Selection of chemical inhibitors of oncoproteins

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DESCRIPTION: (provided by applicant) Human Papilloma Viruses (HPVs) have been conclusively proven as causative agents of ano-genital tumors, and some tumors of the head and neck. A growing body of evidence relates Simian Virus 40 (SV40) with tumors of the mesothelium, brain, and bone. Both HPV and SV40 deregulate the p53 and pRb tumor suppressors pathways through binding of virus-encoded oncoproteins to the cellular p53 and pRb. Antisense technology targeting the HPV and SV40 oncoproteins leads to growth inhibition and apoptosis in cell lines derived from HPV-positive cervical cancers, and from SV40-positive malignant mesotheliomas, respectively. This evidence suggest that the HPV and SV40 oncoproteins represent valuable targets for the treatment of specific types of human cancer. Accordingly, both immuno-therapy and gene-therapy approaches to target HPV E6 and E7 are subjects of pre-clinical or clinical trials for the treatment of cervical cancer, and similar strategies have been proposed for the treatment of SV40-positive mesotheliomas. So far, immuno-therapy approaches have failed to provide a sufficient response in vivo, and genetic approaches are hampered by the lack of an efficient delivery system. We propose an alternative approach: the screening of chemical libraries to identify molecules capable of interfering with the binding of SV 40 and HPV oncoproteins to cellular p53 and pRb in vitro. These strategies require the analysis of a large panel of chemicals, a task feasible only if high-throughput assays to study the interactions of the viral oncoproteins with their cellular targets are available. These assays would require relatively high amounts of viral oncoproteins and tumor suppressors with proper post-translational modifications to ensure biological activity. Such requirement can be fulfilled if the protein substrates are expressed in human cells. However, human cell systems for protein over-expression are presently unavailable. We discovered that SV40-transformed human mesothelial cells (HM) can be used to obtain mg amounts of the SV40 large tumor antigen (Tag) in complex with cellular p53 and pRb. We propose to take advantage of this cell system to identify chemical inhibitors of the SV40 Tag-cellular tumor suppressors interactions. Moreover SV40-transformed mesothelial clones can be used as a basis to propagate "high copy number", episomal expression vectors in actively replicating human mesothelial ce!ls. Such vectors may allow over-expression of proteins requiring post-translational modifications for proper biological activity in human cells. We propose to use this experimental system to overproduce and purify carrier-conjugable HPVl6 E6 and E7. Recombinant E6 and E7 will be subsequently used to develop ELISA-based in vitro assays to study the HPVl6 E6 and E7 binding to p53 and pRb, respectively. Finally, we propose to employ the latter assays for the screening of chemical libraries in order to find inhibitors of the HPV E6 and E7. The identification of putative inhibitors of the SV 40 and HPV oncoproteins may lead to the development of novel anticancer drugs. Furthermore, the experiments proposed may contribute novel technology for the over-expression and purification of potentially any protein in actively replicating human mesothelial cells.