High-Throughput Screening of a 100,000-Compound Library for Inhibitors of Influenza A Virus (H3N2)
Identifieur interne : 000391 ( Istex/Corpus ); précédent : 000390; suivant : 000392High-Throughput Screening of a 100,000-Compound Library for Inhibitors of Influenza A Virus (H3N2)
Auteurs : William E. Severson ; Michael Mcdowell ; Subramaniam Ananthan ; Dong-Hoon Chung ; Lynn Rasmussen ; Melinda I. Sosa ; E. Lucile White ; James Noah ; Colleen B. JonssonSource :
- Journal of Biomolecular Screening [ 1087-0571 ] ; 2008-10.
English descriptors
- Teeft :
- Antimicrob agents chemother, Antiviral, Antiviral activity, Approximate increase, Assay, Assay media, Biomol screen, Biomolecular, Biomolecular screening, Cell control, Cell control signal, Cell culture medium, Cell toxicity, Cell viability, Compound, Control compound, Cytopathic effect, Dmso, Dose response, Final concentration, Final dmso concentration, High humidity, Influenza, Influenza virus, Inhibitor, Inhibitory activities, Inhibitory effects, Madin darby, Mdck, Mdck cells, Molecular biology, Pandemic, Pandemic influenza, Plaque, Plaque assay, Plaque reduction assay, Plaque reduction assays, Plate reader, Postinfection, Primary screen, Ribavirin, Ribavirin triphosphate, Screening, Selective index, Serial dilutions, Severson, Standard deviation, Surface area, Test compound, Time points, Viral, Virus, Virus control signal, Virus life cycle.
Abstract
Using a highly reproducible and robust cell-based high-throughput screening (HTS) assay, the authors screened a 100,000-compound library at 14- and 114-µM compound concentration against influenza strain A/Udorn/72 (H3N2). The “hit” rates (>50% inhibition of the viral cytopathic effect) from the 14- and 114-µM screens were 0.022% and 0.38%, respectively. The hits were evaluated for their antiviral activity, cell toxicity, and selectivity in dose-response experiments. The screen at the lower concentration yielded 3 compounds, which displayed moderate activity (SI50 = 10-49). Intriguingly, the screen at the higher concentration revealed several additional hits. Two of these hits were highly active with an SI50 > 50. Time of addition experiments revealed 1 compound that inhibited early and 4 other compounds that inhibited late in the virus life cycle, suggesting they affect entry and replication, respectively. The active compounds represent several different classes of molecules such as carboxanilides, 1-benzoyl-3-arylthioureas, sulfonamides, and benzothiazinones, which have not been previously identified as having antiviral/anti-influenza activity. (Journal of Biomolecular Screening 2008:879-887)
Url:
DOI: 10.1177/1087057108323123
Links to Exploration step
ISTEX:5540DAECF80FF9959B8AC6489B34831E005EEA81Le document en format XML
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Jonsson</name><affiliation><mods:affiliation>Department of Biochemistry and Molecular Biology</mods:affiliation></affiliation></author></analytic><monogr></monogr><series><title level="j">Journal of Biomolecular Screening</title><idno type="ISSN">1087-0571</idno><idno type="eISSN">1552-454X</idno><imprint><publisher>SAGE Publications</publisher><pubPlace>Sage CA: Los Angeles, CA</pubPlace><date type="published" when="2008-10">2008-10</date><biblScope unit="volume">13</biblScope><biblScope unit="issue">9</biblScope><biblScope unit="page" from="879">879</biblScope><biblScope unit="page" to="887">887</biblScope></imprint><idno type="ISSN">1087-0571</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">1087-0571</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="Teeft" xml:lang="en"><term>Antimicrob agents chemother</term><term>Antiviral</term><term>Antiviral activity</term><term>Approximate increase</term><term>Assay</term><term>Assay media</term><term>Biomol screen</term><term>Biomolecular</term><term>Biomolecular screening</term><term>Cell control</term><term>Cell control signal</term><term>Cell culture medium</term><term>Cell toxicity</term><term>Cell viability</term><term>Compound</term><term>Control compound</term><term>Cytopathic effect</term><term>Dmso</term><term>Dose response</term><term>Final concentration</term><term>Final dmso concentration</term><term>High humidity</term><term>Influenza</term><term>Influenza virus</term><term>Inhibitor</term><term>Inhibitory activities</term><term>Inhibitory effects</term><term>Madin darby</term><term>Mdck</term><term>Mdck cells</term><term>Molecular biology</term><term>Pandemic</term><term>Pandemic influenza</term><term>Plaque</term><term>Plaque assay</term><term>Plaque reduction assay</term><term>Plaque reduction assays</term><term>Plate reader</term><term>Postinfection</term><term>Primary screen</term><term>Ribavirin</term><term>Ribavirin triphosphate</term><term>Screening</term><term>Selective index</term><term>Serial dilutions</term><term>Severson</term><term>Standard deviation</term><term>Surface area</term><term>Test compound</term><term>Time points</term><term>Viral</term><term>Virus</term><term>Virus control signal</term><term>Virus life cycle</term></keywords></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Using a highly reproducible and robust cell-based high-throughput screening (HTS) assay, the authors screened a 100,000-compound library at 14- and 114-µM compound concentration against influenza strain A/Udorn/72 (H3N2). 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Lucile White</name><affiliations><json:string>High-Throughput Screening Center</json:string></affiliations></json:item><json:item><name>James Noah</name><affiliations><json:string>Department of Biochemistry and Molecular Biology</json:string></affiliations></json:item><json:item><name>Colleen B. 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The “hit” rates (>50% inhibition of the viral cytopathic effect) from the 14- and 114-µM screens were 0.022% and 0.38%, respectively. The hits were evaluated for their antiviral activity, cell toxicity, and selectivity in dose-response experiments. The screen at the lower concentration yielded 3 compounds, which displayed moderate activity (SI50 = 10-49). Intriguingly, the screen at the higher concentration revealed several additional hits. Two of these hits were highly active with an SI50 > 50. Time of addition experiments revealed 1 compound that inhibited early and 4 other compounds that inhibited late in the virus life cycle, suggesting they affect entry and replication, respectively. The active compounds represent several different classes of molecules such as carboxanilides, 1-benzoyl-3-arylthioureas, sulfonamides, and benzothiazinones, which have not been previously identified as having antiviral/anti-influenza activity. 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Lucile</forename><surname>White</surname></persName><affiliation>High-Throughput Screening Center</affiliation></author><author xml:id="author-0007"><persName><forename type="first">James</forename><surname>Noah</surname></persName><affiliation>Department of Biochemistry and Molecular Biology</affiliation></author><author xml:id="author-0008"><persName><forename type="first">Colleen B.