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<title>Discovery of Small Molecule Influenza Virus NS1 Antagonist - Probe Reports from the NIH Molecular Libraries Program - NCBI Bookshelf</title>
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<meta name="robots" content="INDEX,FOLLOW,NOARCHIVE" /><meta name="citation_inbook_title" content="Probe Reports from the NIH Molecular Libraries Program [Internet]" /><meta name="citation_title" content="Discovery of Small Molecule Influenza Virus NS1 Antagonist" /><meta name="citation_publisher" content="National Center for Biotechnology Information (US)" /><meta name="citation_date" content="2013/09/03" /><meta name="citation_author" content="Samarjit Patnaik" /><meta name="citation_author" content="Dipanwita Basu" /><meta name="citation_author" content="Seameen Dehdashti" /><meta name="citation_author" content="Wei Zheng" /><meta name="citation_author" content="Marc Ferrer" /><meta name="citation_author" content="Noel Southall" /><meta name="citation_author" content="Mercedes Taylor" /><meta name="citation_author" content="Daniel A. Engel" /><meta name="citation_author" content="Juan Jose Marugan" /><meta name="citation_pmid" content="24260784" /><meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK169451/" /><link rel="schema.DC" href="http://purl.org/DC/elements/1.0/" /><meta name="DC.Title" content="Discovery of Small Molecule Influenza Virus NS1 Antagonist" /><meta name="DC.Type" content="Text" /><meta name="DC.Publisher" content="National Center for Biotechnology Information (US)" /><meta name="DC.Contributor" content="Samarjit Patnaik" /><meta name="DC.Contributor" content="Dipanwita Basu" /><meta name="DC.Contributor" content="Seameen Dehdashti" /><meta name="DC.Contributor" content="Wei Zheng" /><meta name="DC.Contributor" content="Marc Ferrer" /><meta name="DC.Contributor" content="Noel Southall" /><meta name="DC.Contributor" content="Mercedes Taylor" /><meta name="DC.Contributor" content="Daniel A. Engel" /><meta name="DC.Contributor" content="Juan Jose Marugan" /><meta name="DC.Date" content="2013/09/03" /><meta name="DC.Identifier" content="https://www.ncbi.nlm.nih.gov/books/NBK169451/" /><meta name="description" content="The Non Structural 1 (NS1) is a 230–237 amino acid relatively well-conserved viral protein that is usually expressed only during infection [1]. It contains an RNA binding domain (RBD) and an effector domain (ED) that is connected by a variable linker [2]. The NS1 protein has been shown to be a key player that counters the host interferon (IFN) response to viral infection in strain-specific ways [3]. NS1 inhibits the function of OAS (2′–5′-oligoadenylate synthetase) and PKR (protein kinase R) [4] and block IFN-β synthesis at the post-transcriptional level by inhibiting the pre-mRNA splicing and blocking the export of poly(A)-RNAs from the nucleus to the cytoplasm [5]. It also hampers the host’s defense pathways by interfering with the host RNAi pathway, adaptive immune response, and the apoptotic response [6]. NS1 is also a key component in the temporal regulation of viral RNA synthesis, its splicing, and translation [7]. Thus, NS1 enhances the virulence of infection, posing as a compelling target for influenza treatment. These compounds will enable researchers to test how NS1 inhibition impacts the infection cycle and how antagonists can be leveraged alone or in combination with existing agents in the next generation of treatments for influenza infection. We developed and conducted a high-throughput screen using a novel yeast-based phenotypic assay to identify compounds which specifically inhibit NS1 function. Here we report a potent NS1 antagonist chemical series, represented by ML303. The SAR (structure activity relationship) around this chemical series was further developed by measuring the compound’s ability to inhibit replication of the influenza A/PR/8/34 virus in MDCK cells. The ML303 compound series specifically restored the NS1-inhibited expression of the interferon mRNA, inhibited the NS1 function in mechanistic assay and exhibited a potent antiviral activity in culture." /><meta name="og:title" content="Discovery of Small Molecule Influenza Virus NS1 Antagonist" /><meta name="og:type" content="book" /><meta name="og:description" content="The Non Structural 1 (NS1) is a 230–237 amino acid relatively well-conserved viral protein that is usually expressed only during infection [1]. It contains an RNA binding domain (RBD) and an effector domain (ED) that is connected by a variable linker [2]. The NS1 protein has been shown to be a key player that counters the host interferon (IFN) response to viral infection in strain-specific ways [3]. NS1 inhibits the function of OAS (2′–5′-oligoadenylate synthetase) and PKR (protein kinase R) [4] and block IFN-β synthesis at the post-transcriptional level by inhibiting the pre-mRNA splicing and blocking the export of poly(A)-RNAs from the nucleus to the cytoplasm [5]. It also hampers the host’s defense pathways by interfering with the host RNAi pathway, adaptive immune response, and the apoptotic response [6]. NS1 is also a key component in the temporal regulation of viral RNA synthesis, its splicing, and translation [7]. Thus, NS1 enhances the virulence of infection, posing as a compelling target for influenza treatment. These compounds will enable researchers to test how NS1 inhibition impacts the infection cycle and how antagonists can be leveraged alone or in combination with existing agents in the next generation of treatments for influenza infection. We developed and conducted a high-throughput screen using a novel yeast-based phenotypic assay to identify compounds which specifically inhibit NS1 function. Here we report a potent NS1 antagonist chemical series, represented by ML303. The SAR (structure activity relationship) around this chemical series was further developed by measuring the compound’s ability to inhibit replication of the influenza A/PR/8/34 virus in MDCK cells. The ML303 compound series specifically restored the NS1-inhibited expression of the interferon mRNA, inhibited the NS1 function in mechanistic assay and exhibited a potent antiviral activity in culture." /><meta name="og:url" content="https://www.ncbi.nlm.nih.gov/books/NBK169451/" /><meta name="og:site_name" content="NCBI Bookshelf" /><meta name="og:image" content="https://www.ncbi.nlm.nih.gov/corehtml/pmc/pmcgifs/bookshelf/thumbs/th-mlprobe-lrg.png" /><meta name="twitter:card" content="summary" /><meta name="twitter:site" content="@ncbibooks" /><meta name="bk-non-canon-loc" content="/books/n/mlprobe/ml303/" /><link rel="canonical" href="https://www.ncbi.nlm.nih.gov/books/NBK169451/" /><link rel="stylesheet" href="/corehtml/pmc/css/figpopup.css" type="text/css" media="screen" /><link rel="stylesheet" href="/corehtml/pmc/css/bookshelf/2.26/css/books.min.css" type="text/css" /><link rel="stylesheet" href="/corehtml/pmc/css/bookshelf/2.26/css/books_print.min.css" type="text/css" media="print" /><style type="text/css">p a.figpopup{display:inline !important} .bk_tt {font-family: monospace} .first-line-outdent .bk_ref {display: inline} .body-content h2, .body-content .h2 {border-bottom: 1px solid #97B0C8} .body-content h2.inline {border-bottom: none} a.page-toc-label , .jig-ncbismoothscroll a {text-decoration:none;border:0 !important} .temp-labeled-list .graphic {display:inline-block !important} .temp-labeled-list img{width:100%}</style><script type="text/javascript" src="/corehtml/pmc/js/jquery.hoverIntent.min.js"> </script><script type="text/javascript" src="/corehtml/pmc/js/common.min.js?_=3.18"> </script><script type="text/javascript" src="/corehtml/pmc/js/large-obj-scrollbars.min.js"> </script><script type="text/javascript">window.name="mainwindow";</script><script type="text/javascript" src="/corehtml/pmc/js/bookshelf/2.26/book-toc.min.js"> </script><script type="text/javascript" src="/corehtml/pmc/js/bookshelf/2.26/books.min.js"> </script><meta name="book-collection" content="NONE" />
