Small Molecule Screen Targeting p53 proteolysis in HPV positive cancers

描述(申请人提供)：人乳头瘤病毒(HPV)与许多人类癌症有因果关系。目前已发现120多种不同的HPV，其中一部分与有进展为癌症风险的病变有关。几乎所有的宫颈癌病例都可归因于HPV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道癌有关。这些高危HPV与宫颈癌、其他肛门癌和大约20%的头颈癌有关。目前以VLP为基础的疫苗只能预防14种高危HPV类型中的2种感染，年轻女性的疫苗接种率在各国之间差别很大；在美国，年轻女性的疫苗接种率目前仅约为30%。尽管基于VLP的针对HPV16和HPV18(约占宫颈癌的70%)的预防性疫苗已经问世，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。随着每年新诊断的50万宫颈癌病例，以及目前的疫苗对数百万已经感染的妇女没有治疗效果，有必要开发治疗方案两个病毒癌基因，E6和E7，已被牵连到驱动HPV相关的癌症发生。E6最具特征的功能是靶向P53的降解。E6通过劫持细胞泛素连接酶E6相关蛋白(E6AP)形成泛素化P53的复合体来介导P53的降解。抑制HPV阳性细胞中的E6或E6AP导致P53的稳定和随后的E7致癌应激导致的细胞凋亡，证实了抑制E6/E6AP活性是治疗HPV相关癌症和持续感染的潜在治疗方法。这一开发应用的目标是通过高通量筛查确定稳定HPV阳性宫颈癌中P53的小分子。我们已经设计了HPV16和HPV18宫颈癌细胞系，以提供活细胞中P53稳定性的GFP/DsRed荧光比率指示器。建议的小分子筛查将利用P53报告基因SIHA细胞(一种HPV16阳性的宫颈癌细胞株)，并使用使用类似工程的人类骨肉瘤细胞系(SJSA-1)的计数器筛查，其中P53由MDM2而不是E6/E6AP调控，以识别HPV相关癌症和癌前持续感染的特异性小分子。这一开发应用将Peter Howley和Nathanael Gray的实验室与HPV方面的专业知识和Nathanael Gray在药物化学方面的专业知识结合在一起，通过识别可用作工具化合物甚至先导化合物的小分子来满足癌症治疗的重要需求，以开发治疗宫颈癌和HPV阳性癌前病变的疗法。

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.