</forename><surname>Jonsson</surname></persName><affiliation>Department of Biochemistry and Molecular Biology</affiliation></author><idno type="istex">5540DAECF80FF9959B8AC6489B34831E005EEA81</idno><idno type="ark">ark:/67375/M70-8TLW9GJS-J</idno><idno type="DOI">10.1177/1087057108323123</idno><idno type="article-id">10.1177_1087057108323123</idno></analytic><monogr><title level="j">Journal of Biomolecular Screening</title><idno type="pISSN">1087-0571</idno><idno type="eISSN">1552-454X</idno><idno type="publisher-id">JBX</idno><idno type="PublisherID-hwp">spjbx</idno><imprint><publisher>SAGE Publications</publisher><pubPlace>Sage CA: Los Angeles, CA</pubPlace><date type="published" when="2008-10"></date><biblScope unit="volume">13</biblScope><biblScope unit="issue">9</biblScope><biblScope unit="page" from="879">879</biblScope><biblScope unit="page" to="887">887</biblScope></imprint></monogr></biblStruct></sourceDesc></fileDesc><profileDesc><creation><date>2008</date></creation><langUsage><language ident="en">en</language></langUsage><abstract xml:lang="en"><p>Using a highly reproducible and robust cell-based high-throughput screening (HTS) assay, the authors screened a 100,000-compound library at 14- and 114-µM compound concentration against influenza strain A/Udorn/72 (H3N2). The “hit” rates (>50% inhibition of the viral cytopathic effect) from the 14- and 114-µM screens were 0.022% and 0.38%, respectively. The hits were evaluated for their antiviral activity, cell toxicity, and selectivity in dose-response experiments. The screen at the lower concentration yielded 3 compounds, which displayed moderate activity (SI50 = 10-49). Intriguingly, the screen at the higher concentration revealed several additional hits. Two of these hits were highly active with an SI50 > 50. Time of addition experiments revealed 1 compound that inhibited early and 4 other compounds that inhibited late in the virus life cycle, suggesting they affect entry and replication, respectively. The active compounds represent several different classes of molecules such as carboxanilides, 1-benzoyl-3-arylthioureas, sulfonamides, and benzothiazinones, which have not been previously identified as having antiviral/anti-influenza activity. (Journal of Biomolecular Screening 2008:879-887)</p></abstract><textClass><keywords scheme="keyword"><list><head>keywords</head><item><term>influenza</term></item><item><term>HTS</term></item><item><term>high-throughput screening</term></item><item><term>antivirals</term></item></list></keywords></textClass></profileDesc><revisionDesc><change when="2008-10">Published</change></revisionDesc></teiHeader></istex:fulltextTEI><json:item><extension>txt</extension><original>false</original><mimetype>text/plain</mimetype><uri>https://api.istex.fr/ark:/67375/M70-8TLW9GJS-J/fulltext.txt</uri></json:item></fulltext><metadata><istex:metadataXml wicri:clean="corpus sage not found" wicri:toSee="no header"><istex:xmlDeclaration>version="1.0" encoding="UTF-8"</istex:xmlDeclaration><istex:docType PUBLIC="-//NLM//DTD Journal Publishing DTD v2.3 20070202//EN" URI="journalpublishing.dtd" name="istex:docType"></istex:docType><istex:document><article article-type="research-article" dtd-version="2.3" xml:lang="EN"><front><journal-meta><journal-id journal-id-type="hwp">spjbx</journal-id><journal-id journal-id-type="publisher-id">JBX</journal-id><journal-title>Journal of Biomolecular Screening</journal-title><issn pub-type="ppub">1087-0571</issn><publisher>
<publisher-name>SAGE Publications</publisher-name>
<publisher-loc>Sage CA: Los Angeles, CA</publisher-loc>
</publisher></journal-meta><article-meta><article-id pub-id-type="doi">10.1177/1087057108323123</article-id><article-id pub-id-type="publisher-id">10.1177_1087057108323123</article-id><article-categories><subj-group subj-group-type="heading"><subject>Articles</subject></subj-group></article-categories><title-group><article-title>High-Throughput Screening of a 100,000-Compound Library for Inhibitors of Influenza A Virus (H3N2)</article-title></title-group>
<contrib-group><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Severson</surname><given-names>William E.</given-names></name><aff>Department of Biochemistry and Molecular Biology, <email xlink:type="simple">severson@sri.org</email></aff></contrib></contrib-group>
<contrib-group><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>McDowell</surname><given-names>Michael</given-names></name><aff>Department of Biochemistry and Molecular Biology</aff></contrib></contrib-group>
<contrib-group><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Ananthan</surname><given-names>Subramaniam</given-names></name><aff>Department of Chemistry, Southern Research Institute, Birmingham, Alabama</aff></contrib></contrib-group>
<contrib-group><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Chung</surname><given-names>Dong-Hoon</given-names></name><aff>Department of Biochemistry and Molecular Biology</aff></contrib></contrib-group>
<contrib-group><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Rasmussen</surname><given-names>Lynn</given-names></name><aff>High-Throughput Screening Center</aff></contrib></contrib-group>
<contrib-group><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Sosa</surname><given-names>Melinda I.</given-names></name><aff>High-Throughput Screening Center</aff></contrib></contrib-group>
<contrib-group><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>White</surname><given-names>E. Lucile</given-names></name><aff>High-Throughput Screening Center</aff></contrib></contrib-group>
<contrib-group><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Noah</surname><given-names>James</given-names></name><aff>Department of Biochemistry and Molecular Biology</aff></contrib></contrib-group>
<contrib-group><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Jonsson</surname><given-names>Colleen B.</given-names></name><aff>Department of Biochemistry and Molecular Biology</aff></contrib></contrib-group>
<pub-date pub-type="ppub"><month>10</month><year>2008</year></pub-date><volume>13</volume><issue>9</issue><fpage>879</fpage><lpage>887</lpage><abstract><p>Using a highly reproducible and robust cell-based high-throughput screening (HTS) assay, the authors screened a 100,000-compound library at 14- and 114-µM compound concentration against influenza strain A/Udorn/72 (H3N2). The “hit” rates (>50% inhibition of the viral cytopathic effect) from the 14- and 114-µM screens were 0.022% and 0.38%, respectively. The hits were evaluated for their antiviral activity, cell toxicity, and selectivity in dose-response experiments. The screen at the lower concentration yielded 3 compounds, which displayed moderate activity (SI<sub>50</sub> = 10-49). Intriguingly, the screen at the higher concentration revealed several additional hits. Two of these hits were highly active with an SI<sub>50</sub> > 50. Time of addition experiments revealed 1 compound that inhibited early and 4 other compounds that inhibited late in the virus life cycle, suggesting they affect entry and replication, respectively. The active compounds represent several different classes of molecules such as carboxanilides, 1-benzoyl-3-arylthioureas, sulfonamides, and benzothiazinones, which have not been previously identified as having antiviral/anti-influenza activity. (<italic>Journal of Biomolecular Screening</italic> 2008:879-887)</p></abstract><kwd-group><kwd>influenza</kwd>
<kwd>HTS</kwd>
<kwd>high-throughput screening</kwd>
