Structure-activity relationship of small molecule inhibitors of human papillomavirus E6 protein will be tested.

人类乳头瘤病毒(HPV)E6蛋白是病毒复制所必需的。感染“高危”人乳头瘤病毒 这些类型可以进展到称为异型增生的癌前病变，经过几年的时间，可以最终发展为 侵袭性和转移性上皮性恶性肿瘤。HPV E6蛋白与泛素连接酶E6AP结合，这 复合体以P53和其他E6结合蛋白为靶标进行蛋白酶体介导的破坏。这依赖于E6 P53的缺失使HPV能够绕过宿主细胞的防御，促进细胞周期的激活。 E6AP和其他E6相互作用蛋白利用带电的亮氨酸-螺旋LxxLL多肽基序结合到 E6。利用基于E6AP基序结构的分子建模，我们先前鉴定了一系列 抑制HPV-16E6与E6AP结合的新型类黄酮类分子。宫颈癌细胞对这些物质的暴露 化合物诱导P53和p21Cip1/Waf1蛋白表达增加并抑制HPV表达细胞的增殖 台词。我们使用计算模型来预测这些化合物与最近描述的 HPV-16E6与LxxLL多肽的高分辨三维共晶结构。最高的 Score Fits将黄酮类抑制剂放置在疏水口袋中，与亮氨酸形成分子键 E6AP结合口袋和一系列E6的侧翼精氨酸。这些数据支持我们基于结构的 基于抑制E6与E6AP结合的筛选和化合物筛选。重要的是，LxxLL 多肽结合导致P53进入复合体所需的E6蛋白发生变构变化。我们的 抑制剂可能会干扰E6的这种构象变化和/或其与P53的蛋白质-蛋白质相互作用。 黄酮类小分子抑制HPVE6，但水溶性差，药物不利 新陈代谢特性，因此作为治疗药物是次要的。此外，小分子的发展 HPVE6抑制剂疗法将需要一种专利化合物类别。我们利用了黄酮类- 基于构效关系，并对核心结构进行了修改，以建立一种新的、高度可药物的 化学系列。初步研究证明，我们可以改变支架并保留E6抑制活性。虽然威力较小 这项SBIR拨款将允许我们使用已建立的检测方法快速提高效力 竞争性抑制E6AP结合，E6热稳定，以及体外和细胞内P53的降解。一个 破坏E6结合的疏水表面和带电表面的一系列突变的E6蛋白 Pocket让我们探索E6和抑制性化合物之间的结合界面。我们观察到了一些 点突变破坏了E6-E6AP的相互作用，但不改变化合物与E6的结合，反之， 一些突变体保留了E6AP结合，但限制了化合物的结合。这些数据证实了我们的定位 对E6结构上的化合物进行研究，并指导设计更特异和更有效的HPVE6抑制剂。 我们已经证明，我们可以选择和合成E6-E6AP相互作用的抑制剂，因此有可能 为数百万患有癌前和恶性HPV相关癌症的患者提供新的治疗方法。

The human papillomavirus (HPV) E6 protein is needed for viral replication. Infection with “high risk” HPV types can progress to pre-malignant lesions called dysplasias, which over a period of years, can eventuate in invasive and metastatic epithelial malignancies. HPV E6 proteins bind to the ubiquitin ligase E6AP and this complex targets p53 and other E6 binding proteins for proteasome mediated destruction. This E6-dependent loss of p53 enables HPV to bypass host cell defenses and facilitates activation of the cell cycle. E6AP and other E6 interacting proteins utilize a charged leucine -helical LxxLL peptide motif to bind to E6. Using molecular modeling based on the structure of this E6AP motif, we previously identified a series of novel flavone like molecules that inhibit HPV-16 E6 binding to E6AP. Exposure of cervical cancer cells to these compounds led to increases inp53 and p21Cip1/Waf1 proteins and decreased proliferation of HPV expressing cell lines. We used computational modeling to predict where these compounds bind onto the recently described high-resolution three-dimensional co-crystal structure of HPV-16 E6 with an LxxLL peptide. The highest scoring fits placed the flavone inhibitors in a hydrophobic pocket that forms molecular bonds with leucines in the E6AP binding pocket and a series of flanking arginines of E6. These data support our structure-based screening and compound selection based on inhibition of E6 association with E6AP. Importantly, LxxLL peptide binding induces an allosteric change in the E6 protein needed for entry of p53 into the complex. Our inhibitors may interfere with this conformational change in E6 and/or its protein-protein interaction with p53. Flavonoid small molecules inhibit HPV E6 but have poor aqueous solubility and unfavorable drug metabolism properties and thus are suboptimal as therapeutics. Additionally, development of small molecule HPV E6 inhibitor therapeutics will require a proprietary compound class. We took advantage of the flavone- based structure-activity relationships and modified the core structure to establish a novel and highly druggable chemical series. Pilot studies prove we can alter the scaffold and retain E6 inhibitory activity. While less potent than the flavones, this SBIR grant will allow us to rapidly increase potency using established assays of competitive inhibition of E6AP binding, E6 thermo-stabilization, and p53 degradation in vitro and in cells. A focused series of mutated E6 proteins that disrupt the hydrophobic and charged surfaces of the E6 binding pocket let us explore the binding interface between E6 and inhibitory compounds. We have observed some point mutations disrupt the E6-E6AP interaction but do not alter compound binding to E6 and, conversely, some mutants retain E6AP binding but restrict compound association. These data substantiate our positioning of compounds on the E6 structure and instruct design of more specific and potent HPV E6 inhibitors. We have shown we can select and synthesize inhibitors of the E6-E6AP interaction and thus potentially, a novel treatment for the millions afflicted with pre-malignant and malignant HPV associated cancers.