Late stage results for the probe development effort to identify inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3): fluorescence-based biochemical dose response assay for inhibitors of NS3
Name: Late stage results for the probe development effort to identify inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3): fluorescence-based biochemical dose response assay for inhibitors of NS3. ..more
BioActive Compounds: 8
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: NS3HDNA_INH_FLINT_1536_3XIC50 LATE STAGE
Name: Late stage results for the probe development effort to identify inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3): fluorescence-based biochemical dose response assay for inhibitors of NS3.
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, HCV, NS3, NS3 helicase, hepatitis, RNA virus, dose response, HTS, high throughput screen, HTS, 1536, inhibitor, inhibition, FLINT, fluorescence, fluorescence intensity, FLINT, 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 determine dose response curves for powder samples of compounds identified as active in set of previous experiments entitled, "Fluorescence-based biochemical high throughput dose response assay for inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3)" (AID 2173), and inactive in a set of experiments entitled, "Counterscreen for HCV NS3 helicase inhibitors: Fluorescence-based biochemical high-throughput dose response assay for compounds that cause fluorescent intercalator displacement (FID)" (AID 2172). In this assay, 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 at 60 minutes (T60) will prevent hairpin formation and interaction of the Cy5 fluorophore and quencher, thus preventing quenching of well fluorescence. A T0 fluorescence measurement is also performed to identify compounds that quench and enhance Cy5 fluorescence. Compounds were tested in triplicate in a 10-point 1:3 dilution series starting at a nominal test concentration of 109 micromolar.
Prior to the start of the assay, 4 microliters of Assay Buffer (25 mM MOPS pH 6.5, 1.25 mM MgCl2, 0.1 mM DTT, 12.5 mM Tween20, 6 micrograms/mL BSA) containing 13.89 nM NS3 helicase fragment and 5.56 nM NS3 substrate were dispensed into wells of a 1536 microtiter plate. Next, 55 nL of test compound in DMSO, thioflavine S (110 micromolar final concentration), or DMSO alone (0.8% final concentration) were added to the appropriate wells.
The assay was started by dispensing 1 microliter of 5 mM ATP into all wells. Well fluorescence was read after 1 hour of incubation at 25 degrees Celsius on the Viewlux (Perkin-Elmer). Prior to inhibition calculations the ratio between RFU values obtained at t0 (RFU_t0) and t60 (RFU_t60), named Ratio_RFU, was calculated as follows:
Ratio_RFU = RFU_t60/RFU_t0
The percent inhibition for each well was then calculated as follows:
Percent inhibition = ( test_compound_Ratio_RFU - negative_control_Ratio_RFU ) / ( positive_control_Ratio_RFU - negative_control_Ratio_RFU ) * 100
Test_Compound is defined as wells containing test compound.
Negative_Control is defined as wells containing DMSO.
Positive_Control is defined as wells containing Thioflavine S.
For each test compound, percent inhibition was plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Assay Explorer software (Symyx Technologies 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. 109 micromolar) did not result in greater than 50% inhibition, the IC50/ was determined manually as greater than 109 micromolar. Compounds with an IC50 greater than 10 micromolar were considered inactive. Compounds with an IC50 equal to or less than 10 micromolar were considered active.
Any compound with a percent activity value <50% at all test concentrations was assigned an activity score of zero. Any compound with a percent activity value >50% at any test concentration was assigned an activity score greater than zero. Activity score was then ranked by the potency, with the most potent compounds assigned the highest activity scores.
The PubChem Activity Score range for active compounds is 100-1, there are no inactives.
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)
1536-well plates (Greiner, part 789173)
This assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds, or required the calculation of hit cutoff on a plate-by-plate basis within a single HTS campaign. In this case the results of each separate campaign or plate within a single campaign were assigned "Active/Inactive" status based upon that campaign's specific compound activity cutoff value. All data reported were normalized on a per-plate basis. In this assay, Thioflavine S had an IC50 of approximately 10.5 micromolar. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)