Discovery of inhibitors of EBV lytic cycle inducing protein ZTA for therapeutic development

Project Summary: The goal of this SBIR is to discover a small molecule inhibitor to the Epstein-Barr Virus (EBV)-encoded ZTA protein to prevent EBV-associated cancer and immune disorders. EBV is a human herpesvirus that switches between latent and lytic gene expression to escape immune detection and promote oncogenic transformation of diverse cell types. EBV is classified as a class I carcinogen by the WHO and estimated to be responsible for ~200,000 new cancer cases per year. EBV-associated cancers are diverse, and include subtypes of gastric carcinoma, nasopharyngeal carcinoma, Burkitt’s lymphoma, Hodgkin’s lymphoma, NK/T cell lymphoma, and various non-Hodgkin’s lymphomas particularly among immunocompromised populations. EBV is the major causative agent of infectious mononucleosis, and the predominant viral risk factor for multiple sclerosis (MS) and other auto immune diseases. The viral-encoded protein, ZTA (also known as BZLF1, ZEBRA, and Z) is a redox-sensitive DNA-binding transcriptional activator that is essential for EBV lytic cycle gene expression and viral production. The periodic cycling of lytic virus is a critical component for EBV-driven malignancy and auto- immune disease, and ZTA and the downstream viral and cellular genes that it activates, have been implicated in carcinogenesis and immune pathology. Therefore, ZTA is an attractive target for therapeutic intervention. The DNA-binding domain of ZTA has been characterized structurally and biochemically, and provides an ideal target for small molecule inhibition of EBV lytic life cycle. We have developed robust biochemical and cell-based assays for detection of ZTA DNA binding and used the biochemical assays for traditional high throughput screening (HTS). We screened the HTS hits using an unrelated DNA binding protein to eliminate DNA intercalators and tested the most selective compounds using surface plasmon resonance (SPR) to determine direct binding to the target protein. We now propose to use improved counter screening assays to characterize the hits from the previous screening efforts, expand our screening to natural products and an additional library of synthetic compounds designed for infectious disease targets. Natural products have advantageous characteristics such as higher rigidity for targeted protein surfaces outside of active sites like that of ZTA. We will use medicinal chemistry to define chemophore structure-activity relationships of our hits and identify a suitable lead compound for further development. Our goal is to produce lead compounds with nanomolar potency in biochemical assays, low micromolar activity in cell-based assays, and greater than 10-fold selectivity against control counter-screens. In Phase 2, we will develop our advanced hits into a pre-clinical lead candidate. The ultimate goal of this SBIR program is to develop a novel small molecule therapeutic agent to block the EBV lytic cycle and its associated diseases.