Development of an HTS assay for the search of anti-influenza agents targeting the interaction of viral RNA with the NS1 protein

The NS1 protein is a nonstructural protein encoded by the influenza A virus. It is responsible for many alterations produced in the cellular metabolism upon infection by the virus and for modulation of virus virulence. The NS1 protein is able to perform a large variety of functions due to its ability to bind various types of RNA molecules, from both viral and nonviral origin, and to interact with several cell factors. With the aim of exploring whether the binding of NS1 protein to viral RNA (vRNA) could constitute a novel target for the search of anti-influenza drugs, a filter-binding assay measuring the specific interaction between the recombinant His-NS1 protein from influenza A virus and a radiolabeled model vRNA ( 32 P-vNSZ) was adapted to a format suitable for screening and easy automation. Flashplate ® technology (PerkinElmer, Waltham, MA), either in 96- or 384-well plates, was used. The Flashplate ® wells were precoated with the recombinant His-NS1 protein, and the binding of His-NS1 to a 35 S-vNSZ probe was measured. A pilot screening of a collection of 27,520 mixtures of synthetic chemical compounds was run for inhibitors of NS1 binding to vRNA. We found 3 compounds in which the inhibition of NS1 binding to vRNA, observed at submicromolar concentrations, was correlated with a reduction of the cytopathic effect during the infection of cell cultures with influenza virus. These results support the hypothesis that the binding of NS1 to vRNA could be a novel target for the development of anti-influenza drugs. (Journal of Biomolecular Screening 2008:581-590)