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：
10220227
负责人：
ELLIOT J. ANDROPHY
金额：
$ 63.29万
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/10220227
关键词：
Affinity Algorithms Alkynes Antiviral Agents 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 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 base cancer cell cell killing cellular targeting chemical synthesis covalent bond design expectation first-in-human high risk improved infection risk inhibitor/antagonist 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-鉴定出“高风险”人乳头瘤病毒（HPV）类型，例如16（HPV-16）相关的宫颈，肛门生殖和口咽上皮的恶性和恶性病理。 E6蛋白对于病毒复制和致癌转化的细胞模型至关重要。我们假设与E6共价键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并募集p53进行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 E6•E6AP在细胞中的体外和E6介导的p53降解。时间和浓度依赖性与E6结合的四个小分子的质谱和高分辨率共结构结构证实这个假设。该赠款应用的目的是扩展我们对新型E6抑制剂的发现使用计算，生化，晶体学，药物和细胞生物学测定的化学型增加效力和活动。在AIM 1中，我们将预测建模算法与这些X射线结合在一起结构以指导在E6•E6AP接口上接合其他残差的修改。在AIM 2中，强大生化技术将表征这些抑制剂的结合和反应动力学。 X射线晶体学将应用于与HPV E6结合的新化合物的原子坐标。指导AIM 1中提出的基于结构的计算设计。在AIM 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诱导凋亡ORD SENSTIM 表达癌细胞系。将研究E6在细胞中的直接参与，并潜在地靶向靶向将确定细胞蛋白。我们的期望是，有2-3种类似毒品的候选者会有选择地出现抑制HPV-16 E6功能并表现出亚微摩尔IC50活性，并具有合适的药物学特性在人类临床试验中迈进。