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

• 批准号: 8453365
• 负责人: James K. Bashkin
• 金额: $ 33.95万
• 依托单位: UNIVERSITY OF MISSOURI-ST. LOUIS
• 依托单位国家: 美国
• 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/8453365
• 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

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.

描述（由申请人提供）：尽管市场上已有抗 HPV 疫苗，但针对高风险、致癌的人乳头瘤病毒 (HPV) 的抗病毒化合物仍然极其重要。拟议的工作确定了抗病毒聚酰胺 (PA) 与其病毒 DNA 靶标之间相互作用的生物物理学，以确定作用机制。范围已从抗 HPV16 化合物扩展到对三种致癌病毒亚型具有活性的新型抗 HPV 化合物。活性化合物消除人体细胞和组织培养物中的病毒游离体；它们是感染 HPV 后预防宫颈癌的潜在药物。 具有相同的明显 DNA 结合偏好的 PA 表现出显着不同的抗病毒活性。有人提出对这种行为存在一种基本的生物物理学解释，这种解释超出了 PA-DNA 相互作用的已知规则。因此，围绕生物物理光谱学、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已经开始。目标是： 目标 1 通过将病毒 DNA 足迹作为 PA 浓度的函数，确定活性 PA 在 HPV16 基因组上的结合位置和亲和力。将使用毛细管电泳和羟基自由基足迹法将基因组分析为重叠的 500 bp 序列。长 DNA 片段确定的结合常数将与更简单模型（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根据文献进行基准测试并避免伪影。目标 2 使用抗病毒 PA 在体外足迹追踪 HPV18 基因组。确定 HPV16 和 18 的相关结合位点。对 HPV16 和 18 与活性 PA 结合的重要 DNA 序列进行 NMR 结构研究；使用 NMR 结构结果作为其他相互作用的分子建模的基础。目标 3 在人类细胞培养物中体内足迹 HPV16 和 18 基因组上的活性 PA。使用 PCR 生成的约 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共聚焦和流式细胞术方法测量的细胞中 PA 浓度提供绝对结合常数。使用 QPCR 测量病毒 DNA 浓度与时间的关系，以确保体内足迹条件合适。确定 PA 的结构依赖性吸收，可能解释为什么某些异构 PA 的抗病毒药物活性从高活性到无活性。作为对足迹分析结论的另一项测试，在抗病毒 PA 足迹分析过程中确定的 HPV18 位点定点突变将有助于确定特定 PA 结合位点对于抗病毒作用和病毒附加体维持机制的重要性。提供聚酰胺中断高危 HPV 病毒生命周期的新规则。