Discovery of Small Molecule Probes for Influenza NS1A

描述（由申请方提供）：流感病毒是一种负义单链RNA病毒，感染上呼吸道和下呼吸道，每年导致大量发病和死亡。在美国，每年约有36，000人死于流感或其并发症。甲型流感病毒也感染许多禽类和哺乳动物物种，造成相当大的公共卫生负担，具有流行和大流行的潜力。机制等拟议的研究工作旨在确定NS 1A的RNA结合活性的小分子抑制剂。流感病毒NS 1A蛋白是一种高度保守的多功能病毒蛋白，可与宿主RNA和蛋白质相互作用。流感病毒蛋白NS 1A被认为是这些病毒拮抗宿主细胞抗病毒防御的重要病毒因子。我们开发了一种基于AlphaScreen（R）（Perkin Elmer）的NS 1A蛋白的1536孔高通量筛选（HTS），以鉴定NS 1A双链（ds）RNA结合活性的小分子抑制剂。测定的Z值范围为0.82-0.89。鉴定这种活性的探针将为探测其在细胞内的dsRNA结合功能提供有价值的工具。从理论上讲，这个结构域也可以作为抗病毒药物发现的靶点。为此，我们提出两个目标：（1）目标1。协助MLPCN将经验证的NS 1A流感病毒HTS检测试剂盒转移至适当的筛查中心进行初步筛查，并为实施命中的二次检测提供技术支持，以消除假阳性并验证筛查命中的生物学相关性，以及（2）目标2。与MLPCN化学中心合作，通过三级检测分析NS 1A HTS中获得的命中，这些检测将用于命中-探针开发和探针表征（例如选择性、特异性、细胞和组织渗透以及作用机制）。 公共卫生相关性：流感病毒是负义单链RNA病毒，感染上呼吸道和下呼吸道，每年导致大量的发病率和死亡率。在美国，每年约有36，000人死于流感或其并发症。甲型流感病毒也感染许多禽类和哺乳动物物种，造成相当大的公共卫生负担，具有流行和大流行的潜力。拟议的研究工作旨在确定NS 1A的RNA结合活性的小分子抑制剂，NS 1A是病毒生命周期中的关键病毒蛋白。

DESCRIPTION (provided by applicant): Influenza viruses are negative-sense, single-stranded RNA viruses that infect the upper and lower respiratory tracts and cause substantial morbidity and mortality annually. In the United States, approximately 36,000 deaths are attributed to influenza or its complications each year. Influenza A viruses, which also infect a wide number of avian and mammalian species, pose a considerable public health burden with epidemic and pandemic potential. Mechanisms. The proposed research effort seeks to identify small molecule inhibitors for the RNA binding activity of NS1A. The NS1A protein of influenza is a highly conserved, multifunctional viral protein which interacts with host RNA and proteins. The influenza viral protein NS1A is regarded as an important viral factor by which these viruses antagonize host cell anti-viral defense. We have developed an AlphaScreen(R) (Perkin Elmer)-based 1536-well high throughput screen (HTS) for NS1A protein to identify small molecule inhibitors of the NS1A double-stranded (ds) RNA binding activity. The Z-value for the assay ranges from 0.82-0.89. Identification of probes for this activity would provide valuable tools for probing its dsRNA binding function within the cell. Theoretically, this domain could also serve as a target for antiviral drug discovery. For this effort we propose two Aims: (1) Aim 1. Assist the MLPCN in transferring the validated HTS assay for Influenza NS1A to the appropriate screening center for primary screening and provide technical support for implementation of secondary assays of hits to remove false positives and validate biological relevance of the screening hits and (2) Aim 2. Collaborate with the MLPCN Chemistry Center in analyses of the hits acquired in the NS1A HTS with tertiary assays that will be used for the purpose of hit-to-probe development and probe characterizations (e.g. selectivity, specificity, cell and tissue penetration, and mechanisms of action). PUBLIC HEALTH RELEVANCE: Influenza viruses are negative-sense, single-stranded RNA viruses that infect the upper and lower respiratory tracts and cause substantial morbidity and mortality annually. In the United States, approximately 36,000 deaths are attributed to influenza or its complications each year. Influenza A viruses, which also infect a wide number of avian and mammalian species, pose a considerable public health burden with epidemic and pandemic potential. The proposed research effort seeks to identify small molecule inhibitors for the RNA binding activity of NS1A, a key viral protein in the life cycle of the virus.