A pilot study for development of inhibitors of influenza virus

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Abstract The threat of global pandemic influenza is escalating due to the wide spread of the highly pathogenic avian influenza virus in poultry populations through continents and increasing cases of human infection. We devised a plan to develop novel antivirals targeting the nucleoprotein (NP). NP is highly conserved among all influenza virus strains and is indispensable for virus replication, which makes it a more attractive antiviral target than the neuraminidase. Two aims are proposed for the pilot study: Aim 1. Screen for NP inhibitors. We have developed a high throughput (HTP) assay for NP nucleotide binding in a 96-well plate format. A library of 10,000 diversified compounds will be screened to identify inhibitors that block NP binding to nucleotide. Candidates yielded from the HTP screen will be tested for inhibition of NP assembly with nucleotide, and influenza virus replication in cell culture infection. Preliminary results using 800 compounds showed that about 1.4% of the compounds screened affect NP binding with nucleotide. Antiviral studies of these compounds are in progress. Aim 2. Structure of NP in complex with inhibitors. To map the detailed structure of NP interaction with candidate inhibitors, we will determine the 3D structure of NP cocrystallized with inhibitor compounds by Xray crystallography. The crystal structure of NP in complex with inhibitors will help us in design of more potent inhibitors. The goal of the pilot study is to produce proof-of-concept results and a structure of NP-inhibitor complex so we can further improve the efficacy of this class of inhibitors. Design of nucleoprotein inhibitors as antivirals against influenza virus has never been done before. During the previous effort, a potent fusion inhibitor of influenza viruses (EC50 =1.0 nM) was identified. In addition to chemical modification of the inhibitor, other studies will be carried out in the next period to further emploit this class of compounds, including: Test antivital activities with H5N1 and H2N2 strains; Test antiviral activities in a mouse model; Cocrystallization of HA with inhibitors.