Inhibition of HCV NS3/4A protease represents an important avenue for anti- HCV therapy. ProAssay HCV Protease Assay Kit provides a convenient method for high throughput screening of HCV NS3/4A protease inhibitors.
Persistent infection with hepatitis C virus (HCV) is a common cause of chronic liver disease, ranging from chronic hepatitis and cirrhosis to hepatocellular carcinoma (1). HCV is an enveloped, single-stranded RNA virus with a 9.6-kb positive-polarity genome, which encodes a polyprotein precursor of about 3,000 amino acids. The HCV polyprotein is proteolytically processed by cellular and HCV proteases into at least 10 distinct products (reviewed in 2). The HCV NS3/4A protease is responsible for cleavage at four sites within the HCV polyprotein to generate the N termini of the NS4A, NS4B, NS5A, and NS5B proteins (3-6). The order and kinetics of cleavage as well as the extent of precursor processing appear to be critical steps in the generation of fully infectious, appropriately assembled viral particles. Therefore, inhibition of HCV NS3/4A protease represents an important avenue for antiviral therapy (4).
Fluorescence resonance energy transfer (FRET) assays have become a popular and effective means for drug screening. ProAssay HCV Protease Assay Kit provides a convenient method for high throughput screening of HCV NS3/4A protease inhibitors and continuous quantification of HCV protease activity using FRET peptide substrate. The sequence of this FRET peptide is derived from NS3-dependent cleavage site:Asp/GluXaa4Cys/Thr-Ser/Ala (7). In the FRET peptide, the green fluorescence is quenched by appropriate fluorescence quencher until this peptide is cleaved into two separate fragments by HCV NS3/4A protease at the cleavage site. Upon cleavage, the green fluorescence is recovered and can be monitored at excitation/emission = 490 nm/530 nm. The assays can be performed in a black 96-well or 384-well plate format.
Kit Components · HCV NS3/4A protease (5ul or 9ul) · FRET HCV substrate (25uM, 120 or 220ul) · 2X assay buffer (33ml) · 1 M DTT (1 ml) · Fluorescent reference standard (10uM, 20ul) · Control inhibitor (AEBSF, Pefabloc SC, 200mM, 50ul) · Stop solution (10 ml) · Deionized water (50 ml) · Black 96-well plate (1 or 2) · User Manual
System developed for screening of HCV protease inhibitors. Extensive quality control has been conducted. P9001-01 (100 assays / 96-well format) P9001-02 (200 assays / 96-well format) Please see Description for details. Please see Description for details.
References: 1. Alter, M. J. 1997. Epidemiology of hepatitis C. Hepatology 26:62S-65S. 2. Blight, K. J., et al., (1998) Antiviral Ther. 3, Suppl. 3, 71-81 3. Grakoui, A., et al., (1993) J. Virol. 67, 2832-2843 4. Hijikata, M., et al., (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 10773-10777 5. Tomei, L., et al., (1993) J. Virol. 67, 4017-4026 6. Lamarre, D., et al., (2003) Nature 426, 186-189 7. Talianie, M et al., (1996) Anal Biochem, 240, 60-67
DISCLAIMER
This products is recommended For RESEARCH USE ONLY and is Not qualified for Use in Diagnostic or Therapeutic Procedures.
