Beta-lactones as a new class of cysteine proteinase inhibitors: inhibition of hepatitis A virus 3C proteinase by N-Cbz-serine beta-lactone.

[reaction: see text] N-Benzyloxycarbonyl-L-serine beta-lactone (1) is shown to irreversibly inactivate the 3C cysteine proteinase of hepatitis A virus (HAV) with k(inact) = 0.70 min(-1), K(I) = 1.84 x 10(-4) M and k(inact)/K(I) = 3800 M(-1) min(-1) at an enzyme concentration of 0.1 microM. Mass spectrometric and HMQC NMR studies using 13C-labeled 1 show that the active site cysteine (Cys-172) thiol of the HAV 3C proteinase attacks the beta-position (i.e. C-4) of the oxetanone ring, thereby leading to ring opening and alkylation of the sulfur. In contrast, the enantiomer of this beta-lactone, 2, is a reversible competitive inhibitor (Ki = 1.50 x 10(-6) M) at similar enzyme concentrations. The beta-lactone motif represents a new class of inhibitors of cysteine proteinases.

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

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.