<kwd>antivirals</kwd></kwd-group><custom-meta-wrap><custom-meta xlink:type="simple"><meta-name>sagemeta-type</meta-name><meta-value>Journal Article</meta-value></custom-meta><custom-meta xlink:type="simple"><meta-name>search-text</meta-name><meta-value>879 High-Throughput Screening of a 100,000-Compound Library for Inhibitors of Influenza A Virus (H3N2) SAGE Publications, Inc.200810.1177/1087057108323123 William E.Severson Department of Biochemistry and Molecular Biology, severson@sri.org MichaelMcdowell Department of Biochemistry and Molecular Biology SubramaniamAnanthan Department of Chemistry, Southern Research Institute, Birmingham, Alabama Dong-HoonChung Department of Biochemistry and Molecular Biology LynnRasmussen High-Throughput Screening Center Melinda I.Sosa High-Throughput Screening Center E. LucileWhite High-Throughput Screening Center JamesNoah Department of Biochemistry and Molecular Biology Colleen B.Jonsson Department of Biochemistry and Molecular Biology Using a highly reproducible and robust cell-based high-throughput screening (HTS) assay, the authors screened a 100,000-compound library at 14- and 114-µM compound concentration against influenza strain A/Udorn/72 (H3N2). The “hit” rates (>50% inhibition of the viral cytopathic effect) from the 14- and 114-µM screens were 0.022% and 0.38%, respectively. The hits were evaluated for their antiviral activity, cell toxicity, and selectivity in dose-response experiments. The screen at the lower concentration yielded 3 compounds, which displayed moderate activity (SI50 = 10-49). Intriguingly, the screen at the higher concentration revealed several additional hits. Two of these hits were highly active with an SI50 > 50. Time of addition experiments revealed 1 compound that inhibited early and 4 other compounds that inhibited late in the virus life cycle, suggesting they affect entry and replication, respectively. The active compounds represent several different classes of molecules such as carboxanilides, 1-benzoyl-3-arylthioureas, sulfonamides, and benzothiazinones, which have not been previously identified as having antiviral/anti-influenza activity. (Journal of Biomolecular Screening 2008:879-887) influenza HTS high-throughput screening antivirals INTRODUCTION NFLUENZA VIRUSES ARE NEGATIVE-SENSE, single-stranded IRNA viruses that infect the upper and lower respiratory tracts and cause substantial morbidity and mortality annually.1 In the United States, approximately 36,000 deaths are attrib- uted to influenza or its complications each year.2-4 Influenza A viruses, which also infect a wide number of avian and mam- malian species, pose a considerable public health burden with epidemic and pandemic potential.5,6 Approximately 20% to 40% of the world's population became ill during the cata- strophic “Spanish” flu pandemic in 1918, which killed 675,000 people in the United States and an estimated 40 to 50 million people worldwide. The “Asian” flu pandemic of 1957 resulted in the deaths of approximately 69,800 people in the United States and 2.0 to 7.4 million worldwide.7 The current health burden of epidemic influenza, as well as the potential threat of a pandemic, has increased effort toward the discovery and development of antivirals and vaccines for the treatment of influenza disease. Antiviral drugs for the treatment of influenza target 4 of the 10 influenza virus proteins: hemagglutinin (HA), neuraminidase (NA), M2 ion channel protein (M2), and polymerase (PA).8 These include oseltamivir (NA), zanamivir (NA), amantadine (M2), rimantadine (HA), and ribavirin (PA). Of these, only oseltamivir and zanamivir were recommended for the treatment of seasonal influenza in 2007-2008 by the Centers for Disease Control and Prevention.9 Ribavirin 5′-monophosphate resembles guanosine 5′-monophosphate (GMP) and can decrease cellular guanosine 5′-triphosphate (GTP) pools due to the inhibition of the enzyme inosine monophosphate dehydrogenase (IMPDH); however, this decrease does not completely account for the observed antiviral activity for many viruses. Inhibitory effects have also been noted on the capping10 and translation efficiency11 of viral mRNA, as well as a direct suppressive effect on the viral polymerase activity in the case of the influenza virus.10,12,13 Remarkably, the most effective antivirals target each of the 3 surface proteins, M2, HA, and NA. M2 acts as an ion chan- nel during the early stages of entry. The 2 other surface pro- teins, HA and NA, are used to subtype influenza viruses with H1, H2, H3, N1, and N2 associated with human infections. HA binds to host cell sialic acid cell receptors, facilitating penetra- tion of epithelial cells by the virus. NA catalyzes the cleavage of sialic acid residues from glycoproteins, which allows the virus to bud from the plasma membrane unabated and hence aids in virus spread. One drawback of current antivirals is that 1Department of Biochemistry and Molecular Biology, 2High-Throughput Screening Center, and 3Department of Chemistry, Southern Research Institute, Birmingham, Alabama. Received Nov 2, 2007, and in revised form Jun 25, 2008. Accepted for publication Jun 26, 2008. 880 they must be administered within 48 h of the onset of symp- toms to be efficacious. Moreover, oseltamivir and zanamivir are expensive and time-consuming to synthesize. Recently, resis- tant variants to these drugs have also emerged. Whereas antivi- rals have played a key role in treatment, vaccines have and will continue to play a dominant role in the prevention of influenza.8 However, the design of effective influenza vaccines can be hin- dered by antigenic variation of NA and reassortment of HA and NA genes.14 Every year, the influenza vaccine contains pre- dicted influenza virus strain variants that have been derived from the surveillance of characterized virus strains from the previous year. Furthermore, in contrast to antivirals, vaccines cannot be developed until a new viral strain emerges.15,16 Clearly, the continued emergence of new influenza variants, drug-resistant mutants, and potential pandemic strains demands our attention toward the discovery and development of more effective antiviral therapeutics. High-throughput screening (HTS) offers an important tool in accelerating the discovery of new antiviral leads for new and emerging pathogens such as sud- den acute respiratory syndrome (SARS) CoV17 and pandemic influenza. Toward this, we previously reported the development of a cell-based HTS that monitors virus-induced cytopathic effects (CPEs) in Madin Darby canine kidney (MDCK) cells.18 Unlike target-oriented approaches, this CPE HTS approach has the advantage that multiple targets can be screened in a single assay against any target involved in viral infection. Confirmatory assays that consist of dose response in an HTS format are easy to perform and provide valuable cytotoxicity data. In addition, lead compounds or unique targets can be identified for structure- activity relationships (SARs). To date, we have screened 1.6 mil- lion compounds at BSL-2 and more than 200,000 compounds at BSL3 using this cell-based viability assay for influenza. We have employed this assay to screen 100,000 compounds from ChemBridge at 14 and 114 µM. We report the discovery of 5 hits with anti-influenza activity. Four compounds—ARB-06-003174 (AACF-308027), ARB-06-011087 (AACF-316018), ARB- 06-076399 (AACF-381531), and ARB-06-089154 (AACF- 394288)—showed inhibition late in the virus lifecycle (6 h postinfection), suggesting they are affecting replication of the virus. Compound ARB-06-018302 (323316) was efficacious early in the virus life cycle (0-3 h postinfection), indicating that it is affecting entry of the virus. MATERIALS AND METHODS Cell growth conditions and media MDCK cells (ATCC CCL-34, American Tissue Culture Type) were maintained as adherent cell lines in Eagle minimum essen- tial medium with 2 mM L-glutamine and 10% fetal bovine serum (FBS) at 37°C in a humidified 5% CO2 atmosphere as described previously.18 Cells were passaged as needed and harvested from flasks using 0.25% trypsin-EDTA. HTS validation included establishment of the coefficient of variation (CV) for the MDCK cells to ensure assay quality and read stability.18 Thus, cells used for the assay were not used past passage 70. Compound library and controls The positive control drug for this assay, ribavirin12 (#196066, MP Biomedicals, Solon, OH), was solubilized at 8 mg/mL in DMSO (Sigma, St. Louis, MO). The stock solution