Small-Molecule Covalent E6 Antagonists for Treatment of HPV Infection

小分子共价 E6 拮抗剂治疗 HPV 感染

• 批准号: 10397131
• 负责人: ELLIOT J. ANDROPHY
• 金额: $ 64.51万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10397131
• 关键词: Affinity; Algorithms; Alkynes; Apoptosis; Applications Grants; Binding; Binding Sites; Biochemical; Biological; Biological Assay; Cancer cell line; Carcinoma in Situ; Cell Culture Techniques; Cell Line; Cell Proliferation; Cell Survival; Cell model; Cells; Cervical; Charge; Clinical Trials; Complex; Computing Methodologies; Crystallization; Cysteine; DNA; Data; Docking; Drug Design; Episome; Epithelial; Epithelial Cells; Evaluation; Exhibits; FDA approved; Funding; Genome; Goals; Grant; Growth; HPV-High Risk; Human Papillomavirus; Human papilloma virus infection; Human papillomavirus 16; Human papillomavirus 18; In Vitro; Infection; Interferometry; Kinetics; Maintenance; Malignant - descriptor; Malignant Neoplasms; Malignant neoplasm of anus; Malignant neoplasm of cervix uteri; Mass Spectrum Analysis; Mediating; Methods; Modification; Molecular; Neoplasm Metastasis; Oncogenic; Oropharyngeal; Parents; Pathology; Peptides; Persons; Pharmaceutical Chemistry; Pharmaceutical Preparations; Pharmacology; Positioning Attribute; Prevention; Process; Property; Proteins; Reaction; Research Personnel; Resolution; Roentgen Rays; Series; Serine; Site; Specificity; Structure; Surface; TP53 gene; Techniques; Testing; Time; Topical application; Tumor Suppressor Proteins; UBE3A gene; Viral Genome; Viral Proteins; Virus Replication; Work; X-Ray Crystallography; adduct; antagonist; base; cancer cell; cell killing; cellular targeting; chemical synthesis; covalent bond; design; expectation; first-in-human; high risk; improved; infection risk; inhibitor; insight; interdisciplinary approach; malignant oropharynx neoplasm; novel; preclinical evaluation; predictive modeling; premalignant; prevent; protein protein interaction; reaction rate; recruit; scaffold; screening; senescence; small molecule; small molecule libraries; structural biology; tumor progression; ubiquitin ligase

ABSTRACT “High-risk” human papillomavirus (HPV) types such as 16 (HPV-16) are identified in the majority of HPV- associated pre-malignant and malignant pathologies of cervical, anogenital, and oropharyngeal epithelia. The E6 protein is essential for viral replication and cellular models of oncogenic transformation. We hypothesized that small molecules that bind to and form a covalent bond with E6 will antagonize its functions, including the ability to bind the ubiquitin ligase E6AP and recruitment of p53 for proteasomal degradation. Structure-based computational screening followed by design and synthesis of derivatives led to the identification of a series of small molecules that interact with and form a covalent bond to the HPV-16 E6 protein and inhibit both E6•E6AP association in vitro and E6-mediated p53 degradation in cells. Time- and concentration-dependent mass spectrometry and high resolution co-crystal structures of four small molecules bound to E6 confirmed this hypothesis. The objective of this grant application is to extend our discovery of novel E6 inhibitor chemotypes using computational, biochemical, crystallographic, pharmacologic and cell biological assays to increase potency and activity. In Aim 1, we combine predictive modeling algorithms with these X-ray structures to instruct modifications that engage additional residues at the E6•E6AP interface. In Aim 2, robust biochemical techniques will characterize the binding and reaction kinetics of these inhibitors. X-ray crystallography will be applied to resolve atomic coordinates of new compounds bound to HPV E6 and thereby guide the structure-based computational designs proposed in Aim 1. In Aim 3, we test the small-molecule E6 inhibitors for their specific ability to restore p53 levels, and induce apoptosis ord senescence using HPV-16 expressing cancer cell lines. Direct engagement of E6 in cells will be investigated and potential off-target cellular proteins will be identified. Our expectation is that 2-3 drug-like candidates will emerge that selectively inhibit HPV-16 E6 function and exhibit sub-micromolar IC50 activity and suitable pharmacologic properties to advance toward first in human clinical trials.

摘要 “高危”人乳头瘤病毒（HPV）类型，如16（HPV-16），在大多数HPV-16中被鉴定出来。 宫颈、肛门生殖器和口咽上皮的相关癌前病变和恶性病变。的 E6蛋白对于病毒复制和致癌转化的细胞模型是必需的。我们假设 与E6结合并形成共价键的小分子将拮抗其功能，包括 结合泛素连接酶E6 AP和募集p53用于蛋白酶体降解的能力。基于结构 计算筛选，然后设计和合成衍生物，导致了一系列的鉴定， 与HPV-16 E6蛋白相互作用并形成共价键并抑制两者的小分子 体外E6·E6 AP结合和细胞中E6介导的p53降解。时间和浓度依赖性 质谱和高分辨率共晶体结构的四个小分子结合到E6证实 这个假设。这项资助申请的目的是扩大我们对新型E6抑制剂的发现 化学分型使用计算，生物化学，晶体学，药理学和细胞生物学测定， 增加效力和活性。在目标1中，我们将联合收割机预测建模算法与这些X射线 E6·E6 AP结构指示在E6·E6 AP界面处接合额外残基的修饰。在目标2中，稳健 生物化学技术将表征这些抑制剂的结合和反应动力学。x射线 将应用晶体学来解析与HPV E6结合的新化合物的原子坐标，从而 指导目标1中提出的基于结构的计算设计。在目标3中，我们测试了小分子E6 HPV-16抑制剂具有恢复p53水平和诱导细胞凋亡和衰老的特异性能力， 表达癌细胞系。将研究E6在细胞中的直接参与，并可能脱靶 将鉴定细胞蛋白质。我们的期望是，2-3类药物的候选人将出现，选择性 抑制HPV-16 E6功能并显示亚微摩尔IC 50活性和合适药理学性质， 在人类临床试验中取得了第一名。