Biophysics of Drug Interactions with Viral Episomes

药物与病毒附加体相互作用的生物物理学

基本信息

批准号：
8259751
负责人：
James K. Bashkin
金额：
$ 36.11万
依托单位：
UNIVERSITY OF MISSOURI-ST. LOUIS
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-05-01 至 2015-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8259751
关键词：
3-Dimensional Affinity Antiviral Agents Behavior Benchmarking Binding Binding Sites Biological Assay Biophysics Blood capillaries Calibration Calorimetry Cancer Etiology Capillary Electrophoresis Cell Culture Techniques Cell Nucleus Cells Collaborations Complement Complex Confocal Microscopy Contracts DNA DNA Binding DNA Sequence Data Data Analyses Development Dissociation Drug Interactions Dyes Episome Flow Cytometry Fluorescence Fluorescence Anisotropy Fluorescence Microscopy Genome HPV-High Risk High-Risk Cancer Human Human Papilloma Virus Vaccine Human Papillomavirus Human papilloma virus 31 Human papillomavirus 16 Human papillomavirus 18 Hydroxyl Radical Image In Vitro Interruption Investigation Life Cycle Stages Link Literature Location Maintenance Malignant neoplasm of cervix uteri Mammalian Cell Marketing Measurement Measures Medical Methods Modeling Molecular Models Molecular and Cellular Biology Morphologic artifacts Nuclear Nylons Pharmaceutical Preparations Positioning Attribute Preparation Publishing Resolution Risk Shapes Site Site-Directed Mutagenesis Societies Solutions Solvents Specificity Spectrum Analysis Structure Structure-Activity Relationship Study models Surface Surface Plasmon Resonance Synthesis Chemistry Testing Vaccination Viral Viral Genome Virus Diseases Work base calf thymus DNA capillary cervical cancer prevention fighting in vivo interest keratinocyte molecular modeling pre-clinical preference public health relevance time use tissue/cell culture uptake viral DNA virology virus episome maintenance

项目摘要

DESCRIPTION (provided by applicant): Antiviral compounds for high-risk, cancer-causing human papillomavirus (HPV) are extremely important even though anti-HPV vaccines are on the market. The proposed work determines the biophysics of interactions between antiviral polyamides (PAs) and their viral DNA targets, in order to determine the mechanism of action. The scope has been expanded from anti-HPV16 compounds to new anti-HPV compounds active against three cancer-causing viral subtypes. Active compounds eliminate viral episomes from human cell and tissue culture; they are potential drugs for the prevention of cervical cancer after HPV has been contracted. PAs having the same apparent DNA-binding preferences show dramatically different antiviral activities. It is proposed that a fundamental biophysical explanation of this behavior exists, one that goes beyond the known rules for PA-DNA interactions. Therefore, collaboration has begun around biophysical spectroscopy, DNA binding/footprinting assays, synthetic chemistry, virology and cellular-molecular biology. The aims are: Aim 1 Determine the binding position and affinity on the HPV16 genome of active PAs by footprinting the viral DNA as a function of PA concentration. The genome will be analyzed as overlapping 500 bp sequences using capillary electrophoresis and hydroxy radical footprinting. Binding constants determined for long DNA fragments will be compared with those from simpler models (minimal double-stranded or hairpin DNA). Independent physical methods such as fluorescence anisotropy (in competition binding studies), isothermal calorimetry and surface plasmon resonance will allow benchmarking against the literature and avoid artifacts. Aim 2 Footprint the HPV18 genome in vitro with antiviral PAs. Identify related binding sites for both HPV16 and 18. Carry out NMR structural work on DNA sequences of importance to both HPV16 and 18 as bound to active PAs; use NMR structural results as the basis for molecular modeling of additional interactions. Aim 3 Footprint active PAs on the HPV16 and 18 genomes in vivo in human cell culture. Examine the genomes using approximately 500 bp fragments generated by PCR that overlap to cover all sequence space. Determine experimental conditions and provide absolute binding constants with the aid of PA concentrations in cells measured by confocal and flow cytometry methods. Measure viral DNA concentrations vs. time using QPCR to make sure in vivo footprinting conditions are suitable. Identify structure-dependent uptake of PAs, potentially explaining why some isomeric PAs range from highly active to inactive antiviral agents. As an additional test of conclusions from footprinting, the site-directed mutagenesis of HPV18 sites identified during footprinting of antiviral PAs will help determine the importance of specific PA binding sites to the mechanism of antiviral action and viral episome maintenance. Provide new rules for interruption by polyamides of the high-risk HPV viral life cycle. PUBLIC HEALTH RELEVANCE: The studies proposed here are of relevance to society because they relate directly to fighting several medically-important viral diseases. The work is specifically relevant to the prevention of cervical cancer. The medical value of the project is important because effective antiviral medication to fight HPV has not been developed. The antiviral agents we have discovered, and which we study here, have the potential to save lives when used in conjunction with regular check-ups and vaccination, and have the potential to contribute significantly to the control and elimination of cervical cancer.