was diluted to a final concentration of 164 µM in assay media (DMEM without phe- nol red, 1.5% bovine serum albumin [BSA], 4 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin; Gibco, Grand Island, NY) before each experiment and discarded afterwards. Final DMSO concentration in each well was 0.5%. A collection of compounds selected for their conformance with the Lipinski rules21 was purchased solubilized at 1 mg/mL in DMSO from ChemBridge Corporation (San Diego, CA). Before each experiment, all compounds were diluted to approximately 14 or 114 µM in assay media for the screen. Influenza high-throughput screen The high-throughput screen for the identification of poten- tial inhibitors of the influenza virus has been described previ- ously.18 Briefly, MDCK cells (3 × 105 cells/mL) were dispensed into black, clear-bottom, 384-well plates at a density of 6000 cells/well in a 20-µL assay medium, using a WellMate (Matrix, Hudson, NH), and incubated 24 h at 37°C, 5% CO2, with high humidity. The next day, 5 µL of each compound was added to cells using a Biomek FX liquid handler (Beckman Coulter, Fullerton, CA). This resulted in a final drug concentration of 14 µM (0.5% DMSO) or 114 µM (<1% DMSO) for all samples. Within 30 min of compound addition, cells were infected with 5 µL of diluted virus at a concentration of 100 TCID50 doses using a WellMate (multiplicity of infection [MOI] of 0.005 pfu/cell). Virus was diluted from amplified virus stock prepared in egg allantoic fluid into assay media containing 15 mg/mL of N-acetyl trypsin, for a final virus stock dilution of 1:10,000 and a final N-acetyl trypsin concentration of 2.5 µg/mL. Internal controls consisted of wells containing cells only, cells infected with virus, and virus-infected cells treated with ribavirin. Plates were incubated at 37°C, 5% CO2, for 72 h. After incubation, 30 µL of Cell Titer Glo (Promega, Madison, WI) was added to each well using a WellMate and incubated at room temperature (RT) for 10 to 30 min. Luminescence was measured using a Envision plate reader (PerkinElmer, Wellesley, MA). Secondary confirmatory assays For dose-response assays, compounds were diluted serially in serum-free media containing 0.5% DMSO final per well in a plate-to-plate matrix rather than in a well-to-well matrix. This allows 320 compounds in 1 plate to be diluted together, resulting 881 in a 10-point dose-response dilution series. This method is referred to as a stacked plate. It can be visualized as a serial dilution series proceeding vertically through a stack of plates with the high-dose plate on top and the low-dose plate on the bottom (final plate well concentration ranging from 147 to 0.285 µM and a final DMSO concentration of ≤1%). Plate-to-plate variability was controlled by normalizing the compound data using in-plate controls. Cell-only values equal 100% inhibition of CPE, and virus values equal 0% inhibition. For compounds, the percent inhibition is calculated as follows: 100*(Cmpd Lum – Median Virus Ctrl)/(Median Cell Ctrl – Med Virus Ctrl). We control for positional variation during assay development and validation where methods are developed to minimize or eliminate positional artifacts such as edge effects. Plaque assay We developed a plaque assay to confirm the antiviral com- pound effect and determine the potency of “hit” compounds. This was accomplished by indirectly quantifying the amount of virus by staining for the presence of the influenza strain A/Udorn/72 N- protein. Briefly, 1 mL of MDCK cells (3 × 105 cells/mL) was dis- pensed into 12-well plates and incubated 24 h at 37°C, 5% CO2, with high humidity. The next day, media were removed and cells were infected with 200 µL of 100 TCID50, which corresponds to an MOI of 0.005 pfu/cell for 1-h adsorption at 37°C. Media were replaced after infection with 1 mL of media containing com- pounds to a final compound concentration of 57 µM (0.5% DMSO), and plates were returned to 37°C. The infectious prog- eny virus produced in the supernatant was harvested after 48 h and measured with a low-viscosity-overlay plaque assay in con- junction with an immunostaining method. In brief, 50 µL from 10-fold serial dilutions of the supernatants was made in cell cul- ture medium and was added to monolayers of MDCK cells in 96- well plates. The plates were incubated for 1 h at 37°C, and then 50 µL of the cell culture medium containing 2% of Avicel™ RC/CL (FMC Biopolymer, Princeton, NJ) was added into each well and returned to 37°C. Twenty-four hours later, cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) and immunostained for viral N protein as described20 but with some minor modifications. To detect the influenza N- protein, we used a goat polyclonal antibody made against influenza A virus (strain H3N2; Virostat, Inc, Portland, ME) for the primary antibody and a peroxidase-labeled antigoat antibody for the secondary antibody; both were used at a dilution of 1 to 1000. The plaque was developed with the peroxidase substrate, True Blue™ (KPL, Gaithersburg, MD). Measurements were per- formed in triplicate for each dilution of sample tested. Time of addition compound screen MDCK cells were plated in 96-well black tissue culture plates at 15,000 cells per well in 100 µL and incubated 24 h at 37°C, 5% CO2. Lead candidate compounds were diluted in media to give a final concentration of 57 µM and added to plates at –1, 0, 3, 6, 12, and 24 h postinfection. Cells were infected with influenza A/Udorn/72 at an MOI of 0.1 and were incubated at 37°C, 5% CO2. After 72 h, 20 µL MTS was added, and plates were incu- bated an additional 2 h at 37°C, 5% CO2. Plates were read at an absorbance of 490 nm on an Envision plate reader. Ribavirin was used as a control compound at a final concentration of 82 µM. Data analysis Data were analyzed using ActivityBase software (IDBS, Inc, Guildford, UK). Percent CPE inhibition was defined as *100. The definition of *100 is that 100% inhibition of virus is equal to 100 times 1 minus the test compound minus the median of the virus divided by the median of the cell control minus the median of the virus (inhibition of virus = 100*[1 – (test compound – median virus)/(median cells – median virus)]). The cell viability is calcu- lated separately in the dose-response assays. Percent cell viability was defined as (test compound/cell control)*100. An active compound, or “hit,” was defined as a compound that exhibited a %CPE inhibition of >50% without compromising cell viability. Ten concentrations of each drug were added to 384-well plates in triplicate to measure the effec- tive concentration at which the drug inhibited cell death at 50% in the presence (EC50) or absence of virus (IC50), with the IC50 defined as the cytotoxicity of the drug alone at 50%. The selec- tive index (SI) was calculated as SI = IC50/EC50. The Z factor values were calculated from 1 minus (3*stan- dard deviation of cell control (σc) plus 3*standard deviation of the virus control (σv)/[mean cell control signal (µc) minus mean virus control signal (µv)]).19 The signal/background (S/B) was calculated from mean cell control signal (µc) divided by the mean virus control signal (µv). The signal/noise (S/N) was cal- culated from the mean cell control signal (µc) minus the mean virus control signal (µv) divided by the (standard deviation of the cell control signal (σc)2 minus the standard deviation of the virus control signal (σv))1/2. RESULTS AND DISCUSSION Compound screening results In an effort to identify a palette of novel lead compounds act- ing through new mechanisms, we employed a high-throughput cell-based assay that measures the influenza virus-induced CPE in MDCK cells.18 We screened a library of 