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<meta name="ncbi_db" content="books" /><meta name="ncbi_pdid" content="book-part" /><meta name="ncbi_acc" content="NBK179829" /><meta name="ncbi_domain" content="mlprobe" /><meta name="ncbi_report" content="record" /><meta name="ncbi_type" content="fulltext" /><meta name="ncbi_objectid" content="" /><meta name="ncbi_pcid" content="/NBK179829/" /><meta name="ncbi_pagename" content="ML336: Development of Quinazolinone-Based Inhibitors Against Venezuelan Equine Encephalitis Virus (VEEV) - Probe Reports from the NIH Molecular Libraries Program - NCBI Bookshelf" /><meta name="ncbi_bookparttype" content="chapter" /><meta name="ncbi_app" content="bookshelf" />
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<title>ML336: Development of Quinazolinone-Based Inhibitors Against Venezuelan Equine Encephalitis Virus (VEEV) - 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="ML336: Development of Quinazolinone-Based Inhibitors Against Venezuelan Equine Encephalitis Virus (VEEV)" /><meta name="citation_publisher" content="National Center for Biotechnology Information (US)" /><meta name="citation_date" content="2013/11/07" /><meta name="citation_author" content="Donghoon Chung" /><meta name="citation_author" content="Chad E. Schroeder" /><meta name="citation_author" content="Julie Sotsky" /><meta name="citation_author" content="Tuanli Yao" /><meta name="citation_author" content="Sudeshna Roy" /><meta name="citation_author" content="Robert A. Smith" /><meta name="citation_author" content="Nichole A. Tower" /><meta name="citation_author" content="James W. Noah" /><meta name="citation_author" content="Sara McKellip" /><meta name="citation_author" content="Melinda Sosa" /><meta name="citation_author" content="Lynn Rasmussen" /><meta name="citation_author" content="E. Lucile White" /><meta name="citation_author" content="Jeffrey Aubé" /><meta name="citation_author" content="Jennifer E. Golden" /><meta name="citation_pmid" content="24479198" /><meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK179829/" /><link rel="schema.DC" href="http://purl.org/DC/elements/1.0/" /><meta name="DC.Title" content="ML336: Development of Quinazolinone-Based Inhibitors Against Venezuelan Equine Encephalitis Virus (VEEV)" /><meta name="DC.Type" content="Text" /><meta name="DC.Publisher" content="National Center for Biotechnology Information (US)" /><meta name="DC.Contributor" content="Donghoon Chung" /><meta name="DC.Contributor" content="Chad E. Schroeder" /><meta name="DC.Contributor" content="Julie Sotsky" /><meta name="DC.Contributor" content="Tuanli Yao" /><meta name="DC.Contributor" content="Sudeshna Roy" /><meta name="DC.Contributor" content="Robert A. Smith" /><meta name="DC.Contributor" content="Nichole A. Tower" /><meta name="DC.Contributor" content="James W. Noah" /><meta name="DC.Contributor" content="Sara McKellip" /><meta name="DC.Contributor" content="Melinda Sosa" /><meta name="DC.Contributor" content="Lynn Rasmussen" /><meta name="DC.Contributor" content="E. Lucile White" /><meta name="DC.Contributor" content="Jeffrey Aubé" /><meta name="DC.Contributor" content="Jennifer E. Golden" /><meta name="DC.Date" content="2013/11/07" /><meta name="DC.Identifier" content="https://www.ncbi.nlm.nih.gov/books/NBK179829/" /><meta name="description" content="Alphaviruses like Venezuelan Equine Encephalitis Virus (VEEV) are enveloped, positive-sense, single stranded RNA viruses that are geographically widely distributed. They are arthropod-borne viruses that are known to cause rash, arthritis, encephalitis, and death in humans. Of the more than 30 alphavirus pathogens known, about a third contributes to human disease, and currently there are no FDA approved treatments available for any of them. A renewed interest to find effective therapeutic leads for development has emerged due to the lack of effective countermeasures for these pathogens, the increased incidence of their prevalence with global climate changes, and the ease with which they can and have been weaponized as biological threats. VEEV vaccines to date show insufficient efficacy or adverse side effects that limit their use, and disclosed literature compounds possess weak anti-VEEV potency and/or involve host-mediated mechanisms of action, contributing to off-target effects. The high throughput-screen of the MLSMR revealed a subset of scaffolds that inhibited a VEEV-induced cytopathic effect