IN-VITRO AND EX-VIVO INHIBITION OF HEPATITIS-A VIRUS 3C PROTEINASE BYA PEPTIDYL MONOFLUOROMETHYL KETONE

Hepatitis A virus (HAV) 3C proteinase is the enzyme responsible for th e 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 p eptidyl monofluoromethyl ketone (peptidyl-FMK) based on the preferred peptide substrates for HAV 3C proteinase was generated by first coupli ng the precursor, N,N-dimethylglutamine fluoromethylalcohol, to the tr ipeptide, Ac-Leu-Ala-Ala-OH, and then oxidizing the product to the cor responding peptidyl-FMK (Ac-LAAQ'-FMK). This molecule was found to be an irreversible inactivator of HAV 3C with a second-order rate constan t of 3.3 x 10(2) M-1 s(-1). F-19 NMR spectroscopy indicates the displa cement of fluoride on inactivation of the enzyme by the fluoromethyl k etone. NMR spectroscopy of the complex between the C-13-labeled inhibi tor and the HAV 3C proteinase indicates that an (alkylthio)methyl keto ne is formed. Studies of polyprotein processing, using various substra tes generated by in vitro transcription/translation, demonstrated effi cient blocking of even the most rapid proteolytic events such as cleav age of the 2A-2B and 2C-3A junctions. Subsequent ex vivo studies, to t est for antiviral activity, show a 25-fold reduction in progeny virus production as the result of treatment with 5 mu M inhibitor 24 h posti nfection. (C) 1997 Elsevier Science Ltd.