title: Biotin-labelled peptidyl diazomethane inhibitors derived from the substrate-like sequence of cystatin: targeting of the active site of cruzipain, the major cysteine proteinase of Trypanosoma cruzi.
abstract: Biotin-labelled peptidyl diazomethane inhibitors of cysteine proteinases, based on the N-terminal substrate-like segment of human cystatin C, a natural inhibitor of cysteine proteinases, were synthesized. These synthetic derivatives were tested as irreversible inhibitors of cruzipain, the major cysteine proteinase of Trypanosoma cruzi, to compare the kinetics of the inhibition of the parasite proteinase with that of the mammalian cathepsins B and L. The accessibility of the active sites of these proteinases to these probes was also investigated. The inhibition of cruzipain by Biot-LVG-CHN2 (where Biot represents biotinyl and L,V and G are single-letter amino acid residue abbreviations) and Biot-Ahx-LVG-CHN2 (where Ahx represents 6-aminohexanoic acid) was similar to that of unlabelled inhibitor. Biotin labelling of the inhibitor slowed the inhibition of both cathepsin B and cathepsin L. Adding a spacer arm (Ahx) between the biotin and the peptide moiety of the derivative increased the inhibition of cathepsin B but not that of cathepsin L. The discrimination provided by this spacer is probably due to differences in the topologies of the binding sites of proteinases, a feature that can be exploited to improve targeting of individual cysteine proteinases. Analysis of the blotted proteinases revealed marked differences in the accessibility of extravidin-peroxidase conjugate to the proteinase-bound biotinylated inhibitor. Cruzipain molecules exposed to Biot-LVG-CHN2 or Biot-Ahx-LVG-CHN2 were readily identified, but the reaction was much stronger when the enzyme was treated with the spacer-containing inhibitor. In contrast with the parasite enzyme, rat cathepsin B and cathepsin L treated with either Biot-LVG-CHN2 or Biot-Ahx-LVG-CHN2 produced no detectable bands. Papain, the archetype of this family of proteinases, was poorly labelled with Biot-LVG-CHN2, but strong staining was obtained with Biot-Ahx-LVG-CHN2. These findings suggest that optimized biotinylated diazomethanes might considerably improve their selectivity for the T. cruzi target enzyme.
authors.affiliation: Enzymologie et Chimie des Protéines, CNRS URA 1334, Faculté de Médecine, Université François Rabelais, Tours, France.
authors.forename: G
authors.initials: G
authors.lastname: Lalmanach
authors.forename: R
authors.initials: R
authors.lastname: Mayer
authors.forename: C
authors.initials: C
authors.lastname: Serveau
authors.forename: J
authors.initials: J
authors.lastname: Scharfstein
authors.forename: F
authors.initials: F
authors.lastname: Gauthier
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001710:Biotin; D002401:Cathepsin B / Q000037:antagonists & inhibitors; D056668:Cathepsin L; D002403:Cathepsins / Q000037:antagonists & inhibitors; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D003978:Diazomethane; D010450:Endopeptidases; D006801:Humans; D007700:Kinetics; D008322:Mammals; D008961:Models, Structural; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D011487:Protein Conformation; D015800:Protozoan Proteins; D051381:Rats; D014349:Trypanosoma cruzi / Q000201:enzymology*
chemical_list: D015853:Cysteine Proteinase Inhibitors; D009842:Oligopeptides; D015800:Protozoan Proteins; D003978:Diazomethane; D001710:Biotin; D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D002401:Cathepsin B; C534281:CTSL protein, human; D056668:Cathepsin L; C534280:Ctsl protein, rat; C063214:cruzipain