Late stage probe development counterscreen for inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3): real-time florescence-based biochemical assay to determine whether compounds inhibit the helicase encoded by one or more HCV strains: Set 3
Name: Late stage probe development counterscreen for inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3): real-time florescence-based biochemical assay to determine whether compounds inhibit the helicase encoded by one or more HCV strains: Set 3. ..more
Depositor Specified Assays
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_QFRET_96_IC50_SET 3
Name: Late stage probe development counterscreen for inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3): real-time florescence-based biochemical assay to determine whether compounds inhibit the helicase encoded by one or more HCV strains: Set 3.
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.
late stage, late stage AID, powders, University of Kansas, University of Kansas Specialized Chemistry Center, KUSCC, KU, HCV, NS3, NS3 helicase, hepatitis, genotype, HCV genotype, 2a(JFH1), 1b(con1), RNA virus, dose response, counterscreen, triplicate, 96, assay provider, inhibitor, inhibition, inhibit, fluorescence, FRET, QFRET, ssDNA oligonucleotide, hairpin, oligonucleotide molecular beacon, quench, quencher, Cy5 fluorophore, 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 in a kinetic assay 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 genotypes NS3h_1b[con1] or NS3h_2a[JFH1]. In these assays a ssDNA oligonucleotide molecular beacon substrate featuring a 5' fluorescent Cy5 moiety and a 3' quencher is annealed to a second longer DNA oligonucleotide. Upon strand separation by NS3 helicase and ATP, the beacon strand forms an intramolecular hairpin that brings the tethered fluorophore and quencher molecules into juxtaposition, quenching fluorescence. As designed, compounds that inhibit helicase activity will prevent hairpin formation and interaction of the Cy5 fluorophore and quencher, thus preventing quenching of well fluorescence.
Assays are initiated by rapidly mixing 10% of reaction volume of 10 mM ATP into 90% volume reaction mix such that the final reaction contained 25 mM MOPS pH 6.5, 1.25 mM MgCl2, 5 nM [Cy5-labeled]substrate, 12.5 nM enzyme (NS3h isolation from genotype 1b(con1)) or 5 nM enzyme (NS3h isolation from genotype 2a(JFH1)), 1.0 mM ATP and 5% (v/v) DMSO (final reaction volume 60 uL). Compounds are diluted in DMSO at 20x concentration and added as 5% of the reaction mixture; control (no inhibitor) reactions include DMSO only. Enzyme is diluted in buffer containing 25 mM MOPS pH 6.5, 1 mM DTT, 0.1 mg/mL BSA and 0.2% tween20 to 20x final concentration and comprises 5% (v/v) of the reaction mixture. Reactions are performed at 23 C in low volume white 96-well microplates and monitored with a Varioskan Flash (Thermo Fisher Scientific, Inc.) or FLUOstar Omega (BMG Labtech, Inc). Cy5 -labeled substrates are measured at excitation wavelength 643 nm (12 nm slit) and emission wavelength 667 nm (12 nm slit) for Varioskan Flash, or excitation wavelength 640 nm (10 nm slit) and emission wavelength 680 (10 nm slit) for FLUOstar.
Initial rates of fluorescence decrease after ATP addition were plotted versus compound concentration to calculate IC50 values. For each test compound, reaction velocity (relative fluorescence units (RFU)/min) was plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted using SkanIt Software v 2.4.3 (Thermo Fisher Scientific, Inc.). The reported IC50 values were generated from fitted curves by solving for the X-intercept value at the 50% inhibition level of the Y-intercept value. In cases where the highest concentration tested (i.e. 100 uM) did not result in greater than 50% inhibition, the IC50 was determined manually as greater than 100 uM.
For each assay, each compound was tested in 1 to 5 independent experiments.
PubChem Activity Outcome and Score:
The following applies to each panel in this assay:
Compounds with an IC50 greater than 50 uM were considered inactive. Compounds with an IC50 equal to or less than 50 uM were considered active.
Inactive compounds were scored as 0. Active compounds were scored as follows:
Score = ( 1 - ( ( Ave_IC50 - Min_IC50) / ( ( 100 - Min_IC50 ) / 2 ) ) * 100
Ave_IC50 is the calculated average IC50 for that compound
Min_IC50 is the minimum Ave_IC50 observed for all compounds in the Assay data set
NS3h 1b[con1] Score: The PubChem Activity Score range for active compounds is 100-10, and for inactive compounds 0-0.
NS3h 2a[JFH1] Score: The PubChem Activity Score range for active compounds is 100-1, and for inactive compounds 0-0.
Overall Outcome and Score:
Compounds that were active in both experiments were considered active, otherwise they were considered inactive.
The PubChem Activity Score is assigned a value of 100 for the probe compound, 50 for active compounds, and 0 for inactive compounds.
The PubChem Activity Score range for active compounds is 100-50, and for inactive compounds 0-0.
List of Reagents:
NS3 helicase fragment (supplied by Assay Provider)
Cy5/quencher-labeled molecular beacon (Integrated DNA Technologies Inc, custom synthesized)
Thioflavine S (Sigma-Aldrich, part T1892)
MOPS (Fisher-Biotech, part BP308-100)
ATP (Fisher-BioReagents, part BP413-25)
Magnesium Chloride (Fisher-Biotech, part BP214-500)
Assay Buffer (supplied by Assay Provider)
96-well plates (Corning Costar, white half volume, part 3693)
This assay was performed by the assay provider, and submitted to PubChem by the Scripps Research Institute Molecular Screening Center (SRIMSC)on behalf of the University of Kansas Specialized Chemistry Center. Compounds tested in this assay were purchased and/or synthesized by the University of Kansas Specialized Chemistry Center. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that quench or emit fluorescence.
BAO: version: 1.4b1090
BAO: bioassay specification: assay stage: lead optimization
BAO: bioassay specification: assay biosafety level: bsl1
BAO: assay format: biochemical format: protein format: single protein format
BAO: bioassay specification: assay measurement type: kinetic 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: transcription factor
BAO: meta target: biological process target: viral genome replication
BAO: detection technology: fluorescence: fret: htrf
* Activity Concentration. § Panel component ID.