Center for Inflammation, Immunity & Infection, Georgia State University, Atlanta, Georgia, USA.
J Virol. 2013 Oct;87(20):11076-87. doi: 10.1128/JVI.01425-13. Epub 2013 Aug 7.
As we are confronted with an increasing number of emerging and reemerging viral pathogens, the identification of novel pathogen-specific and broad-spectrum antivirals has become a major developmental objective. Targeting of host factors required for virus replication presents a tangible approach toward obtaining novel hits with a broadened indication range. However, the identification of developable host-directed antiviral candidates remains challenging. We describe a novel screening protocol that interrogates the myxovirus host-pathogen interactome for broad-spectrum drug candidates and simultaneously probes for conventional, pathogen-directed hits. With resource efficiency and pan-myxovirus activity as the central developmental parameters, we explored coscreening against two distinct, independently traceable myxoviruses in a single-well setting. Having identified a pair of unrelated pathogenic myxoviruses (influenza A virus and measles virus) with comparable replication kinetics, we observed unimpaired coreplication of both viruses, generated suitable firefly and Renilla luciferase reporter constructs, respectively, and validated the protocol for up to a 384-well plate format. Combined with an independent counterscreen using a recombinant respiratory syncytial virus luciferase reporter, implementation of the protocol identified candidates with a broadened antimyxovirus profile, in addition to pathogen-specific hits. Mechanistic characterization revealed a newly discovered broad-spectrum lead that does not block viral entry but stimulates effector pathways of the innate cellular antiviral response. In summary, we provide proof of concept for the efficient discovery of broad-spectrum myxovirus inhibitors in parallel to para- and orthomyxovirus-specific hit candidates in a single screening campaign. The newly identified compound provides a basis for the development of a novel broad-spectrum small-molecule antiviral class.
当我们面临越来越多的新兴和重现的病毒病原体时，寻找新型的病原体特异性和广谱抗病毒药物已成为一个主要的发展目标。针对病毒复制所需的宿主因子是获得具有广泛适应证范围的新型药物的可行方法。然而，鉴定可开发的宿主定向抗病毒候选物仍然具有挑战性。我们描述了一种新的筛选方案，该方案用于研究副黏液病毒宿主-病原体相互作用组，以寻找广谱药物候选物，并同时探测传统的、针对病原体的药物。以资源效率和泛副黏液病毒活性为核心开发参数，我们在单个孔中针对两种不同的、可独立追踪的副黏液病毒进行了共筛选。我们发现了一对不相关的致病性副黏液病毒（流感病毒和麻疹病毒），它们具有相似的复制动力学，观察到两种病毒的核心复制不受影响，分别生成了合适的萤火虫荧光素酶和海肾荧光素酶报告构建体，并验证了该方案最多可用于 384 孔板格式。与使用重组呼吸道合胞病毒荧光素酶报告基因的独立对照筛选相结合，该方案的实施除了鉴定出针对特定病原体的药物外，还确定了具有广谱抗副黏液病毒特性的候选药物。机制表征揭示了一种新发现的广谱先导化合物，它不阻断病毒进入，但能刺激先天细胞抗病毒反应的效应途径。总之，我们提供了在单个筛选活动中同时发现广谱副黏液病毒抑制剂和副黏液病毒、正黏液病毒和负黏液病毒特异性药物的有效方法的概念验证。新鉴定的化合物为开发新型广谱小分子抗病毒药物提供了基础。
