Identification of SV40 T Antigen Inhibitors:  A Route to Novel Anti-Viral Reagent

Brodsky, Jeffrey

Abstract

The Polyomaviruses (Py) normally form latent infections in the kidney but upon re-activation can trigger the onset of incurable diseases. For example, re-activation of JC virus (JCV) results in Progressive Multifocal Leukoencephalopathy, which kills ~5% of AIDS patients, and re-activation of BK virus (BKV) leads to BKV Associated Nephropathy, which is the leading cause of rejection in kidney transplant recipients. Another Py is SV40, whose natural hosts are monkeys, and ~10% of the human population is infected with SV40 because the first polio vaccines were inadvertently contaminated with the virus; the long-term effects of SV40 infection in humans are unclear, although SV40 has been linked to several cancers. Each member of the Py family encodes the large tumor antigen (TAg), which is essential for viral replication and tumorigenesis. Previous work in the PI's laboratory contributed to the discovery that TAg possesses a chaperone-like domain, and that the interaction between this domain and the cellular Hsp70 chaperone is essential to orchestrate viral replication and cellular transformation. Furthermore, small molecule modulators that compromise TAg-Hsp70 interaction have been identified. One modulator (MAL2-11B) inhibits TAg's endogenous ATPase activity, an activity that is also essential for viral replication and cellular transformation. Indeed, MAL2-11B inhibits SV40 DNA synthesis in vitro and viral replication in tissue culture cells. Through these efforts, a significant battery of in vitro methods have been developed that have been re-configured for a high throughput screen to identify new, more potent TAg and Py inhibitors. Specifically, TAg's ATPase activity has recently been assessed in a 96-well format. Thus, the hypothesis underlying these studies is that chemical modulators of TAg ATPase activity might represent a new avenue to combat the catastrophic effects of Polyomavirus-associated diseases.
The Specific Aims of this application are: (1) To perform high throughput screens to isolate TAg inhibitors using small molecule libraries at the resident University of Pittsburgh MLSCN, and (2) To utilize established assays as secondary and counter screens to assess the effects of """"""""hits"""""""" from Aim 1. In both aims, MAL2-11B will serve as a positive control. Further tests will be conducted with the MLSCN and resident medicinal chemists to obtain more refined chemical probes. The Relevance of this study is underscored by the lack of approved and efficacious methods to treat diseases that arise from Polyomavirus infection. The described efforts employ a novel target, a positive control, and local collaborators, which will aid the completion of the stated goals. ? ? ?