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

将测试人乳头瘤病毒E6蛋白小分子抑制剂的构效关系。

• 批准号: 9200070
• 负责人: Alison M Strack
• 金额: $ 22.5万
• 依托单位: SUNNYLIFE PHARMA, INC.
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-14 至 2017-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9200070
• 关键词: Accounting; Affinity; Amino Acids; Apoptosis; Arginine; Binding; Binding Proteins; Biological Assay; Bypass; Cancer cell line; Cell Cycle; Cell Line; Cells; Cellular Assay; Characteristics; Charge; Chemicals; Collection; Complex; Computer Simulation; Country; Data; Development; Digit structure; Down-Regulation; Dysplasia; Economically Deprived Population; Epithelial; Exhibits; Flavones; Flavonoids; Genetic study; Grant; High-Risk Cancer; Homologous Gene; Human; Human Papilloma Virus Vaccine; Human Papillomavirus; Human papillomavirus 16; In Vitro; Infection; Lead; Left; Lesion; Leucine; Ligands; Malignant - descriptor; Malignant Neoplasms; Malignant neoplasm of cervix uteri; Measures; Mediating; Medical; Molecular; Molecular Bank; Molecular Models; Mutate; Mutation; Names; Neoplasm Metastasis; Oncogenic; Pathology; Peptides; Pharmaceutical Preparations; Phase; Pilot Projects; Point Mutation; Positioning Attribute; Premalignant; Property; Protein p53; Proteins; Resolution; Series; Side; Small Business Innovation Research Grant; Solubility; Structure; Structure-Activity Relationship; Surface; TP53 gene; Technology; Testing; Therapeutic; Time; Tumor-Derived; Ubiquitination; Vaccines; Virus Replication; Woman; Work; aqueous; base; cancer cell; design; drug discovery; drug metabolism; expectation; high risk; in vivo; inhibitor/antagonist; men; molecular modeling; multicatalytic endopeptidase complex; mutant; nanomolar; neoplastic; neoplastic cell; novel; pharmacophore; prevent; programs; protein protein interaction; research study; scaffold; screening; senescence; small molecule; small molecule inhibitor; tool; ubiquitin ligase

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.

人乳头瘤病毒（HPV）E6蛋白是病毒复制所必需的。感染“高危”HPV 类型可以进展到称为发育不良的癌前病变，在一段时间内， 侵袭性和转移性上皮恶性肿瘤。HPV E6蛋白与泛素连接酶E6 AP结合， 复合物靶向p53和其他E6结合蛋白，用于蛋白酶体介导的破坏。E6依赖型 p53的缺失使得HPV能够绕过宿主细胞防御并促进细胞周期的激活。 E6 AP和其他E6相互作用蛋白利用带电亮氨酸β-螺旋LxxLL肽基序结合E6 AP和其他E6相互作用蛋白。 E6.使用基于E6 AP基序结构的分子建模，我们先前鉴定了一系列 抑制HPV-16 E6与E6 AP结合的新型黄酮样分子。子宫颈癌细胞暴露于这些 化合物导致p53和p21 Cip 1/Waf 1蛋白表达增加，HPV表达细胞增殖降低 线我们使用计算机建模来预测这些化合物与最近描述的 HPV-16 E6与LxxLL肽高分辨率三维共晶体结构。最高 将黄酮抑制剂置于疏水口袋中，与亮氨酸形成分子键， E6 AP结合口袋和E6的一系列侧翼序列。这些数据支持我们基于结构的 基于E6与E6 AP结合的抑制进行筛选和化合物选择。重要的是，LxxLL 肽结合诱导了p53进入复合物所需的E6蛋白的变构变化。我们 抑制剂可以干扰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HPV E6，但水溶性差， 代谢特性，因此作为治疗剂是次优的。此外，小分子的发展 HPV E6抑制剂治疗将需要一个专有的化合物类。我们利用了黄酮- 基于构效关系和修改的核心结构，建立一个新的和高度可药用的 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。虽然效力较低 比黄酮，这SBIR赠款将使我们能够迅速提高效力使用建立的测定， 体外和细胞中E6 AP结合、E6热稳定和p53降解的竞争性抑制。一 聚焦一系列突变的E6蛋白，这些蛋白破坏E6结合的疏水和带电表面， 口袋让我们探索E6和抑制性化合物之间的结合界面。我们观察到一些 点突变破坏E6-E6 AP相互作用，但不改变化合物与E6的结合，相反， 一些突变体保留E6 AP结合，但限制化合物缔合。这些数据证实了我们的定位 E6结构上的化合物，并指导设计更特异和有效的HPV E6抑制剂。 我们已经表明，我们可以选择和合成E6-E6 AP相互作用的抑制剂，因此可能是一种新的抑制剂。 为数百万患有癌前病变和恶性HPV相关癌症的患者提供新的治疗方法。