in the low micromolar range. Medicinal chemistry optimization resulted in the development of ML336, a first-in-class probe that inhibited a VEEV-induced cytopathic effect in three strains of the virus (TC-83, V3526, and Trinidad donkey) in the low nanomolar range without showing cytotoxicity (> 50 μM, selectivity index > 1500). Furthermore, ML336 dramatically reduced viral titer (> 7.2 log) below a 1 μM compound concentration and features a favorable in vitro pharmacokinetic profile which includes moderate blood-brain barrier permeability. Importantly, ML336 appears to target the VEEV non-structural protein 2 (nsP2) which is necessary for transcription and replication of viral RNA. This finding alone distinguishes ML336 from all other compounds described to date, and in combination with its overall profile, makes it an ideal candidate for further in vivo development." /><meta name="og:title" content="ML336: Development of Quinazolinone-Based Inhibitors Against Venezuelan Equine Encephalitis Virus (VEEV)" /><meta name="og:type" content="book" /><meta name="og:description" content="Alphaviruses like Venezuelan Equine Encephalitis Virus (VEEV) are enveloped, positive-sense, single stranded RNA viruses that are geographically widely distributed. They are arthropod-borne viruses that are known to cause rash, arthritis, encephalitis, and death in humans. Of the more than 30 alphavirus pathogens known, about a third contributes to human disease, and currently there are no FDA approved treatments available for any of them. A renewed interest to find effective therapeutic leads for development has emerged due to the lack of effective countermeasures for these pathogens, the increased incidence of their prevalence with global climate changes, and the ease with which they can and have been weaponized as biological threats. VEEV vaccines to date show insufficient efficacy or adverse side effects that limit their use, and disclosed literature compounds possess weak anti-VEEV potency and/or involve host-mediated mechanisms of action, contributing to off-target effects. The high throughput-screen of the MLSMR revealed a subset of scaffolds that inhibited a VEEV-induced cytopathic effect in the low micromolar range. Medicinal chemistry optimization resulted in the development of ML336, a first-in-class probe that inhibited a VEEV-induced cytopathic effect in three strains of the virus (TC-83, V3526, and Trinidad donkey) in the low nanomolar range without showing cytotoxicity (> 50 μM, selectivity index > 1500). Furthermore, ML336 dramatically reduced viral titer (> 7.2 log) below a 1 μM compound concentration and features a favorable in vitro pharmacokinetic profile which includes moderate blood-brain barrier permeability. Importantly, ML336 appears to target the VEEV non-structural protein 2 (nsP2) which is necessary for transcription and replication of viral RNA. This finding alone distinguishes ML336 from all other compounds described to date, and in combination with its overall profile, makes it an ideal candidate for further in vivo development." /><meta name="og:url" content="https://www.ncbi.nlm.nih.gov/books/NBK179829/" /><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/ml336/" /><link rel="canonical" href="https://www.ncbi.nlm.nih.gov/books/NBK179829/" /><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="#__NBK179829_dtls__">Show details</a><div style="display:none" class="ui-widget" id="__NBK179829_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/ml337/" title="Previous page in this title">< Prev</a><a class="active page_link next" href="/books/n/mlprobe/ml335/" 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="_NBK179829_"><span class="title" itemprop="name">ML336: Development of Quinazolinone-Based Inhibitors Against Venezuelan Equine Encephalitis Virus (VEEV)</span></h1><p class="contrib-group"><span itemprop="author">Donghoon Chung</span>, <span itemprop="author">Chad E. Schroeder</span>, <span itemprop="author">Julie Sotsky</span>, <span itemprop="author">Tuanli Yao</span>, <span itemprop="author">Sudeshna Roy</span>, <span itemprop="author">Robert A. Smith</span>, <span itemprop="author">Nichole A. Tower</span>, <span itemprop="author">James W. Noah</span>, <span itemprop="author">Sara McKellip</span>, <span itemprop="author">Melinda Sosa</span>, <span itemprop="author">Lynn Rasmussen</span>, <span itemprop="author">E. Lucile White</span>, <span itemprop="author">Jeffrey Aubé</span>, and <span itemprop="author">Jennifer E. Golden</span>.