Project Summary/Abstract Epstein-Barr Virus (EBV), a human tumor virus, is the causative agent of mononucleosis and immunoblastic lymphomas. EBV is strongly associated with Burkitt’s lymphoma, Hodgkin’s lymphoma, and nasopharyngeal carcinoma (NPC). Additionally, recent studies suggest that EBV has a pathogenic role with the onset of Multiple Sclerosis (MS). Approximately 90% of the world’s population is infected with EBV, but most do not present disease. However, for those that do develop EBV-related illness there remains no directed small molecule therapy. EBV-triggered disease, causing debilitating illness and death, remains a world-wide problem. Interestingly, EBV expresses a unique protein (BPLF1) that possesses deubiquitinating activity. BPFL1 is known to regulate both cellular and viral target activities, yet it remains largely unstudied. Our work has implicated BPLF1 in a wide range of viral and cellular processes including infectivity (90% reduction with knockout of BPLF1), viral DNA replication, and DNA repair. Also we recently reported surprising new findings that knockout of BPLF1 delays and reduces human B-cell immortalization and lymphoma formation in humanized mice. The aim of this proposal is to discover and characterize the first-in-world small molecule inhibitors of BPLF1 deubiquitinating activity. The goals of this proposal are to 1) use high throughput screening to identify novel chemotypes for a lead optimization effort and 2) produce further validation that small molecule inhibition of BPLF1 deubiquitinating activity is a promising avenue for treating diseases caused by EBV infection. This work will lay the foundation for a chemical probe and drug discovery effort to combat EBV-associated disease.