title: Bifunctional inhibitors of the trypsin-like activity of eukaryotic proteasomes.
abstract: BACKGROUND
  The 20S proteasome is a multicatalytic protease complex that exhibits trypsin-like, chymotrypsin-like and post-glutamyl-peptide hydrolytic activities associated with the active sites of the beta2, beta5 and beta1 subunits, respectively. Modulation of these activities using inhibitors is essential for a better understanding of the proteasome's mechanism of action. Although there are highly selective inhibitors of the proteasome's chymotryptic activity, inhibitors of similar specificity have not yet been identified for the other activities.
  
  
  RESULTS
  The X-ray structure of the yeast proteasome reveals that the sidechain of Cys118 of the beta3 subunit protrudes into the S3 subsite of the beta2 active site. The location of this residue was exploited for the rational design of bidentated inhibitors containing a maleinimide moiety at the P3 position for covalent linkage to the thiol group and a carboxy-terminal aldehyde group for hemiacetal formation with the Thr1 hydroxyl group of the active site. Structure-based modelling was used to determine the optimal spacing of the maleinimide group from the P2-P1 dipeptide aldehydes and the specificity of the S1 subsite was exploited to limit the inhibitory activity to the beta2 active site. X-ray crystallographic analysis of a yeast proteasome-inhibitor adduct confirmed the expected irreversible binding of the inhibitor to the P3 subsite.
  
  
  CONCLUSIONS
  Maleoyl-beta-alanyl-valyl-arginal is a new type of inhibitor that is highly selective for the trypsin-like activity of eukaryotic proteasomes. Despite the reactivity of the maleinimide group towards thiols, and therefore the limited use of this inhibitor for in vitro studies, it might represent an interesting new biochemical tool.
authors: Max-Planck-Institut für Biochemie, 82152 Martinsried, Germany.
authors: G
authors: G
authors: Loidl
authors: M
authors: M
authors: Groll
authors: H J
authors: HJ
authors: Musiol
authors: L
authors: L
authors: Ditzel
authors: R
authors: R
authors: Huber
authors: L
authors: L
authors: Moroder
mesh_terms: D001665:Binding Sites; D018360:Crystallography, X-Ray; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D004151:Dipeptides / Q000138:chemical synthesis / Q000494:pharmacology*; D015195:Drug Design; D008958:Models, Molecular; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D014357:Trypsin / Q000737:chemistry; D014361:Trypsin Inhibitors / Q000138:chemical synthesis* / Q000494:pharmacology
chemical_list: D004151:Dipeptides; D009097:Multienzyme Complexes; D014361:Trypsin Inhibitors; D014357:Trypsin; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex