title: Probing cathepsin S activity in whole blood by the activity-based probe BIL-DMK: cellular distribution in human leukocyte populations and evidence of diurnal modulation.
abstract: Using the cell-permeable, radioiodinated, irreversible inhibitor BIL-DMK, we probed active cysteine cathepsins in blood. Incubation of the probe in human whole blood followed by separation of white blood cells by dextran sedimentation led to the labeling of one major band at 24kDa. Two-dimensional gel electrophoresis showed that the band resolved in a single protein spot and corresponded to cathepsin S based on its molecular mass, isoelectric point, and Western blot analysis using anti-human cathepsin S antibodies. Cathepsin S activity in human whole blood was dependent on the time of blood collection, suggesting that cathepsin S activity is subject to circadian variations. Separation of white blood cell populations using a magnetic cell sorter and further characterization by FACS (fluorescent-activated cell sorting) analysis demonstrated that the majority of active cathepsin S resided in the monocyte and neutrophil populations, whereas on a cell basis cathepsin S activity in granulocytes is 10-fold lower than that in monocytes. A whole blood cathepsin S assay was developed and used to measure cathepsin S inhibition in both in vitro and ex vivo conditions. To determine the correlation between the in vitro and ex vivo assays, a reversible cathepsin S inhibitor was dosed intravenously to a rhesus monkey. The inhibitor concentration required to inhibit 50% of the cathepsin S activity ex vivo correlated well with the concentration required to inhibit the enzyme in rhesus monkey whole blood in vitro. The results reported here demonstrate the utility of the activity-based probe BIL-DMK for the ex vivo assessment of cathepsin S inhibition.
authors.affiliation: Merck Frosst Centre for Therapeutic Research, Kirkland, Quebec, Canada.
authors.forename: Alain
authors.initials: A
authors.lastname: Veilleux
authors.forename: W Cameron
authors.initials: WC
authors.lastname: Black
authors.forename: Jacques Yves
authors.initials: JY
authors.lastname: Gauthier
authors.forename: Christophe
authors.initials: C
authors.lastname: Mellon
authors.forename: M David
authors.initials: MD
authors.lastname: Percival
authors.forename: Paul
authors.initials: P
authors.lastname: Tawa
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Falgueyret
mesh_terms: D000818:Animals; D001713:Biphenyl Compounds / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D015153:Blotting, Western; D056657:Cathepsin K / Q000037:antagonists & inhibitors / Q000378:metabolism; D002403:Cathepsins / Q000097:blood*; D002469:Cell Separation; D002940:Circadian Rhythm / Q000187:drug effects; D003545:Cysteine / Q000378:metabolism; D006801:Humans; D007930:Leucine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D007962:Leukocytes / Q000201:enzymology*; D008253:Macaca mulatta; D021381:Protein Transport / Q000187:drug effects; D013997:Time Factors
chemical_list: D001713:Biphenyl Compounds; C527129:L-873724; C503724:N-((1S)-1-(((1-(2-diazoacetyl)butyl)amino)carbonyl)-3-methylbutyl)-4'-iodo-(1,1'-biphenyl)-4-carboxamide; D002403:Cathepsins; C028217:cathepsin S; D056657:Cathepsin K; D007930:Leucine; D003545:Cysteine