title: The application of a novel, cell permeable activity-based probe for the detection of cysteine cathepsins.
abstract: Cysteine cathepsins, such as cathepsin S (CTSS), are implicated in the pathology of a wide range of diseases and are of potential utility as diagnostic and prognostic biomarkers. In previous work, we demonstrated the potency and efficiency of a biotinylated diazomethylketone (DMK)-based activity-based probe (ABP), biotin-PEG-LVG-DMK, for disclosure of recombinant CTSS and CTSS in cell lysates. However, the limited cell permeability of both the biotin and spacer groups restricted detection of CTSS to cell lysates. The synthesis and characterisation of a cell permeable ABP to report on intracellular CTSS activity is reported. The ABP, Z-PraVG-DMK, a modified peptidyl diazomethylketone, was based on the N-terminus of human cystatin motif (Leu-Val-Gly). The leucine residue was substituted for the alkyne-bearing proparcylglycine to facilitate conjugation of an azide-tagged reporter group using click chemistry, following irreversible inhibition of CTSS. When incubated with viable Human Embryonic Kidney 293 cells, Z-PraVG-DMK permitted disclosure of CTSS activity following cell lysis and rhodamine azide conjugation, by employing standard click chemistry protocols. Furthermore, the fluorescent tag facilitated direct detection of CTSS using in-gel fluorescent scanning, obviating the necessity for downstream biotin-streptavidin conjugation and detection procedures.
authors.affiliation: School of Pharmacy, Queen's University Belfast, 97 Lisburn Road, Belfast, BT9 7BL, UK.
authors.forename: Caroline S
authors.initials: CS
authors.lastname: Hughes
authors.affiliation: School of Pharmacy, Queen's University Belfast, 97 Lisburn Road, Belfast, BT9 7BL, UK.
authors.forename: George
authors.initials: G
authors.lastname: Shaw
authors.affiliation: School of Pharmacy, Queen's University Belfast, 97 Lisburn Road, Belfast, BT9 7BL, UK.
authors.forename: Roberta E
authors.initials: RE
authors.lastname: Burden
authors.affiliation: School of Pharmacy, Queen's University Belfast, 97 Lisburn Road, Belfast, BT9 7BL, UK.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Scott
authors.affiliation: School of Pharmacy, Queen's University Belfast, 97 Lisburn Road, Belfast, BT9 7BL, UK. Electronic address: b.gilmore@qub.ac.uk.
authors.forename: Brendan F
authors.initials: BF
authors.lastname: Gilmore
mesh_terms: D002403:Cathepsins / Q000032:analysis*; D002463:Cell Membrane Permeability; D057930:Click Chemistry / Q000379:methods*; D003545:Cysteine / Q000032:analysis*; D004789:Enzyme Activation; D057809:HEK293 Cells; D006801:Humans; D015336:Molecular Probe Techniques; D015335:Molecular Probes / Q000737:chemistry*
chemical_list: D015335:Molecular Probes; D002403:Cathepsins; C028217:cathepsin S; D003545:Cysteine
keywords: Affinity-binding probe; Cathepsin; Click chemistry