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

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

展开

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.

人乳头瘤病毒（HPV）E6蛋白需要进行病毒复制。感染“高风险” HPV 类型可以发展为称为增生性的恶性病变，多年来，这种病变可能会在侵入性和转移性上皮性疟疾。 HPV E6蛋白与泛素连接酶E6AP结合，这复杂的靶标P53和其他E6结合蛋白用于蛋白酶体介导的破坏。这种依赖于E6 p53的丢失使HPV绕过宿主细胞防御并促进细胞周期的激活。 E6AP和其他E6相互作用的蛋白使用带电的亮氨酸螺旋lxxllial lxxll pepperide基序与结合 E6。使用基于该E6AP基序的结构的分子建模，我们先前鉴定了一系列新颖的黄酮类似于抑制HPV-16 E6与E6AP结合的分子。宫颈癌细胞暴露于这些化合物导致INP53和P21CIP1/WAF1蛋白增加，并改善了HPV表达细胞的增殖线。我们使用计算建模来预测这些化合物在哪里结合到最近所描述的 HPV-16 E6的高分辨率三维共晶结构与LXXLL Pepperide。最高评分拟合将黄酮抑制剂放在疏水口袋中 E6AP结合口袋和一系列E6的侧翼精氨酸。这些数据支持我们的基于结构的数据基于对E6与E6AP的关联的抑制和化合物选择。重要的是，lxxll 肽结合诱导p53进入复合物所需的E6蛋白的变构变化。我们的抑制剂可能会干扰E6中的这种会议变化和/或其蛋白质 - 蛋白质与p53的相互作用。类黄酮小分子抑制HPV E6，但水溶性差和不利的药物代谢特性，因此是次优的治疗。另外，小分子的发展 HPV E6抑制剂疗法将需要专有化合物类别。我们利用了黄酮 - 基于结构活性关系，并修改了核心结构，以建立一种新颖且高度吸毒的化学系列。试点研究证明我们可以改变支架并保留E6抑制活性。虽然有效比黄酮，这项SBIR赠款将使我们能够使用既定测定法 E6AP结合，E6热稳定性和p53降解的竞争抑制作用在体外和细胞中。一个聚焦的一系列突变的E6蛋白破坏E6结合的疏水表面和带电的表面口袋让我们探索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，但不会改变与E6的化合物结合，相反，一些突变体保留E6AP结合，但限制了复合关联。这些数据证实了我们的定位 E6结构上的化合物和更具体和潜在的HPV E6抑制剂的指导设计。我们已经表明我们可以选择和合成E6-E6AP相互作用的抑制剂，因此可能是A 对数百万的新型治疗方法，患有恶性和恶性HPV相关的取消器。