Stage-Specific Inhibitors of Orthopoxviruses

描述(申请人提供)：我们建议筛选MLPCN文库以确定新的正痘病毒抑制剂。正痘病毒是包括猴痘、天花(天花的病原体)和牛痘在内的一类病毒。牛痘是一种典型的正痘病毒，曾用于根除天花的全球疫苗接种计划。天花曾经是人类最致命的病原体，据估计已导致超过3亿人死亡。在根除天花后，1970年代停止了常规疫苗接种，因此，人们对天花和其他正痘病毒的免疫力急剧下降。目前还没有FDA许可的药物来治疗感染了任何痘病毒的个人。鉴于天花武器化的威胁，以及人类感染猴痘的报告增加，治疗天花病毒感染是一个重要的优先事项。猴痘是中非和西非的地方病，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部机构具有重大意义。为了识别新的病毒复制抑制物，我们将使用正畸病毒的原型疫苗作为筛选病毒。我们开发了一种基于疫苗依赖表达的快速成熟荧光蛋白的HTS筛查方法。该检测方法的信噪比大于300：1，并允许对复制进行动力学和终点测试。我们已经通过对两个小规模文库的筛选验证了这一方法。我们将与布罗德研究所的合作者合作，将这一测试与一种简单的细胞活性测试相结合，这将成为我们大规模筛选在不杀死细胞的情况下阻止病毒复制的化合物的基础。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分析方法，这将允许验证和表征初始筛选的“命中”。这些额外的化验报告了疫苗生命周期的各个阶段，并将使我们能够将已识别的痘病毒抑制剂作为复制的早期、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，开发和优化我们识别的最有趣的化合物(可能是那些在病毒DNA复制后抑制作用的化合物)，以获得高效、低毒的抗痘病毒化合物。这超出了R03应用程序的范围，但我的实验室和社区中的其他人非常感兴趣，我们将通过我的实验室与USAMRIID的现有合作来测试它们抑制其他正痘病毒(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畸抑制剂，用于作为探针和治疗剂。 公共卫生相关性：项目叙述天花和猴痘等天花病毒会导致严重的人类疾病。目前还没有FDA批准的治疗这些感染的药物。我们将使用这些病毒的一个高度减毒的表亲疫苗病毒来筛选MLPCN化合物文库，以寻找阻止痘病毒复制的药物。将开发有效的探针，目的是提供新的工具，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来预防痘病毒感染。

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.