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:
- 金额:$ 36.11万
- 依托单位:
- 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:
- 关键词:3-DimensionalAffinityAntiviral AgentsBehaviorBenchmarkingBindingBinding SitesBiological AssayBiophysicsBlood capillariesCalibrationCalorimetryCancer EtiologyCapillary ElectrophoresisCell Culture TechniquesCell NucleusCellsCollaborationsComplementComplexConfocal MicroscopyContractsDNADNA BindingDNA SequenceDataData AnalysesDevelopmentDissociationDrug InteractionsDyesEpisomeFlow CytometryFluorescenceFluorescence AnisotropyFluorescence MicroscopyGenomeHPV-High RiskHigh-Risk CancerHumanHuman Papilloma Virus VaccineHuman PapillomavirusHuman papilloma virus 31Human papillomavirus 16Human papillomavirus 18Hydroxyl RadicalImageIn VitroInterruptionInvestigationLife Cycle StagesLinkLiteratureLocationMaintenanceMalignant neoplasm of cervix uteriMammalian CellMarketingMeasurementMeasuresMedicalMethodsModelingMolecular ModelsMolecular and Cellular BiologyMorphologic artifactsNuclearNylonsPharmaceutical PreparationsPositioning AttributePreparationPublishingResolutionRiskShapesSiteSite-Directed MutagenesisSocietiesSolutionsSolventsSpecificitySpectrum AnalysisStructureStructure-Activity RelationshipStudy modelsSurfaceSurface Plasmon ResonanceSynthesis ChemistryTestingVaccinationViralViral GenomeVirus DiseasesWorkbasecalf thymus DNAcapillarycervical cancer preventionfightingin vivointerestkeratinocytemolecular modelingpre-clinicalpreferencepublic health relevancetime usetissue/cell cultureuptakeviral DNAvirologyvirus 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):针对高危、致癌的人乳头瘤病毒(HPV)的抗病毒化合物非常重要,即使抗HPV疫苗已经上市。这项工作旨在确定抗病毒聚酰胺(PAs)与其病毒DNA靶点之间相互作用的生物物理学,以确定其作用机制。范围已从抗hpv16化合物扩展到抗三种致癌病毒亚型的新型抗hpv化合物。活性化合物消除人体细胞和组织培养中的病毒片段;它们是在感染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表现出明显不同的抗病毒活性。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PA-DNA相互作用的规则。因此,在生物物理光谱、DNA结合/足迹分析、合成化学、病毒学和细胞分子生物学方面的合作已经开始。目的1通过足迹病毒DNA作为PA浓度的函数来确定活性PAs在HPV16基因组上的结合位置和亲和力。基因组将被分析为重叠的500 bp序列,使用毛细管电泳和羟基自由基足迹。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对文献进行基准测试,并避免伪影。目的2用抗病毒PAs在体外足迹HPV18基因组。确定HPV16和18的相关结合位点。对HPV16和hpv18结合活性PAs的重要DNA序列进行核磁共振结构分析;利用核磁共振结构结果作为附加相互作用的分子建模的基础。目的3在人细胞培养中在HPV16和hpv18基因组上足迹活性PAs。使用PCR产生的约500 bp的片段检查基因组,这些片段重叠以覆盖所有序列空间。通过共聚焦和流式细胞术方法测量细胞中的PA浓度,确定实验条件并提供绝对结合常数。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是合适的。鉴定结构依赖的pa摄取,可能解释为什么一些同分异构体pa从高活性到无活性的抗病毒药物。作为足迹分析结论的另一项测试,抗病毒PA足迹分析过程中发现的HPV18位点定向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片段维持的重要性。提供聚酰胺阻断高危HPV病毒生命周期的新规则。
项目成果
期刊论文数量(0)
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James K. Bashkin其他文献
Polyamides pour le traitement du virus du papillome humain
人乳头状病毒性状聚酰胺
- DOI:
- 发表时间:
2007 - 期刊:
- 影响因子:0
- 作者:
James K. Bashkin;C. Fisher;Kevin J. Koeller - 通讯作者:
Kevin J. Koeller
James K. Bashkin的其他文献
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{{ truncateString('James K. Bashkin', 18)}}的其他基金
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:
7987715 - 财政年份:2010
- 资助金额:
$ 36.11万 - 项目类别:
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:
8646850 - 财政年份:2010
- 资助金额:
$ 36.11万 - 项目类别:
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:
8063932 - 财政年份:2010
- 资助金额:
$ 36.11万 - 项目类别:
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 - 财政年份:2010
- 资助金额:
$ 36.11万 - 项目类别:
HYDROLYTIC CLEAVAGE OF RNA BY METAL COMPLEXES, FUNDAMENTAL STUDY IN CATALYSIS
金属配合物对 RNA 的水解裂解,催化基础研究
- 批准号:
6665799 - 财政年份:2002
- 资助金额:
$ 36.11万 - 项目类别:
MECHANISM & CHARGE CONTROL OF METAL CATALYZED RNA TRANSESTERIFICATION
机制
- 批准号:
6665800 - 财政年份:2002
- 资助金额:
$ 36.11万 - 项目类别:
HYDROLYTIC CLEAVAGE OF RNA BY METAL COMPLEXES, FUNDAMENTAL STUDY IN CATALYSIS
金属配合物对 RNA 的水解裂解,催化基础研究
- 批准号:
6486679 - 财政年份:2001
- 资助金额:
$ 36.11万 - 项目类别:
MECHANISM & CHARGE CONTROL OF METAL CATALYZED RNA TRANSESTERIFICATION
机制
- 批准号:
6486680 - 财政年份:2001
- 资助金额:
$ 36.11万 - 项目类别:
HYDROLYTIC CLEAVAGE OF RNA BY METAL COMPLEXES, FUNDAMENTAL STUDY IN CATALYSIS
金属配合物对 RNA 的水解裂解,催化基础研究
- 批准号:
6336749 - 财政年份:2000
- 资助金额:
$ 36.11万 - 项目类别:
MECHANISM & CHARGE CONTROL OF METAL CATALYZED RNA TRANSESTERIFICATION
机制
- 批准号:
6336750 - 财政年份:2000
- 资助金额:
$ 36.11万 - 项目类别:
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