Kao_NatBiotech_2010 - LETTERS Identification of influenza A...

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© 2010 Nature America, Inc. All rights reserved. NATURE BIOTECHNOLOGY ADVANCE ONLINE PUBLICATION 1 LETTERS Influenza A remains a significant public health challenge because of the emergence of antigenically shifted or highly virulent strains 1–5 . Antiviral resistance to available drugs such as adamantanes or neuraminidase inhibitors has appeared rapidly 6–9 , creating a need for new antiviral targets and new drugs for influenza virus infections. Using forward chemical genetics, we have identified influenza A nucleoprotein (NP) as a druggable target and found a small-molecule compound, nucleozin, that triggers the aggregation of NP and inhibits its nuclear accumulation. Nucleozin impeded influenza A virus replication in vitro with a nanomolar median effective concentration (EC 50 ) and protected mice challenged with lethal doses of avian influenza A H5N1. Our results demonstrate that viral NP is a valid target for the development of small-molecule therapies. The propensity of influenza virus to develop resistance to commonly used drugs requires continued development of new therapeutics. In the 2008–2009 flu season, almost 100% of the seasonal influenza H1N1 viruses circulating in the United States were resistant to the neuraminidase inhibitor oseltamivir (Tamiflu), and all isolates of the H3N2 viruses were resistant to adamantanes 1–6 . Over half of the indi- viduals infected by the H5N1 subtype died irrespective of treatment with both classes of drug 7–9 . In our previous studies on SARS corona- virus, we demonstrated that a forward chemical genetics approach using a chemical library of 50,240 compounds with diverse structures could interrogate most known targets for viral infection 10,11 . Here we screened the same library using Madin-Darby canine kidney (MDCK) cell–based influenza A infection assays and identified 950 compounds that showed protective effects as primary hits. We evaluated the selected compounds in a secondary screen using a cytopathic effect assay and selected 39 compounds for further studies based on their potency. To investigate the modes of action of these bioactive compounds, we focused on processes crucial for successful establishment of influenza infection. Influenza NP is the most abundantly expressed protein during the course of infection with multiple functionalities 12 . NP accumulates in the nucleus in the early phases of infection and is exclusively dis- tributed in cytoplasm later during viral assembly and maturation 12–15 . We examined the effects of the 39 compounds on NP nuclear traf- ficking by fluorescence microscopy and identified 5 compounds that blocked the nuclear accumulation of NP. Compound FA-1 showed the best potency with EC 50 < 1 N M in a plaque reduction assay (PRA) on MDCK cells infected with influenza A/WSN/33 (H1N1) virus. The schematic representation of the procedures and results of the primary, secondary and subsequent fluorescence microscopy screens are sum- marized in Supplementary Figure 1 .
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This note was uploaded on 11/01/2010 for the course A B taught by Professor C during the Spring '10 term at HKU.

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Kao_NatBiotech_2010 - LETTERS Identification of influenza A...

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