possible quenching phenomenon is currently underway. With low background signal, low cross-reactivity for CTB and CTV, and an optimal readout window within 5-10 min of sample incubation, CTLAP has possible applications in sensitive, selective, and rapid detection of CTL activity in clinical samples.

Fluorogenic probe for selective detection of cathepsin L

Macromolecular Chemistry: The Second Century

[Author Benefits](https://scimeetings.acs.org/?utm_source=pubs_content_marketing&utm_medium=website&utm_campaign=0320_MCF_NPI_Launch_Spring_Homepage&ref=pubs_content_marketing)

[How to use SciMeetings](https://storage.googleapis.com/dl.morressier.com/acs/SciMeetings-ACSSpring2020-HowToGuide_M3020-Final.pdf)

[Frequently Asked Questions](https://storage.googleapis.com/dl.morressier.com/acs/SciMtng_ACSNMSp2020_FAQ_M3120_Final.pdf)

ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.