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<div class="pre-content"><div><div class="bk_prnt"><p class="small">NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health.</p><p>Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-. </p></div><div class="iconblock clearfix whole_rhythm no_top_margin bk_noprnt"><a class="img_link icnblk_img" title="Table of Contents Page" href="/books/n/mlprobe/"><img class="source-thumb" src="/corehtml/pmc/pmcgifs/bookshelf/thumbs/th-mlprobe-lrg.png" alt="Cover of Probe Reports from the NIH Molecular Libraries Program" height="100px" width="80px" /></a><div class="icnblk_cntnt eight_col"><h2>Probe Reports from the NIH Molecular Libraries Program [Internet].</h2><a data-jig="ncbitoggler" href="#__NBK169451_dtls__">Show details</a><div style="display:none" class="ui-widget" id="__NBK169451_dtls__"><div>Bethesda (MD): National Center for Biotechnology Information (US); 2010-.</div></div><div class="half_rhythm"><ul class="inline_list"><li style="margin-right:1em"><a class="bk_cntns" href="/books/n/mlprobe/">Contents</a></li></ul></div><div class="bk_noprnt"><form method="get" action="/books/n/mlprobe/" id="bk_srch"><div class="bk_search"><label for="bk_term" class="offscreen_noflow">Search term</label><input type="text" title="Search this book" id="bk_term" name="term" value="" data-jig="ncbiclearbutton" /> <input type="submit" class="jig-ncbibutton" value="Search this book" submit="false" style="padding: 0.1em 0.4em;" /></div></form></div></div><div class="icnblk_cntnt two_col"><div class="pagination bk_noprnt"><a class="active page_link prev" href="/books/n/mlprobe/ml304/" title="Previous page in this title">< Prev</a><a class="active page_link next" href="/books/n/mlprobe/ml302/" title="Next page in this title">Next ></a></div></div></div></div></div>
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<div class="main-content lit-style" itemscope="itemscope" itemtype="http://schema.org/CreativeWork"><div class="meta-content fm-sec"><h1 id="_NBK169451_"><span class="title" itemprop="name">Discovery of Small Molecule Influenza Virus NS1 Antagonist</span></h1><p class="contrib-group"><span itemprop="author">Samarjit Patnaik</span>, <span itemprop="author">Dipanwita Basu</span>, <span itemprop="author">Seameen Dehdashti</span>, <span itemprop="author">Wei Zheng</span>, <span itemprop="author">Marc Ferrer</span>, <span itemprop="author">Noel Southall</span>, <span itemprop="author">Mercedes Taylor</span>, <span itemprop="author">Daniel A. Engel</span>, and <span itemprop="author">Juan Jose Marugan</span>.</p><a data-jig="ncbitoggler" href="#__NBK169451_ai__" style="border:0;text-decoration:none">Author Information and Affiliations</a><div style="display:none" class="ui-widget" id="__NBK169451_ai__"><p class="contrib-group"><h4>Authors</h4><span itemprop="author">Samarjit Patnaik</span>,<sup>a</sup> <span itemprop="author">Dipanwita Basu</span>,<sup>b</sup> <span itemprop="author">Seameen Dehdashti</span>,<sup>a</sup> <span itemprop="author">Wei Zheng</span>,<sup>a</sup> <span itemprop="author">Marc Ferrer</span>,<sup>a</sup> <span itemprop="author">Noel Southall</span>,<sup>a</sup> <span itemprop="author">Mercedes Taylor</span>,<sup>a</sup> <span itemprop="author">Daniel A. Engel</span>,<sup>b</sup> and <span itemprop="author">Juan Jose Marugan</span><sup>a</sup><sup>,*</sup>.</p><h4>Affiliations</h4><div class="affiliation"><sup>a</sup>
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NIH Chemical Genomics Center, National Human Genome Research Institute, National Institutes of Health, 9800 Medical Center Drive, Rockville, MD, 20850.</div><div class="affiliation"><sup>b</sup>
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Department of Microbiology, University of Virginia School of Medicine, 1300 Jefferson Park Ave., Charlottesville, VA 22908.</div><div class="affiliation">
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<sup>*</sup> To whom correspondence should be addressed: Email:
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<a href="mailto:dev@null" data-email="vog.hin.liam@jnaguram" class="oemail">vog.hin.liam@jnaguram</a></div></div><p class="small">Received: <span itemprop="datePublished">April 13, 2012</span>; Last Update: <span itemprop="dateModified">September 3, 2013</span>.</p></div><div class="jig-ncbiinpagenav body-content whole_rhythm" data-jigconfig="allHeadingLevels: ['h2'],smoothScroll: false" itemprop="text"><div id="_abs_rndgid_" itemprop="description"><p>The Non Structural 1 (NS1) is a 230–237 amino acid relatively well-conserved viral protein that is usually expressed only during infection [<a class="bk_pop" href="#ml303.r1">1</a>]. It contains an RNA binding domain (RBD) and an effector domain (ED) that is connected by a variable linker [<a class="bk_pop" href="#ml303.r2">2</a>]. The NS1 protein has been shown to be a key player that counters the host interferon (IFN) response to viral infection in strain-specific ways [<a class="bk_pop" href="#ml303.r3">3</a>]. NS1 inhibits the function of OAS (2′–5′-oligoadenylate synthetase) and PKR (protein kinase R) [<a class="bk_pop" href="#ml303.r4">4</a>] and block IFN-β synthesis at the post-transcriptional level by inhibiting the pre-mRNA splicing and blocking the export of poly(A)-RNAs from the nucleus to the cytoplasm [<a class="bk_pop" href="#ml303.r5">5</a>]. It also hampers the host’s defense pathways by interfering with the host RNAi pathway, adaptive immune response, and the apoptotic response [<a class="bk_pop" href="#ml303.r6">6</a>]. NS1 is also a key component in the temporal regulation of viral RNA synthesis, its splicing, and translation [<a class="bk_pop" href="#ml303.r7">7</a>]. Thus, NS1 enhances the virulence of infection, posing as a compelling target for influenza treatment. These compounds will enable researchers to test how NS1 inhibition impacts the infection cycle and how antagonists can be leveraged alone or in combination with existing agents in the next generation of treatments for influenza infection. We developed and conducted a high-throughput screen using a novel yeast-based phenotypic assay to identify compounds which specifically inhibit NS1 function. Here we report a potent NS1 antagonist chemical series, represented by <a href="/pcsubstance/?term=ML303[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML303</a>. The SAR (structure activity relationship) around this chemical series was further developed by measuring the compound’s ability to inhibit replication of the influenza A/PR/8/34 virus in MDCK cells. The <a href="/pcsubstance/?term=ML303[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML303</a> compound series specifically restored the NS1-inhibited expression of the interferon mRNA, inhibited the NS1 function in mechanistic assay and exhibited a potent antiviral activity in culture.</p></div><div class="h2"></div><p><b>Assigned Assay Grant #:</b> R03-MH085680</p><p><b>Version #:</b> 1</p><p><b>Date Submitted:</b> 4/13/2012</p><p><b>Screening Center Name & PI:</b> NIH Chemical Genomics Center, Christopher P. Austin</p><p><b>Chemistry Center Name & PI:</b> NIH Chemical Genomics Center, Christopher P. Austin</p><p><b>Assay Submitter & Institution:</b> Daniel A. Engel, University of Virginia</p><p><b>PubChem Summary Bioassay Identifier (AID):</b>
