Late stage assay provider counterscreen results for the probe development effort to identify inhibitors of Hepatitis C Virus (HCV) core protein dimerization: Absorbance-based cell-based assay to identify compounds that are cytotoxic to Huh-7.5 cells
Name: Late stage assay provider counterscreen results for the probe development effort to identify inhibitors of Hepatitis C Virus (HCV) core protein dimerization: Absorbance-based cell-based assay to identify compounds that are cytotoxic to Huh-7.5 cells. ..more
Depositor Specified Assays
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: HUH7.5-CYTOX_INH_ABS_0096_3XIC50 MDCSRUN
Name: Late stage assay provider counterscreen results for the probe development effort to identify inhibitors of Hepatitis C Virus (HCV) core protein dimerization: Absorbance-based cell-based assay to identify compounds that are cytotoxic to Huh-7.5 cells.
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 cytotoxic to Huh-7.5 cells. The assay utilizes compound XTT (2,3-bis[2-Methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide inner salt) with addition of an electron coupling agent phenazine methosulfate (PMS) to measure mitochondrial dehydrogenases activity in living cells. Dehydrogenases in mitochondria of living cells will cleave the tetrazolium ring of XTT changing the color of the solution from clear to orange. As designed, compounds that reduce cell viability will reduce activity of the mitochondrial dehydrogenases, thereby reducing the orange color in solution and well absorbance. Compounds were tested in triplicate in a 7-point 1:3 dilution series starting at a nominal test concentration of 320 uM.
The Huh-7.5 cell line was routinely cultured in 15-cm dishes at 37 C and 95% relative humidity (RH). The growth media consisted of DMEM supplemented with 10% v/v certified fetal bovine serum, 4 mM L-glutamine, 1 mM sodium pyruvate, 1X antibiotic mix (penicillin, streptomycin, and glutamine), and 1X non-essential amino acids.
Prior to the start of the assay 8000 cells in 100 uL volume of colorless growth media were dispensed into each well of 96-well tissue culture-treated microtiter plates and incubated overnight at 37 C (5% CO2, 95% RH) to allow cells to adhere to the plate. The next day, 100 uL of test compound in DMSO (1.25 % final DMSO concentration) and DMSO only were added to wells. Wells with only test compound in media without cells were used to normalize the data (to account for the color a test compound may have). Next, the plates were incubated for 72 hours at 37 C (5% CO2, 95% RH). Solution of XTT-PMS was prepared in 1X PBS and added to the cells in 50 uL volume to each well and incubated for 4 hours at 37 C (5% CO2, 95% RH). After equilibrating the plates to room temperature for 10 minutes, optical density at 450 nM and 650 nM was measured on Biotek plate reader.
The percent inhibition for each compound was calculated as follows:
% Inhibition = ( ( DMSO_Control - Test_Compound ) / DMSO_Control ) * 100
Test Compound is defined as wells containing test compound.
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 Microsoft Excel software. The reported CC50 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. 320 uM) did not result in greater than 50% inhibition, the CC50 value was determined manually as greater than 320 uM.
PubChem Activity Outcome and Score:
Compounds with a CC50 value > 24 uM were considered inactive. Compounds with a CC50 value <= 24 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 inactive compounds is 100-1. There are no active compounds.
List of Reagents:
Huh-7.5 cell line (APATH)
DMEM medium (Invitrogen, 31053-028)
100X L-Glutamine (Invitrogen, 25030-081)
100X Sodium pyruvate (Invitrogen, 11360-070)
100X Penicillin-Streptomycin-Glutamine (Invitrogen, 10378-016)
Trypsin-EDTA solution (Invitrogen, part 25200-056)
Fetal Bovine Serum (Akron, FBS-400-3D)
100X Non-essential amino acids (Invitrogen, 11140-050)
XTT (Sigma Chemical, X4626)
PMS (Sigma Chemical, P9625)
DMSO (Sigma Chemical, D8418)
96-well flat-bottom plates (Fisher, 08-772-3)
This assay was performed by the assay provider. In this assay an Inactive assignment indicates that the compound is not cytotoxic, while an Active assignment indicates that a compound is cytotoxic. 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.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)