Small molecule and peptide-mediated inhibition of Epstein-Barr virus nuclear antigen 1 dimerization.
Latent Epstein-Barr virus (EBV) infection is associated with human B cell lymphomas and certain carcinomas. EBV episome persistence, replication, and gene expression are dependent on EBV-encoded nuclear antigen 1 (EBNA1)'s DNA binding domain (DBD)/dimerization domain (DD)-mediated sequence-specific DNA binding activity. Homodimerization of EBNA1 is essential for EBNA1 DNA binding and transactivation. In this study, we characterized a novel small molecule EBNA1 inhibitor EiK1, screened from the previous high throughput screening (HTS). The EiK1 compound specifically inhibited the EBNA1-dependent, OriP-enhanced transcription, but not EBNA1-independent transcription. A Surface Plasmon Resonance Biacore assay revealed that EiK1 associates with EBNA1 amino acid 459-607 DBD/DD. Consistent with the SPR data, in vitro gel shift assays showed that EiK1 suppressed the activity of EBNA1 binding to the cognate familial repeats (FR) sequence, but not control RBP-Jκ binding to the Jκ site. Subsequently, a cross-linker-mediated in vitro multimerization assay and EBNA1 homodimerization-dependent yeast two-hybrid assay showed that EiK1 significantly inhibited EBNA1 dimerization. In an attempt to identify more highly specific peptide inhibitors, small peptides encompassing the EBNA1 DBD/DD were screened for inhibition of EBNA1 DBD-mediated DNA binding function. The small peptide P85, covering EBNA1 a.a. 560-574, significantly blocked EBNA1 DNA binding activity in vitro, prevented dimerization in vitro and in vivo, associated with EBNA1 in vitro, and repressed EBNA1-dependent transcription in vivo. Collectively, this study describes two novel inhibitors of EBNA1 dimerization. This study demonstrates that EBNA1 homodimerization can be effectively targeted by a small molecule or peptide.
Samsung Advanced Institute for Health Sciences and Technology, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea.
Sun Young
SY
Kim
Kyung-A
KA
Song
Elliott
E
Kieff
Myung-Soo
MS
Kang
D000998:Antiviral Agents / Q000737:chemistry / Q000494:pharmacology*; D001565:Benzoates / Q000737:chemistry / Q000494:pharmacology*; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D019309:Epstein-Barr Virus Nuclear Antigens / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology; D055503:Protein Multimerization / Q000187:drug effects*; D018741:Replication Origin; D013858:Thiobarbiturates / Q000737:chemistry / Q000494:pharmacology*
D000998:Antiviral Agents; D001565:Benzoates; D004268:DNA-Binding Proteins; C576164:EiK1 compound; D019309:Epstein-Barr Virus Nuclear Antigens; D010455:Peptides; D013858:Thiobarbiturates; D004247:DNA; C417029:EBV-encoded nuclear antigen 1