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<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2336" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2336</a></p><div id="ml303.s1"><h2 id="_ml303_s1_">Probe Structure & Characteristics</h2><div id="ml303.fu1" class="figure bk_fig"><div class="graphic"><img src="/books/NBK169451/bin/ml303fu1.jpg" alt="ML303." /></div><h3><span class="title">ML303</span></h3></div><div id="ml303.tu1" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK169451/table/ml303.tu1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml303.tu1_lrgtbl__"><table><thead><tr><th id="hd_h_ml303.tu1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">CID/ML#</th><th id="hd_h_ml303.tu1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Target Name</th><th id="hd_h_ml303.tu1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">IC50/EC50 (nM) [SID, AID]</th><th id="hd_h_ml303.tu1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Anti-target Name(s)</th><th id="hd_h_ml303.tu1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">IC50/EC50 (μM) [SID, AID]</th><th id="hd_h_ml303.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Fold Selective</th><th id="hd_h_ml303.tu1_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Secondary Assay(s) Name: IC50/EC50 (nM) [SID, AID]</th></tr></thead><tbody><tr><td headers="hd_h_ml303.tu1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">CID 53316412/<a href="/pcsubstance/?term=ML303[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML303</a></td><td headers="hd_h_ml303.tu1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">NS1</td><td headers="hd_h_ml303.tu1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Anti-Viral (A/PR/38) IC<sub>90</sub>: 155 nM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/124954998" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 124954998</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602450" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 602450</a>]</td><td headers="hd_h_ml303.tu1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Cytotoxicity (MDCK)</td><td headers="hd_h_ml303.tu1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">>100 μM IC<sub>50</sub> [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/124954998" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 124954998</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/623997" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 623997</a>]</td><td headers="hd_h_ml303.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">>100 fold</td><td headers="hd_h_ml303.tu1_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Restored interferon-beta mRNA expression [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/124954998" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 124954998</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602456" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 602456</a>]</td></tr></tbody></table></div></div></div><div id="ml303.s2"><h2 id="_ml303_s2_">1. Recommendations for Scientific Use of the Probe</h2><p><a href="/pcsubstance/?term=ML303[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML303</a> exemplifies a pyrazolopyridine chemical series, where several compounds show potent reduction of viral titer in MDCK cells infected with the Influenza A/PR/8/34 strain. This antiviral small molecule addresses the weak activity limitations of the prior art compounds as it showed improved potency of IC<sub>90</sub> of 155nM without overt cytotoxicity to the host cells. <a href="/pcsubstance/?term=ML303[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML303</a> can therefore be used by virologists as a tool to inhibit the function of the influenza NS1 viral protein to study its role in the antagonism of the host interferon system and its impacts on the viral infection cycle. Given the reasonable pharmacokinetics and ADME profile of <a href="/pcsubstance/?term=ML303[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML303</a>, this probe can also enabling research clinicians to test how it can be leveraged (alone or in combination) with existing agents in the next generation of treatments for influenza and other viral infections where the NS1 protein is crucial.</p></div><div id="ml303.s3"><h2 id="_ml303_s3_">2. Materials and Methods</h2><p><b>Mammalian cells and viruses</b>. All media and sera were from Invitrogen. MDCK cells were maintained in Iscove medium supplemented with 10% fetal bovine serum (FBS) and 2 mM L-glutamine. For infections, viral stocks were diluted in growth medium supplemented with 0.3% bovine serum albumin (BSA), 0.22% sodium bicarbonate, and 0.25 units of tolylsulfonyl phenylalanyl chloromethyl ketone (TPCK) -trypsin per mL. Influenza viruses A/PR/8/34 (PR) were gifts from Adolfo Garcia-Sastre. The viruses were propagated in 10-day-old embryonated chicken eggs at 37 °C. Titers of influenza virus stocks were determined by hemagglutination and analyzed via 50% tissue culture infective dose (TCID<sub>50</sub>) [<a class="bk_pop" href="#ml303.r8">8</a>].</p><p><b>Yeast strains and growth</b>. Strain 9526-6-2 (<i>MAT</i><b>a</b><i>his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 pdr1::KanMX4 pdr3::KanMX4</i>) was a gift from Dan Burke. It was derived by tetrad dissection from two parent strains that had been modified by one step gene replacements. The <i>pdr1::KanMX4</i> was constructed in BY4741 (<i>MAT</i><b>a</b><i>his3</i>Δ<i>1 leu2</i>Δ<i>0 met15</i>Δ<i>0 ura3</i>Δ<i>0</i>) and the <i>pdr3::KanMX4</i> was constructed in BY4742 (<i>MAT</i>α<i>his3</i>Δ<i>1 leu2</i>Δ<i>0 met15</i>Δ<i>0 ura3</i>Δ<i>0</i>). PCR-mediated one-step gene replacements, matings and tetrad dissections were performed as described previously [<a class="bk_pop" href="#ml303.r9">9</a>]. The strain 9526-6-2/pYES-NS1 was generated by transformation of 9526-6-2 with the plasmid pYES-NS1 and maintained on synthetic complete medium lacking uracil (SC). For growth experiments and library screening, a single transformed colony was grown overnight, and the cell number was determined by using a coulter counter (Beckman Coulter Corp). The cells were diluted to 5 × 10<sup>5</sup> cells/mL in SC and containing raffinose and 2% galactose. A portion of this culture was added to preplated test compounds such that the final dimethyl sulfoxide (DMSO) concentration was 1%. Optical density readings at 600 nm (OD<sub>600</sub>) were taken using a Thermomax microplate reader (Molecular Devices).</p><p><b>General Methods for Chemistry</b>. The reagents and solvents used were commercial anhydrous grade and used without further purification. The column chromatography was carried out over silica gel (100–200 mesh). <sup>1</sup>H NMR spectra were recorded with a Varian 400 MHz spectrometer from solutions in CDCl<sub>3</sub> and DMSO-d<sub>6</sub>. Chemical shifts in <sup>1</sup>H NMR spectra are reported in parts per million (ppm, δ) with solvent peaks as internal. Molecular weight confirmation was performed using an Agilent 6224 Time-Of-Flight Mass Spectrometer (TOF, Agilent Technologies, Santa Clara, CA). A 3 minute gradient from 5 to 100% Acetonitrile in water (0.03% formic acid) was used with a 5.1 minute run time at a flow rate of 0.4 mL/min. A Waters Atlantis T3 C18 column (1.8 micron, 2.1 × 50 mm) was used at a temperature of 25 °C. Confirmation of molecular formula was confirmed using electrospray ionization in the positive mode with the Agilent Masshunter software (version B.02) while stability of the probe was assess using the liquid chromatography – mass spectrometry (LC/MS/MS) system.</p><div id="ml303.s4"><h3>2.1. Assays</h3><p><b>Quantitative High-troughput Screen (qHTS) for Antagonists of Influenza NS1 Protein Function (BioAssay <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2326" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 2326</a>).</b> The primary yeast assay was developed where the expression of NS1 induces a slow-growth phenotype that could be observed by measuring optical density of the yeast. One hundred milliliter of yeast culture medium was inoculated with 1 mL of frozen yeast aliquot and incubated overnight at 30 ºC, mixed at 250 RPM. The culture was harvested through spinning at 4000 RPM and the pellet was washed with 15 mL H<sub>2</sub>O three times. The washed pellet was re-suspended in 10 mL of fresh yeast assay medium. A 1:100 dilution of the re-suspended culture was prepared and 3.5 μL of this yeast suspension was dispensed into 1536 black, clear-bottom assay plates (columns 2–48). Compounds were transferred via the Kalypsys pintool robotics equipped with a 23 nL pinheads. Additional 3.5 μL of yeast assay medium was added and read using the ViewLux plate reader at OD<sub>600</sub>.</p><p><b>Influenza Virus Replication Assay in MDCK Cells (BioAssay <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504647" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504647</a> and <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602454" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 602454</a>).