Stage-Specific Inhibitors of Orthopoxviruses

正痘病毒阶段特异性抑制剂

• 批准号: 8136883
• 负责人: John H Connor
• 金额: $ 4.08万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-15 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8136883
• 关键词: A549; Biological; Biological Assay; Biological Models; Bromides; Cell Survival; Cells; Central Africa; Chemicals; Collaborations; Combined Modality Therapy; Communities; DNA biosynthesis; Data; Development; Fluorescence; Gene Expression; Human; Immunity; Individual; Infection; Institutes; Kinetics; Laboratories; Libraries; Licensing; Life Cycle Stages; Marketing; Monitor; Monkeypox; Mosses; Noise; Orthopoxvirus; Pharmaceutical Preparations; Phase; Population; Poxviridae; Poxviridae Infections; Production; Proteins; Public Health; Reporter; Reporting; Screening procedure; Security; Signal Transduction; Smallpox; Staging; Structure-Activity Relationship; System; Testing; Tetrazolium; Therapeutic Agents; Toxic effect; Vaccination; Vaccines; Vaccinia; Vaccinia virus; Vaccinium; Validation; Venus; Viral; Viral Genes; Virus; Virus Inhibitors; Virus Replication; Western Africa; Work; base; cell killing; cytotoxic; cytotoxicity; high throughput screening; improved; inhibitor/antagonist; interest; killings; novel; pathogen; programs; prototype; recombinant virus; small molecule; viral DNA

DESCRIPTION (provided by applicant): We propose screening the MLPCN library to identify novel inhibitors of orthopoxviruses. Orthopoxviruses are a genus of viruses that include monkeypox, variola (the causative agent of smallpox) and vaccinia. Vaccinia is the prototypical orthopoxvirus which was used in the world-wide vaccination program that eradicated smallpox. Smallpox was once the most deadly human pathogen, and is estimated to have killed more than 300 million people. Following the eradication of smallpox, routine vaccination was discontinued in the 1970s and there has consequently been a precipitous decline in population immunity to smallpox and other orthopoxviruses. There are currently no FDA-licensed drugs to treat individuals infected with any poxvirus. Therapies for poxvirus infection are a significant priority given the threat of smallpox weaponization and the rise in reports of humans infected with monkey pox, which is endemic to Central and Western Africa and was exported to the US in 2003. The identification and development of new antipoxviral compounds will be of significant interest both to the public health community and to the Defense and Homeland Security agencies To identify new inhibitors of viral replication we will use the prototype orthopox virus, vaccine, as a screening virus. We have developed an HTS-ready screening approach based on vaccine-dependent expression of a rapidly maturing fluorescent protein. This assay has a signal-to-noise ratio of greater than 300:1 and allows kinetic as well as endpoint testing of replication. We have validated this assay through screening of two small- scale libraries. We will work with our collaborators at the Broad institute to couple this assay with a simple cell- viability assay that will be the basis of our large-scale screen for compounds that block virus replication without killing cells. To further classify relevant compounds we have developed and implemented a suite of assays using different reporter viruses for secondary screening that will allow validation and characterization of the initial screen "hits". These additional assays report on the individual stages of the vaccines life cycle and will allow us to "bin" the identified poxvirus inhibitors as inhibitors of early, intermediate or late stages of replication. We will work with our collaborators at the Broad Institute to develop and optimize the most interesting of our identified compounds (likely those that inhibit after viral DNA replication) towards high-efficacy low-toxicity antipoxviral compounds. Beyond the scope of this R03 application but of great interest to my lab and others in the community, probes will be tested for their abilities to inhibit the replication of other orthopox viruses (Monkey pox and Smallpox) through my laboratory's existing collaboration with USAMRIID. Through these efforts we will develop novel orthopox inhibitors capable of blocking Vaccine, Monkey pox, and Smallpox infection for application both as probes and as a therapeutic agent. PUBLIC HEALTH RELEVANCE: Project Narrative Poxviruses such as Smallpox and Monkeypox cause serious human disease. There are currently no FDA- approved drugs for treatment of these infections. We will use a highly attenuated cousin of these viruses called Vaccines virus to screen the MLPCN compound library for drugs that block poxvirus replication. Effective probes will be developed with the aim of providing new tools to further dissect the mechanism of viral infection and spread, and may also lead to one or more new drugs to protect against poxvirus infection.

描述（由申请人提供）：我们建议筛选MLPCN文库，以识别正托病毒的新型抑制剂。正托病毒是包括Monkeypox，Variola（天花的病因）和离甲酸的病毒属。 Vaccinia是原型的正质病毒，它在世界范围内消除了天花的疫苗接种计划中使用。天花曾经是最致命的人类病原体，据估计已杀死超过3亿人。根除天花后，常规疫苗接种在1970年代停产，因此人口对天花和其他正oxoxviruse的人口免疫力急剧下降。目前尚无FDA许可的药物来治疗感染任何痘病毒的个体。鉴于天花武器化的威胁和人类感染了猴子痘的报道的兴起，蛇毒感染的疗法是重中之重，猴子痘（中部和西非是特有的），并于2003年出口到美国。公共卫生社区以及国防和国土安全机构都会引起人们的重大关注，以鉴定病毒复制的新抑制剂，我们将使用原型的正托病毒疫苗作为筛查病毒。我们基于迅速成熟的荧光蛋白的疫苗依赖性表达开发了一种可HTS的筛选方法。该测定的信噪比大于300：1，并且可以进行动力学和终点测试。我们通过筛选两个小型库来验证了该测定法。我们将与广泛研究所的合作者合作，将此测定法与简单的细胞活力测定法进行，这将是我们大规模筛选的基础，用于阻止病毒复制而无需杀死细胞的化合物。为了进一步对相关化合物进行分类，我们已经开发并实施了使用不同的记者病毒进行二级筛查的一组测定法，这将允许验证和表征初始屏幕“ hits”。这些额外的测定报告在疫苗生命周期的各个阶段报告，将使我们可以将已鉴定的痘病毒抑制剂“ bin”为早期，中间或晚期复制阶段的抑制剂。我们将与广大研究所的合作者合作开发和优化我们确定的最有趣的化合物（可能在病毒DNA复制后抑制的化合物）朝着高效率低毒性的抗氧化病毒化合物。除了该R03应用的范围外，但我的实验室和社区中的其他应用程序的范围非常重要，还将通过我的实验室与usamriid的现有合作来测试探测器的能力。通过这些努力，我们将开发出能够阻止疫苗，猴子痘和天花感染的新型东拓抑制剂，以作为探针和作为治疗剂的应用。 公共卫生相关性：天花和猴子等项目叙事毒病毒会导致严重的人类疾病。目前尚无FDA批准的药物来治疗这些感染。我们将使用称为疫苗病毒的这些病毒的高度减毒表弟来筛选MLPCN化合物库，以阻止Poxvirus复制的药物。将开发有效的探针，目的是提供新工具，以进一步剖析病毒感染和传播的机制，并可能导致一种或多种新药以防止痘病毒感染。