Late stage probe development assay for inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3): absorbance-based biochemical assay to determine whether compounds inhibit ATPase activity
Name: Late stage probe development assay for inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3): absorbance-based biochemical assay to determine whether compounds inhibit ATPase activity. ..more
BioActive Compounds: 13
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Center Affiliation: The Scripps Research Institute (TSRI)
Assay Provider: David Frick, New York Medical College
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R03 MH085690-01
Grant Proposal PI: David Frick, New York Medical College
External Assay ID: HCV NS3_INH_ABS_96_IC50
Name: Late stage probe development assay for inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3): absorbance-based biochemical assay to determine whether compounds inhibit ATPase activity.
The flavivirus Hepatitis C Virus (HCV) is a major cause of liver failure and hepatocellular cancer, with about 170 million people infected worldwide (1). The 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). The HCV NS3 helicase mediates the "active" form of duplex unwinding, and thus is dependent upon NTP and at least two nucleic acid binding sites on the NS3 surface (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.
8. Lanzetta,P.A., Alvarez,L.J., Reinach,P.S. and Candia,O.A. (1979) An improved assay for nanomole amounts of inorganic phosphate. Anal. Biochem. 100, 95-97.
late stage, late stage AID, powders, University of Kansas, University of Kansas Specialized Chemistry Center, KUSCC, KU, HCV, NS3, NS3 helicase, hepatitis, NS3h_1b(Con1), 1b(Con1), RNA virus, ATPase, malachite green, malachite green dye, molydbate, dose response, counterscreen, 96, assay provider, inhibitor, inhibition, inhibit, Scripps, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
The purpose of this assay is to verify enzyme activity and to examine specificity by examining whether powder samples of purchased or synthesized compounds identified as possible probe candidates inhibit the helicase encoded by the HCV genotype 1b(Con1). The assay monitors the amount of ATP hydrolysis by NS3h_1b(Con1). A modified colorimetric "malachite green" assay was used (8). The hydrolysis of ATP releases an inorganic phosphate molecule that forms a complex with molybdate. This complex then forms a subsequent colored complex with the malachite green dye under acidic conditions and absorbance of the colored complex is read in a plate reader.
Reactions contained 25 mM MOPS pH 6.5, 1.25 mM MgCl2 and 1 mM ATP in 30 uL total volume. The colorimetric reagent was prepared fresh by mixing 3 volumes 0.045% (w/v) malachite green, 1 volume 4.2% ammonium molybdate in 4N HCl, and 0.05 volume of 20% Tween 20. For SAR profiling, reactions were performed in 10% DMSO with 8 concentrations of each compound ranging from 200 uM to 1.6 uM in the absence of RNA with 50 uM of NS3h_1b(Con1). Reactions were initiated by adding ATP, incubated for 30 minutes at 23 C, and terminated by adding 200 ul of the malachite green reagent, followed by 30 uL of 35% sodium citrate. The color was allowed to develop for 30 minutes and absorbance at 630 nm was read. Percent inhibition was calculated by normalizing the data to reactions without enzyme (100% inhibition) and reactions with DMSO only (0% inhibition). Average IC50 values were then calculated from a normalized dose response curve of replicate assays using GraphPad Prism (v. 5).
PubChem Activity Outcome and Score:
Compounds with an IC50 greater than 100 uM were considered inactive. Compounds with an IC50 equal to or less than 100 uM were considered active.
Activity score was then ranked by the potency of the compounds with fitted curves, with the most potent compounds assigned the highest activity scores.
The PubChem Activity Score range for active compounds is 100-64, and for inactive compounds 32-0.
List of Reagents:
NS3 helicase fragment (supplied by Assay Provider)
MOPS (Fisher Scientific, part BP308-100)
ATP (Fisher Scientific, part BP413-25)
Magnesium Chloride (Fisher Scientific, part BP214-500)
Malachite Green (Acros, 41349-0250)
Ammonium Molybdate (Fisher Scientific, A674-500)
HCl (Fisher Scientific, A142-212)
Tween 20 (Fisher Scientific, BP337-500)
Assay Buffer (supplied by Assay Provider)
96-well plates (Corning Costar, clear, part 9017)
This assay was performed by the assay provider, and submitted to PubChem by the Scripps Research Institute Molecular Screening Center (SRIMSC). Compounds tested in this assay were purchased or synthesized by the University of Kansas Specialized Chemistry Center. Details of protocols, compound structures, and results from the original assays can be found in PubChem at the respective AIDS listed in the Related Bioassays section of this AID.
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 readout content: assay readout method: regular screening
BAO: bioassay specification: assay readout content: content readout type: single readout
BAO: bioassay specification: assay stage: secondary: alternate confirmatory
BAO: detection technology: spectrophotometry: absorbance
BAO: meta target: biological process target: viral genome replication
BAO: meta target: molecular target: protein target: enzyme regulator
BAO: version: 1.4b1090
Assay Format: Biochemical
* Activity Concentration. ** Test Concentration.
Data Table (Concise)