In vitro and ex vivo inhibition of hepatitis a virus 3C proteinase by a peptidyl monofluoromethyl ketone

Article abstract of DOI:10.1016/S0968-0896(97)88649-X

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 10² 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 μM inhibitor 24 h postinfection.