Small Molecule Screen Targeting p53 proteolysis in HPV positive cancers

描述（由申请人提供）：人类乳头瘤病毒（hpv）与许多人类癌症有因果关系。已经鉴定出超过120种不同的人乳头瘤病毒，其中一部分与有进展为癌症风险的病变有关。几乎所有子宫颈癌病例都可归因于人乳头瘤病毒感染。在许多不同的HPV类型中，有14种被称为“高风险”，因为它们与肛门生殖器癌有关。这些高风险的人乳头瘤病毒与宫颈癌、其他肛门生殖器癌症以及大约20%的头颈癌有关。目前基于vlp的疫苗只能预防14种高危HPV类型中的2种感染，各国年轻女性的疫苗接种率差异很大；在美国，年轻女性的疫苗接种率目前大约只有30%。尽管有针对HPV16和HPV18的基于vlp的预防性疫苗（约占宫颈癌的70%），但仍需要针对HPV相关癌症和导致这些癌症的持续感染的治疗选择。由于每年新诊断出50万例宫颈癌病例，而目前的疫苗对数百万已感染的妇女没有治疗效果，因此需要开发治疗方案。两种病毒致癌基因E6和E7与hpv相关的致癌作用有关。E6最具特色的功能是靶向p53降解。E6通过劫持细胞泛素连接酶E6相关蛋白（E6AP）来形成泛素化p53的复合物，从而介导p53的降解。在hpv阳性细胞中抑制E6或E6AP可导致p53稳定并随后因e7诱导的癌性应激而凋亡，从而验证了抑制E6/E6AP活性作为治疗hpv相关癌症和持续性感染的潜在治疗方法。这种发展应用的目标是通过高通量筛选确定稳定HPV阳性宫颈癌中p53的小分子。我们设计了HPV16和HPV18宫颈癌细胞系，以提供活细胞中p53稳定性的GFP/DsRed荧光比例指标。提出的小分子筛选将利用p53报告细胞SiHa细胞（一种HPV16阳性的宫颈癌细胞系），并将使用类似工程的人骨肉瘤细胞系（SJSA-1）进行反筛选，其中p53受Mdm2而不是E6/E6AP调节，以识别对hpv相关癌症和癌前持续性感染具有特异性的小分子。这一发展应用程序汇集了Peter Howley在HPV方面的专业知识和Nathanael Gray在药物化学方面的专业知识，通过识别小分子来解决癌症治疗中的重要需求，这些小分子可以作为工具化合物甚至是先导化合物，用于治疗宫颈癌和HPV阳性肿瘤前病变的治疗。

DESCRIPTION (provided by applicant): The human papillomaviruses (HPVs) are causally linked to a number of human cancers. Over 120 different HPVs have been identified and a subset of them are associated with lesions that are at risk for progression to cancer. Virtually al cases of cervical cancer are attributable to infection by HPV. Of the many different HPV types, 14 are referred to as "high risk" because of their association with anogenital cancers. These high risk HPVs are associated with cervical cancer, other anogenital cancers and approximately 20% of head and neck cancers. The current VLP-based vaccines prevent infection by only 2 of the 14 high-risk HPV types and vaccination rates in young women vary greatly among countries; in the United States the rate of vaccination among young women is presently approximately only 30%. Despite the availability of VLP-based preventive vaccines against HPV16 and HPV18 (that account for roughly 70% of cervical cancers), there is a need for therapeutic options targeting HPV- associated cancers and the persistent infections that give rise to these cancers. With 500,000 new cases of cervical cancer diagnosed annually and current vaccines having no therapeutic benefit for the millions of already infected women, there is a need for the development of treatment options Two viral oncogenes, E6 and E7, have been implicated in driving HPV-associated carcinogenesis. The best characterized function of E6 is the targeting of p53 for degradation. E6 mediates p53 degradation by hijacking the cellular ubiquitin ligase E6-associated protein (E6AP) to form a complex that ubiquitylates p53. Inhibition of E6 or E6AP in HPV-positive cells leads to p53 stabilization and subsequent apoptosis due to E7-induced oncogenic stress, validating the inhibition of E6/E6AP activity as a potential therapeutic approach for treating HPV-associated cancers and persistent infections. The goal of this developmental application is to identify, via a high throughput screen, small molecules that stabilize p53 in HPV positive cervical cancer. We have engineered HPV16 and HPV18 cervical cancer cell lines to provide a GFP/DsRed fluorescent ratio indicator of p53 stability in living cells. The small molecule screen proposed will utilize the p53 reporter SiHa cells (an HPV16 positive cervical cancer cell line) and would employ a counter screen using a similarly engineered human osteosarcoma cell line (SJSA-1) in which p53 is regulated by Mdm2 rather than E6/E6AP to identify small molecules that would be specific for HPV-associated cancers and precancerous persistent infections. This developmental application brings together the laboratories of Peter Howley with expertise in HPV and Nathanael Gray with expertise in medicinal chemistry to address an important need in cancer treatment by identifying small molecules that could serve as tool compounds or even lead compounds for development of therapies to treat cervical cancer and HPV-positive preneoplastic lesions.