Biophysics of Drug Interactions with Viral Episomes

描述(由申请人提供)：尽管市场上有抗HPV疫苗，但针对高危、致癌的人类乳头瘤病毒(HPV)的抗病毒化合物极其重要。这项拟议的工作确定了抗病毒多胺(PA)与其病毒DNA靶标之间相互作用的生物物理，以确定其作用机制。范围已从抗HPV16化合物扩大到新的抗HPV化合物，对三种致癌病毒亚型具有活性。活性化合物从人类细胞和组织培养中消除病毒表型；它们是感染HPV后预防宫颈癌的潜在药物。具有相同表观DNA结合偏好的PA表现出显著不同的抗病毒活性。有人提出，这种行为存在一个基本的生物物理解释，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/足迹分析、合成化学、病毒学和细胞-分子生物学展开了合作。其目的是：1通过足迹病毒DNA确定活性PAS在HPV16基因组上的结合位置和亲和力随PA浓度的变化。基因组将用毛细管电泳法和羟基自由基足迹法分析为重叠的500个碱基序列。长DNA片段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，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与文献进行基准比较，并避免人工制品。目的2用抗病毒药物Pas在体外对HPV18基因组进行印迹。确定HPV16和18的相关结合位点。对与活性PAS结合的对HPV16和18都重要的DNA序列进行核磁共振结构工作；使用核磁共振结构结果作为额外相互作用的分子建模的基础。目的人乳头瘤病毒(HPV16)和人乳头瘤病毒(HPV18)基因组在体外培养中的3个足迹活性PAS。使用由聚合酶链式反应产生的大约500个碱基的片段来检查基因组，这些片段重叠覆盖所有的序列空间。通过共聚焦和流式细胞术测量细胞中PA浓度，确定实验条件并提供绝对结合常数。使用定量聚合酶链式反应测量病毒DNA浓度与时间的关系，以确保体内足迹条件合适。确定PAS的结构依赖摄取，潜在地解释为什么一些异构体PAS从高活性到非活性抗病毒药物。作为对足迹试验结论的另一种检验，在抗病毒PA足迹过程中发现的HPV18位点的定点突变将有助于确定特定PA结合位点对抗病毒作用机制和病毒Episome维持的重要性。为聚酰胺中断高危HPV病毒生命周期提供新的规则。

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.