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Development of a high-throughput screen for inhibitors of Epstein-Barr virus EBNA1.

ABSTRACT: Latent infection with Epstein-Barr virus (EBV) is a carcinogenic cofactor in several lymphoid and epithelial cell malignancies. At present, there are no small-molecule inhibitors that specifically target EBV latent infection or latency-associated oncoproteins. EBNA1 is an EBV-encoded sequence-specific DNA binding protein that is consistently expressed in EBV-associated tumors and required for stable maintenance of the viral genome in proliferating cells. EBNA1 is also thought to provide cell survival function in latently infected cells. In this work, the authors describe the development of a biochemical high-throughput screening (HTS) method using a homogeneous fluorescence polarization (FP) assay monitoring EBNA1 binding to its cognate DNA binding site. An FP-based counterscreen was developed using another EBV-encoded DNA binding protein, Zta, and its cognate DNA binding site. The authors demonstrate that EBNA1 binding to a fluorescent-labeled DNA probe provides a robust assay with a Z factor consistently greater than 0.6. A pilot screen of a small-molecule library of ~14,000 compounds identified 3 structurally related molecules that selectively inhibit EBNA1 but not Zta. All 3 compounds had activity in a cell-based assay specific for the disruption of EBNA1 transcription repression function. One of the compounds was effective in reducing EBV genome copy number in Raji Burkitt lymphoma cells. These experiments provide a proof of concept that small-molecule inhibitors of EBNA1 can be identified by biochemical HTS of compound libraries. Further screening in conjunction with medicinal chemistry optimization may provide a selective inhibitor of EBNA1 and EBV latent infection.

SUBMITTER: Thompson S

PROVIDER: S-EPMC3310380 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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Development of a high-throughput screen for inhibitors of Epstein-Barr virus EBNA1.

Thompson Scott S Messick Troy T Schultz David C DC Reichman Melvin M Lieberman Paul M PM

Journal of biomolecular screening 20101001 9

Latent infection with Epstein-Barr virus (EBV) is a carcinogenic cofactor in several lymphoid and epithelial cell malignancies. At present, there are no small-molecule inhibitors that specifically target EBV latent infection or latency-associated oncoproteins. EBNA1 is an EBV-encoded sequence-specific DNA binding protein that is consistently expressed in EBV-associated tumors and required for stable maintenance of the viral genome in proliferating cells. EBNA1 is also thought to provide cell sur ...[more]

PMID: 20930215

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Project description:Epstein-Barr virus (EBV) establishes a stable latent infection that can persist for the life of the host. EBNA1 is required for the replication, maintenance, and segregation of the latent episome, but the structural features of EBNA1 that confer each of these functions are not completely understood. Here, we have solved the X-ray crystal structure of an EBNA1 DNA-binding domain (DBD) and discovered a novel hexameric ring oligomeric form. The oligomeric interface pivoted around residue T585 as a joint that links and stabilizes higher-order EBNA1 complexes. Substitution mutations around the interface destabilized higher-order complex formation and altered the cooperative DNA-binding properties of EBNA1. Mutations had both positive and negative effects on EBNA1-dependent DNA replication and episome maintenance with OriP. We found that one naturally occurring polymorphism in the oligomer interface (T585P) had greater cooperative DNA binding in vitro, minor defects in DNA replication, and pronounced defects in episome maintenance. The T585P mutant was compromised for binding to OriP in vivo as well as for assembling the origin recognition complex subunit 2 (ORC2) and trimethylated histone 3 lysine 4 (H3K4me3) at OriP. The T585P mutant was also compromised for forming stable subnuclear foci in living cells. These findings reveal a novel oligomeric structure of EBNA1 with an interface subject to naturally occurring polymorphisms that modulate EBNA1 functional properties. We propose that EBNA1 dimers can assemble into higher-order oligomeric structures important for diverse functions of EBNA1.IMPORTANCE Epstein-Barr virus is a human gammaherpesvirus that is causally associated with various cancers. Carcinogenic properties are linked to the ability of the virus to persist in the latent form for the lifetime of the host. EBNA1 is a sequence-specific DNA-binding protein that is consistently expressed in EBV tumors and is the only viral protein required to maintain the viral episome during latency. The structural and biochemical mechanisms by which EBNA1 allows the long-term persistence of the EBV genome are currently unclear. Here, we have solved the crystal structure of an EBNA1 hexameric ring and characterized key residues in the interface required for higher-order complex formation and long-term plasmid maintenance.

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Development of a high-throughput screen for inhibitors of Epstein-Barr virus EBNA1.

Publications

Development of a high-throughput screen for inhibitors of Epstein-Barr virus EBNA1.

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