Late stage assay provider results for the probe development effort to identify inhibitors of the Epstein-Barr virus nuclear antigen 1 (EBNA-1): Cell-based luciferase-based dose response assay for EBNA-1 inhibitors
Name: Late stage assay provider results for the probe development effort to identify inhibitors of the Epstein-Barr virus nuclear antigen 1 (EBNA-1): Cell-based luciferase-based dose response assay for EBNA-1 inhibitors. ..more
BioActive Compound: 1
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Center Affiliation: The Scripps Research Institute (TSRI)
Assay Provider: Paul Lieberman, Wistar Institute
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: I R21 NS063906-01
Grant Proposal PI: Paul Lieberman, Wistar Institute
External Assay ID: EBNA1_INH_LUMI_IC50 MDRUN Assay Provider
Name: Late stage assay provider results for the probe development effort to identify inhibitors of the Epstein-Barr virus nuclear antigen 1 (EBNA-1): Cell-based luciferase-based dose response assay for EBNA-1 inhibitors.
During each cell cycle in eukaryotes, the genome must be completely replicated and this replication must begin at the correct time and site (initiation site or origin) (1). Pathogenic viruses often take advantage of this cellular precision to maintain replication of their own genome. The Epstein-Barr virus (EBV) is an orally-transmitted herpesvirus associated with numerous human neoplasms (2) that infects over 90% of the world's population (3). Latent EBV infection stimulates B cell proliferation in vitro, and can lead to B cell transformation (4). Following infection, the EBV genome is maintained in the host cell as a plasmid that is replicated by machinery comprised of several host proteins and a sole EBV protein, the EBV nuclear antigen 1 (EBNA-1). This EBNA-1 protein is required for replication of the EBV DNA genome. EBNA-1 is a DNA-binding protein that binds to an EBV sequence called the viral origin of replication plasmid, OriP (5). Studies demonstrating a role for EBV in Burkitt's lymphoma and Hodgkin's disease (6), multiple sclerosis (7), lupus (8), infectious mononucleosis (9), and nasopharyngeal carcinoma (10), combined with the recent discovery that EBNA-1 induces DNA double strand breaks (11), suggest that inhibitors of EBNA-1 may serve useful for understanding virus-cell interactions, virus-mediated cellular transformation, and as therapies for EBV-associated pathologies.
1. Norseen, J, Thomae, A, Sridharan, V, Aiyar, A, Schepers, A and Lieberman, PM, RNA-dependent recruitment of the origin recognition complex. EMBO J, 2008. 27(22): p. 3024-35.
2. Carbone, A, Gloghini, A and Dotti, G, EBV-associated lymphoproliferative disorders: classification and treatment. Oncologist, 2008. 13(5): p. 577-85.
3. Schulz, TF and Cordes, S, Is the Epstein-Barr virus EBNA-1 protein an oncogene? Proc Natl Acad Sci U S A, 2009. 106(7): p. 2091-2.
4. Thorley-Lawson, DA and Babcock, GJ, A model for persistent infection with Epstein-Barr virus: the stealth virus of human B cells. Life Sci, 1999. 65(14): p. 1433-53.
5. Kirchmaier, AL and Sugden, B, Dominant-negative inhibitors of EBNA-1 of Epstein-Barr virus. J Virol, 1997. 71(3): p. 1766-75.
6. Hammerschmidt, W and Sugden, B, Epstein-Barr virus sustains Burkitt's lymphomas and Hodgkin's disease. Trends Mol Med, 2004. 10(7): p. 331-6.
7. Lunemann, JD, Kamradt, T, Martin, R and Munz, C, Epstein-barr virus: environmental trigger of multiple sclerosis? J Virol, 2007. 81(13): p. 6777-84.
8. Harley, JB, Harley, IT, Guthridge, JM and James, JA, The curiously suspicious: a role for Epstein-Barr virus in lupus. Lupus, 2006. 15(11): p. 768-77.
9. Vetsika, EK and Callan, M, Infectious mononucleosis and Epstein-Barr virus. Expert Rev Mol Med, 2004. 6(23): p. 1-16.
10. Raab-Traub, N, Epstein-Barr virus in the pathogenesis of NPC. Semin Cancer Biol, 2002. 12(6): p. 431-41.
11. Gruhne, B, Sompallae, R, Marescotti, D, Kamranvar, SA, Gastaldello, S and Masucci, MG, The Epstein-Barr virus nuclear antigen-1 promotes genomic instability via induction of reactive oxygen species. Proc Natl Acad Sci U S A, 2009. 106(7): p. 2313-8.
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The purpose of the cell-based luciferase assay is to determine dose response curves for powder samples of compounds identified as possible EBNA-1 inhibitor probe candidates. This assay assesses the ability of compounds to inhibit the binding of EBNA-1 to the Qp promoter of the luciferase reporter construct. In the virus, the Qp promoter controls the expression of EBNA-1. In an EBV-infected cell, low EBNA-1 levels stimulate the expression from the Qp promoter of the EBNA-1 gene. When EBNA-1 levels are high, EBNA-1 binds to the Qp promoter and represses expression of EBNA-1 transcripts. EBNA-1 levels are thus auto-regulated using this feedback mechanism. For this assay, we cloned the Qp promoter upstream of the luciferase reporter construct, pGL3-Basic (Promega), N1852. Full length Flag-tagged EBNA-1 is expressed using p3xFLAG-Myc-CMV-24 Expression Vector (Sigma), N803. For control experiments, empty (no insert) p3xFLAG-Myc-CMV-24 Expression Vector (N799) is used. These vectors are (transiently) transfected using lipofectamine into 293T cells. As designed, compounds that act as EBNA-1 inhibitors will prevent the repression of EBNA-1 at the Qp promoter, thereby increasing the expression of luciferase in the well. The amount of luciferase is measured using the Steady-Glo Luciferase Assay kit (Promega). This assay was performed by the assay provider.
