Stage-Specific Inhibitors of Orthopoxviruses

描述（由申请人提供）：我们建议筛选MLPCN文库以鉴定正痘病毒的新型抑制剂。正痘病毒是包括猴痘、天花（天花的病原体）和牛痘的病毒属。牛痘是典型的正痘病毒，用于根除天花的世界范围的疫苗接种计划。天花曾经是最致命的人类病原体，据估计已造成3亿多人死亡。在天花被消灭后，常规疫苗接种在20世纪70年代停止，因此人群对天花和其他正痘病毒的免疫力急剧下降。目前没有FDA许可的药物来治疗感染任何痘病毒的个体。考虑到天花武器化的威胁和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，猴痘是中非和西非的地方病，并于2003年出口到美国。新的抗痘病毒化合物的鉴定和开发将对公共卫生界以及国防和国土安全机构都具有重大意义。为了鉴定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。我们已经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。该检测试剂盒的信噪比大于300：1，可进行复制动力学和终点检测。我们已经通过筛选两个小规模文库验证了该测定。我们将与布罗德研究所的合作者合作，将这种检测与一种简单的细胞活力检测结合起来，这将是我们大规模筛选阻断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，这将允许验证和表征初始筛选"命中"。这些额外的试验报告了疫苗生命周期的各个阶段，并将使我们能够将鉴定出的痘病毒抑制剂"分组"为复制早期、中期或晚期的抑制剂。我们将与布罗德研究所的合作者合作，开发和优化我们鉴定的最有趣的化合物（可能是那些抑制病毒DNA复制的化合物），以获得高效低毒的抗痘病毒化合物。在R03应用范围之外，但我的实验室和社区中的其他人对此非常感兴趣，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，我们将开发新的orthopox抑制剂，能够阻断疫苗，猴痘和天花感染的应用程序作为探针和治疗剂。 公共卫生相关性：项目叙述痘病毒，如天花和猴痘，导致严重的人类疾病。目前没有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疫苗病毒来筛选MLPCN化合物库中阻断痘病毒复制的药物。有效的探针将被开发，目的是提供新的工具，以进一步剖析病毒感染和传播的机制，也可能导致一种或多种新的药物，以防止痘病毒感染。

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.