</p><a data-jig="ncbitoggler" href="#__NBK179829_ai__" style="border:0;text-decoration:none">Author Information and Affiliations</a><div style="display:none" class="ui-widget" id="__NBK179829_ai__"><p class="contrib-group"><h4>Authors</h4><span itemprop="author">Donghoon Chung</span>,<sup>1</sup><sup>,*</sup> <span itemprop="author">Chad E. Schroeder</span>,<sup>2</sup> <span itemprop="author">Julie Sotsky</span>,<sup>1</sup> <span itemprop="author">Tuanli Yao</span>,<sup>2</sup> <span itemprop="author">Sudeshna Roy</span>,<sup>2</sup> <span itemprop="author">Robert A. Smith</span>,<sup>2</sup> <span itemprop="author">Nichole A. Tower</span>,<sup>3</sup> <span itemprop="author">James W. Noah</span>,<sup>3</sup> <span itemprop="author">Sara McKellip</span>,<sup>3</sup> <span itemprop="author">Melinda Sosa</span>,<sup>3</sup> <span itemprop="author">Lynn Rasmussen</span>,<sup>3</sup> <span itemprop="author">E. Lucile White</span>,<sup>3</sup> <span itemprop="author">Jeffrey Aubé</span>,<sup>2,4</sup> and <span itemprop="author">Jennifer E. Golden</span><sup>2</sup><sup>,*</sup>.</p><h4>Affiliations</h4><div class="affiliation"><sup>1</sup>
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Department of Microbiology and Immunology, College of Medicine, University of Louisville, KY 40202</div><div class="affiliation"><sup>2</sup>
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University of Kansas Specialized Chemistry Center, Lawrence, KS 66049</div><div class="affiliation"><sup>3</sup>
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Southern Research Institute, 2000 9<sup>th</sup> Ave. S. Birmingham, AL 35205</div><div class="affiliation"><sup>4</sup>
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Department of Medicinal Chemistry, University of Kansas, Lawrence, KS 66047</div><div class="affiliation">
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<sup>*</sup> Corresponding authors:
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<span class="before-email-separator"></span><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="ude.ellivsiuol@gnuhc.nooh" class="oemail">ude.ellivsiuol@gnuhc.nooh</a> (virology);
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<span class="before-email-separator"></span><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="ude.uk@nedlognej" class="oemail">ude.uk@nedlognej</a> (chemistry)</div></div><p class="small">Received: <span itemprop="datePublished">December 17, 2012</span>; Last Update: <span itemprop="dateModified">November 7, 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>Alphaviruses like Venezuelan Equine Encephalitis Virus (VEEV) are enveloped, positive-sense, single stranded RNA viruses that are geographically widely distributed. They are arthropod-borne viruses that are known to cause rash, arthritis, encephalitis, and death in humans. Of the more than 30 alphavirus pathogens known, about a third contributes to human disease, and currently there are no FDA approved treatments available for any of them. A renewed interest to find effective therapeutic leads for development has emerged due to the lack of effective countermeasures for these pathogens, the increased incidence of their prevalence with global climate changes, and the ease with which they can and have been weaponized as biological threats. VEEV vaccines to date show insufficient efficacy or adverse side effects that limit their use, and disclosed literature compounds possess weak anti-VEEV potency and/or involve host-mediated mechanisms of action, contributing to off-target effects. The high throughput-screen of the MLSMR revealed a subset of scaffolds that inhibited a VEEV-induced cytopathic effect in the low micromolar range. Medicinal chemistry optimization resulted in the development of <a href="/pcsubstance/?term=ML336[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML336</a>, a first-in-class probe that inhibited a VEEV-induced cytopathic effect in three strains of the virus (TC-83, V3526, and Trinidad donkey) in the low nanomolar range without showing cytotoxicity (> 50 μM, selectivity index > 1500). Furthermore, <a href="/pcsubstance/?term=ML336[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML336</a> dramatically reduced viral titer (> 7.2 log) below a 1 μM compound concentration and features a favorable <i>in vitro</i> pharmacokinetic profile which includes moderate blood-brain barrier permeability. Importantly, <a href="/pcsubstance/?term=ML336[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML336</a> appears to target the VEEV non-structural protein 2 (nsP2) which is necessary for transcription and replication of viral RNA. This finding alone distinguishes <a href="/pcsubstance/?term=ML336[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML336</a> from all other compounds described to date, and in combination with its overall profile, makes it an ideal candidate for further <i>in vivo</i> development.