Structure-based design of small-molecule inhibitors of EBNA1 DNA binding blocks Epstein-Barr virus latent infection and tumor growth

Abstract

Epstein-Barr virus (EBV) is a DNA tumor virus responsible for 1 to 2% of human cancers including subtypes of Burkitt’s lymphoma, Hodgkin’s lymphoma, gastric carcinoma, and nasopharyngeal carcinoma (NPC). Persistent latent infection drives EBV-associated tumorigenesis. Epstein-Barr nuclear antigen 1 (EBNA1) is the only viral protein consistently expressed in all EBV-associated tumors and is therefore an attractive target for therapeutic intervention. It is a multifunctional DNA binding protein critical for viral replication, genome maintenance, viral gene expression, and host cell survival. Using a fragment-based approach and x-ray crystallography, we identify a 2,3-disubstituted benzoic acid series that selectively inhibits the DNA binding activity of EBNA1. We characterize these inhibitors biochemically and in cell-based assays, including chromatin immunoprecipitation and DNA replication assays. In addition, we demonstrate the potency of EBNA1 inhibitors to suppress tumor growth in several EBV-dependent xenograft models, including patient-derived xenografts for NPC. These inhibitors selectively block EBV gene transcription and alter the cellular transforming growth factor–β (TGF-β) signaling pathway in NPC tumor xenografts. These EBNA1-specific inhibitors show favorable pharmacological properties and have the potential to be further developed for the treatment of EBV-associated malignancies.

Biophysical Screens Identify Fragments That Bind to the Viral DNA-Binding Proteins EBNA1 and LANA

“…During the EBNA1 screen, we used fragment VK0044/CC34301 as a control. This fragment was identified from a previous X-ray crystal screen [7]. VK0044 was used to optimize and validate the method and demonstrate protein stability and signal quality over 5 days.…”

“…However, efforts to discover small-molecule inhibitors that specifically target these protein-DNA interactions remain in a relatively early stage [5]. Many viruses control viral gene expression and DNA replication using site-specific DNA-binding proteins [6], which can be targeted by small molecules [7].…”

“…Previously, we reported the discovery of an EBNA1 inhibitor series using FBDD from a pilot fragment screen of 100 fragments using X-ray crystallography [7]. The 100-fragment library was chosen based on in silico docking to a site adjacent to the site identified by X-ray crystallography [23].…”

Xeno‐Free Reprogramming of Peripheral Blood Mononuclear Erythroblasts on Laminin‐521

“…It is also possible to culture Sendai virus RNA-positive hiPSCs at elevated temperatures (for 5 days at 39°C instead of 37°C) to further decrease the activity of the temperature-sensitive Sendai viral RNA polymerase. Similarly, loss of episomal plasmids could potentially be accelerated using recently described EBNA1 inhibitors (Messick et al, 2019).…”

Mechanistic Insights into Chemoresistance Mediated by Oncogenic Viruses in Lymphomas

“…Although there are complex factors that contribute to the chemoresistance of virus-associated lymphomas, the intrinsic viral proteins or components play a pivotal role in the chemoresistant phenotype of tumor cells. For instance, recent efforts to target EBNA1 with small molecules and inhibit its DNA binding activity proved useful in suppressing tumor growth in lymphoblastoid and patient-derived nasopharyngeal carcinoma models [38]. Resolving the mechanisms through which oncogenic viruses mediate chemoresistance may bring new insights and promising strategies to improve chemotherapy efficacy and allow for dose reduction to minimize the toxicity of chemotherapeutic agents in cancer patients.…”