Small Molecule Screen Targeting p53 proteolysis in HPV positive cancers

描述（由申请人提供）：人乳头瘤病毒（HPV）与许多人类癌症存在因果关系。已经鉴定了超过120种不同的HPV，其中一部分与有进展为癌症风险的病变相关。几乎所有的宫颈癌病例都是由HPV感染引起的。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。这些高危HPV与宫颈癌、其他肛门生殖器癌和约20%的头颈癌有关。目前基于VLP的疫苗仅预防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异很大;在美国，年轻女性的疫苗接种率目前约为30%。尽管有针对HPV 16和HPV 18（约占宫颈癌的70%）的基于VLP的预防性疫苗可用，但仍需要靶向HPV相关癌症和引起这些癌症的持续感染的治疗选择。每年诊断出50万例宫颈癌新病例，而目前的疫苗对数百万已感染的妇女没有治疗益处，因此需要开发治疗方案。E6的最佳表征功能是靶向p53降解。E6通过劫持细胞泛素连接酶E6相关蛋白（E6 AP）形成使p53泛素化的复合物来介导p53降解。HPV阳性细胞中E6或E6 AP的抑制导致p53稳定和随后的由于E7诱导的致癌应激而引起的细胞凋亡，从而验证了E6/E6 AP活性的抑制作为治疗HPV相关癌症和持续性感染的潜在治疗方法。该开发应用的目标是通过高通量筛选鉴定稳定HPV阳性宫颈癌中p53的小分子。我们已经改造了HPV 16和HPV 18宫颈癌细胞系，以提供活细胞中p53稳定性的GFP/DsRed荧光比率指示剂。提出的小分子筛选将利用p53报告SiHa细胞（HPV 16阳性宫颈癌细胞系），并将采用使用类似工程化的人骨肉瘤细胞系（SJSA-1）的计数器筛选，其中p53由Mdm 2而不是E6/E6 AP调节，以鉴定对HPV相关癌症和癌前持续感染特异的小分子。这种开发应用汇集了Peter Howley的实验室，他们在HPV方面具有专业知识，Nathanael Gray在药物化学方面具有专业知识，通过鉴定可以作为工具化合物甚至先导化合物的小分子来解决癌症治疗的重要需求，用于开发治疗宫颈癌和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.