Prior to the start of the assay, culture 293T cells in antibiotic free DMEM+10% FBS in 10 cm tissue culture plates.
Day 1: Prepare 10 cm tissue culture plates of 293T cells. Harvest cells from a confluent 10 cm plate and seed at least 2 10 cm tissue culture plate 1:3 with antibiotic free media and incubate plates at 37 C, 5% CO2 overnight. One plate is for the control plasmid and the other plate is for the EBNA-1 expression plasmid.
Day 2: Transfection of the cells. Prepare the Lipofectamine Mix (Mix A) by adding 40 uL Lipofectamine to 1.0 mL of Optimem per plate. Mix the reaction well by inverting tube several times and incubate for at least 5 minute at room temperature. Next, prepare the Luciferase Mix (Mix B) by adding 4 ug of a luciferase expression plasmid (N1852) to 1.0 mL of Optimem per plate. Prepare the Control Mix by adding 0.125 ug of a control plasmid not containing EBNA-1 (N799) to 1.0 mL of Mix B. Prepare the EBNA-1 Mix by adding 0.125 ug of an EBNA-1 expressing plasmid (N803) to 1.0 mL of Mix B. Incubate the Control and EBNA-1 Mix for at least 5 minutes at room temperature. After incubation, add 1.0 mL Mix A to the Control Mix and the EBNA-1 Mix. Mix well by inverting tube several times and incubate for 20 minutes at room temperature. After incubation, add 2.0 mL of the Control Mix in a drop-wise manner to a prepared 10 cm plate of 293T cells. Repeat with the EBNA-1 mix. Incubate plates overnight at 37 C, 5% CO2.
Day 3: Harvesting and plating in 384-well plates. Harvest transfected cells without trypsin. The cells easily detach from the plate by pipetting the media over the bottom of the plate. Transfer the cells to a 50 mL conical tube and pellet by centrifuging at 300 x g for 5 minutes at room temperature. Aspirate the supernatant and resuspend cells in 5.0 mL of media. Perform a cell count and resuspend to 1.0 x10E5 cells/mL. Plate a 384-well plate with 40 uL of cells using a Biotek Microflo. Incubate 384-well plate at 37 C, 5% CO2 for 48 hours.
Day 5: Developing and reading the plate using Steady-Glo Luciferase Assay System. Add 20 uL of the Steady-Glo reagent to each well of 384-well plate. Incubate for 5 minutes at room temperature and read luminescence using the Envision.
For each test compound, bioluminescence levels were plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Prism software (GraphPad). The reported EC50 values were generated from fitted curves by solving for the X-intercept value at the 50% stimulation level of the maximum Y-intercept value. In cases where the highest concentration tested (i.e. 148 uM) did not result in greater than 50% inhibition, the IC50 was determined manually as greater than 148 uM.
PubChem Activity Outcome and Score:
Compounds with an EC50 greater than 10 uM were considered inactive. Compounds with an EC50 equal to or less than 10 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 active compounds is 100-100, and for inactive compounds 89-0.
List of Reagents:
EBNA-1 expression vector (p3xFLAG-Myc-CMV-24-full length EBNA-1), N803 (supplied by Assay Provider)
Control expression vector, (p3xFLAG-Myc-CMV-24), N799 (supplied by Assay Provider)
Qp luciferase reporter vector, (pGL3-Basic-Qp-luciferase), N1852 (supplied by Assay Provider)
293T (American Type Culture Collection (ATCC), catalog number CRL-11268)
DMEM (Cellgro, Catalog number 10-013-CM)
Gibco FBS (Invitrogen, Catalog number 16000)
Optimem (Invitrogen, Catalog number 31985-070)
Lipofectamine 2000 (Invitrogen, Catalog number 11668-019)
Steady-Glo Luciferase Assay System (Promega, Catalog number E2550)
10-cm cell culture plates (Corning, Catalog number 430167)
Tissue culture treated 384-well plates (Corning, part 3570)
This assay was performed by the assay provider. Replicate values and raw data were not provided. This assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. Possible artifacts of this assay can include, but are not limited to: dust or lint and compounds that modulate luminescence. 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 by the Kansas University Specialized Chemistry Center.
Categorized Comment - additional comments and annotations
From BioAssay Depositor:
BAO: assay design: inducible reporter: luciferase induction
BAO: assay format: biochemical format: protein format: single protein format
BAO: bioassay specification: assay biosafety level: bsl1
BAO: bioassay specification: assay measurement type: endpoint assay
BAO: bioassay specification: assay readout content: assay readout method: regular screening
BAO: bioassay specification: assay readout content: content readout type: single readout
BAO: detection technology: luminescence: chemiluminescence
BAO: meta target detail: binding reporter specification: interaction: protein-dna
BAO: meta target: biological process target: regulation of gene expression
BAO: meta target: molecular target: nucleic acid: dna
BAO: version: 1.4b1090
Assay Format: Cell-based
Assay Cell Type: 293T
* Activity Concentration.
Data Table (Concise)