Small Molecule E6 Inhibitors to Treat Oropharyngeal Cancers Caused by HPV Infections

致癌性人乳头瘤病毒(HPV)，如HPV16型，是50%-70%癌症的病原体 口咽部的。在美国，HPV相关性口咽癌的发病率正在上升。这是 部分原因是目前还没有确定的口服HPV筛查试验。在1-8%的人中，通过聚合酶链式反应检测到HPV-16DNA 口腔拭子取决于接受调查的人群，其中艾滋病毒携带者的频率最高。只有一个 这些感染中的一小部分将发展为直白的恶性肿瘤。此外，早期病变很难定位。 而患者在患上晚期肿瘤之前基本上是没有症状的。 科维纳治疗公司正在开发选择性结合HPV-16E6癌蛋白的抗病毒疗法 并阻止与其细胞目标的相互作用。E6是病毒基因组复制和维护所必需的 并且总是在HPV相关的异型增生和恶性肿瘤中表达。我们在硅胶结构中表演 筛选适合E6对接部位的分子的化学库，以供其几个细胞伙伴使用。我们的 分析的重点是E6蛋白与E6AP的相互作用，E6AP将泛素转移到肿瘤上 抑制蛋白P53，并导致其被蛋白酶体破坏。我们设计并合成了 1)与HPV-16E6结合的化合物和2)与HPV-16E6中的特定半胱氨酸在其 蛋白质-蛋白质与E6AP以及其他细胞相互作用蛋白质相互作用。识别的方法 装有反应性弹头的分子可以与半胱氨酸进行共价和不可逆的结合 在几种新的临床可用药物上取得了成功，这一领域正在迅速发展到靶向蛋白质，这些蛋白质 之前被认为是不可下药的。我们发现这些化合物共价结合到HPV-16E6，阻断 E6与E6AP相互作用，恢复P53功能，促使HPV感染细胞发生凋亡。多元生化 包括X射线结晶学在内的分析证明，这些抑制剂占据了E6口袋，形成了单一的加合物 用这种半胱氨酸。在共晶数据的指导下进行迭代优化，设计并合成了新化合物 &gt；200导致活性增强的新型化学类型。这项为期一年的第一阶段SBIR计划将选择销售线索 在此基础上进行药代动力学表征。最有希望的E6抑制剂将在体内进行测试和验证 在肿瘤异种移植模型中使用人类口咽癌来源细胞的概念性实验。成功 这些第一阶段实验的结果将有力地为我们提供第二阶段SBIR赠款，以实现IND-Enabling 学习。

Oncogenic human papillomaviruses (HPV) such as HPV type 16 are the etiologic agent of 50-70% of cancers of the oropharynx. In the USA, the incidence of HPV-associated oropharyngeal cancers is increasing. This is partly because there is no established screening test for oral HPV. HPV-16 DNA is detected by PCR in 1-8% of oral swabs depending on the surveyed populations, with the highest frequencies in HIV+ individuals. Only a small fraction of these infections will progress to frank malignancy. Moreover, early lesions are difficult to locate and patients are largely asymptomatic until they have advanced tumors. Kovina Therapeutics is developing anti-viral therapeutics that selectively bind to the HPV-16 E6 oncoprotein and prevent interactions with its cellular targets. E6 is necessary for viral genome replication and maintenance and is always expressed in HPV-associated dysplasia and malignancies. We performed in silico structural screening of chemical libraries for molecules that fit the E6 docking site for several of its cellular partners. Our assays focused on the E6 protein-protein interaction with E6AP, which transfers ubiquitin onto the tumor suppressor protein p53 and leads to its destruction by the proteasome. We designed and synthesized compounds that 1) bind to HPV-16 E6 and 2) form a covalent bond with a specific cysteine in HPV-16 E6 at its protein-protein interface with E6AP as well as other cellular interacting proteins. The approach to identify molecules equipped with reactive ‘warheads’ that mediate covalent and irreversible binding to cysteine has been successful for several new clinically available drugs, and this field is rapidly progressing to target proteins that were previously considered undruggable. We show that these compounds covalently bind to HPV-16 E6, block E6 interaction with E6AP, restore P53 function, and drive the HPV infected cell to apoptosis. Multiple biochemical analyses including X-ray crystallography prove these inhibitors occupy this E6 pocket and form a single adduct with this cysteine. Iterative optimization guided by the co-crystal data resulted in the design and synthesis of >200 novel chemotypes that resulted in increased activity. This one-year Phase I SBIR proposal will select leads based on for pharmacokinetic characterization. The most promising E6 inhibitors will then be test in vivo proof of concept experiments using human oropharyngeal cancer derived cells in a tumor xenograft model. Successful results in these Phase I experiments will strongly position us for a Phase II SBIR grant to carry out IND-enabling studies.