描述（由申请人提供）：即使抗HPV疫苗在市场上也是如此，引起癌症的人乳头瘤病毒（HPV）的抗病毒化合物也极为重要。提出的工作决定了抗病毒聚酰胺（PA）及其病毒DNA靶标之间相互作用的生物物理学，以确定作用机理。该范围已从抗HPV16化合物扩展到针对三种引起癌症病毒亚型的新型抗HPV化合物。活性化合物消除了人类细胞和组织培养的病毒发作。它们是HPV收缩后预防宫颈癌的潜在药物。具有相同明显的DNA结合偏好的PA显示出明显不同的抗病毒活性。有人提出，存在对这种行为的基本生物物理解释，它超出了已知的PA-DNA相互作用规则。因此，围绕生物物理光谱，DNA结合/足迹测定，合成化学，病毒学和细胞分子生物学的合作开始了。目的是：AIM 1通过将病毒DNA作为PA浓度的函数来确定活性PA的HPV16基因组的结合位置和亲和力。基因组将使用毛细管电泳和羟基自由基足迹分析为500 bp序列。针对长DNA片段确定的结合常数将与简单模型（最小双链或发夹DNA）的结合常数进行比较。独立的物理方法，例如荧光各向异性（在竞争结合研究中），等温量热法和表面等离子体共振将允许对文献进行基准测试并避免文物。 AIM 2足迹在体外用抗病毒PAS进行HPV18基因组。识别HPV16和18的相关结合位点。对HPV16和18的DNA序列进行NMR结构性工作，与活性PA结合；使用NMR结构结果作为其他相互作用的分子建模的基础。在人类细胞培养中，在体内HPV16和18个基因组上的AIM 3足迹活性PA。使用PCR生成的大约500 bp片段检查基因组，这些片段重叠以覆盖所有序列空间。确定实验条件，并借助于共焦和流式细胞仪方法测量的细胞中PA浓度提供绝对结合常数。使用QPCR测量病毒DNA浓度与时间，以确保体内足迹条件合适。识别结构依赖于PA的摄取，有可能解释为什么某些同类PA从高度活跃到无活跃的抗病毒剂。作为对足迹的结论的额外测试，在抗病毒药PAS足迹期间鉴定出的HPV18位点的位置指导诱变将有助于确定特定PA结合位点对抗病毒作用作用和病毒偶发的机制的重要性。通过聚酰胺的高风险HPV病毒生命周期的聚酰胺中断提供新规则。公共卫生相关性：这里提出的研究与社会有关，因为它们与几种医学上重要的病毒疾病直接相关。这项工作与预防宫颈癌特别相关。该项目的医疗价值很重要，因为尚未开发出有效的与HPV作斗争的抗病毒药物。我们发现的抗病毒剂和我们在这里研究的抗病毒剂有可能与定期检查和疫苗接种一起挽救生命，并有可能为控制和消除宫颈癌的控制和消除做出重大贡献。