100,000 compounds, which were selected for diversity and drug likeness using the Lipinski criteria for drug-like compounds.21 A majority of this library of compounds, for example, had molecular weight ~350, CLogP value ~3.5, number of rotatable bonds ~4, topological polar surface area (tPSA)22 ~60 Å2, hydrogen bond donors <3, and hydrogen bond acceptors <5, and they were devoid of com- pounds with reactive functional groups. The selected library 882 contained a variety of heterocyclic compounds such as pyrroles, furans, thiophenes, indoles and their benzo analogs, isoindolines, imidazoles, pyrazoles, triazoles, isoxazoles, thiazoles, oxadia- zoles, thiadiazoles, pyridines, quinolines, pyridazines, pyrim- idines, pyrazines, quinazolines, quinoxalines, pyrrolidines, piperazines, and morpholines. The initial screening was per- formed in duplicate at a concentration of 14 µM (Fig. 1A). We identified 22 compounds that showed >50% inhibition of the influenza virus strain A/Udorn/72 for a “hit” rate of 0.022%. The screen at 14 µM gave very few hits, and hence a subset of 16,000 compounds was run at a 114-µM (Fig. 1B) concentration to ascertain if our screening window lacked sensitivity for detection of hits. Indeed, we identified an additional 74 compounds that dis- played >50% inhibition of the influenza virus for a hit rate of 0.46% in this pilot. This is an approximate 21-fold increase in the hit rate as compared with the screen at 14 µM (Fig. 1A). Given the greater success, we screened the remaining 84,000 compounds at 114 µM with the recognition that our false-positive rate would likely increase in the primary screen. The additional screening activity resulted in an additional 307 hits. In total, we identified an additional 381 compounds that displayed >50% inhibition of CPE by the influenza virus for a hit rate of 0.38%. This was an approxi- mate 17-fold increase in the hit rate as compared with screening at 14 µM. Four of the 381 compounds identified were previously dis- covered in the 14-µM screen: ARB-06-01916 (AACF-315846), ARB-06-067453 (AACF-372583), ARB-06-076399 (AACF- 381531), and ARB-06-089154 (AACF-394288). The hits were evaluated by measuring their antiviral activity, cell toxicity, and selectivity in dose-response experiments. We set our selective index (SI50) as follows: <4, not active; SI50 = 4-9, slightly active; SI = 10-49, moderately active; and SI50 >50, highly active. Seven compounds were slightly active, 17 were moderately active, and 2 were highly active. The EC50 curves of all the compounds, except for 2, never reached the 90% effec- tive range due to the observed cellular toxicity of these com- pounds at higher concentrations. The inhibitory activities of selected compounds are shown in Table 1 and Figure 2. Among these compounds, 2—the bissulfonamide, ARB-06- 076399, and the pyridothiazinone, ARB-06-089154—were cho- sen as interesting lead compounds on the basis of several considerations, including the fact that these 2 displayed at least a 3-log reduction from the control (pfu/mL) in a preliminary plaque reduction assay. A substructure search for analogs of these 2 compounds led to the identification of a total of 45 commercially available compounds as closely related analogs. Samples of these 45 compounds were procured and evaluated in dose-response and toxicity assays against the H3N2 virus. Two of these compounds were moderately active (SI50= 10-49). Interestingly, 2 of the com- pounds—the 1-benzoyl-3-arylthioureas, ARB-06-070333 (AACF- 375463) and ARB-06-046310 (AACF-351438)—displayed SI90 values of >3.7 and > 32.2, respectively (Table 1 and Fig. 2). Overall, we observed a 0.4% hit rate for compounds that inhibited CPE by >40%, with an 8% confirmation of hits by dose response FIG. 1. ChemBridge library screen of 16,000 compounds. (A) Average of duplicate ChemBridge compound screen at 14 µM. (B) ChemBridge screen at 114 µM. Compounds from ChemBridge, along with the influenza A/Udorn/72, were added to 6 × 103 Madin Darby canine kidney (MDCK) cells per well in 384-well plates. Inhibitory effects were assessed after 72 h, as described in Materials and Methods. Control drug used was rib- avirin. CPE, cytopathic effect. based on the criteria that a hit is confirmed if the compound has an SI50 value of greater than 4. We have developed a plaque reduction assay for the influenza virus in a 96-well format, which was used to screen 21 selected compounds from Table 1 as a confirmatory screen. The plaque reduction assays were performed in duplicate per compound. As shown in Figure 3 and Table 2, 6 of these compounds (33%) exhibited a 2-log reduction or >100-fold difference from the control in pfu/mL. These compounds are the car- boxanilides ARB-06-003174 (AACF-308027) and ARB-06- 011087 (AACF-316018), the 1-benzoyl-3-arylthioureas ARB-06- 047279 (AACF-352407) and ARB-06-100378 (AACF-405513), the pyridothiazinone ARB-06-089154 (AACF-394288), and the sulfonamides ARB-06-018302 (AACF-323316) and ARB-06- 076399 (AACF-381531). Finally, we employed an assay to determine the point in the influenza virus life cycle that the dose-response and plaque assay hits inhibited. This screen allowed us to ascertain if the inhibition activity of the compound was early (entry) or late (replication) in the virus life cycle. In this screen, compounds were added in trip- licate to plates at time points –1, 0, 3, 6, 12, and 24 h postinfec- tion (Fig. 4). Four of these 7 compounds—ARB-06-003174 883 Table 1. Structures and Inhibitory Activities of Selected Compounds (µM) 884 (AACF-308027), ARB-06-011087 (AACF-316018), ARB-06- 076399 (AACF-381531), and ARB-06-089154 (AACF- 394288)—showed activity 6 h postinfection, suggesting they affect replication. Compound 06-018302 (323316) was effica- cious early in the virus life cycle (0-3 h postinfection), indicating it is affecting entry of the virus. The results of compound ARB- 06-047279 (AACF-352407) are inconclusive. Compound ARB- 06-100378 (AACF-405513) showed a flat curve over the time course, indicating no antiviral effect. The aim of this study was to discover new potential influenza antiviral lead compounds using our high-throughput cell-based assay as the primary screen. In addition, we compared the effec- tiveness at a low (14 µM) and high concentration (114 µM) of compound. Of the total number of compounds screened in dose response and cell toxicity, only 26 compounds met our criteria of activity: the efficacy EC50 value of <25 µM and with toxicity to efficacy SI50 of >10. We performed SAR analysis of each of these hits to identify and choose the most promising lead com- pounds. We selected 21 compounds for further analysis. In the plaque reduction assay, we identified 6 compounds that exhib- ited a 2-log reduction or >100-fold difference from the control in pfu/mL. These compounds are grouped by structural class 885 FIG. 2. Dose-response confirmation of influenza inhibitor compounds. The EC50 values (circles) are shown for ARB 06-070333 (AACF- 375463) and ARB 06-046310 (AACF-351438). In parallel experiments, median inhibitory concentrations, IC50 values, (triangles), and selective indices (SI = IC/EC) at 72 h postinfection were also determined and are indicated. Table 2. Influenza Virus (H3N2) Plaque Assay FIG. 3. Plaque reduction assay in Madin Darby canine kidney (MDCK) cells. Influenza A/Udorn/72 (H3N2) was assayed for its sensi- tivity to 21 compounds. Supernatants were harvested from test (57 µM) or control compound (82 µM) treated influenza-infected MDCK cells at a multiplicity of infection (MOI) of 0.1. Serial 10-fold dilutions of the supernatants were added to monolayers of MDCK cells in 96-well plates. Cells were fixed after 24 h and