Fluorescence-based counterscreen assay for HCV NS3 helicase inhibitors of selected ChemBridge compounds: biochemical high-throughput screening assay to identify compounds that enhance or quench fluorescence of a Cy5-labeled oligonucleotide bound to E. coli SSB
The hepatitis C virus (HCV) is a major cause of liver failure and hepatocellular cancer, with about 170 million people infected worldwide (1). HCV has a small RNA genome that is directly translated by the infected host cell into a single precursor polyprotein that is processed by enzymatic cleavage into 10 proteins of diverse function. The non-structural proteins include p7, NS2, NS3, NS4A, more ..
BioActive Compounds: 22
The hepatitis C virus (HCV) is a major cause of liver failure and hepatocellular cancer, with about 170 million people infected worldwide (1). HCV has a small RNA genome that is directly translated by the infected host cell into a single precursor polyprotein that is processed by enzymatic cleavage into 10 proteins of diverse function. The non-structural proteins include p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B, and are responsible for the replication and packaging of the HCV genome into capsids formed by the structural proteins (core, E1, E2)(2). Replication of HCV in human cells requires the action of the HCV non-structural protein 3 (NS3). This enzyme exhibits dual NTPase/helicase activities and functions to unwind DNA/DNA, RNA/RNA, and RNA/DNA duplexes by disrupting hydrogen bonds that hold the two strands together (3). HCV NS3 is able to target homotypic and heterotypic duplexes because the interaction between the enzyme and the DNA or RNA substrate is mediated by phosphate groups and not by the nucleotide base or sugar moieties (4). The current absence of a vaccine to prevent HCV infection (5), along with knockout studies showing that the helicase and/or NTPase activities are essential for viral replication (6), and the lack of HCV genotype-specific differences in helicase residues and activities (7), support a role for NS3 as an important pathogenic component of HCV. The identification of specific inhibitors of HCV NS3 helicase will add insights into the biology of HCV infection and replication, and serve as valuable tools for inhibiting HCV replication in human cells.
1. Hoofnagle, J.H., Course and outcome of hepatitis C. Hepatology, 2002. 36(5 Suppl 1): p. s21-s29.
2. Frick, D.N., The hepatitis C virus NS3 protein: a model RNA helicase and potential drug target. Curr Issues Mol Biol, 2007. 9(1): p. 1-20.
3. Borowski, P., Schalinski, S., and Schmitz, H., Nucleotide triphosphatase/helicase of hepatitis C virus as a target for antiviral therapy. Antiviral Res, 2002. 55(3): p. 397-412.
4. Kim, J.L., Morgenstern, K.A., Griffith, J.P., Dwyer, M.D., Thomson, J.A., Murcko, M.A., Lin, C., and Caron, P.R., Hepatitis C virus NS3 RNA helicase domain with a bound oligonucleotide: the crystal structure provides insights into the mode of unwinding. Structure, 1998. 6(1): p. 89-100.
5. Yang, J.P., Zhou, D., and Wong-Staal, F., Screening of small-molecule compounds as inhibitors of HCV entry. Methods Mol Biol, 2009. 510: p. 295-304.
6. Gu, B., Liu, C., Lin-Goerke, J., Maley, D.R., Gutshall, L.L., Feltenberger, C.A., and Del Vecchio, A.M., The RNA helicase and nucleotide triphosphatase activities of the bovine viral diarrhea virus NS3 protein are essential for viral replication. J Virol, 2000. 74(4): p. 1794-800.
7. Cho, H.S., Ha, N.C., Kang, L.W., Chung, K.M., Back, S.H., Jang, S.K., and Oh, B.H., Crystal structure of RNA helicase from genotype 1b hepatitis C virus. A feasible mechanism of unwinding duplex RNA. J Biol Chem, 1998. 273(24): p. 15045-52.
powders, HCV, NS3, NS3 helicase, hepatitis, RNA virus, 384, assay provider, inhibitor, inhibition, inhibit, 5' Cy5, Cy5-dT15, fluorescence intensity, University of Wisconin-Milwauke, UW-Milwaukee, UW-M
The purpose of this biochemical assay is to confirm the ability of selected compounds in a ChemBridge compound library to enhance or quench the fluorescence signal of a Cy5-labeled substrate oligonucliotide bound to E. coli single stranded binding protein (SSB). The entire library was screened in an assay called "Fluorescence-based counterscreen assay for HCV NS3 helicase inhibitors of a ChemBridge library: biochemical high-throughput screening assay to identify compounds that cause fluorescent interference." In this biochemical assay, the fluorescence of a single strand of DNA with a 5' Cy5 fluorophore (Cy5-dT15) incubated with SSB and test compound is monitored.
Assays were performed in 20 uL in 384-well black small-volume microplates. All assays contained 5 nM Cy5-dT15 DNA (5'- Cy5 TT TTT TTT TTT TTT T -3'), 20 nM SSB, 25 mM MOPS, pH 7.5, 1.25 mM MgCl2, 0.0025 mg/ml BSA, 0.005% (v/v) Tween 20, and 0.025 mM DTT. Compounds dissolved in DMSO were added at final concentration of 0.1mM and a final DMSO concentration of 1% (v/v). Cy5 fluorescence was read. Interference was calculated according to the equation below.
Interference = Fc / F[-]
Fc is the fluorescence in the presence of the compound
F[-] is the average fluorescence of negative controls
Any compound that exhibited an interference value greater than 0.8 or less than 1.2 was considered inactive. Any compound that exhibited an interference value greater than or equal to 1.2 was considered active (a fluorescent enhancer) in the assay and will not be pursued further. Any compound that exhibited an interference value less than or equal to 0.8 was considered active (a fluorescent quencher) in the assay and will not be pursued further.
List of Reagents:
Cy5-dT15 (Integrated DNA Technologies Inc, custom synthesized)
dT20 (Integrated DNA Technologies Inc, custom synthesized)
MOPS (Fisher Scientific, part BP308-100)
Magnesium Chloride (Fisher Scientific, part BP214-500)
384-well plates (Greiner Bio-One, black, part 784076)
The assay was performed and submitted by the Frick Lab at the University of Wisconsin-Milwaukee. The compounds tested in this assay were from a ChemBridge compound library.
Categorized Comment - additional comments and annotations
From BioAssay Depositor:
BAO: assay format: biochemical format: protein format: single protein format
BAO: bioassay specification: assay biosafety level: bsl1
BAO: bioassay specification: assay measurement type: endpoint assay
BAO: bioassay specification: assay stage: Primary
BAO: detection technology: fluorescence: fluorescence intensity
BAO: meta target: molecular target: protein target: enzyme regulator
BAO: bioassay specification: assay readout content: assay readout method:: regular screening
BAO: bioassay specification: assay readout content: content readout type:: single readout
BAO: meta target: biological process target:: viral genome replication
Assay Format: Biochemical
Data Table (Concise)