</b> The replication assay was developed to test the compounds ability to slow growth of the PR virus in the MDCK mammalian cells. Titers of influenza virus stocks were determined by 50% TCID<sub>50</sub> analysis on MDCK cells, maintained in Iscove medium supplemented with 10% FBS and 2 mM L-glutamine. Confluent MDCK monolayers were infected with the PR virus at a multiplicity of infection (MOI) of 0.1 for 48 hr in the presence or absence of 5 or 10 μM compounds. Compounds were added at the beginning of infection and were present throughout the infection. After 48 hr virus titers were determined using the by standard TCID<sub>50</sub> described in details in Basu <i>et al</i> publication [<a class="bk_pop" href="#ml303.r10">10</a>].</p><p><b>Mammalian Cell Viability Assay (BioAssay <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/623997" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 623997</a>).</b> This viability assay was developed to determine general toxicity of the identified inhibitors. Opaque-walled 96-well plates were seeded with 10<sup>4</sup> MDCK cells per well in 100 μL medium and incubated overnight at 37 ºC. The compound was added in different concentrations (in triplicate) and incubated 37 ºC. After 48 hours, the plates were equilibrated for 30 min at room temperature and 100 μL CellTiter Glo reagent (Promega<sup>®</sup>) was added according to the manufacturer’s protocol. The plates were shaken for 2 min and then incubated for 10 min at room temperature. Luminescence was recorded in an LMAX 384 reader (Molecular Devices) with 1 sec integration time.</p><p><b>TCID<sub>50</sub> Assay (BioAssay <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602450" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 602450</a>).</b> This assay was used to determine the virus titer in the MDCK cell line. Cell monolayers were infected at an MOI of 0.1 for 48 hr in the presence or absence of the compound added at the beginning of infection. Virus titers were determined by standard TCID<sub>50</sub> analysis.</p><p><b>RT-PCR based secondary assay to measure induction of interferon-beta mRNA (BioAssay <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602456" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 602456</a>).</b> This secondary assay was developed to confirm the antiviral activity of the identified probe by assessing the interferon beta (IFN-β) expression of the MDCK cells in response to virus infection. The cells were infected for 6 hr with PR virus at MOI of 0.1 in the presence or absence of drug. Total RNA was isolated using RNeasy (Qiagen). For first-strand cDNA synthesis, 2 μg of total RNA was primed with random nanomers (New England Biolabs) at a final concentration of 2 μM. Reverse transcription was performed with 10 U/μL of Moloney murine leukemia virus reverse transcriptase (New England Biolabs) in the presence of 1 U/μL of RNase inhibitor (New England Biolabs). Thereafter, 1/20 volume of cDNA was used as a template for PCR (30 cycles). IFN-β mRNA (accession number <a href="/nuccore/209417931" class="bk_tag" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=nuccore">NM_001135787</a>) expression levels were quantitated using the 5′CCAGTTCCAGAAGGAGGACA3′ and 5′CCTGTTGTCCCAGGTGAAGT3′ primer pair; and normalized to β-actin (<a href="/nuccore/73951812" class="bk_tag" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=nuccore">XM_536230</a>) expression levels quantitated using the primer pair 5′GGCATCCTGACCCT GAAGTA3′ and 5′GGGGTGTTGAAAGTCTCGAA3′.</p></div><div id="ml303.s5"><h3>2.2. Probe Chemical Characterization</h3><div id="ml303.fu2" class="figure bk_fig"><div class="graphic"><img src="/books/NBK169451/bin/ml303fu2.jpg" alt="Probe ML303 (CID 53316412)." /></div><h3><span class="title">Probe ML303 (CID 53316412)</span></h3><div class="caption"><p></p></div><div><dl class="temp-labeled-list small"><dt>*</dt><dd><div id="ml303.fn2"><p class="no_top_margin">Purity 100% as determined by LC/MS and <sup>1</sup>H NMR analyses</p></div></dd></dl></div></div><p><b>2-Methoxy-4-(3-methyl-1-(4-(trifluoromethyl)phenyl)-1H-pyrazolo[3,4-</b><b><i>b</i></b><b>]pyridin-4-yl)phenol</b>: LC/MS (Agilent system) Retention time t<sub>1</sub> (long) = 6.97 min; <sup>1</sup>H NMR (400 MHz, DMSO-<i>d</i><sub>6</sub>) δ ppm 2.35 (s, 3 H), 3.85 (s, 3 H), 6.96 (m, 1 H), 7.02 (m, 1 H), 7.17 (d, <i>J</i>=2.3 Hz, 1 H), 7.28 (d, <i>J</i>=4.7 Hz, 1 H), 7.94 (d, <i>J</i>=9.0 Hz, 2 H), 8.63 (d, <i>J</i>=9.0 Hz, 2 H), 8.66 (d, <i>J</i>=4.7 Hz, 1 H), 9.44 (s, 1 H); HRMS (ESI) m/z calculated for C<sub>21</sub>H<sub>17</sub>F<sub>3</sub>N<sub>3</sub>O<sub>2</sub> [M + H]<sup>+</sup> 400.1267 found 400.1270.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml303f2" co-legend-rid="figlgndml303f2"><a href="/books/NBK169451/figure/ml303.f2/?report=objectonly" target="object" title="Figure 2" class="img_link icnblk_img figpopup" rid-figpopup="figml303f2" rid-ob="figobml303f2"><img class="small-thumb" src="/books/NBK169451/bin/ml303f2.gif" src-large="/books/NBK169451/bin/ml303f2.jpg" alt="Figure 2. Structures of the ML303 and five analogs (with corresponding CID) that have been submitted to the MLSMR compound repository." /></a><div class="icnblk_cntnt" id="figlgndml303f2"><h4 id="ml303.f2"><a href="/books/NBK169451/figure/ml303.f2/?report=objectonly" target="object" rid-ob="figobml303f2">Figure 2</a></h4><p class="float-caption no_bottom_margin">Structures of the ML303 and five analogs (with corresponding CID) that have been submitted to the MLSMR compound repository. </p></div></div><div id="ml303.t1" class="table"><h3><span class="label">Table 1</span><span class="title">List of the NS1 inhibitor probe ML303 and related analogs and their corresponding identification numbers</span></h3><div class="caption"><p>Their corresponding molecular structures are shown above in <a class="figpopup" href="/books/NBK169451/figure/ml303.f2/?report=objectonly" target="object" rid-figpopup="figml303f2" rid-ob="figobml303f2">Figure 2</a>.</p></div><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK169451/table/ml303.t1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml303.t1_lrgtbl__"><table><thead><tr><th id="hd_h_ml303.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Internal ID</th><th id="hd_h_ml303.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">MLS ID</th><th id="hd_h_ml303.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">SID</th><th id="hd_h_ml303.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">CID</th><th id="hd_h_ml303.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">ML#</th><th id="hd_h_ml303.t1_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Type</th><th id="hd_h_ml303.t1_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Source</th></tr></thead><tbody><tr><td headers="hd_h_ml303.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00248767</td><td headers="hd_h_ml303.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS004256089</td><td headers="hd_h_ml303.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/124954998" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">124954998</a></td><td headers="hd_h_ml303.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">53316412</td><td headers="hd_h_ml303.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="/pcsubstance/?term=ML303[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML303</a></td><td headers="hd_h_ml303.t1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Probe</td><td headers="hd_h_ml303.t1_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC</td></tr><tr><td headers="hd_h_ml303.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00248768</td><td headers="hd_h_ml303.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS004256090</td><td headers="hd_h_ml303.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/124954999" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">124954999</a></td><td headers="hd_h_ml303.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">53316413</td><td headers="hd_h_ml303.