Development of HTS Assays for Inhibitors of Viral-Cell Interactions

病毒-细胞相互作用抑制剂的 HTS 检测方法的开发

• 批准号: 8049411
• 负责人: TIONE BURANDA
• 金额: $ 15.06万
• 依托单位: UNIVERSITY OF NEW MEXICO HEALTH SCIS CTR
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-27 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8049411
• 关键词: Actins; Adhesions; Aerosols; Affinity; Bacteria; Binding; Biological Assay; Biological Models; Biological Warfare; CD55 Antigens; Cardiopulmonary; Case Fatality Rates; Categories; Cell Adhesion; Cell Communication; Cell surface; Cells; Development; Disease; Dissection; Dose; Event; Flow Cytometry; Focal Adhesions; GPI Membrane Anchors; Glycoproteins; Glycosylphosphatidylinositols; Goals; Hantavirus; Hantavirus Infections; Hemorrhagic Fever with Renal Syndrome; Human; Image; Infection; Integrins; Intervention; Label; Laboratories; Lead; Life; Measurement; Modeling; Molecular; Monitor; Pathogenesis; Pathway interactions; Pharmaceutical Preparations; Positioning Attribute; Process; Proteins; Reagent; Regulation; Route; Screening procedure; Signal Transduction; Sin Nombre virus; Surface; Syndrome; Technology; Therapeutic Intervention; Titrations; Translating; Ultraviolet Rays; Vaccines; Viral; Viral Proteins; Virion; Virus; Virus Inhibitors; Virus-Cell Membrane Interaction; Work; abstracting; biosafety level 2 facility; drug discovery; experience; high throughput screening; inhibitor/antagonist; killings; particle; pathogen; prevent; programs; receptor; research study; small molecule; therapeutic vaccine; tool; uptake; viral RNA; virology

DESCRIPTION (provided by applicant): Hantaviruses cause severe diseases in humans: hemorrhagic fever with renal syndrome (HFRS) and Hantavirus cardiopulmonary syndrome (HCPS). The lack of vaccines or specific drugs to prevent or treat HCPS, and the requirement for conducting experiments in a biosafety level 3 laboratory (BSL-3) limit the ability to probe the mechanism of infection and disease pathogenesis. Case fatality ratios generally range from 30 to 50%. No vaccines or specific therapy are currently available for use in the US. The high case-fatality rate, the absence of vaccines and therapeutics, and the ability of Hantaviruses to be transmitted via the aerosol route have led to their categorization as Category A pathogens, and potential agents of biological warfare. We therefore initiated a program to study Sin Nombre virus (SNV) killed with a calibrated dose of UV radiation as a model system to dissect its mechanism of cellular entry in BSL-2 facilities. We demonstrated that UV-killed SNV attaches to the glycosylphosphatidylinositol (GPI)-anchored protein decay accelerating factor (DAF/CD55) and low affinity state avb3 integrins in a manner that parallels live virions. We propose to screen for small molecule inhibitors of hantavirus infection by blocking the binding of hantaviruses to their surface co- receptor DAF and cellular entry through ?v?? integrins. To achieve these goals, we will pursue the following aims: Aim 1. To develop HTS primary screens for small molecule inhibitors of binding of Sin Nombre Viruses to DAF and ?v??. Aim 2. To develop secondary HT screening assays for small molecule inhibitors of SNV cell interactions. PUBLIC HEALTH RELEVANCE: The proposed work uses a unique interdisciplinary combination of virology and quantitative biophysical tools to identify small molecule inhibitors of pathogenic hantaviruses, which cause hantavirus cardiopulmonary syndrome (HCPS) and hemorrhagic fever with renal syndrome (HFRS). There are currently no vaccines or specific therapy that are available for use in the US to treat HCPS or HFRS. The high case-fatality rate, the absence of vaccines and therapeutics has led to their categorization as Category A pathogens, and potential agents of biological warfare. Our preliminary studies have shown that UV-killed viruses bind to the same receptors used by live viruses. Thus we will develop flow cytometry-compatible high throughput screening assays for small molecule inhibitors of the binding of hantaviruses to its surface co-receptor Decay Accelerating factor and entry receptor ?v?? integrin. Successful completion of this project will lead to the development of therapeutic intervention-probes for hantavirus infection and other pathogens that rely on these receptors for infection.

描述（由申请方提供）：汉坦病毒在人类中引起严重疾病：肾综合征出血热（HFRS）和汉坦病毒心肺综合征（HCPS）。缺乏预防或治疗HCPS的疫苗或特定药物，以及在生物安全3级实验室（BSL-3）进行实验的要求限制了探索感染机制和疾病发病机制的能力。病死率一般在30%至50%之间。目前在美国没有疫苗或特定的治疗方法可供使用。高病死率、缺乏疫苗和治疗方法以及汉坦病毒通过气溶胶途径传播的能力导致其被归类为A类病原体和潜在的生物战剂。因此，我们启动了一项计划，以研究用校准剂量的UV辐射杀死的Sin Nombre病毒（SNV）作为模型系统，以剖析其在BSL-2设施中进入细胞的机制。我们证明，紫外线杀死的SNV重视糖基磷脂酰肌醇（GPI）锚定的蛋白质衰变加速因子（CD 45/CD 55）和低亲和力状态的avb 3整合素的方式，平行活病毒粒子。我们建议通过阻断汉坦病毒与其表面辅助受体结合和细胞进入来筛选汉坦病毒感染的小分子抑制剂。v？？整合素为了实现这些目标，我们将努力实现以下目标：目标1。开发HTS初步筛选Sin Nombre病毒与病毒结合的小分子抑制剂？v？。目标2.开发SNV细胞相互作用小分子抑制剂的二次HT筛选试验。 公共卫生相关性：拟议的工作使用病毒学和定量生物物理学工具的独特跨学科组合，以确定致病性汉坦病毒的小分子抑制剂，导致汉坦病毒心肺综合征（HCPS）和肾综合征出血热（HFRS）。目前在美国没有疫苗或特定疗法可用于治疗HCPS或HFRS。高病死率、缺乏疫苗和治疗剂导致将其归类为A类病原体和潜在的生物战剂。我们的初步研究表明，紫外线杀死的病毒与活病毒使用的受体结合。因此，我们将开发流式细胞术兼容的高通量筛选检测小分子抑制剂的结合汉坦病毒的表面共受体衰变加速因子和进入受体？v？？整联蛋白该项目的成功完成将导致汉坦病毒感染和其他依赖这些受体进行感染的病原体的治疗干预探针的开发。