High Throughput Screening-Based Identification of Measles Virus Probes

描述(申请人提供)：麻疹病毒(MV)是副粘病毒家族的成员，尽管存在减毒活疫苗，但仍是人类十大最致命的病原体之一。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。没有可用于管理严重麻疹病例或快速控制局部暴发的治疗方法。因此，开发新型MV抑制剂是本项目的长期目标。为实现这一目标，在试点研究中建立了多种化验方法，并将实施一项有三个具体目标的研究计划。第一个特定目的是通过高通量筛选(HTS)为MLSCN提供一种可靠的检测方法，用于HIT鉴定。已经建立了一种基于细胞的自动化检测方法，该方法使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。测试评估显示有利的z‘值为0.8，Emory的34,000个内部化合物文库的中试筛选已产生了一个有希望的新抑制剂类别，为该方法提供了概念验证。第二个具体目的是用三种独立的手动检测来支持自动HIT发现，这三种检测方法量化了在化合物存在时对病毒诱导的细胞病变的抑制和病毒产量的减少，并确定了化合物固有的细胞毒性。总而言之，这些分析允许确认命中、计算有效浓度和评估化合物的选择性。第三个特定目的决定了与副粘病毒家族其他成员相比，针对MV的有效靶标的特异性，并对选定的候选对象进行了抗病毒活性机制的初步表征。对MV具有高生物学活性的候选探针将接受针对密切相关的犬瘟热病毒和远缘的人类副流感病毒2型的测试。在个别测试中，将考察选定的化合物对受体结合、膜融合、病毒基因表达和基因组复制的影响。综上所述，这项筛选工作将使我们能够确定最有希望的小分子探针，这些小分子探针适合未来对接模式的生化特性，通过建立定量构效关系和药效团提取，以及使用棉鼠小动物模型进行体内毒性和有效性测试，进一步进行体外优化。总体而言，副粘病毒在世界范围内造成了严重的发病率和死亡率。仅麻疹病毒(MV)就是该病毒家族的一员，每年造成约50万人死亡，使其成为最致命的人类病原体之一，尽管存在疫苗。这在一定程度上是由于麻疹的传染性极高，而且没有治疗方法来处理严重的麻疹病例或迅速控制局部暴发。因此，基于高通量筛选的新的干扰病毒复制和传播的生物探针的识别，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.