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。

基本信息

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
项目类别：
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

项目摘要

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.

“高危”HPV 病毒复制需要人乳头瘤病毒 (HPV) E6 蛋白。类型可以进展为称为不典型增生的癌前病变，经过数年的时间，最终可能会导致侵袭性和转移性上皮恶性肿瘤。HPV E6 蛋白与泛素连接酶 E6AP 结合。复合物针对 p53 和其他 E6 结合蛋白进行蛋白酶体介导的破坏。 p53 的缺失使 HPV 能够绕过宿主细胞防御并促进细胞周期的激活。 E6AP 和其他 E6 相互作用蛋白利用带电荷的亮氨酸 α-螺旋 LxxLL 肽基序结合 E6.使用基于该E6AP基序结构的分子模型，我们之前鉴定了一系列新型黄酮类分子可抑制 HPV-16 E6 与 E6AP 的结合。化合物导致 p53 和 p21Cip1/Waf1 蛋白增加，并减少 HPV 表达细胞的增殖我们使用计算模型来预测这些化合物与最近描述的结合的位置。 HPV-16 E6 与 LxxLL 肽的高分辨率三维共晶结构。评分拟合将黄酮抑制剂置于疏水袋中，该疏水袋与亮氨酸形成分子键 E6AP结合口袋和E6的一系列侧翼精氨酸这些数据支持我们基于结构的数据。重要的是，基于抑制 E6 与 E6AP 的 LxxLL 进行筛选和化合物选择。肽结合诱导 E6 蛋白发生变构变化，这是 p53 进入复合物所需的。抑制剂可能会干扰 E6 的构象变化和/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，药物不利代谢特性，因此作为治疗药物不是最理想的。 HPV E6 抑制剂疗法需要专有的化合物类别，我们利用了黄酮。基于结构-活性关系并修改核心结构以建立一种新颖且高度可成药的药物初步研究证明我们可以改变支架并保留 E6 抑制活性，同时效果较弱。与黄酮相比，这项 SBIR 资助将使我们能够利用已建立的检测方法快速提高功效体外和细胞内 E6AP 结合、E6 热稳定性和 p53 降解的竞争性抑制。一系列集中突变的 E6 蛋白，破坏 E6 结合的疏水性和带电表面 pocket让我们探索E6和抑制化合物之间的结合界面，我们观察到了一些。点突变破坏 E6-E6AP 相互作用，但不会改变化合物与 E6 的结合，相反，一些突变体保留 E6AP 结合但限制化合物关联。这些数据证实了我们的定位。化合物对 E6 结构的影响，并指导设计更特异和有效的 HPV E6 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。