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 文库以鉴定新型正痘病毒抑制剂。正痘病毒是病毒的一个属，包括猴痘、天花（天花的病原体）和牛痘。痘苗病毒是典型的正痘病毒，用于消灭天花的全球疫苗接种计划。天花曾经是最致命的人类病原体，估计已导致超过 3 亿人死亡。天花被消灭后，常规疫苗接种在 20 世纪 70 年代停止，导致人群对天花和其他正痘病毒的免疫力急剧下降。目前尚无 FDA 许可的药物可用于治疗感染任何痘病毒的个体。鉴于天花武器化的威胁以及人类感染猴痘的报告增加，痘病毒感染的治疗是一个重要的优先事项，猴痘在中非和西非流行，并于 2003 年出口到美国。新型抗痘病毒药物的鉴定和开发化合物将引起公共卫生界以及国防和国土安全机构的重大兴趣。为了识别病毒复制的新抑制剂，我们将使用原型正痘病毒疫苗作为筛选病毒。我们开发了一种基于快速成熟荧光蛋白的疫苗依赖性表达的 HTS 就绪筛选方法。该测定的信噪比大于 300:1，并允许进行动力学以及复制终点测试。我们通过筛选两个小规模文库验证了该测定法。我们将与布罗德研究所的合作者合作，将该测定与简单的细胞活力测定结合起来，这将成为我们大规模筛选阻止病毒复制而不杀死细胞的化合物的基础。为了进一步对相关化合物进行分类，我们开发并实施了一套使用不同报告病毒进行二次筛选的测定方法，这将允许对初始筛选“命中”进行验证和表征。这些额外的检测报告了疫苗生命周期的各个阶段，并使我们能够将已识别的痘病毒抑制剂“分类”为早期、中期或晚期复制阶段的抑制剂。我们将与布罗德研究所的合作者合作，开发和优化我们已识别的最有趣的化合物（可能是那些在病毒 DNA 复制后抑制的化合物），以实现高效低毒的抗痘病毒化合物。除了 R03 应用的范围之外，但我的实验室和社区其他人对此非常感兴趣，我们将通过我的实验室与 USAMRIID 的现有合作来测试探针抑制其他正痘病毒（猴痘和天花）复制的能力。通过这些努力，我们将开发能够阻断疫苗、猴痘和天花感染的新型正痘抑制剂，以用作探针和治疗剂。 公共卫生相关性：项目叙事 痘病毒（如天花和猴痘）会导致严重的人类疾病。目前尚无 FDA 批准的药物用于治疗这些感染。我们将使用这些病毒的高度减毒表亲（称为疫苗病毒）来筛选 MLPCN 化合物库，寻找阻止痘病毒复制的药物。将开发有效的探针，旨在提供新的工具来进一步剖析病毒感染和传播的机制，并且还可能产生一种或多种预防痘病毒感染的新药物。