TY - JOUR
T1 - Synthetic depsipeptide substrates for the assay of human hepatitis C virus protease
AU - Bianchi, Elisabetta
AU - Steinkühler, Christian
AU - Taliani, Marina
AU - Urbani, Andrea
AU - De Francesco, Raffaele
AU - Pessi, Antonello
PY - 1996/6/1
Y1 - 1996/6/1
N2 - 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.
AB - 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.
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U2 - 10.1006/abio.1996.0235
DO - 10.1006/abio.1996.0235
M3 - Article
C2 - 8660572
AN - SCOPUS:0342624795
VL - 237
SP - 239
EP - 244
JO - Analytical Biochemistry
JF - Analytical Biochemistry
SN - 0003-2697
IS - 2
ER -