Selection of chemical inhibitors of oncoproteins

描述：（申请人提供） 人类乳头状瘤病毒（HPV）已被最终证明是致病的 肛门生殖器肿瘤和一些头颈部肿瘤的病原体。越来越 大量证据表明，猿猴病毒40（SV40）与 间皮瘤、脑和骨。HPV和SV40均使p53和pRb失调控 通过病毒编码的癌蛋白结合肿瘤抑制因子途径， 细胞p53和pRb。靶向HPV和SV40的反义技术 癌蛋白导致细胞系的生长抑制和凋亡 HPV阳性宫颈癌和SV40阳性恶性宫颈癌 间皮瘤，分别。这一证据表明，HPV和SV40 癌蛋白代表了治疗特定类型肿瘤的有价值的靶点， 人类癌症因此，免疫治疗和基因治疗方法， 靶向HPV E6和E7是临床前或临床试验的对象， 宫颈癌的治疗，并提出了类似的策略， SV40阳性间皮瘤的治疗到目前为止，免疫疗法 这些方法未能在体内提供足够的反应，并且遗传学上， 由于缺乏有效的交付系统，这些办法受到阻碍。我们 提出一种替代办法：筛选化学品文库， 鉴定能够干扰SV 40和HPV结合的分子 癌蛋白与细胞p53和pRb的结合。这些战略要求 一个大的化学品面板的分析，一个任务可行，只有当 研究病毒癌蛋白相互作用的高通量测定 与他们的细胞目标是可用的。这些分析将需要 相对大量的病毒癌蛋白和肿瘤抑制因子， 适当的翻译后修饰以确保生物活性。等 如果蛋白质底物在人类中表达，则可以满足要求。 细胞然而，用于蛋白质过表达的人细胞系统目前还不成熟。 不可用.我们发现SV40转化的人间皮细胞（HM） 可用于获得mg量的SV40大肿瘤抗原（Tag）， 与细胞p53和pRb复合。我们建议利用这个牢房 用于鉴定SV40标签细胞肿瘤的化学抑制剂的系统 抑制剂相互作用。此外，SV40转化的中皮细胞克隆可被 用作繁殖"高拷贝数"附加型表达载体的基础 活跃地复制人类间皮细胞LS.这样的载体可以允许 需要翻译后修饰的蛋白质的过表达， 人体细胞中适当的生物活性。我们建议使用这个实验 过量生产和纯化可结合载体HPV 16 E6和E7的系统。 重组E6和E7随后将用于开发基于ELISA的 研究HPV 16 E6和E7与p53和pRb结合的体外测定， 分别最后，我们建议采用后一种检测方法， 筛选化学文库以发现HPV E6的抑制剂， e7. SV 40和HPV的推定抑制剂的鉴定 癌蛋白可能导致开发新的抗癌药物。 此外，所提出的实验可能有助于新的技术， 过表达和纯化潜在的任何蛋白质， 复制人类间皮细胞

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.