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

Anal Biochem. 2004 Dec 15;335(2):218-27. doi: 10.1016/j.ab.2004.09.005.


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.

Publication types

  • Comparative Study

MeSH terms

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


  • 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