</p></div><div class="h2"></div><p><b>Assigned Assay Grant #:</b> 1 R03 MH087448-01A1</p><p><b>Screening Center & PI:</b> Southern Research Biocontainment Specialized Screening Center, E.Lucile White</p><p><b>Chemistry Center & PI:</b> University of Kansas Specialized Chemistry Center, Dr. Jeffrey Aubé</p><p><b>Assay Submitter & Institution:</b> Dr. Donghoon Chung, University of Louisville</p><p><b>PubChem Summary Bioassay Identifier (AID):</b> Summary <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/588723" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 588723</a></p><div id="ml336.s1"><h2 id="_ml336_s1_">Probe Structure & Characteristics</h2><div id="ml336.f1" class="figure"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Image%20ml336f1&p=BOOKS&id=179829_ml336f1.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/NBK179829/bin/ml336f1.jpg" alt="Image ml336f1" class="tileshop" title="Click on image to zoom" /></a></div></div><div id="ml336.t1" class="table"><h3><span class="label">Table 1</span><span class="title">ML336 Data Summary</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK179829/table/ml336.t1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml336.t1_lrgtbl__"><table class="no_margin"><thead><tr><th id="hd_h_ml336.t1_1_1_1_1" rowspan="2" colspan="1" headers="hd_h_ml336.t1_1_1_1_1" style="text-align:center;vertical-align:middle;">CID ML#</th><th id="hd_h_ml336.t1_1_1_1_2" rowspan="2" colspan="1" headers="hd_h_ml336.t1_1_1_1_2" style="text-align:center;vertical-align:middle;">Target Name</th><th id="hd_h_ml336.t1_1_1_1_3" rowspan="2" colspan="1" headers="hd_h_ml336.t1_1_1_1_3" style="text-align:center;vertical-align:middle;">VEEV TC-83 CPE IC<sub>50</sub> (nM)<br /><br /><a href="https://pubchem.ncbi.nlm.nih.gov/substance/144087340" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 144087340</a><br /><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651734" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651734</a></th><th id="hd_h_ml336.t1_1_1_1_4" rowspan="2" colspan="1" headers="hd_h_ml336.t1_1_1_1_4" style="text-align:center;vertical-align:middle;">VEEV V3526 CPE IC<sub>50</sub> (nM)<br /><br /><a href="https://pubchem.ncbi.nlm.nih.gov/substance/144087340" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 144087340</a><br /><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651884" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651884</a></th><th id="hd_h_ml336.t1_1_1_1_5" rowspan="2" colspan="1" headers="hd_h_ml336.t1_1_1_1_5" style="text-align:center;vertical-align:middle;">VEEV Wild Type<sup><a class="bk_pop" href="#ml336.tfn2">**</a></sup> CPE IC<sub>50</sub> (nM)<br /><br /><a href="https://pubchem.ncbi.nlm.nih.gov/substance/144087340" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 144087340</a><br /><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651874" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651874</a></th><th id="hd_h_ml336.t1_1_1_1_6" colspan="5" rowspan="1" style="text-align:center;vertical-align:middle;">Cytotoxicity and Alphavirus Selectivity</th><th id="hd_h_ml336.t1_1_1_1_7" rowspan="2" colspan="1" headers="hd_h_ml336.t1_1_1_1_7" style="text-align:center;vertical-align:middle;">VEEV TC-83<sup><a class="bk_pop" href="#ml336.tfn3">⊥</a></sup> Plaque Assay at 5 μM, Virus Titer Reduction (log)<br /><a href="https://pubchem.ncbi.nlm.nih.gov/substance/144087340" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 144087340</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651886" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651886</a></th><th id="hd_h_ml336.t1_1_1_1_8" rowspan="2" colspan="1" headers="hd_h_ml336.t1_1_1_1_8" style="text-align:center;vertical-align:middle;">VEEV Wild Type<sup><a class="bk_pop" href="#ml336.tfn2">**</a></sup> Plaque Assay at 5 μM, Virus Titer Reduction (log)<br /><a href="https://pubchem.ncbi.nlm.nih.gov/substance/144087340" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 144087340</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651883" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651883</a></th></tr><tr><th headers="hd_h_ml336.t1_1_1_1_6" id="hd_h_ml336.t1_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Anti-target Name(s)</th><th headers="hd_h_ml336.t1_1_1_1_6" id="hd_h_ml336.t1_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CC<sub>50</sub> or EC<sub>50</sub> (μM)<br /><a href="https://pubchem.ncbi.nlm.nih.gov/substance/144087340" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 144087340</a></th><th headers="hd_h_ml336.t1_1_1_1_6" id="hd_h_ml336.t1_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">VEEV TC-83 Fold Selective<sup><a class="bk_pop" href="#ml336.tfn1">*</a></sup></th><th headers="hd_h_ml336.t1_1_1_1_6" id="hd_h_ml336.t1_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">VEEV V3526 Fold Selective<sup><a class="bk_pop" href="#ml336.tfn1">*</a></sup></th><th headers="hd_h_ml336.t1_1_1_1_6" id="hd_h_ml336.t1_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">VEEV Wild type Fold Selective<sup><a class="bk_pop" href="#ml336.tfn1">*</a></sup></th></tr></thead><tbody><tr><td headers="hd_h_ml336.t1_1_1_1_1" rowspan="3" colspan="1" style="text-align:center;vertical-align:middle;">CID 71301451<br /><a href="/pcsubstance/?term=ML336[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML336</a></td><td headers="hd_h_ml336.t1_1_1_1_2" rowspan="3" colspan="1" style="text-align:center;vertical-align:middle;">Venezuelan equine encephalitis virus (VEEV)</td><td headers="hd_h_ml336.t1_1_1_1_3" rowspan="3" colspan="1" style="text-align:center;vertical-align:middle;">32</td><td headers="hd_h_ml336.t1_1_1_1_4" rowspan="3" colspan="1" style="text-align:center;vertical-align:middle;">20</td><td headers="hd_h_ml336.t1_1_1_1_5" rowspan="3" colspan="1" style="text-align:center;vertical-align:middle;">41</td><td headers="hd_h_ml336.t1_1_1_1_6 hd_h_ml336.t1_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Mammalian cell toxicity, Vero76 cells</td><td headers="hd_h_ml336.t1_1_1_1_6 hd_h_ml336.t1_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CC<sub>50</sub> > 50<br /><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/65173" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 65173</a></td><td headers="hd_h_ml336.t1_1_1_1_6 hd_h_ml336.t1_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">> 1563</td><td headers="hd_h_ml336.t1_1_1_1_6 hd_h_ml336.t1_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">> 2500</td><td headers="hd_h_ml336.t1_1_1_1_6 hd_h_ml336.t1_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">> 1219</td><td headers="hd_h_ml336.t1_1_1_1_7" rowspan="3" colspan="1" style="text-align:center;vertical-align:middle;">> 7.20</td><td headers="hd_h_ml336.t1_1_1_1_8" rowspan="3" colspan="1" style="text-align:center;vertical-align:middle;">No viral replication observed at 5 μM to 0.50 μM (lower limit of assay)</td></tr><tr><td headers="hd_h_ml336.t1_1_1_1_6 hd_h_ml336.t1_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Chikungunya virus (alphavirus)</td><td headers="hd_h_ml336.t1_1_1_1_6 hd_h_ml336.t1_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">EC<sub>50</sub> > 50<br /><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651738" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651738</a></td><td headers="hd_h_ml336.t1_1_1_1_6 hd_h_ml336.t1_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">> 1563</td><td headers="hd_h_ml336.t1_1_1_1_6 hd_h_ml336.t1_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">> 2500</td><td headers="hd_h_ml336.t1_1_1_1_6 hd_h_ml336.t1_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">> 1219</td></tr><tr><td headers="hd_h_ml336.t1_1_1_1_6 hd_h_ml336.t1_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Respiratory Syncytial Virus (non-alphavirus)</td><td headers="hd_h_ml336.t1_1_1_1_6 hd_h_ml336.t1_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">EC<sub>50</sub> > 25<br /><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651932" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651932</a></td><td headers="hd_h_ml336.t1_1_1_1_6 hd_h_ml336.t1_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">> 781</td><td headers="hd_h_ml336.t1_1_1_1_6 hd_h_ml336.t1_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">> 