title: In vitro and ex vivo inhibition of hepatitis A virus 3C proteinase by a peptidyl monofluoromethyl ketone.
abstract: Hepatitis A virus (HAV) 3C proteinase is the enzyme responsible for the processing of the viral polyprotein. Although a cysteine proteinase, it displays an active site configuration like those of the mammalian serine proteinases (Malcolm, B. A. Protein Science 1995, 4, 1439). A peptidyl monofluoromethyl ketone (peptidyl-FMK) based on the preferred peptide substrates for HAV 3C proteinase was generated by first coupling the precursor, N,N-dimethylglutamine fluoromethylalcohol, to the tripeptide, Ac-Leu-Ala-Ala-OH, and then oxidizing the product to the corresponding peptidyl-FMK (Ac-LAAQ'-FMK). This molecule was found to be an irreversible inactivator of HAV 3C with a second-order rate constant of 3.3 x 10(2) M-1 s-1. 19F NMR spectroscopy indicates the displacement of fluoride on inactivation of the enzyme by the fluoromethyl ketone. NMR spectroscopy of the complex between the 13C-labeled inhibitor and the HAV 3C proteinase indicates that an (alkylthio)methyl ketone is formed. Studies of polyprotein processing, using various substrates generated by in vitro transcription/translation, demonstrated efficient blocking of even the most rapid proteolytic events such as cleavage of the 2A-2B and 2C-3A junctions. Subsequent ex vivo studies, to test for antiviral activity, show a 25-fold reduction in progeny virus production as the result of treatment with 5 microM inhibitor 24 h post-infection.
authors.affiliation: Hepatitis Viruses Section, National Institute of Allergies and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-0740, USA.
authors.forename: T S
authors.initials: TS
authors.lastname: Morris
authors.forename: S
authors.initials: S
authors.lastname: Frormann
authors.forename: S
authors.initials: S
authors.lastname: Shechosky
authors.forename: C
authors.initials: C
authors.lastname: Lowe
authors.forename: M S
authors.initials: MS
authors.lastname: Lall
authors.forename: V
authors.initials: V
authors.lastname: Gauss-Müller
authors.forename: R H
authors.initials: RH
authors.lastname: Purcell
authors.forename: S U
authors.initials: SU
authors.lastname: Emerson
authors.forename: J C
authors.initials: JC
authors.lastname: Vederas
authors.forename: B A
authors.initials: BA
authors.lastname: Malcolm
mesh_terms: D000086042:3C Viral Proteases; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000187:drug effects* / Q000235:genetics; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D006507:Hepatovirus / Q000187:drug effects* / Q000201:enzymology* / Q000502:physiology; D007700:Kinetics; D008253:Macaca mulatta; D009842:Oligopeptides / Q000494:pharmacology*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000378:metabolism; D014779:Virus Replication / Q000187:drug effects
chemical_list: D000998:Antiviral Agents; D015853:Cysteine Proteinase Inhibitors; D009842:Oligopeptides; D011994:Recombinant Proteins; D014764:Viral Proteins; C107860:acetyl-leucyl-alanyl-alanyl-N,N-dimethylglutamine fluoromethyl ketone; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases