Late stage assay provider counterscreen results for the probe development effort to identify inhibitors of Hepatitis C Virus (HCV) core protein dimerization: Fluorescence-based cell-based Quantitative Polymerase Chain Reaction (QPCR) assay to identify inhibitors of HCV infectivity (2 timepoints)
Name: Late stage assay provider counterscreen results for the probe development effort to identify inhibitors of Hepatitis C Virus (HCV) core protein dimerization: Fluorescence-based cell-based Quantitative Polymerase Chain Reaction (QPCR) assay to identify inhibitors of HCV infectivity (2 timepoints). ..more
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center
Affiliation: The Scripps Research Institute, TSRI
Assay Provider: A.D. Strosberg, TSRI
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1-X01-MH085709-01
Grant Proposal PI: A.D. Strosberg, TSRI
External Assay ID:CORE_INH_FLUOR_0096_SAR_ROUND_1 MDCSRUN QPCR T1 T2
Name: Late stage assay provider counterscreen results for the probe development effort to identify inhibitors of Hepatitis C Virus (HCV) core protein dimerization: Fluorescence-based cell-based Quantitative Polymerase Chain Reaction (QPCR) assay to identify inhibitors of HCV infectivity (2 timepoints).
The 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 most N-terminal 21kDa protein of this HCV polyprotein is the HCV core (C) protein, which is a highly basic, RNA-binding structural protein essential for assembly and packaging of the viral genome (2). Core protein is cleaved by a host peptidase and anchored to the host cell endoplasmic reticulum, where it undergoes further processing into its mature form (3). The N terminal portion of this mature C protein mediates viral assembly through homodimerization and formation of higher order complexes with viral RNA to form the nucleocapsid, while the hydrophobic C terminal interacts with envelope glycoproteins to form the infectious particle (4). The conserved nature of the HCV protein and absence of a vaccine to prevent HCV infection (5), along with studies demonstrating that C protein contributes to host cell oncogenesis (6), apoptosis inhibition (7), and suppression of host T cell responses (8), support a role for core protein as a major pathogenic component of HCV. The identification of specific inhibitors of HCV core dimerization will provide valuable tools for inhibiting HCV assembly without host cell effects (9).
1. Hoofnagle, J.H., Course and outcome of hepatitis C. Hepatology, 2002. 36(5 Suppl 1): p. s21-s29.
2. Lin, C., Lindenbach, B.D., Pragai, B.M., McCourt, D.W., and Rice, C.M., Processing in the hepatitis C virus E2-NS2 region: identification of p7 and two distinct E2-specific products with different C termini. J Virol, 1994. 68(8): p. 5063-73.
3. Moradpour, D. and Blum, H.E., A primer on the molecular virology of hepatitis C. Liver Int, 2004. 24(6): p. 519-25.
4. Majeau, N., Gagne, V., Boivin, A., Bolduc, M., Majeau, J.A., Ouellet, D., and Leclerc, D., The N-terminal half of the core protein of hepatitis C virus is sufficient for nucleocapsid formation. J Gen Virol, 2004. 85(Pt 4): p. 971-81.
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. Ray, R.B., Lagging, L.M., Meyer, K., and Ray, R., Hepatitis C virus core protein cooperates with ras and transforms primary rat embryo fibroblasts to tumorigenic phenotype. J Virol, 1996. 70(7): p. 4438-43.
7. Marusawa, H., Hijikata, M., Chiba, T., and Shimotohno, K., Hepatitis C virus core protein inhibits Fas- and tumor necrosis factor alpha-mediated apoptosis via NF-kappaB activation. J Virol, 1999. 73(6): p. 4713-20.
8. Large, M.K., Kittlesen, D.J., and Hahn, Y.S., Suppression of host immune response by the core protein of hepatitis C virus: possible implications for hepatitis C virus persistence. J Immunol, 1999. 162(2): p. 931-8.
9. Kota S, Coito C, Mousseau G, Lavergne JP, Strosberg AD. Peptide inhibitors of hepatitis C virus core oligomerization and virus production. J Gen Virol. 2009 Jun;90(Pt 6):1319-28.
