An activity-based probe for the determination of cysteine cathepsin protease activities in whole cells.

Abstract

We describe a novel diazomethylketone-containing irreversible inhibitor (BIL-DMK) which is specific for a subset of pharmaceutically important cysteine cathepsin proteases. BIL-DMK rapidly inactivates cathepsins B, F, K, L, S, and V in isolated enzyme assays and labels cathepsins in whole cells. The presence of catalytically active cathepsins B, L, and K or S was demonstrated using radioiodinated BIL-DMK in HepG2 (hepatoma), HIG82 (rabbit synoviocyte), and Ramos (B lymphoma) cell lines, respectively. The identity of each protein labeled was confirmed from the isoelectric point and molecular mass of the radioactive spots on two-dimensional gel and by comigration with each cathepsin as identified by immunoblotting. These cell lines were used to establish whole-cell enzyme occupancy assays to determine the potency of both irreversible and reversible inhibitors against each cathepsin in their native cellular lysosomal or endosomal environment. These whole-cell enzyme occupancy assays are useful to determine the cellular permeability of competing inhibitors and have the advantage of not requiring specific substrates for each cathepsin of interest.

• Authors: Jean-Pierre Falgueyret, W Cameron Black, Wanda Cromlish, Sylvie Desmarais, Sonia Lamontagne, Christophe Mellon, Denis Riendeau, Sevgi Rodan, Paul Tawa, Gregg Wesolowski, Kathryn E Bass, Shankar Venkatraman, M David Percival
• Journal: Analytical biochemistry (Anal Biochem) Vol. 335 Issue 2 Pg. 218-27 (Dec 15 2004) ISSN: 0003-2697 [Print] United States
• PMID: 15556560 (Publication Type: Comparative Study, Journal Article)

Chemical References

• Biphenyl Compounds
• Cysteine Proteinase Inhibitors
• Iodine Radioisotopes
• N-((1S)-1-(((1-(2-diazoacetyl)butyl)amino)carbonyl)-3-methylbutyl)-4'-iodo-(1,1'-biphenyl)-4-carboxamide
• diazomethyl ketones
• Diazomethane
• Cathepsins
• Cysteine Endopeptidases
• Cathepsin B
• CTSL protein, human
• Cathepsin L
• cathepsin S
• CTSK protein, human
• Cathepsin K
• Leucine

Topics

• Animals
• Autoradiography
• Biphenyl Compounds (pharmacology)
• Blotting, Western
• Cathepsin B (analysis, antagonists & inhibitors)
• Cathepsin K
• Cathepsin L
• Cathepsins (analysis, antagonists & inhibitors, metabolism)
• Cell Line, Tumor
• Cysteine Endopeptidases (analysis)
• Cysteine Proteinase Inhibitors (pharmacology)
• Diazomethane (analogs & derivatives, chemical synthesis, pharmacology)
• Humans
• Iodine Radioisotopes
• Leucine (analogs & derivatives, pharmacology)
• Rabbits