immunostained. Plaque reduction assays were performed in triplicate per compound. CC, uninfected cell control; VC, untreated virus-infected control; RBV, ribavirin. FIG. 4. Time of addition compound screen against influenza A/Udorn/72 (H3N2). Madin Darby canine kidney (MDCK) cells were plated in 96-well black tissue plates at 15,000 cells per well and incu- bated 24 h at 37°C, 5% CO2. Test compounds were diluted in media to give a final concentration of 57 µM and added to plates at time points –1, 0, 3, 6, 12, and 24 h postinfection. Cells were infected with influenza A/Udorn/72 at a multiplicity of infection (MOI) of 0.1 and incubated 72 h at 37°C, 5% CO2. MTS was added and plates were incubated for an additional 2 h at 37°C, 5% CO2. The plates were read at an absorbance of 490 nm on an Envision plate reader. Ribavirin was used as a control compound. and the structures given in Figure 5. In view of these data, we believe that the design, synthesis, and evaluation of targeted analogs of these various scaffolds in an iterative fashion should lead us toward the identification of compounds possessing greatly improved potency and selectivity that can be developed into clinically useful therapeutic agents. ACKNOWLEDGMENTS We appreciate the technical assistance of Sara McKellip, Clinton Maddox, Lakshmi Reddy, and Anna Manouvakhova. 886 FIG. 5. Structures of lead candidates. This work was supported by National Institutes of Health contract N01-AI-30047 (Dr. Michael Murray, principle investigator [PI]; Dr. Colleen B. Jonsson [Co-PI]). The compounds were made available for screening through NIAID N01-AI-15449 HTS Tuberculosis Drug Screening, Robert C. Goldman, project officer. REFERENCES Lamb RA, Krug RM: Orthomyxoviridae: the viruses and their replication. In Fields BN, Knipe DM, Howley PM (eds): Fundamental Virology. Philadelphia: Lippincott-Raven, 1996:605-647. Thompson WW, Shay DK, Weintraub E., Brammer L., Bridges CB, Cox NJ, et al: Influenza-associated hospitalizations in the United States. JAMA 2004;292:1333-1340. Thompson WW, Shay DK, Weintraub E., Brammer L., Cox N., Anderson LJ, et al: Mortality associated with influenza and respiratory syncytial virus in the United States. JAMA 2003;289:179-186. Ford SM, Grabenstein JD: Pandemics, avian influenza A (H5N1), and a strategy for pharmacists . Pharmacotherapy 2006;26:312-322. Webby RJ, Webster RG: Are we ready for pandemic influenza? Science 2003;302:1519-1522. Morens DM, Fauci AS: The 1918 influenza pandemic: insights for the 21st century. J Infect Dis 2007;195:1018-1028. Rogers DE, Louria DB, Kilbourne ED: The syndrome of fatal influenza virus pneumonia. Trans Assoc Am Physicians 1958;71:260-273. De Clercq E. : Antiviral agents active against influenza A viruses. Nat Rev Drug Discov 2006;5:1015-1025. Fiore AE, Shay DK, Haber P., Iskander JK, Uyeki TM, Mootrey G., et al: Prevention and control of influenza: recommendations of the Advisory Committee on Immunization Practices (ACIP), 2007. MMWR Recomm Rep 2007;56(RR-6):1-54. Fernandez-Larsson R., O'Connell K., Koumans E., Patterson JL: Molecular analysis of the inhibitory effect of phosphorylated ribavirin on the vesicular stomatitis virus in vitro polymerase reaction. Antimicrob Agents Chemother 1989 ;33:1668-1673. Toltzis P., Huang AS: Effect of ribavirin on macromolecular synthesis in vesicular stomatitis virus-infected cells. Antimicrob Agents Chemother 1986;29:1010-1016. Eriksson B. , Helgstrand E., Johansson NG, Larsson A., Misiorny A., Noren JO, et al: Inhibition of influenza virus ribonucleic acid polymerase by ribavirin triphosphate. Antimicrob Agents Chemother 1977;11: 946-951. Wray SK, Gilbert BE, Knight V.: Effect of ribavirin triphosphate on primer generation and elongation during influenza virus transcription in vitro. Antiviral Res 1985;5:39-48. Romanova J. : The fight against new types of influenza virus . Biotechnol J 2006;1:1381-1392. 887 Bright RA, Shay DK, Shu B., Cox NJ, Klimov AI: Adamantane resistance among influenza A viruses isolated early during the 2005-2006 influenza season in the United States. JAMA 2006;295:891-894. Luke C., Subbarao K.: Vaccines for pandemic influenza. Emerg Infect Dis 2006;12:66-72. Severson WE, Shindo N., Sosa M., Fletcher T. III, White EL, Ananthan S., et al: Development and validation of a high-throughput screen for inhibitors of SARS CoV and its application in screening of a 100,000-compound library . J Biomol Screen 2007;12:33-40. Noah JW, Severson W., Noah DL, Rasmussen L., White EL, Jonsson CB: A cell-based luminescence assay is effective for high-throughput screening of potential influenza antivirals. Antiviral Res 2007;73:50-59. Zhang JH, Chung TD, Oldenburg KR: A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 1999;4:67-73. Matrosovich M., Matrosovich T., Garten W., Klenk HD: New low-viscosity overlay medium for viral plaque assays. Virol J 2006;3:63. Lipinski CA , Lombardo F., Dominy BW, Feeney PJ: Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv Drug Delivery Rev 1997;23:3-25. Ertl P., Rohde B., Selzer P.: Fast calculation of molecular polar surface area as a sum of fragment-based contributions and its application to the prediction of drug transport properties . J Med Chem 2000;43:3714-3717. Address correspondence to: William E. Severson Department of Biochemistry and Molecular Biology Southern Research Institute 2000 9th Avenue South Birmingham, AL 35205 E-mail: severson@sri.org</meta-value></custom-meta></custom-meta-wrap></article-meta></front><back><ref-list>
<ref>
<citation citation-type="book" xlink:type="simple">
<name name-style="western"><surname>Lamb RA</surname></name>, <name name-style="western"><surname>Krug RM</surname></name>: <source>Orthomyxoviridae: the viruses and their replication</source>. In <name name-style="western"><surname>Fields BN</surname></name>, <name name-style="western"><surname>Knipe DM</surname></name>, <name name-style="western"><surname>Howley PM</surname></name> (eds): <source>Fundamental Virology</source>. <publisher-loc>Philadelphia</publisher-loc>: <publisher-name>Lippincott-Raven</publisher-name>, <year>1996</year>:<fpage>605</fpage>-<lpage>647</lpage>.</citation>
</ref>
<ref>
<citation citation-type="journal" xlink:type="simple">
<name name-style="western"><surname>Thompson WW</surname></name>, <name name-style="western"><surname>Shay DK</surname></name>, <name name-style="western"><surname>Weintraub E.</surname></name>, <name name-style="western"><surname>Brammer L.</surname></name>, <name name-style="western"><surname>Bridges CB</surname></name>, <name name-style="western"><surname>Cox NJ</surname></name>, et al: <article-title>Influenza-associated hospitalizations in the United States</article-title>. <source>JAMA</source>
<year>2004</year>;<volume>292</volume>:<fpage>1333</fpage>-<lpage>1340</lpage>.</citation>
</ref>
<ref>
<citation citation-type="journal" xlink:type="simple">
<name name-style="western"><surname>Thompson WW</surname></name>, <name name-style="western"><surname>Shay DK</surname></name>, <name name-style="western"><surname>Weintraub E.</surname></name>, <name name-style="western"><surname>Brammer L.</surname></name>, <name name-style="western"><surname>Cox N.</surname></name>, <name name-style="western"><surname>Anderson LJ</surname></name>, et al: <article-title>Mortality associated with influenza and respiratory syncytial virus in the United States</article-title>. <source>JAMA</source>
<year>2003</year>;<volume>289</volume>:<fpage>179</fpage>-<lpage>186</lpage>.</citation>
</ref>
<ref>
<citation citation-type="journal" xlink:type="simple">