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The purpose of this assay is to determine whether powder samples of compounds identified as possible HCV core probe candidates are able to block HCV infectivity of Huh-7.5 cells. In this assay HCV infectivity is measured using real-time RT-PCR to monitor changes in expression of HCV 2a J6/JFH-1 RNA. Cells are incubated with test compound in the presence of HCV, followed by isolation of RNA, conversion to cDNA, and Taqman-based QPCR. As designed, a compound that inhibits HCV infectivity will reduce HCV RNA expression, leading to decreased production of the PCR amplicon, thereby reducing fluorescence, and increasing Ct. Compounds were tested in triplicate in a 6-point 1:10 dilution series starting at a nominal test concentration of 100 uM. The range of activity is normalized based on measurement of total RNA.
Cells were plated the day before the assay. After allowing the cells to adhere overnight, test compound was prepared in HCV supernatant by making 1:10 serial dilutions from 100 uM down to 0.001 uM. Doses of test compound in virus were added to cells and incubated for 24 hours. The next day, cell culture media was removed from each well and replaced with same dilutions of compound in complete media were added to cells and incubated for another 48 hours for T1 timepoint. Then cells were lysed and RNA was isolated using the RNeasy kit (QIAGEN, Valencia, CA). Supernatant from T1 was transferred to fresh naive cells and incubated for 24 hours. Culture media were removed from cells and replaced with complete media and incubated for 48 hours for T2 timepoint. Cells for T2 timepoint were lysed and RNA was isolated using the RNeasy kit (QIAGEN, Valencia, CA). DNA was generated using the Taqman reverse transcription kit (Applied Biosystems, Foster City, CA). Quantitative real-time polymerase chain reaction (PCR) was performed in triplicate using LightCycler RNA Amplification Kit HybProbe master mix (Roche) with Taqman MGB Probe 6FAM-TATGAGTGTCGTGCAGCCTC-MGBNFQ on a model LightCycler480 real time PCR system (Roche). Primers used were forward CTTCACGCAGAAAGCGTCTA and reverse CAAGCACCCTATCAGGCAGT.
The range of activity was normalized based on measurement of total RNA.
As a control a known RNA standard was included in the assays. The Ct values were converted to copy number of RNA using the known internal control. The RNA copy number for the inhibition doses of the compound was then compared to the RNA copy number from un-treated but infected Huh-7.5 cells to calculate the percent inhibition for each dose of the compound.
Percent inhibition at each dose of compound was plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using GraphPad software. 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. 50 uM) did not result in greater than 50% inhibition, the IC50 was determined manually as greater than 50 uM.
PubChem Activity Outcome and Score:
The following applies to each panel in this assay:
Compounds with a IC50 value greater than 20 uM were considered inactive. Compounds with a IC50 value equal to or less than 20 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.
Timepoint 1 Score: The PubChem Activity Score range for active compounds is 100-1. There are no inactive compounds.
Timepoint 2 Score: The PubChem Activity Score range for active compounds is 100-47, and for inactive compounds 1-1.
Overall Outcome and Score:
If tested at both timepoints, a compound must have been active at both T1 and T2 timepoints to be considered active.
The overall score is 0 if the compound was inactive and 100 if the compound was active.
The PubChem Activity Score range for active compounds is 100-100, and for inactive compounds 0-0.
List of Reagents:
Huh-7.5 cell line (APATH)
DMEM medium (Invitrogen, 11995-073)
100X Penicillin-Streptomycin-Glutamine (Invitrogen, 10378-016)
Trypsin-EDTA solution (Invitrogen, 25200-056)
Fetal Bovine Serum (Akron, FBS-400-3D)
QiaShredder (Qiagen, 79656)
RNeasy mini kit (Qiagen, 74104)
LightCylcer RNA Amplification Kit HybProbe (Roche, 12015145001)
Forward primer (Lifetech Applied Biosystems, CTTCACGCAGAAAGCGTCTA)
Reverse primer (Lifetech Applied Biosystems, CAAGCACCCTATCAGGCAGT)
MGBProbe (Lifetech Applied Biosystems, 4304971, 6FAM-TATGAGTGTCGTGCAGCCTC-MGBNFQ)
LightCycler480 multiwell plate 96 (Roche, 04729692001)
LightCycler480 sealing foil (Roche, 04729757001)
This assay was performed in the assay provider's lab. This assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. 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.
Categorized Comment - additional comments and annotations
* Activity Concentration. § Panel component ID.