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

致癌性人乳头瘤病毒（HPV）如HPV 16型是50-70%癌症的病原体 口咽部在美国，HPV相关口咽癌的发病率正在增加。这是 部分原因是没有建立口腔HPV的筛查测试。HPV-16 DNA通过PCR在1-8%的 口腔拭子的感染率取决于被调查人群，艾滋病毒阳性者的感染率最高。只有 这些感染中的一小部分将发展为明显的恶性肿瘤。而且，早期病变很难定位 并且患者在患晚期肿瘤之前基本上没有症状。 Kovenant Therapeutics正在开发选择性结合HPV-16 E6癌蛋白的抗病毒治疗剂 并阻止其与细胞靶点的相互作用。E6是病毒基因组复制和维持所必需的 并且总是在HPV相关的异型增生和恶性肿瘤中表达。我们进行了计算机结构分析， 筛选化学文库中适合E6对接位点的分子，用于其几种细胞伴侣。我们 分析集中在E6蛋白与E6 AP的相互作用，E6 AP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。我们设计合成了 化合物1）结合HPV-16 E6和2）与HPV-16 E6中的特异性半胱氨酸在其 与E6 AP以及其他细胞相互作用蛋白质的蛋白质-蛋白质界面。识别方法 配备有反应性“弹头”的分子介导了与半胱氨酸的共价和不可逆结合， 成功地用于几种新的临床可用药物，并且该领域正在迅速发展到靶向蛋白质， 以前被认为是不可用的我们表明，这些化合物共价结合HPV-16 E6，阻断HPV-16 E6， E6与E6 AP相互作用，恢复P53功能，驱动HPV感染细胞凋亡。多个生物化学 包括X射线晶体学在内的分析证明这些抑制剂占据了E6口袋并形成单一加合物 这个半胱氨酸。在共晶数据的指导下进行迭代优化，设计和合成了 >200种新的化学型，导致活性增加。这份为期一年的第一阶段SBIR提案将选择潜在客户 基于药代动力学表征。最有前途的E6抑制剂将在体内进行测试证明 在肿瘤异种移植模型中使用人口咽癌衍生细胞的概念实验。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拨款，以实现IND 问题研究

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.