High Throughput Screening-Based Identification of Measles Virus Probes

性状（由申请方提供）：麻疹病毒（MV）是副粘病毒家族的一员，尽管存在减毒活疫苗，但仍是人类十大致死性病原体之一。该病毒在非洲、亚洲和欧洲部分地区流行，美国每年都有病例报告。没有治疗方法可用于管理严重的麻疹病例或快速控制局部疫情。因此，本项目的长期目标是开发新型MV抑制剂。为了实现这一目标，在试点研究中建立了多种分析方法，并将实施一项具有三个具体目标的研究计划。第一个具体目标是通过高通量筛选（HTS）为MLSCN提供一种稳健的测定法，用于命中鉴定。已经建立了基于细胞的自动化测定，其采用稳定表达绿色荧光蛋白的荧光MV报告基因作为额外的转录单位。测定评价显示有利的z '值为0.8，并且在Emory对34，000种化合物的内部文库进行的中试筛选已经产生了一种有希望的新抑制剂类别，为该方法提供了概念验证。第二个具体目标是通过三种独立的手动测定来支持自动命中发现，这些测定在化合物存在下量化病毒诱导的细胞病变的抑制和病毒产量的降低，并确定固有的化合物细胞毒性。总的来说，这些测定允许命中确认、活性浓度的计算和化合物选择性的评估。第三个特定目的确定了与副粘病毒家族的其他成员相比，对MV的有效命中的靶特异性，并使所选择的候选物经受抗病毒活性机制的初步表征。对MV具有高生物活性的候选探针将进行针对密切相关的犬瘟热病毒和远相关的人副流感病毒2型的检测。在单个试验中，然后将检查选定化合物对受体结合、膜融合和病毒基因表达和基因组复制的影响。结合，这种筛选工作将使我们能够确定最有前途的小分子探针，值得未来的生物化学表征的对接模式，进一步在体外优化，通过生成的定量结构-活性关系和药效团提取，并在体内毒性和效力测试使用棉鼠小动物模型MV感染。总的来说，副粘病毒是世界范围内显著发病率和死亡率的原因。麻疹病毒（MV）是该病毒家族的一员，每年造成约50万人死亡，尽管存在疫苗，但仍将其置于最致命的人类病原体之列。部分原因是麻疹病毒的传染性特别高，而且没有治疗方法可用于管理严重的麻疹病例或快速控制局部疫情。因此，基于高通量筛选的干扰MV复制和传播的新型生物探针的鉴定以及基于这些探针的适用药物的进一步开发是本项目的最终目标。

DESCRIPTION (provided by applicant): Measles virus (MV), a member of the paramyxovirus family, remains one of the ten most lethal human pathogens despite the existence of a live-attenuated vaccine. The virus is endemic in Africa, Asia and parts of Europe, and cases are reported annually in the US. No therapy is available for management of severe cases of measles or rapid control of local outbreaks. It is therefore the long-term objective of this project to develop novel inhibitors of MV. Towards this goal multiple assays have been established in pilot studies and a research plan with three specific aims will be implemented. The first specific aim provides a robust assay to the MLSCN for hit identification through high throughput screening (HTS). A cell-based automated assay has been established that employs a fluorescent MV reporter stably expressing green fluorescent protein as additional transcription unit. Assay evaluation has revealed favorable z'-values of 0.8 and a pilot screen of a 34,000 compound in- house library at Emory has yielded amongst others a promising new inhibitor class, providing proof-of-concept for the approach. The second specific aim backs up the automated hit discovery with three independent manual assays that quantify suppression of virus-induced cytopathicity and reduction of virus yields in the presence of compound, and determine inherent compound cytotoxicity. Collectively, these assays allow hit confirmation, calculation of active concentrations, and assessment of compound selectivity. The third specific aim determines the target specificity of potent hits towards MV in comparison with other members of the paramyxovirus family, and subjects selected candidates to an initial characterization of the mechanism of antiviral activity. Candidate probes with high biologic activity towards MV will be subjected to testing against closely related canine distemper virus and distantly related human parainfluenza virus type 2. In individual assays, the effect of selected compounds on receptor binding, membrane fusion, and viral gene expression and genome replication will then be examined. Combined, this screening exercise will enable us to identify the most promising small molecule probes that merit future biochemical characterization of docking modes, further in vitro optimization through generation of quantitative structure-activity relationships and pharmacophore extraction, and in vivo toxicity and efficacy testing using the cotton rat small animal model for MV infection. Collectively, paramyxoviruses are responsible for significant morbidity and mortality worldwide. Measles virus (MV) alone, a member of this virus family, accounts for approximately 500,000 deaths annually, placing it among the most lethal human pathogens despite the existence of a vaccine. This is partially due to the exceptionally high infectivity of MV and the fact that no therapy is available for management of severe cases of measles or rapid control of local outbreaks. The high-throughput screening-based identification of novel biological probes that interfere with MV replication and spread, and the further development of applicable drugs based on these probes is therefore the ultimate objective of this project.