Fluorescence polarization-based biochemical high throughput dose response assay for inhibitors of the Epstein-Barr virus nuclear antigen 1 (EBNA-1)
Name: Fluorescence polarization-based biochemical high throughput dose response assay for inhibitors of the Epstein-Barr virus nuclear antigen 1 (EBNA-1). ..more
BioActive Compounds: 61
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_FP_1536_3XIC50
Name: Fluorescence polarization-based biochemical high throughput dose response assay for inhibitors of the Epstein-Barr virus nuclear antigen 1 (EBNA-1).
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.
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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.
EBNA-1, EBNA1, EBNA, Epstein-Barr virus, EBV, herpesvirus, DNA virus, human herpesvirus 4, HHV4, lymphoma, cancers, inhibitors, inhibition, antagonists, fluorescence polarization, FP, dose response, HTS, high throughput screen, 1536, 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 compounds identified as active in a previous set of experiments entitled, "Fluorescence polarization-based biochemical high throughput confirmation assay to identify inhibitors of the Epstein-Barr virus nuclear antigen 1 (EBNA-1)." (AID 2292), and inactive in a set of experiments entitled, "Counterscreen for inhibitors of EBNA-1: fluorescence polarization-based biochemical high throughput assay for inhibitors of the Epstein-Barr virus-encoded protein, ZTA, in triplicate" (AID 2328). This assay assesses the ability of compounds to inhibit the binding of EBNA-1 protein to DNA. In this biochemical assay, recombinant EBNA-1 DNA binding domain (DBD) (aa 448-610) purified from E. coli is incubated with a Cyanine (Cy5)-labeled 36-bp hairpin DNA oligonucleotide probe in the presence of test compounds. The reaction is excited with linear polarized light and the intensity of the emitted light is measured as the polarization value (mP). As designed, compounds that act as EBNA inhibitors will prevent EBNA-DNA binding, thereby increasing the proportion of free DNA in the well, resulting in low fluorescence polarization in the well. Compounds were tested in triplicate in a 10-point 1:3 dilution series starting at a nominal test concentration of 148 micromolar.
Prior to the start of the assay, Assay Buffer (20 mM Tris HCl, pH 7.4; 200 mM NaCl; 1 mM DTT; 10 micrograms/ml BSA; 10 nM Cy5 DNA probe) containing 246 nM of EBNA-1 protein was prepared and incubated for 30 minutes at room temperature. Next, 4.0 microliters of Assay Buffer were dispensed into 1536-well microtiter plates.
The assay started by dispensing 60 nL of test compound in DMSO or DMSO alone (1.5% final concentration) to the appropriate wells. Plates were then centrifuged and incubated for 1 hour at room temperature. Fluorescence polarization was read on an Envision microplate reader (PerkinElmer, Turku, Finland) using a Cy5 FP filter set (Excitation = 620nm, Emission = 688nm) and a Cy5 dichroic mirror. The well Fluorescence Polarization value (mP) was calculated from the parallel (S) and perpendicular (P) polarization values corrected with the G factor value (G) using the following formula:
mP = 1000 * ( S - G * P ) / ( S + G * P )
The percent inhibition for each compound was calculated as follows:
Percent inhibition = ( Test_Compound_mP - median_High_Control_mP ) / ( median_Low_Control_mP - median_High_Control_mP ) * 100
Test_Compound is defined as wells containing EBNA-1 in the presence of test compound.
Low_Control is defined as wells containing EBNA-1.
High_Control is defined as wells containing no EBNA-1.
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 (MDL Information Systems). 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. 148 micromolar) did not result in greater than 50% inhibition, the IC50 was determined manually as greater than 148 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 of the compounds with fitted curves, with the most potent compounds assigned the highest activity scores.
The PubChem Activity Score for active compounds is 100-84, for inactive 83-0.
List of Reagents:
Recombinant EBNA-1 (supplied by Assay Provider)
Cy5 DNA probe (supplied by Assay Provider)
Tris HCl (Sigma, part T1503)
NaCl (Fisher, part S640-10)
DTT (Sigma, part D9779)
BSA (Calbiochem, part 126609)
1536-well plates (Greiner, part 789176)
Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. In this case the results of each separate 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. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and 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 by the MLSMR. The MLSMR was not able to provide all compounds selected for testing in this AID.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)