title: Synthetic depsipeptide substrates for the assay of human hepatitis C virus protease.
abstract: Hepatitis C virus (HCV) is the major etiological agent of both parenterally transmitted and sporadic non-A, non-B hepatitis. The disease is a major health problem with an estimated 50 million people infected worldwide, a high percentage of whom become chronically infected and are at high risk for liver cirrhosis. The serine protease contained within the N-terminal region of the nonstructural protein 3 (NS3 protease) of HCV is considered a promising target for the development of an antiviral therapy. A prime requisite to study in detail the biochemistry of the protease as well as develop inhibitors is the availability of a fast and sensitive in vitro assay of enzyme activity. However, due to their low kcat/Km values, synthetic peptide substrates based on the natural cleavage sites appear unsuitable for this purpose. We show here that appropriate substrates can be obtained by substituting the scissile amide bond with an ester linkage. The resulting depsipeptides show >100-fold improvement in kcat/Km values, up to 13,000 M-1 s-1, enabling detection of activity with subnanomolar NS3 concentrations. The ester substrates are obtained in high yield entirely by solid-phase synthesis using commercially available materials, without the need for any preassembled building blocks.(c) 1996 Academic Press, Inc.
authors.affiliation: Istituto di Ricerche di Biologia Molecolare P. Angeletti (IRBM), Via Pontina Km 30,600, Rome, Italy.
authors.forename: E
authors.initials: E
authors.lastname: Bianchi
authors.forename: C
authors.initials: C
authors.lastname: Steinkühler
authors.forename: M
authors.initials: M
authors.lastname: Taliani
authors.forename: A
authors.initials: A
authors.lastname: Urbani
authors.forename: R
authors.initials: R
authors.lastname: De Francesco
authors.forename: A
authors.initials: A
authors.lastname: Pessi
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D016174:Hepacivirus / Q000201:enzymology*; D006801:Humans; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry; D010455:Peptides / Q000138:chemical synthesis* / Q000737:chemistry; D012697:Serine Endopeptidases / Q000032:analysis* / Q000378:metabolism; D013379:Substrate Specificity; D017361:Viral Nonstructural Proteins / Q000032:analysis / Q000378:metabolism; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D009842:Oligopeptides; D010455:Peptides; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases