Counterscreen for inhibitors of ZTA: fluorescence polarization-based biochemical high throughput assay to identify inhibitors of the Epstein-Barr virus nuclear antigen 1 (EBNA-1)
Name: Counterscreen for inhibitors of ZTA: fluorescence polarization-based biochemical high throughput assay to identify inhibitors of the Epstein-Barr virus nuclear antigen 1 (EBNA-1). ..more
BioActive Compounds: 4
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_3X%INH ZTA CSRUN
Name: Counterscreen for inhibitors of ZTA: fluorescence polarization-based biochemical high throughput assay to identify 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 and other proteins essential for EBV replication such as Zta (12), 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.
12. Lieberman PM, Hardwick JM, Sample J, Hayward GS, Hayward SD. The zta transactivator involved in induction of lytic cycle gene expression in Epstein-Barr virus-infected lymphocytes binds to both AP-1 and ZRE sites in target promoter and enhancer regions. J Virol. 1990 Mar;64(3):1143-55.
EBNA-1, EBNA1, EBNA, Zta, BZLF1, K8, ZEBRA, Epstein-Barr virus, EB1, EBV, herpesvirus, DNA virus, human herpesvirus 4, HHV4, lymphoma, cancers, transcription factor, B-Zip, inhibitors, inhibition, antagonists, fluorescence polarization, FP, counterscreen, triplicate, 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 counterscreen assay is to determine whether compounds identified as active in a set of experiments entitled, "Counterscreen for inhibitors of EBNA-1: fluorescence polarization-based biochemical high throughput primary assay to identify inhibitors of the Epstein-Barr virus-encoded protein, ZTA (AID 2234) are nonselective inhibitors due to inhibition of EBNA-1. 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 are tested in triplicate at a final nominal concentration of 11.1 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 45 nL of test compound in DMSO or DMSO alone (1.1% 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.
The average percent inhibition and standard deviation of each compound tested were calculated. Any compound that exhibited an average percent inhibition greater than the hit cutoff calculated for the primary screen (AID 1950) was declared active.
The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.
The PubChem Activity Score for active compounds is 100-46, for inactive 34-0.
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
** Test Concentration.
Data Table (Concise)