title: Development and validation of a simple cell-based fluorescence assay for dipeptidyl peptidase 1 (DPP1) activity.
abstract: Dipeptidyl peptidase 1 (DPP1) (EC 3.4.14.1; also known as cathepsin C, cathepsin J, dipeptidyl aminopeptidase, and dipeptidyl aminotransferase) is a lysosomal cysteinyl protease of the papain family involved in the intracellular degradation of proteins. Isolated enzyme assays for DPP1 activity using a variety of synthetic substrates such as dipeptide or peptide linked to amino-methyl-coumarin (AMC) or other fluorophores are well established. There is, however, no report of a simple whole-cell-based assay for measuring lysosomal DPP1 activity other than the use of flow cytometry (fluorescence-activated cell sorting) or the use of invasive activity-based probes or the production of physiological products such as neutrophil elastase. The authors investigated a number of DPP1 fluorogenic substrates that have the potential to access the lysosome and enable the measurement of DPP1 enzyme activity in situ. They describe the development and evaluation of a simple noninvasive fluorescence assay for measuring DPP1 activity in fresh or cryopreserved human THP-1 cells using the substrate H-Gly-Phe-AFC (amino-fluoro-coumarin). This cell-based fluorescence assay can be performed in a 96-well plate format and is ideally suited for determining the cell potency of potential DPP1 enzyme inhibitors.
authors.affiliation: Bioscience Department, AstraZeneca R&D Charnwood, Loughborough, Leicestershire, UK. bob.thong@astrazeneca.com
authors.forename: Bob
authors.initials: B
authors.lastname: Thong
authors.forename: James
authors.initials: J
authors.lastname: Pilling
authors.forename: Edward
authors.initials: E
authors.lastname: Ainscow
authors.forename: Raj
authors.initials: R
authors.lastname: Beri
authors.forename: John
authors.initials: J
authors.lastname: Unitt
mesh_terms: D049933:Aminocoumarins / Q000378:metabolism*; D000818:Animals; D001681:Biological Assay; D020789:Cathepsin C / Q000032:analysis*; D045744:Cell Line, Tumor; D004151:Dipeptides / Q000378:metabolism*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism*; D006801:Humans; D042541:Intracellular Space / Q000201:enzymology; D008247:Lysosomes / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D049933:Aminocoumarins; D004151:Dipeptides; D005456:Fluorescent Dyes; C029660:glycylphenylalanine; D020789:Cathepsin C