Design and Optimization of Benzoic Acid Inhibitors Targeting the EBNA1 DNA-Binding Interface

Epstein–Barr nuclear antigen 1 (EBNA1) is an essential viral DNA-binding protein required for maintenance of Epstein–Barr virus (EBV) episomes and is expressed in all EBV-associated malignancies, making it an attractive therapeutic target. Using fragment-based screening and X-ray crystallography, we identified a 2,3-disubstituted benzoic acid scaffold that binds at the EBNA1 DNA-binding interface. Structure-guided optimization revealed that the carboxylic acid pharmacophore engages Asn519 and Thr590, while electron-rich heterocycles are positioned between Lys477 and Lys586, forming a cation−π–cation “lysine sandwich” that drives potency. Iterative medicinal chemistry improved binding affinity and physicochemical stability, leading to compound 35 (VK-2019), a submicromolar EBNA1 to DNA binding inhibitor. Compound 35 demonstrated favorable drug-like properties and robust antitumor efficacy in EBV-positive xenograft models and has advanced to clinical evaluation. These studies define the structural determinants of EBNA1 inhibition and establish a framework for targeting viral genome-maintenance proteins with small molecules.