The identification of alpha-ketoamides as potent inhibitors of hepatitis C virus NS3-4A proteinase.

Peptides based upon the non-prime side residues of the NS4A-4B cleavage site of hepatitis C virus (HCV) NS3-4A proteinase containing an alpha-ketoamide moiety in place of the scissile amide bond are potent inhibitors of this enzyme.

The design and synthesis of potent inhibitors of hepatitis C virus NS3-4A proteinase.

Hepatitis C virus (HCV) is the cause of the majority of transfusion-associated hepatitis and a significant proportion of community-acquired hepatitis worldwide. Infection by HCV frequently leads to persistent infections that result in a range of clinical conditions including an asymptomatic carrier state, severe chronic active hepatitis, cirrhosis and, in some cases, hepatocellular carcinoma. The HCV genome consists of a single-stranded, positive sense RNA containing an open reading frame of approximately 9060 nucleotides. This is translated into a single polyprotein of approximately 3020 amino acids (C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B), which in turn is processed by a series of host and viral proteinases into at least 10 cleavage products. The N-terminal portion of the NS3 protein encodes a serine proteinase that is responsible for the cleavage at the NS3-4A, NS4A-4B, NS4B-5A and NS5A-5B junctions. The 54 amino acid NS4A protein is a cofactor that binds to the NS3 protein and enhances its proteolytic activity. This report describes the expression of a recombinant NS3-4A proteinase fusion protein in Escherichia coli and the in vitro characterization of the enzyme activity using synthetic peptide substrates. It then demonstrates how these results were employed to guide the design of potent inhibitors of this enzyme.

Expression of endothelin-converting enzyme in both neuroblastoma and glial cell lines and its localization in rat hippocampus.

Endothelin-converting enzyme is a phosphoramidon-sensitive metalloprotease that cleaves big endothelin to the potent vasoconstrictor peptide, endothelin. The converting enzyme is expressed in endothelial cells in a variety of tissues and in some secretory cells. In the present study, phosphoramidon-sensitive endothelin-converting enzyme activity has been demonstrated by radioimmunoassay in the neuroblastoma cell line, SH-SY5Y, and in Bu17 and C6 glioma lines. The identity of the activity was confirmed by immunoblotting, revealing a polypeptide of approximately 120 kDa in each of these lines, in D384 glioma cells, and in primary astrocytes. Immunofluorescence revealed the cell-surface location of endothelin-converting enzyme in the neuronal and glial cell lines and in primary astrocytes. Pretreatment of SH-SY5Y and Bu17 cells with phosphoramidon resulted in an apparent concentration of the enzyme protein in an intracellular compartment. Immunoperoxidase-staining of rat brain sections located this metalloprotease to the pyramidal cells of the hippocampus. Endothelin-converting enzyme-1 was revealed by in situ hybridisation in the neuronal and glial cell lines.

Studies on fatty acid-binding proteins. The binding properties of rat liver fatty acid-binding protein.

1. The fluorescent fatty acid probe 11-(dansylamino)undecanoic acid binds to rat liver fatty acid-binding protein with a 1:1 stoichiometry. 2. The binding of the fluorescent probe is competitive with long-chain fatty acids. 3. Binding displacement studies were performed with a wide range of fatty acids and other ligands and identified C16 and C18 fatty acids as the preferred fatty acids for rat liver fatty acid-binding protein. No preference was observed for unsaturated fatty acids within this group. 4. Fatty acyl-CoA binds less well than the corresponding fatty acid.

Studies on fatty acid-binding proteins. The diurnal variation shown by rat liver fatty acid-binding protein.

The concentration of fatty acid-binding protein in rat liver was examined by SDS/polyacrylamide-gel electrophoresis, by Western blotting and by quantifying the fluorescence enhancement achieved on the binding of the fluorescent probe 11-(dansylamino)undecanoic acid. A 2-3-fold increase in the concentration of this protein produced by treatment of rats with the peroxisome proliferator tiadenol was readily detected; however, only a small variation in the concentration of the protein due to a diurnal rhythm was observed. This result contradicts the 7-10-fold variation previously reported for this protein [Hargis, Olson, Clarke & Dempsey (1986) J. Biol. Chem. 261, 1988-1991].

Studies on fatty acid-binding proteins. Changes in the concentration of hepatic fatty acid-binding protein during development in the rat.

The concentration of hepatic fatty acid-binding protein was determined in the livers of rats at various stages of development from foetus to young adult. Fatty acid-binding protein concentrations were determined by quantifying the fluorescence enhancement on the binding of the fluorescent probe 11-(dansylamino)-undecanoic acid. A 20-fold increase in the concentration of the protein was observed between the foetus and adult, and this increase was confirmed by immuno-blotting. No other protein in the 14,000-Mr range was observed in the foetus. Possible alternative fatty acid-binding proteins could not be detected in h.p.l.c.-fractionated foetal cytosol by the fluorescence-enhancement method.

Studies on fatty acid-binding proteins. The detection and quantification of the protein from rat liver by using a fluorescent fatty acid analogue.

Fatty acid-binding protein from rat liver is shown to bind the fluorescent fatty acid probe dansyl undecanoic acid. Binding is accompanied by a shift in the fluorescence emission maximum from 550 nm to 500 nm and a 60-fold fluorescence enhancement at 500 nm. These spectral properties have allowed the use of this probe to detect and quantify microgram amounts of liver fatty acid-binding protein during purification procedures. In conjunction with h.p.l.c. the method allows the rapid estimation of liver fatty acid-binding protein in biological samples. The validity of the method is demonstrated by measuring the concentration of fatty acid-binding protein in livers from control and hypolipidaemic-drug-treated rats. The dramatic diurnal rhythm previously reported for this protein [Dempsey (1984) Curr. Top. Cell. Regul. 24, 63-86] was not observed with this method.