Biophysics of Drug Interactions with Viral Episomes

描述（由申请人提供）：尽管抗HPV疫苗已上市，但针对高危、致癌人乳头瘤病毒（HPV）的抗病毒化合物极为重要。拟开展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HPV 16化合物扩展到对三种致癌病毒亚型有效的新型抗HPV化合物。活性化合物从人体细胞和组织培养中消除病毒游离体;它们是预防HPV感染后宫颈癌的潜在药物。 具有相同表观DNA结合偏好的PA显示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，一个超越了已知的规则PA-DNA相互作用。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的合作已经开始。其目标是：目的1通过对HPV 16病毒DNA的足迹分析，确定活性PA在HPV 16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分析重叠的500 bp序列的基因组。将长DNA片段的结合常数与简单模型（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），等温量热法和表面等离子体共振将允许基准对文献和避免文物。目的2用抗病毒多聚腺苷体外印迹HPV 18基因组。确定HPV 16和18的相关结合位点。对HPV 16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基础。目的3在人细胞培养中，在HPV 16和18基因组上留下活性PA的体内足迹。使用PCR产生的约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，并通过共聚焦和流式细胞术方法测量细胞中的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，以确保体内足迹法条件合适。确定PA的结构依赖性摄取，可能解释为什么一些异构体PA的范围从高活性到非活性的抗病毒剂。作为足迹法结论的额外检验，在抗病毒PA足迹法期间鉴定的HPV 18位点的定点诱变将有助于确定特异性PA结合位点对抗病毒作用机制和病毒附加体维持的重要性。为聚酰胺阻断高危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.