<name name-style="western"><surname>Ford SM</surname></name>, <name name-style="western"><surname>Grabenstein JD</surname></name>: <article-title>Pandemics, avian influenza A (H5N1), and a strategy for pharmacists</article-title> . <source>Pharmacotherapy</source>
<year>2006</year>;<volume>26</volume>:<fpage>312</fpage>-<lpage>322</lpage>.</citation>
</ref>
<ref>
<citation citation-type="journal" xlink:type="simple">
<name name-style="western"><surname>Webby RJ</surname></name>, <name name-style="western"><surname>Webster RG</surname></name>: <article-title>Are we ready for pandemic influenza</article-title>? <source>Science</source>
<year>2003</year>;<volume>302</volume>:<fpage>1519</fpage>-<lpage>1522</lpage>.</citation>
</ref>
<ref>
<citation citation-type="journal" xlink:type="simple">
<name name-style="western"><surname>Morens DM</surname></name>, <name name-style="western"><surname>Fauci AS</surname></name>: <article-title>The 1918 influenza pandemic: insights for the 21st century</article-title>. <source>J Infect Dis</source>
<year>2007</year>;<volume>195</volume>:<fpage>1018</fpage>-<lpage>1028</lpage>.</citation>
</ref>
<ref>
<citation citation-type="journal" xlink:type="simple">
<name name-style="western"><surname>Rogers DE</surname></name>, <name name-style="western"><surname>Louria DB</surname></name>, <name name-style="western"><surname>Kilbourne ED</surname></name>: <article-title>The syndrome of fatal influenza virus pneumonia</article-title>. <source>Trans Assoc Am Physicians</source>
<year>1958</year>;<volume>71</volume>:<fpage>260</fpage>-<lpage>273</lpage>.</citation>
</ref>
<ref>
<citation citation-type="journal" xlink:type="simple">
<name name-style="western"><surname>De Clercq E.</surname></name> : <article-title>Antiviral agents active against influenza A viruses</article-title>. <source>Nat Rev Drug Discov</source>
<year>2006</year>;<volume>5</volume>:<fpage>1015</fpage>-<lpage>1025</lpage>.</citation>
</ref>
<ref>
<citation citation-type="journal" xlink:type="simple">
<name name-style="western"><surname>Fiore AE</surname></name>, <name name-style="western"><surname>Shay DK</surname></name>, <name name-style="western"><surname>Haber P.</surname></name>, <name name-style="western"><surname>Iskander JK</surname></name>, <name name-style="western"><surname>Uyeki TM</surname></name>, <name name-style="western"><surname>Mootrey G.</surname></name>, et al: <article-title>Prevention and control of influenza: recommendations of the Advisory Committee on Immunization Practices (ACIP), 2007</article-title>. <source>MMWR Recomm Rep</source>
<year>2007</year>;<volume>56</volume>(RR-<issue>6</issue>):<fpage>1</fpage>-<lpage>54</lpage>.</citation>
</ref>
<ref>
<citation citation-type="journal" xlink:type="simple">
<name name-style="western"><surname>Fernandez-Larsson R.</surname></name>, <name name-style="western"><surname>O'Connell K.</surname></name>, <name name-style="western"><surname>Koumans E.</surname></name>, <name name-style="western"><surname>Patterson JL</surname></name>: <article-title>Molecular analysis of the inhibitory effect of phosphorylated ribavirin on the vesicular stomatitis virus in vitro polymerase reaction</article-title>. <source>Antimicrob Agents Chemother</source>
<year>1989</year> ;<volume>33</volume>:<fpage>1668</fpage>-<lpage>1673</lpage>.</citation>
</ref>
<ref>
<citation citation-type="journal" xlink:type="simple">
<name name-style="western"><surname>Toltzis P.</surname></name>, <name name-style="western"><surname>Huang AS</surname></name>: <article-title>Effect of ribavirin on macromolecular synthesis in vesicular stomatitis virus-infected cells</article-title>. <source>Antimicrob Agents Chemother</source>
<year>1986</year>;<volume>29</volume>:<fpage>1010</fpage>-<lpage>1016</lpage>.</citation>
</ref>
<ref>
<citation citation-type="journal" xlink:type="simple">
<name name-style="western"><surname>Eriksson B.</surname></name> , <name name-style="western"><surname>Helgstrand E.</surname></name>, <name name-style="western"><surname>Johansson NG</surname></name>, <name name-style="western"><surname>Larsson A.</surname></name>, <name name-style="western"><surname>Misiorny A.</surname></name>, <name name-style="western"><surname>Noren JO</surname></name>, et al: <article-title>Inhibition of influenza virus ribonucleic acid polymerase by ribavirin triphosphate</article-title>. <source>Antimicrob Agents Chemother</source>
<year>1977</year>;<volume>11</volume>: <fpage>946</fpage>-<lpage>951</lpage>.</citation>
</ref>
<ref>
<citation citation-type="journal" xlink:type="simple">
<name name-style="western"><surname>Wray SK</surname></name>, <name name-style="western"><surname>Gilbert BE</surname></name>, <name name-style="western"><surname>Knight V.</surname></name>: <article-title>Effect of ribavirin triphosphate on primer generation and elongation during influenza virus transcription in vitro</article-title>. <source>Antiviral Res</source>
<year>1985</year>;<volume>5</volume>:<fpage>39</fpage>-<lpage>48</lpage>.</citation>
</ref>
<ref>
<citation citation-type="journal" xlink:type="simple">
<name name-style="western"><surname>Romanova J.</surname></name> : <article-title>The fight against new types of influenza virus</article-title> . <source>Biotechnol J</source>
<year>2006</year>;<volume>1</volume>:<fpage>1381</fpage>-<lpage>1392</lpage>.</citation>
</ref>
<ref>
<citation citation-type="journal" xlink:type="simple">
<name name-style="western"><surname>Bright RA</surname></name>, <name name-style="western"><surname>Shay DK</surname></name>, <name name-style="western"><surname>Shu B.</surname></name>, <name name-style="western"><surname>Cox NJ</surname></name>, <name name-style="western"><surname>Klimov AI</surname></name>: <article-title>Adamantane resistance among influenza A viruses isolated early during the 2005-2006 influenza season in the United States</article-title>. <source>JAMA</source>
<year>2006</year>;<volume>295</volume>:<fpage>891</fpage>-<lpage>894</lpage>.</citation>
</ref>
<ref>
<citation citation-type="journal" xlink:type="simple">
<name name-style="western"><surname>Luke C.</surname></name>, <name name-style="western"><surname>Subbarao K.</surname></name>: <article-title>Vaccines for pandemic influenza</article-title>. <source>Emerg Infect Dis</source>
<year>2006</year>;<volume>12</volume>:<fpage>66</fpage>-<lpage>72</lpage>.</citation>
</ref>
<ref>
<citation citation-type="journal" xlink:type="simple">
<name name-style="western"><surname>Severson WE</surname></name>, <name name-style="western"><surname>Shindo N.</surname></name>, <name name-style="western"><surname>Sosa M.</surname></name>, <name name-style="western"><surname>Fletcher T. III</surname></name>, <name name-style="western"><surname>White EL</surname></name>, <name name-style="western"><surname>Ananthan S.</surname></name>, et al: <article-title>Development and validation of a high-throughput screen for inhibitors of SARS CoV and its application in screening of a 100,000-compound library</article-title> . <source>J Biomol Screen</source>
<year>2007</year>;<volume>12</volume>:<fpage>33</fpage>-<lpage>40</lpage>.</citation>
</ref>
<ref>
<citation citation-type="journal" xlink:type="simple">
<name name-style="western"><surname>Noah JW</surname></name>, <name name-style="western"><surname>Severson W.</surname></name>, <name name-style="western"><surname>Noah DL</surname></name>, <name name-style="western"><surname>Rasmussen L.</surname></name>, <name name-style="western"><surname>White EL</surname></name>, <name name-style="western"><surname>Jonsson CB</surname></name>: <article-title>A cell-based luminescence assay is effective for high-throughput screening of potential influenza antivirals</article-title>. <source>Antiviral Res</source>
<year>2007</year>;<volume>73</volume>:<fpage>50</fpage>-<lpage>59</lpage>.</citation>
