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 2
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 2. ..more
BioActive Compounds: 16
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 2
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 2.
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] (Assay 1) or NS3h_2a[JFH1] (Assay 2). 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 5 uL of 10 mM ATP into a 45 uL reaction mix such that the final 50 uL reaction contains 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. 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.). Cy5-labeled substrate is measured at excitation wavelength 643 nm (12 nm slit) and emission wavelength 667 nm (12 nm slit).
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.
Each compound was tested in 1 to 5 independent experiments in Assay 1, and in 1 to 4 independent experiments in Assay 2.
PubChem Activity Outcome and Score:
The following applies to each panel in this assay:
Compounds with an IC50 greater than 100 uM in any experiment were considered inactive in that Assay. For compounds with IC50s less than 100 uM in all experiments, the average IC50 was calculated. If the average IC50 was greater than 10 uM the compound was considered inactive in that Assay. If the average IC50 was equal to or less than 10 uM the compound was considered active in that Assay.
Activity score was then ranked by the potency of the compounds with fitted curves, with the most potent compounds assigned the highest activity scores.
NS3h 1b (con1) Score: The PubChem Activity Score range for active compounds is 100-79, and for inactive compounds 74-0.
NS3h 2a (JFH1) Score: The PubChem Activity Score range for active compounds is 100-87, and for inactive compounds 68-0.
Overall Outcome and Score:
The overall outcome was active if the compound was active in at least one panel, inactive otherwise.
The overall score is 0 if the compound was inactive, otherwise the score is taken as the fraction of panels where the compound is active, multiplied by 100.
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.
Categorized Comment - additional comments and annotations
* Activity Concentration. § Panel component ID.