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml303.t1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Analog</td><td headers="hd_h_ml303.t1_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC</td></tr><tr><td headers="hd_h_ml303.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00244898</td><td headers="hd_h_ml303.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS004256091</td><td headers="hd_h_ml303.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/124951246" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">124951246</a></td><td headers="hd_h_ml303.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">53316317</td><td headers="hd_h_ml303.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml303.t1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Analog</td><td headers="hd_h_ml303.t1_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC</td></tr><tr><td headers="hd_h_ml303.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00244896</td><td headers="hd_h_ml303.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS004256092</td><td headers="hd_h_ml303.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/124951244" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">124951244</a></td><td headers="hd_h_ml303.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">53316315</td><td headers="hd_h_ml303.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml303.t1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Analog</td><td headers="hd_h_ml303.t1_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC</td></tr><tr><td headers="hd_h_ml303.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00244897</td><td headers="hd_h_ml303.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS004256093</td><td headers="hd_h_ml303.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/124951245" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">124951245</a></td><td headers="hd_h_ml303.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">53316316</td><td headers="hd_h_ml303.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml303.t1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Analog</td><td headers="hd_h_ml303.t1_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC</td></tr><tr><td headers="hd_h_ml303.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00248765</td><td headers="hd_h_ml303.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS004256094</td><td headers="hd_h_ml303.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/124954996" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">124954996</a></td><td headers="hd_h_ml303.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">53316410</td><td headers="hd_h_ml303.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml303.t1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Analog</td><td headers="hd_h_ml303.t1_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC</td></tr></tbody></table></div></div></div><div id="ml303.s6"><h3>2.3. Probe Preparation</h3><p>Preparation of 2-methoxy-4-(3-methyl-1-(4-(trifluoromethyl)phenyl)-1<i>H</i>-pyrazolo[3,4-<i>b</i>]pyridin-4-yl)phenol (<a href="/pcsubstance/?term=ML303[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML303</a>) is a multi-step process with 6 compound intermediates illustrated above (<a class="figpopup" href="/books/NBK169451/figure/ml303.f7/?report=objectonly" target="object" rid-figpopup="figml303f7" rid-ob="figobml303f7">Scheme 1</a>) and summarized below.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml303f7" co-legend-rid="figlgndml303f7"><a href="/books/NBK169451/figure/ml303.f7/?report=objectonly" target="object" title="Scheme 1" class="img_link icnblk_img figpopup" rid-figpopup="figml303f7" rid-ob="figobml303f7"><img class="small-thumb" src="/books/NBK169451/bin/ml303f7.gif" src-large="/books/NBK169451/bin/ml303f7.jpg" alt="Scheme 1. Synthetic route to ML303 is a multi-step process with 6 compound intermediates A – F." /></a><div class="icnblk_cntnt" id="figlgndml303f7"><h4 id="ml303.f7"><a href="/books/NBK169451/figure/ml303.f7/?report=objectonly" target="object" rid-ob="figobml303f7">Scheme 1</a></h4><p class="float-caption no_bottom_margin">Synthetic route to ML303 is a multi-step process with 6 compound intermediates A – F. </p></div></div><ol><li class="half_rhythm"><div>A mixture of (<i>E</i>)-3-aminobut-2-enenitrile and (4-(trifluoromethyl)phenyl)hydrazine was stirred in ethanol (EtOH) and heated to reflux for 16 hr. The mixture was cooled and poured into water and extracted with ethyl acetate (EtOAc). The organic phase was washed with brine, dried over MgSO<sub>4</sub>, filtered and then concentrated to yield 6.7 g (99% yield) 3-methyl-1-(4-(trifluoromethyl)phenyl)-1<i>H</i>-pyrazol-5-amine (intermediate <b>A</b>), a dark red oil which was used in the next step without further purification.</div></li><li class="half_rhythm"><div>Intermediate <b>A</b> and diethyl(ethoxymethylene)malonate were taken in 1,4-dioxane and heated to 120 °C for 16 hr. Analysis by LCMS indicated completion of the reaction. The mixture was concentrated to give the intermediate <b>B</b>, dark oil which crystallized on standing.</div></li><li class="half_rhythm"><div>The crude material (<b>B</b>) was dissolved in phosphoryl chloride (POCl<sub>3</sub>) and heated at 120 °C for 16 hr. The reaction mixture was cooled, poured over ice, and extracted with EtOAc. The organic layer was washed carefully with sat. aq. sodium bicarbonate (NaHCO<sub>3</sub>), then brined, dried over magnesium sulfate (MgSO<sub>4</sub>), filtered, concentrated, and purified via flash silicon dioxide (SiO<sub>2</sub>) chromatography to provide 2.7 g (25% yield over 2 steps) of ethyl 4-chloro-3-methyl-1-(4-(trifluoromethyl)phenyl)-1<i>H</i>-pyrazolo[3,4-<i>b</i>]pyridine-5-carboxylate (intermediate <b>C</b>).</div></li><li class="half_rhythm"><div>Intermediate <b>C</b> was dissolved in 1,4-dioxane, treated with excess 2 N lithium hydroxide (LiOH) and heated at 100 °C for 36 hr until full conversion to intermediate <b>D</b> was observed by HPLC. The reaction was cooled and extracted with EtOAc. The pH of the aqueous phase was adjusted to ~5 and was extracted with EtOAc.</div></li><li class="half_rhythm"><div>The organic layers were dried over MgSO<sub>4</sub>, filtered, and concentrated to provide <b>D</b> which was dissolved in ethylene glycol, treated with solid sodium hydroxide (NaOH), and heated at 180 °C for 4 hr until complete conversion to intermediate <b>E</b> was observed by LCMS. The reaction was cooled to room temperature, diluted with water, and the pH was adjusted to 6.5. The mixture was extracted with EtOAc.</div></li><li class="half_rhythm"><div>The organic layers were combined, dried over MgSO<sub>4</sub>, filtered, concentrated, and purified via flash SiO<sub>2</sub> chromatography to provide 1.84 g (90% yield over 2 steps) of <b>E</b>. Phenol <b>E</b> was dissolved in POCl<sub>3</sub> and heated at 120 °C for 16 hr. The reaction mixture was cooled, poured over ice, and extracted with EtOAc.</div></li><li class="half_rhythm"><div>The organic layer was washed carefully with sat. aq. NaHCO<sub>3</sub>, then brine, dried over MgSO<sub>4</sub>, filtered, concentrated, and purified via flash SiO<sub>2</sub> chromatography to provide an 86% yield of 4-chloro-3-methyl-1-(4-(trifluoromethyl)phenyl)-1<i>H</i>-pyrazolo[3,4-<i>b</i>]pyridine (intermediate <b>F</b>).</div></li><li class="half_rhythm"><div>Intermediate <b>F</b>, 4-hydroxy-3-methoxyphenylboronic acid, potassium phosphate (K<sub>3</sub>PO<sub>4</sub>), and bis(triphenylphosphine)palladium(II) dichloride (PdCl<sub>2</sub>(PPh<sub>3</sub>)<sub>2</sub>) were taken in dimethylformamide (DMF) and water. The mixture was heated at 100 °C for 10 hr, cooled, concentrated, diluted with dichloromethane (DCM), and filtered through a PL-Thiol MP Resin cartridge (Agilent). The filtrate was concentrated and purified by flash SiO<sub>2</sub> chromatography to obtain 2-methoxy-4-(3-methyl-1-(4-(trifluoromethyl)phenyl)-1<i>H</i>-pyrazolo[3,4-<i>b</i>]pyridin-4-yl)phenol (probe <a href="/pcsubstance/?term=ML303[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML303</a>).