RT Journal Article
SR Electronic
T1 Internally quenched fluorogenic probe provides selective and rapid detection of cathepsin L activity
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2020.03.28.012708
DO 10.1101/2020.03.28.012708
A1 Kelton A. Schleyer
A1 Ben Fetrow
A1 Peter Zannes Fatland
A1 Jun Liu
A1 Maya Chaaban
A1 Biwu Ma
A1 Lina Cui
YR 2020
UL http://biorxiv.org/content/early/2020/03/29/2020.03.28.012708.abstract
AB Cathepsin L (CTL) is a cysteine protease that demonstrates upregulated activity and/or altered trafficking during 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. Despite 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 others. In this vein, we present 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 probes. CTLAP exhibits intrinsically low background fluorescence, which we attribute to possible self-quenching mechanisms. CTLAP demonstrates markedly higher turn-on ratios (24-fold) and moderately improved enzyme selectivity compared to Z-FR-AMC (10-fold turn-on ratio), a commercially available CTL-selective probe commonly used to detect CTL activity in mixed samples. Optimum selectivity for CTL is achieved within 10 min of incubation with the enzyme, suggesting that CTLAP is amenable for rapid detection of CTL, even in the presence of competing cathepsins.