Late stage results for the probe development effort to identify inhibitors of Hepatitis C Virus (HCV) core protein dimerization: TR-FRET-based biochemical dose response assay for HCV core inhibitors
Name: Late stage results for the probe development effort to identify inhibitors of Hepatitis C Virus (HCV) core protein dimerization: TR-FRET-based biochemical dose response assay for HCV core inhibitors. ..more
BioActive Compound: 1
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: HCVCORE_INH_HTRF_1536_3XIC50 LATE STAGE
Name: Late stage results for the probe development effort to identify inhibitors of Hepatitis C Virus (HCV) core protein dimerization: TR-FRET-based biochemical dose response assay for HCV core inhibitors.
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.
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.
late stage, HCV, core protein, core 106, core, hepatitis, hepatitis C, RNA virus, protein-protein interaction, dimerization, dose response, HTS, high throughput screen, 1536, inhibitor, inhibition, inhibit, HTRF, TR-FRET, time resolved fluorescence resonance energy transfer, fluorescence, 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 a set of previous experiments entitled, "TR-FRET-based primary biochemical high-throughput screening assay to identify inhibitors of Hepatitis C Virus (HCV) core protein dimerization" (AID 1899), "TR-FRET-based biochemical high-throughput confirmation assay for inhibitors of Hepatitis C Virus (HCV) core protein dimerization" (AID 2152), and "TR-FRET-based biochemical high-throughput dose response assay to identify inhibitors of Hepatitis C Virus (HCV) core protein dimerization" (AID 2159). In this assay, test compounds are incubated with N-terminally tagged GST-core106 and Flag-core106 peptides, followed by addition of a Europium cryptate-tagged anti-GST antibody and a XL-665-tagged anti-Flag antibody. Dimerization of the core106 peptides and subsequent antibody binding brings the antibody tags into close proximity, allowing FRET from the Europium donor to the XL-665 acceptor, resulting in an increase in well FRET. As designed, compounds that inhibit core106 dimerization will prevent the interaction of the tagged antibodies and energy transfer from Europium to XL-665, leading to inhibition of well FRET. Compounds were tested in triplicate in a 10-point 1:3 dilution series starting at a nominal test concentration of 50 uM.
Prior to the start of the assay, 5 microliters of Assay Buffer (100 mM HEPES, 0.5 mM EDTA, 2.5 mM DTT, 200 mM Potassium Fluoride, 0.05% CHAPS, 0.05% BSA, pH 7.45, filtered at 0.22 micrometer) were dispensed into columns 1 and 2 of 1536-well assay plates. The remaining 46 columns were filled with 2 microliters of Assay Buffer supplemented with 225 ng/mL of Eu(K)-anti GST antibody and 42.5 nM of Flag-Core106. Next, 1 microliter of inhibition controls were dispensed into column 3 (12.5 uM unlabelled core106 protein, 100% inhibition), column 46 (200 nM core106 protein, 50% inhibition), and columns 4 to 45, 47 and 48 (Assay Buffer alone). Twenty-five nL of 10-points serial dilutions of test compounds or DMSO alone (0.5% final concentration) were then added to the appropriate wells. Next, 2 microliters of Assay Buffer supplemented with 2.5 micrograms/mL XL665-anti FLAG antibody and 33.75 nM GST-Core106 were dispensed to columns 3 to 48. The assay plates were centrifuged for 30 seconds at 300g and incubated for 4 hours at 22.5 degrees Celsius. At the end of the incubation time, TR-FRET was measured by exciting the plates at 340 nM, and monitoring well fluorescence at 617 nm (Eu) and 671 nm (XL665) with the ViewLux microplate reader (PerkinElmer). All wells had a final volume of 5 microliters. The final reagent concentrations were 90 ng/mL Eu(K)-anti GST antibody, 1 microgram/mL XL665-anti FLAG, 17 nM Flag-Core106 and 13.5 nM GST-Core106. Final control concentrations were 2.5 uM and 40 nM for the 100% and 50% inhibition controls, respectively.
To normalize data, values measured from both fluorescence emission wavelengths were used to calculate a ratio for each well, according to the following mathematical expression:
Ratio = ( I671nm / I617nm ) x 10,000
I represents the measured fluorescence emission intensity at the enumerated wavelength in nanometers.
The percent inhibition for each compound is reported as the average and the standard deviation of three replicate wells, calculated as follows:
% Inhibition = ( 1 - ( ( Ratio_TestCompound - Median_Ratio_HighControl ) / ( Median_Ratio_LowControl - Median_Ratio_HighControl ) ) ) * 100
Test_Compound is defined as wells containing test compound.
Negative_Control is defined as wells containing no GST-core106.
Positive_Control is defined as wells containing 1 uM of unlabelled Core106 protein.
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. 50 uM) did not result in greater than 50% inhibition, the IC50 was determined manually as greater than 50 uM. Compounds with an IC50 greater than 10 uM were considered inactive. Compounds with an IC50 equal to or less than 10 uM 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 100-100, for inactives 60-0.
List of Reagents:
GST-core106 (supplied by Assay Provider)
Flag-core106 (supplied by Assay Provider)
Unlabelled core106 peptide (supplied by Assay Provider)
Anti-Flag Antibody (XL-665 labeled) (Cisbio, part 61FG2XLB)
Anti-GST Antibody (Europium labeled) (Cisbio, part 61GSTKLB)
1536-well plates (Greiner, part 789175)
HEPES (Fisher Scientific, part BP299-500)
EDTA (Sigma, part E7889)
Potassium Fluoride (Sigma, part 402931)
Bovine Serum Albumin (Sigma, part A9647)
CHAPS (Sigma, part C5070)
DTT (Sigma, part 43815)
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)