Discovery of Small Molecule Probes for Influenza NS1A

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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.