The goal of this R21 is to develop a robust high-throughput screen for identification of small molecule inhibitors of Kaposi's Sarcoma Associated Herpes virus (KSHV) and associated diseases. KSHV is a human herpes virus and the causative agent of Kaposi's sarcoma's (KS). KS is a lymphatic-endothelial cell malignancy that occurs most frequently and severely in HIV-AIDS. KSHV has also been implicated in several lymphoid disorders, including pleural effusion lymphoma and Castlemen's disease. Currently, no therapies exist that effectively block KSHV infection or latency, and therefore it remains impossible to effectively treat or prevent KSHV-associated disease. Latent infection and viral pathogenesis is absolutely dependent on one viral-encoded protein, LANA. LANA is a DNA binding protein required for viral replication and episome maintenance during latency. Disruption of LANA by siRNA depletion or gene deletion blocks viral genome persistence during latent infection. The LANA DNA binding domain (DBD) has been characterized biochemically, and serves as an attractive target for small molecule inhibition of KSHV infection. Our preliminary data indicates that highly purified LANA DBD is active in DNA binding and that robust cell-based validation assays are in place. We have recently completed a high-throughput screen (HTS) for small molecule inhibitors of EBNA1, a functionally related protein encoded by Epstein-Barr virus (EBV). In this R21 proposal, we plan to develop a similar strategy for identification of inhibitors of LANA. The risk of this proposal is substantially mitigated by our previous successful development of a screen for EBNA1.
The significance of this proposal to human health will be the development of new therapies for eliminating KSHV latent infection and preventing or treating KSHV-associated disease. Kaposi's Sarcoma-Associated Herpes virus (KSHV), also referred to as human herpes virus 8 (HHV8), was originally isolated from Kaposi's sarcoma (KS) tissue and infects ~6% of the adult population world-wide. Its prevalence increases to greater than 60% in sub-Saharan Africa, where HIV is endemic. Numerous molecular and epidemiological studies support the conclusion that KSHV is the causative agent of KS. KSHV has also been associated with several lymphoid disorders, including primary effusion lymphoma (PELs), multicentric Castleman's disease, and post-transplant lymphoproliferative disorders. KSHV is consistently detected in all forms of KS, including HIV negative KS found predominantly in elderly males of Mediterranean descent. Risk and severity of KSHV-associated disease is significantly elevated in the context of HIV-AIDS, as well as in other forms of immunosuppression, including organ and bone-marrow transplant. The immediate goal of this proposal is to establish a platform to identify and characterize small molecule inhibitors of LANA. LANA is required for the KSHV latency and host-cell survival. The candidate inhibitors will target the DNA binding function of LANA, which is essential for all of its known functions in viral persistence and pathogenesis. Direct, specific inhibitors of LANA will provide new methods to treat KSHV associated disease. These studies will also provide new reagents for manipulation of LANA protein and KSHV latent infection. Finally, this research may provide therapeutic strategies for related tumor viruses, especially EBV and human papillomavirus (HPV), which possess LANA-like orthologues.