</div></li></ol></div></div><div id="ml303.s7"><h2 id="_ml303_s7_">3. Results</h2><div id="ml303.s8"><h3>3.1. Dose Response Curves for Probe</h3><div id="ml303.f3" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%203.%20Dose%20response%20activity%20of%20ML303%20on%20viral%20titer.&p=BOOKS&id=169451_ml303f3.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img src="/books/NBK169451/bin/ml303f3.jpg" alt="Figure 3. Dose response activity of ML303 on viral titer." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 3</span><span class="title">Dose response activity of ML303 on viral titer</span></h3><div class="caption"><p>Triplicate cultures were infected with PR virus at MOI of 0.1% and treated with different concentrations of <a href="/pcsubstance/?term=ML303[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML303</a>. After 48 hour, the supernatants were collected and analyzed to determine the reduction in virus by assaying the supernatant infectivity. The data is presented as a percent decrease in titer <b>(A)</b> which is calculated from TCID<sub>50</sub> where the probe IC<sub>90</sub> is 155 nM and as the actual TCID<sub>50</sub><b>(B)</b> where 3 log units of viral suppression are observed at 1.6 μM concentration (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602450" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 602450</a>).</p></div></div></div><div id="ml303.s9"><h3>3.2. Cellular Activity</h3><div id="ml303.f4" class="figure bk_fig"><div class="graphic"><img src="/books/NBK169451/bin/ml303f4.jpg" alt="Figure 4. Compound-dependent restoration of yeast growth." /></div><h3><span class="label">Figure 4</span><span class="title">Compound-dependent restoration of yeast growth</span></h3><div class="caption"><p>Growth of the control yeast strain pYES (without NS1) and the NS1 expressing stain NS1-PYES were measured as OD<sub>600</sub> nm at 22, 42 and 60 hours. Results showed the slow growth phenotype exhibited by the NS1-pYes strain is reversed by the addition of 50 μM <a href="/pcsubstance/?term=ML303[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML303</a> and after 60 hour the growth phenotype was comparable to that of the control treatments.</p></div></div><div id="ml303.f5" class="figure bk_fig"><div class="graphic"><img src="/books/NBK169451/bin/ml303f5.jpg" alt="Figure 5. Compound-dependent restoration of IFN-β mRNA levels in MDCK cells." /></div><h3><span class="label">Figure 5</span><span class="title">Compound-dependent restoration of IFN-β mRNA levels in MDCK cells</span></h3><div class="caption"><p>Cells were mock infected <b>(lane 2)</b> or infected with influenza strain A/PR/8 at an MOI of 0.1 <b>(lanes 4–9)</b>. Infected cells were treated with negative control 1% DMSO or different compounds (20 μM) including the probe <a href="/pcsubstance/?term=ML303[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML303</a>
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<b>(lane 7)</b>. After 6 hour, cells were harvested for RT-PCR analysis of IFN-β and β-actin mRNA levels. Results showed treatment with these compound restored IFN-β levels comparable to the positive control treatment poly(I:C) which directly induces IFN-β <b>(lane 3)</b> (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602456" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 602456</a>).</p></div></div></div><div id="ml303.s10"><h3>3.3. Profiling Assays</h3><p>Preliminary ADME profiling revealed that <a href="/pcsubstance/?term=ML303[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML303</a> and relative analogs have good stability in mouse live microsomes (MLL) (45% remaining after 30 minutes) and good permeability with low efflux in Caco-2 monolayer of cells (<a class="figpopup" href="/books/NBK169451/table/ml303.t2/?report=objectonly" target="object" rid-figpopup="figml303t2" rid-ob="figobml303t2">Table 2</a>). <a href="/pcsubstance/?term=ML303[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML303</a> and members of this series showed low solubility (< 50 μM), but this didn’t translate to poor <i>in vivo</i> pharmacokinetics as <a href="/pcsubstance/?term=ML303[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML303</a> exhibited high exposure in plasma and lung after a single 30 mg/kg intra-peritoneal (IP) dose in mice (<a class="figpopup" href="/books/NBK169451/table/ml303.t3/?report=objectonly" target="object" rid-figpopup="figml303t3" rid-ob="figobml303t3">Table 3</a>) and after 48 hours of probe treatment (<a class="figpopup" href="/books/NBK169451/figure/ml303.f6/?report=objectonly" target="object" rid-figpopup="figml303f6" rid-ob="figobml303f6">Figure 6</a>). It is well known that low solubility impacts permeability in Caco-2 assays, but the use of an appropriate formulation often results in reasonable <i>in vivo</i> bioavailability. Additionally the compound showed good stability in mouse plasma and PBS buffer (<a class="figpopup" href="/books/NBK169451/figure/ml303.f1/?report=objectonly" target="object" rid-figpopup="figml303f1" rid-ob="figobml303f1">Figure 1</a>).</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml303t2"><a href="/books/NBK169451/table/ml303.t2/?report=objectonly" target="object" title="Table 2" class="img_link icnblk_img figpopup" rid-figpopup="figml303t2" rid-ob="figobml303t2"><img class="small-thumb" src="/books/NBK169451/table/ml303.t2/?report=thumb" src-large="/books/NBK169451/table/ml303.t2/?report=previmg" alt="Table 2. In vitro ADME data for ML303 and relative active analogs." /></a><div class="icnblk_cntnt"><h4 id="ml303.t2"><a href="/books/NBK169451/table/ml303.t2/?report=objectonly" target="object" rid-ob="figobml303t2">Table 2</a></h4><p class="float-caption no_bottom_margin"><i>In vitro</i> ADME data for ML303 and relative active analogs. </p></div></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml303t3"><a href="/books/NBK169451/table/ml303.t3/?report=objectonly" target="object" title="Table 3" class="img_link icnblk_img figpopup" rid-figpopup="figml303t3" rid-ob="figobml303t3"><img class="small-thumb" src="/books/NBK169451/table/ml303.t3/?report=thumb" src-large="/books/NBK169451/table/ml303.t3/?report=previmg" alt="Table 3. Pharmacokinetics (PK) data showing the mean plasma, live, and lung concentration of ML303 in IP treated male C57BL/6 mice (n=3)." /></a><div class="icnblk_cntnt"><h4 id="ml303.t3"><a href="/books/NBK169451/table/ml303.t3/?report=objectonly" target="object" rid-ob="figobml303t3">Table 3</a></h4><p class="float-caption no_bottom_margin">Pharmacokinetics (PK) data showing the mean plasma, live, and lung concentration of ML303 in IP treated male C57BL/6 mice (n=3). </p></div></div><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml303f6" co-legend-rid="figlgndml303f6"><a href="/books/NBK169451/figure/ml303.f6/?report=objectonly" target="object" title="Figure 6" class="img_link icnblk_img figpopup" rid-figpopup="figml303f6" rid-ob="figobml303f6"><img class="small-thumb" src="/books/NBK169451/bin/ml303f6.gif" src-large="/books/NBK169451/bin/ml303f6.jpg" alt="Figure 6. Time course PK plot of the ML303 concentration in plasma, liver and lung of IP treated mice." /></a><div class="icnblk_cntnt" id="figlgndml303f6"><h4 id="ml303.f6"><a href="/books/NBK169451/figure/ml303.f6/?report=objectonly" target="object" rid-ob="figobml303f6">Figure 6</a></h4><p class="float-caption no_bottom_margin">Time course PK plot of the ML303 concentration in plasma, liver and lung of IP treated mice. Each point represents the mean +/− SD of n=3. </p></div></div><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml303f1" co-legend-rid="figlgndml303f1"><a href="/books/NBK169451/figure/ml303.f1/?report=objectonly" target="object" title="Figure 1" class="img_link icnblk_img figpopup" rid-figpopup="figml303f1" rid-ob="figobml303f1"><img class="small-thumb" src="/books/NBK169451/bin/ml303f1.gif" src-large="/books/NBK169451/bin/ml303f1.jpg" alt="Figure 1. Solubility and stability data of probe ML303 in different media as evaluated by LC/MS/MS." /></a><div class="icnblk_cntnt" id="figlgndml303f1"><h4 id="ml303.f1"><a href="/books/NBK169451/figure/ml303.f1/?report=objectonly" target="object" rid-ob="figobml303f1">Figure 1</a></h4><p class="float-caption no_bottom_margin">Solubility and stability data of probe ML303 in different media as evaluated by LC/MS/MS. <i>(A)</i> Dot plot of the stability of the probe in D-PBS pH 7.4 at room temperature in a 24 hour period. <i>(B)</i> Dot plot of the stability of the probe in mouse plasma at <a href="/books/NBK169451/figure/ml303.f1/?report=objectonly" target="object" rid-ob="figobml303f1">(more...)