Small-Molecule Covalent E6 Antagonists for Treatment of HPV Infection

小分子共价 E6 拮抗剂治疗 HPV 感染

基本信息

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
项目类别：
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

项目摘要

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.

抽象的大多数 HPV 中都发现了“高危”人乳头瘤病毒 (HPV) 类型，例如 16 型 (HPV-16) 宫颈、肛门生殖器和口咽上皮的相关癌前和恶性病理。 E6 蛋白对于病毒复制和致癌转化的细胞模型至关重要。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 E6AP 和招募 p53 进行蛋白酶体降解的能力。计算筛选，然后设计和合成衍生物，从而鉴定出一系列与 HPV-16 E6 蛋白相互作用并形成共价键并抑制两者的小分子 E6•E6AP 体外关联和 E6 介导的细胞内 p53 降解具有时间和浓度依赖性。质谱和高分辨率共晶结构证实了四种与 E6 结合的小分子这一假设的目的是扩展我们对新型 E6 抑制剂的发现。使用计算、生物化学、晶体学、药理学和细胞生物学测定来确定化学型在目标 1 中，我们将预测建模算法与这些 X 射线结合起来。指导在 E6·E6AP 界面上进行额外残基的修饰的结构，目标 2，稳健。生化技术将表征这些抑制剂的结合和反应动力学。晶体学将用于解析与 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 抑制剂具有恢复 p53 水平的特定能力，并使用 HPV-16 诱导细胞凋亡或衰老将研究 E6 在细胞中的直接参与以及潜在的脱靶情况。我们预计将会有 2-3 种类似药物的候选药物被选择性地鉴定出来。抑制 HPV-16 E6 功能并表现出亚微摩尔 IC50 活性和合适的药理学特性迈向人体临床试验第一的进展。