1250</td><td headers="hd_h_ml336.t1_1_1_1_6 hd_h_ml336.t1_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">> 610</td></tr></tbody></table></div><div><div><dl class="temp-labeled-list small"><dt>*</dt><dd><div id="ml336.tfn1"><p class="no_margin">Selectivity = anti-target IC<sub>50</sub> or CC<sub>50</sub> (mammalian toxicity or CHIKV or RSV)/VEEV strain CPE IC<sub>50</sub></p></div></dd><dt>**</dt><dd><div id="ml336.tfn2"><p class="no_margin">Wild type = Trinidad Donkey strain of VEEV virus</p></div></dd><dt>⊥</dt><dd><div id="ml336.tfn3"><p class="no_margin"><a href="/pcsubstance/?term=ML336[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML336</a> evaluated at multiple concentrations. Results were >7.2 log at 5 μM and 1 μM, and 5.8 log at 0.5 μM.</p></div></dd></dl></div></div></div></div><div id="ml336.s2"><h2 id="_ml336_s2_">1. Recommendations for Scientific Use of the Probe</h2><p><b>What limitations in current state of the art is the probe addressing?</b> There are no specific inhibitors or therapeutic countermeasures for any alphavirus infection. All compounds with some reported degree of VEEV activity are derived from other viral applications. Importantly, their mechanisms of action are dependent on inhibition of cellular metabolism, which is not selective for the virus and thus, can be potentially toxic to the host. Moreover most are weakly efficacious at best, hence rendering them unsatisfactory as starting points for development as potential alphavirus therapeutics. We have shown that probe <a href="/pcsubstance/?term=ML336[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML336</a> potently inhibits a VEEV-induced cytopathic effect in three strains of the virus (TC-83, V3526, and wild type Trinidad donkey) in the low nanomolar range without showing cytotoxicity (> 50 μM). Furthermore, <a href="/pcsubstance/?term=ML336[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML336</a> dramatically reduced viral titers below a 1 μM compound concentration and features a favorable <i>in vitro</i> pharmacokinetic profile which includes moderate blood-brain barrier permeability. Importantly, <a href="/pcsubstance/?term=ML336[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML336</a> appears to target the non-structural protein 2 (nsP2) which is necessary for transcription and replication of viral RNA. This finding alone distinguishes <a href="/pcsubstance/?term=ML336[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML336</a> from all other compounds described to date, and in combination with its overall profile, makes it an ideal candidate for further <i>in vivo</i> development.</p><p><b>What will the probe be used for?</b> Probe <a href="/pcsubstance/?term=ML336[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML336</a> and its analogs will be used primarily as anti-VEEV lead compounds with which further <i>in vivo</i> lead optimization work will be done, with the ultimate goal of delivering viable VEEV antiviral drug candidates. The compounds will be assessed for efficacy against serotypes of VEEV, other alphaviruses such as WEEV and EEEV, and they will be used as a platform to develop a PET imaging agent to track how effectively the probe penetrates and concentrates in infected brain tissue. The probe will also be used for advanced mechanism of action studies aimed at supporting our preliminary findings that the probe targets viral nsP2.</p><p><b>Who in the research community will use the probe</b>? The lack of effective treatments for alphavirus infection, coupled with the historical weaponization of VEEV as a bioterrorism agent, makes the development of efficacious, safe and readily available antiviral leads for VEEV an interest to national security and protection of military personnel. The Department of Defense has sponsored repeated initiatives that specifically address the need for VEEV antivirals and agents effective against other alphaviruses (see solicitation: CBO-ALPHA-01 and <a href="http://globalbiodefense.com/2012/03/20/dod-seeks-new-drug-for-alphaviruses/" ref="pagearea=body&targetsite=external&targetcat=link&targettype=uri">http://globalbiodefense.com/2012/03/20/dod-seeks-new-drug-for-alphaviruses/</a>). Effective VEEV leads will be used as tools to elucidate mechanisms of action and provide insight into the alphavirus life cycle. <a href="/pcsubstance/?term=ML336[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML336</a> is a first in class VEEV inhibitor with a unique mechanism of action targeting viral nsP2 and it possesses an attractive physiochemical and pharmacokinetic profile. As such, probe <a href="/pcsubstance/?term=ML336[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML336</a> will be of broad interest to the antiviral community as a standard point of reference for future leads and development.