</a></p></div></div></div></div><div id="ml303.s11"><h2 id="_ml303_s11_">4. Discussion</h2><div id="ml303.s12"><h3>4.1. Comparison to Existing Art and How the New Probe is an Improvement</h3><p>Influenza continues to pose a global public health problem. The development of novel antivirals is imperative due to the ability of the virus to develop drug resistance. A patent disclosing eight chemical series of NS1 influenza protein inhibitors was revealed in 2009 but their chemical structures were not disclosed to public [<a class="bk_pop" href="#ml303.r11">11</a>]. The Engle lab also identified the small molecule JJ3297 that indirectly perturb NS1with potency of 5μM. However, the precise mechanism of action remains to be elucidated. Moreover, treatment with JJ329 is limited to infections at low multiplicity since it showed no effect on virus spread or medium titer during infections at high multiplicity [<a class="bk_pop" href="#ml303.r12">12</a>]. <a href="/pcsubstance/?term=ML303[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML303</a> is a new NS1 inhibitor that exhibits improved antiviral activity. Compared to the prior art, <a href="/pcsubstance/?term=ML303[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML303</a> has better potency of 155nM (IC<sub>90</sub>) against the NS1 without an overt cytotoxicity to the host cells. Furthermore, <a href="/pcsubstance/?term=ML303[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML303</a> showed good stability and <i>in vivo</i> bioavailability. Hence the <a href="/pcsubstance/?term=ML303[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML303</a> probe poses as a tool compound for evaluation of NS1 antagonism in advance in vitro assay and animal studies as a therapeutic strategy against influenza.</p></div></div><div id="ml303.s13"><h2 id="_ml303_s13_">5. References</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="ml303.r1">Palese P, Shaw ML. <span class="ref-journal">Fields Virology.</span> 5<sup>th</sup> edition. 2007. Orthomyxoviridae: the viruses and their replication; pp. 1647–1689.</div></dd><dt>2.</dt><dd><div class="bk_ref" id="ml303.r2">Bornholdt AA, Carrilli B, Venkataraman Prasad BV. <span class="ref-journal">Influenza: Molecular Virology.</span> 2<sup>nd</sup> edition. 2010. Structural Insights into the Function of the Influenza NS1.</div></dd><dt>3.</dt><dd><div class="bk_ref" id="ml303.r3">Talon J, Salvatore M, O’Neill RE, Nakaya Y, Zheng H, Muster T, Garcia-Sastre A, Palese P. Influenza A and B viruses expressing altered NS1 proteins: a vaccine approach. <span><span class="ref-journal">Proc. Natl. Acad. Sci. </span>2000;<span class="ref-vol">97</span>:4309–4314.</span> [<a href="/pmc/articles/PMC18238/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC18238</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/10725408" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 10725408</span></a>]</div></dd><dt>4.</dt><dd><div class="bk_ref" id="ml303.r4">Silverman RH. Viral encounters with 2′,5′-oligoadenylate synthetase and RNase L during the interferon antiviral response. <span><span class="ref-journal">J. Virol. </span>2007;<span class="ref-vol">81</span>:12720–12729.</span> [<a href="/pmc/articles/PMC2169107/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2169107</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/17804500" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17804500</span></a>]</div></dd><dt>5.</dt><dd><div class="bk_ref" id="ml303.r5">Qiu Y, Krug RM. The influenza virus NS1 protein is a poly(A)-binding protein that inhibits nuclear export of mRNAs containing poly(A). <span><span class="ref-journal">J. Virol. </span>1994;<span class="ref-vol">68</span>:2425–2432.</span> [<a href="/pmc/articles/PMC236720/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC236720</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/7908060" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 7908060</span></a>]</div></dd><dt>6.</dt><dd><div class="bk_ref" id="ml303.r6">Nemeroff ME, Barabino SML, Li Y, Keller W, Krug RM. Influenza virus NS1 protein interacts with the cellular 30 kDa subunit of CPSF and inhibits 3′end formation of cellular premRNAs. <span><span class="ref-journal">Molecular Cell. </span>1998;<span class="ref-vol">1</span>:991–1000.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/9651582" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 9651582</span></a>]</div></dd><dt>7.</dt><dd><div class="bk_ref" id="ml303.r7">Chen ZLY, Krug RM. Influenza A virus NS1 protein targets poly(A)-binding protein II of the cellular 3′-end processing machinery. <span><span class="ref-journal">EMBO J. </span>1999;<span class="ref-vol">18</span>:2273–2283.</span> [<a href="/pmc/articles/PMC1171310/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC1171310</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/10205180" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 10205180</span></a>]</div></dd><dt>8.</dt><dd><div class="bk_ref" id="ml303.r8">Reed L, Muench H. A simple method for estimating fifty percent endpoints. <span><span class="ref-journal">Am J Hyg. </span>1938;<span class="ref-vol">27</span>:943–947.</span></div></dd><dt>9.</dt><dd><div class="bk_ref" id="ml303.r9">Amberg D, Burke DJ, Strathern JN. <span class="ref-journal">Methods in yeast genetics.</span> 2005. </div></dd><dt>10.</dt><dd><div class="bk_ref" id="ml303.r10">Basu D, Walkiewicz MP, Frieman M, Baric RS, Auble DT, Engel DA. Novel Influenza Virus NS1 Antagonists Block Replication and Restore Innate Immune Function. <span><span class="ref-journal">J. Virol. </span>2009;<span class="ref-vol">83</span>:1881–1891.</span> [<a href="/pmc/articles/PMC2643796/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2643796</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/19052087" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19052087</span></a>]</div></dd><dt>11.</dt><dd><div class="bk_ref" id="ml303.r11">Roth M, Fontoura B, Wei S, Satterly N. US20090170840A120090702. <span><span class="ref-journal">US Pat Appl Publ. </span>2009</span></div></dd><dt>12.</dt><dd><div class="bk_ref" id="ml303.r12">Walkiewicz MP, Dipanwita B, Jablonski JJ, Geysen HM, Engel DA. Novel inhibitor of influenza non-structural protein 1 blocks multi-cycle replication in an RNase L-dependent manner. <span><span class="ref-journal">J Gen Virology. </span>2011;<span class="ref-vol">92</span>:60–70.</span> [<a href="/pmc/articles/PMC3052532/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3052532</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/20881091" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 20881091</span></a>]</div></dd></dl></div><div id="bk_toc_contnr"></div></div></div>
