Fluorescence polarization based primary biochemical high throughput screening assay of ChemBridge compounds to identify inhibitors of hepatitis C virus non-structural protein 3 helicase
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 ..
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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.
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 polarization, University of Wisconin-Milwauke, UW-Milwaukee, UW-M
The purpose of this biochemical assay is to determine the ability of compounds in a ChemBridge compound library to displace the hepatitis C non-structural protein 3 helicase (NS3h) from its substrate oligonucleotide. In this biochemical assay, the fluorescence polarization of a single strand of DNA with a 5' Cy5 fluorophore (Cy5-dT15) incubated with NS3h and test compound is monitored. Fluorescence polarization increases when NS3h binds the substrate. As designed, compounds that interfere with binding decrease the fluorescence polarization.
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'), 15 nM NS3h, 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 polarization was read using a G-factor calculated from a well lacking NS3h or test compounds. Percent inhibition was calculated according to the equation below.
%_Inhibition = 100 - ( ( Fc - F[+]) / (F[-] - F[+] ) ) * 100
Fc is the fluorescence polarization in the presence of the compound
F[-] is the average fluorescence polarization of no enzyme negative controls
F[+] is the average fluorescence polarization of positive controls (200 nM dT20).
Two values were calculated: (1) the average percent inhibition of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter. Any compound that exhibited greater percent inhibition than the cutoff parameter was considered active.
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 Library set.
BAO: bioassay specification: assay stage: Primary
BAO: bioassay specification: assay biosafety level: bsl1
BAO: assay format: biochemical format: protein format: Single protein format
BAO: bioassay specification: assay measurement type: endpoint assay
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: molecular target: protein target: enzyme regulator
BAO: meta target: biological process target:: viral genome replication
BAO: detection technology: fluorescence: fluorescence polarization
Data Table (Concise)