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Synthetic depsipeptide substrates for the assay of human hepatitis C virus protease
- Source :
- Analytical biochemistry. 237(2)
- Publication Year :
- 1996
-
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 k cat / K m 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 k cat / K m 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.
- Subjects :
- Hepatitis C virus
medicine.medical_treatment
Molecular Sequence Data
Biophysics
Peptide
Hepacivirus
Viral Nonstructural Proteins
medicine.disease_cause
Biochemistry
Substrate Specificity
medicine
Humans
Amino Acid Sequence
Molecular Biology
Chromatography, High Pressure Liquid
Hepatitis
chemistry.chemical_classification
Serine protease
Depsipeptide
NS3
Protease
Binding Sites
biology
Molecular Structure
Settore BIO/12
Serine Endopeptidases
Cell Biology
medicine.disease
Virology
NS2-3 protease
chemistry
biology.protein
Peptides
Oligopeptides
Subjects
Details
- ISSN :
- 00032697
- Volume :
- 237
- Issue :
- 2
- Database :
- OpenAIRE
- Journal :
- Analytical biochemistry
- Accession number :
- edsair.doi.dedup.....ed5fdf01eddd3b270296a25d5fb3a1eb