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<div xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance"></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Views</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="PDF_download" id="Shutter"></a></div><div class="portlet_content"><ul xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="simple-list"><li><a href="/books/NBK169451/?report=reader">PubReader</a></li><li><a href="/books/NBK169451/?report=printable">Print View</a></li><li><a data-jig="ncbidialog" href="#_ncbi_dlg_citbx_NBK169451" data-jigconfig="width:400,modal:true">Cite this Page</a><div id="_ncbi_dlg_citbx_NBK169451" style="display:none" title="Cite this Page"><div class="bk_tt">Patnaik S, Basu D, Dehdashti S, et al. Discovery of Small Molecule Influenza Virus NS1 Antagonist. 2012 Apr 13 [Updated 2013 Sep 3]. In: Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-. <span class="bk_cite_avail"></span></div></div></li></ul></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>In this Page</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="page-toc" id="Shutter"></a></div><div class="portlet_content"><ul xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="simple-list"><li><a href="#ml303.s1" ref="log$=inpage&link_id=inpage">Probe Structure & Characteristics</a></li><li><a href="#ml303.s2" ref="log$=inpage&link_id=inpage">Recommendations for Scientific Use of the Probe</a></li><li><a href="#ml303.s3" ref="log$=inpage&link_id=inpage">Materials and Methods</a></li><li><a href="#ml303.s7" ref="log$=inpage&link_id=inpage">Results</a></li><li><a href="#ml303.s11" ref="log$=inpage&link_id=inpage">Discussion</a></li><li><a href="#ml303.s13" ref="log$=inpage&link_id=inpage">References</a></li></ul></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Related information</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="discovery_db_links" id="Shutter"></a></div><div class="portlet_content"><ul><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pmc&DbFrom=books&Cmd=Link&LinkName=books_pmc_refs&IdsFromResult=3060698" ref="log$=recordlinks">PMC</a><div class="brieflinkpop offscreen_noflow">PubMed Central citations</div></li><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pcassay&DbFrom=books&Cmd=Link&LinkName=books_pcassay_probe&IdsFromResult=3060698" ref="log$=recordlinks">PubChem BioAssay for Chemical Probe</a><div class="brieflinkpop offscreen_noflow">PubChem BioAssay records reporting screening data for the development of the chemical probe(s) described in this book chapter</div></li><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pcsubstance&DbFrom=books&Cmd=Link&LinkName=books_pcsubstance&IdsFromResult=3060698" ref="log$=recordlinks">PubChem Substance</a><div class="brieflinkpop offscreen_noflow">Related PubChem Substances</div></li><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pubmed&DbFrom=books&Cmd=Link&LinkName=books_pubmed_refs&IdsFromResult=3060698" ref="log$=recordlinks">PubMed</a><div class="brieflinkpop offscreen_noflow">Links to PubMed</div></li></ul></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Similar articles in PubMed</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="PBooksDiscovery_RA" id="Shutter"></a></div><div class="portlet_content"><ul><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/8806538" ref="ordinalpos=1&linkpos=1&log$=relatedarticles&logdbfrom=pubmed">The RNA-binding and effector domains of the viral NS1 protein are conserved to different extents among influenza A and B viruses.</a><span class="source">[Virology. 1996]</span><div class="brieflinkpop offscreen_noflow">The RNA-binding and effector domains of the viral NS1 protein are conserved to different extents among influenza A and B viruses.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Wang W, Krug RM. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Virology. 1996 Sep 1; 223(1):41-50. </em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/16987984" ref="ordinalpos=1&linkpos=2&log$=relatedarticles&logdbfrom=pubmed">Double-stranded RNA binding of influenza B virus nonstructural NS1 protein inhibits protein kinase R but is not essential to antagonize production of alpha/beta interferon.</a><span class="source">[J Virol. 2006]</span><div class="brieflinkpop offscreen_noflow">Double-stranded RNA binding of influenza B virus nonstructural NS1 protein inhibits protein kinase R but is not essential to antagonize production of alpha/beta interferon.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Dauber B, Schneider J, Wolff T. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">J Virol. 2006 Dec; 80(23):11667-77. 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Epub 2015 Jan 29.</em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/17944383" ref="ordinalpos=1&linkpos=5&log$=relatedreviews&logdbfrom=pubmed"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> [Study progress of NS1 protein of influenza A virus].</a><span class="source">[Wei Sheng Wu Xue Bao. 2007]</span><div class="brieflinkpop offscreen_noflow"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> [Study progress of NS1 protein of influenza A virus].<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Li JL, Zhang WP, Bi DR. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Wei Sheng Wu Xue Bao. 2007 Aug; 47(4):729-33. </em></div></div></li></ul><a class="seemore" href="/sites/entrez?db=pubmed&cmd=link&linkname=pubmed_pubmed_reviews&uid=24260784" ref="ordinalpos=1&log$=relatedreviews_seeall&logdbfrom=pubmed">See reviews...</a><a class="seemore" href="/sites/entrez?db=pubmed&cmd=link&linkname=pubmed_pubmed&uid=24260784" ref="ordinalpos=1&log$=relatedarticles_seeall&logdbfrom=pubmed">See all...</a></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Recent Activity</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="recent_activity" id="Shutter"></a></div><div class="portlet_content"><div xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" id="HTDisplay" class=""><div class="action"><a href="javascript:historyDisplayState('ClearHT')">Clear</a><a href="javascript:historyDisplayState('HTOff')" class="HTOn">Turn Off</a><a href="javascript:historyDisplayState('HTOn')" class="HTOff">Turn On</a></div><ul id="activity"><li class="ra_rcd ralinkpopper two_line"><a class="htb ralinkpopperctrl" ref="log$=activity&linkpos=1" href="/portal/utils/pageresolver.fcgi?recordid=67d66b762f30673f7bf66b41">Discovery of Small Molecule Influenza Virus NS1 Antagonist - Probe Reports from ...</a><div class="ralinkpop offscreen_noflow">Discovery of Small Molecule Influenza Virus NS1 Antagonist - Probe Reports from the NIH Molecular Libraries Program<div class="brieflinkpopdesc"></div></div><div class="tertiary"></div></li><li class="ra_rcd ralinkpopper two_line"><a class="htb ralinkpopperctrl" ref="log$=activity&linkpos=2" href="/portal/utils/pageresolver.fcgi?recordid=67d66b7567c23b31e0b17954">A 2nd Selective Inhibitor of Plasmodium falciparum Glucose-6-Phosphate Dehydroge...</a><div class="ralinkpop offscreen_noflow">A 2nd Selective Inhibitor of Plasmodium falciparum Glucose-6-Phosphate Dehydrogenase (PfG6PDH) - Probe 2 - Probe Reports from the NIH Molecular Libraries Program<div class="brieflinkpopdesc"></div></div><div class="tertiary"></div></li><li class="ra_rcd ralinkpopper two_line"><a class="htb ralinkpopperctrl" ref="log$=activity&linkpos=3" href="/portal/utils/pageresolver.fcgi?recordid=67d66b7467c23b31e0b176a9">Discovery of Novel General Anesthetics Using Apoferritin as a Surrogate System -...</a><div class="ralinkpop offscreen_noflow">Discovery of Novel General Anesthetics Using Apoferritin as a Surrogate System - Probe Reports from the NIH Molecular Libraries Program<div class="brieflinkpopdesc"></div></div><div class="tertiary"></div></li><li class="ra_rcd ralinkpopper two_line"><a class="htb ralinkpopperctrl" ref="log$=activity&linkpos=4" href="/portal/utils/pageresolver.fcgi?recordid=67d66b7367c23b31e0b174e0">Selective UBC 13 Inhibitors - Probe Reports from the NIH Molecular Libraries Pro...</a><div class="ralinkpop offscreen_noflow">Selective UBC 13 Inhibitors - Probe Reports from the NIH Molecular Libraries Program<div class="brieflinkpopdesc"></div></div><div class="tertiary"></div></li><li class="ra_rcd ralinkpopper two_line"><a class="htb ralinkpopperctrl" ref="log$=activity&linkpos=5" href="/portal/utils/pageresolver.fcgi?recordid=67d66b7284f3725e592b88c9">Development of a Selective Chemical Inhibitor for the Two-Pore Potassium Channel...</a><div class="ralinkpop offscreen_noflow">Development of a Selective Chemical Inhibitor for the Two-Pore Potassium Channel, KCNK9 - Probe Reports from the NIH Molecular Libraries Program<div class="brieflinkpopdesc"></div></div><div class="tertiary"></div></li></ul><p class="HTOn">Your browsing activity is empty.</p><p class="HTOff">Activity recording is turned off.</p><p id="turnOn" class="HTOff"><a href="javascript:historyDisplayState('HTOn')">Turn recording back on</a></p><a class="seemore" href="/sites/myncbi/recentactivity">See more...</a></div></div></div>
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