</ref>
<ref>
<citation citation-type="journal" xlink:type="simple">
<name name-style="western"><surname>Zhang JH</surname></name>, <name name-style="western"><surname>Chung TD</surname></name>, <name name-style="western"><surname>Oldenburg KR</surname></name>: <article-title>A simple statistical parameter for use in evaluation and validation of high throughput screening assays</article-title>. <source>J Biomol Screen</source>
<year>1999</year>;<volume>4</volume>:<fpage>67</fpage>-<lpage>73</lpage>.</citation>
</ref>
<ref>
<citation citation-type="journal" xlink:type="simple">
<name name-style="western"><surname>Matrosovich M.</surname></name>, <name name-style="western"><surname>Matrosovich T.</surname></name>, <name name-style="western"><surname>Garten W.</surname></name>, <name name-style="western"><surname>Klenk HD</surname></name>: <article-title>New low-viscosity overlay medium for viral plaque assays</article-title>. <source>Virol J</source>
<year>2006</year>;<volume>3</volume>:<fpage>63</fpage>.</citation>
</ref>
<ref>
<citation citation-type="journal" xlink:type="simple">
<name name-style="western"><surname>Lipinski CA</surname></name> , <name name-style="western"><surname>Lombardo F.</surname></name>, <name name-style="western"><surname>Dominy BW</surname></name>, <name name-style="western"><surname>Feeney PJ</surname></name>: <article-title>Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings</article-title>. <source>Adv Drug Delivery Rev</source>
<year>1997</year>;<volume>23</volume>:<fpage>3</fpage>-<lpage>25</lpage>.</citation>
</ref>
<ref>
<citation citation-type="journal" xlink:type="simple">
<name name-style="western"><surname>Ertl P.</surname></name>, <name name-style="western"><surname>Rohde B.</surname></name>, <name name-style="western"><surname>Selzer P.</surname></name>: <article-title>Fast calculation of molecular polar surface area as a sum of fragment-based contributions and its application to the prediction of drug transport properties</article-title> . <source>J Med Chem</source>
<year>2000</year>;<volume>43</volume>:<fpage>3714</fpage>-<lpage>3717</lpage>.</citation>
</ref>
</ref-list></back></article></istex:document></istex:metadataXml><mods version="3.6"><titleInfo lang="en"><title>High-Throughput Screening of a 100,000-Compound Library for Inhibitors of Influenza A Virus (H3N2)</title></titleInfo><titleInfo type="alternative" lang="en" contentType="CDATA"><title>High-Throughput Screening of a 100,000-Compound Library for Inhibitors of Influenza A Virus (H3N2)</title></titleInfo><name type="personal"><namePart type="given">William E.</namePart><namePart type="family">Severson</namePart><affiliation>Department of Biochemistry and Molecular Biology,</affiliation><affiliation>E-mail: severson@sri.org</affiliation></name><name type="personal"><namePart type="given">Michael</namePart><namePart type="family">McDowell</namePart><affiliation>Department of Biochemistry and Molecular Biology</affiliation></name><name type="personal"><namePart type="given">Subramaniam</namePart><namePart type="family">Ananthan</namePart><affiliation>Department of Chemistry, Southern Research Institute, Birmingham, Alabama</affiliation></name><name type="personal"><namePart type="given">Dong-Hoon</namePart><namePart type="family">Chung</namePart><affiliation>Department of Biochemistry and Molecular Biology</affiliation></name><name type="personal"><namePart type="given">Lynn</namePart><namePart type="family">Rasmussen</namePart><affiliation>High-Throughput Screening Center</affiliation></name><name type="personal"><namePart type="given">Melinda I.</namePart><namePart type="family">Sosa</namePart><affiliation>High-Throughput Screening Center</affiliation></name><name type="personal"><namePart type="given">E. Lucile</namePart><namePart type="family">White</namePart><affiliation>High-Throughput Screening Center</affiliation></name><name type="personal"><namePart type="given">James</namePart><namePart type="family">Noah</namePart><affiliation>Department of Biochemistry and Molecular Biology</affiliation></name><name type="personal"><namePart type="given">Colleen B.</namePart><namePart type="family">Jonsson</namePart><affiliation>Department of Biochemistry and Molecular Biology</affiliation></name><typeOfResource>text</typeOfResource><genre type="research-article" displayLabel="research-article" authority="ISTEX" authorityURI="https://content-type.data.istex.fr" valueURI="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</genre><originInfo><publisher>SAGE Publications</publisher><place><placeTerm type="text">Sage CA: Los Angeles, CA</placeTerm></place><dateIssued encoding="w3cdtf">2008-10</dateIssued><copyrightDate encoding="w3cdtf">2008</copyrightDate></originInfo><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><abstract lang="en">Using a highly reproducible and robust cell-based high-throughput screening (HTS) assay, the authors screened a 100,000-compound library at 14- and 114-µM compound concentration against influenza strain A/Udorn/72 (H3N2). The “hit” rates (>50% inhibition of the viral cytopathic effect) from the 14- and 114-µM screens were 0.022% and 0.38%, respectively. The hits were evaluated for their antiviral activity, cell toxicity, and selectivity in dose-response experiments. The screen at the lower concentration yielded 3 compounds, which displayed moderate activity (SI50 = 10-49). Intriguingly, the screen at the higher concentration revealed several additional hits. Two of these hits were highly active with an SI50 > 50. Time of addition experiments revealed 1 compound that inhibited early and 4 other compounds that inhibited late in the virus life cycle, suggesting they affect entry and replication, respectively. The active compounds represent several different classes of molecules such as carboxanilides, 1-benzoyl-3-arylthioureas, sulfonamides, and benzothiazinones, which have not been previously identified as having antiviral/anti-influenza activity. (Journal of Biomolecular Screening 2008:879-887)</abstract><subject><genre>keywords</genre><topic>influenza</topic><topic>HTS</topic><topic>high-throughput screening</topic><topic>antivirals</topic></subject><relatedItem type="host"><titleInfo><title>Journal of Biomolecular Screening</title></titleInfo><genre type="journal" authority="ISTEX" authorityURI="https://publication-type.data.istex.fr" valueURI="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</genre><identifier type="ISSN">1087-0571</identifier><identifier type="eISSN">1552-454X</identifier><identifier type="PublisherID">JBX</identifier><identifier type="PublisherID-hwp">spjbx</identifier><part><date>2008</date><detail type="volume"><caption>vol.</caption><number>13</number></detail><detail type="issue"><caption>no.</caption><number>9</number></detail><extent unit="pages"><start>879</start><end>887</end></extent></part></relatedItem><identifier type="istex">5540DAECF80FF9959B8AC6489B34831E005EEA81</identifier><identifier type="ark">ark:/67375/M70-8TLW9GJS-J</identifier><identifier type="DOI">10.1177/1087057108323123</identifier><identifier type="ArticleID">10.1177_1087057108323123</identifier><recordInfo><recordContentSource authority="ISTEX" authorityURI="https://loaded-corpus.data.istex.fr" valueURI="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-0J1N7DQT-B">sage</recordContentSource></recordInfo></mods><json:item><extension>json</extension><original>false</original><mimetype>application/json</mimetype><uri>https://api.istex.fr/ark:/67375/M70-8TLW9GJS-J/record.json</uri></json:item></metadata><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
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