</p><p><b>What is the relevant biology to which the probe can be applied</b>? There are more than 30 alphavirus pathogens known, about a third of these contribute to human disease, and currently there are no FDA approved treatments available for any of them. Alphavirus infections, spread mostly by mosquitoes, can enter the CNS where they grow within neurons, resulting in degradation and acute inflammation in brain tissues, or encephalitis, which can lead to death. Vaccines to date show insufficient efficacy or adverse side effects that limit their use, and disclosed literature compounds possess weak anti-VEEV potency and/or involve host-mediated mechanisms of action, contributing to off-target effects. Importantly, <a href="/pcsubstance/?term=ML336[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML336</a> appears to specifically target the viral non-structural protein 2 (nsP2) which is necessary for transcription and replication of viral RNA which makes it a first-in-class probe. <a href="/pcsubstance/?term=ML336[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML336</a> has a virus-specific mechanism of action, shows <i>in vitro</i> blood brain barrier (BBB) permeability, maintains cell viability by potently inhibiting VEEV infection and reduces viral titer at low concentrations of compound without inducing toxicity. <a href="/pcsubstance/?term=ML336[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML336</a> and its analogs can be fluorinated without significantly altering the activity profile and as such, the probe is a suitable scaffold for incorporation of an <sup>18</sup>F tracer for positron emission tomography (PET) for imaging of the real time bioavailability and localization of BBB penetrant anti-VEEV therapeutics.</p></div><div id="ml336.s3"><h2 id="_ml336_s3_">2. Materials and Methods</h2><div id="ml336.s4"><h3>2.1. Assays</h3><p>The details of the primary HTS and additional assays can be found in the <a href="#ml336.s21">“Assay Description” section</a> in the PubChem BioAssay view under the AIDs as listed (<a class="figpopup" href="/books/NBK179829/table/ml336.t2/?report=objectonly" target="object" rid-figpopup="figml336t2" rid-ob="figobml336t2">Table 2</a>). Additionally the details for the primary HTS are provided in the <a href="#ml336.ack">Appendix</a> at the end of this probe report.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml336t2"><a href="/books/NBK179829/table/ml336.t2/?report=objectonly" target="object" title="Table 2" class="img_link icnblk_img figpopup" rid-figpopup="figml336t2" rid-ob="figobml336t2"><img class="small-thumb" src="/books/NBK179829/table/ml336.t2/?report=thumb" src-large="/books/NBK179829/table/ml336.t2/?report=previmg" alt="Table 2. Summary of assays, listed by title and AID, used for the development of ML336." /></a><div class="icnblk_cntnt"><h4 id="ml336.t2"><a href="/books/NBK179829/table/ml336.t2/?report=objectonly" target="object" rid-ob="figobml336t2">Table 2</a></h4><p class="float-caption no_bottom_margin">Summary of assays, listed by title and AID, used for the development of ML336. </p></div></div></div><div id="ml336.s5"><h3>2.2. Probe Chemical Characterization</h3><div id="ml336.s6"><h4>A. Probe chemical name, structure and physiochemical data</h4><p>The IUPAC name of the probe <a href="/pcsubstance/?term=ML336[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML336</a> is (<i>E</i>)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-5-nitro-N-phenylbenzamide. The batch prepared and submitted to the MLSMR is archived as <a href="https://pubchem.ncbi.nlm.nih.gov/substance/144087340" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 144087340</a>, corresponding to CID 71301451.</p><div id="ml336.f2" class="figure"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Image%20ml336f2&p=BOOKS&id=179829_ml336f2.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/NBK179829/bin/ml336f2.jpg" alt="Image ml336f2" class="tileshop" title="Click on image to zoom" /></a></div></div></div><div id="ml336.s7"><h4>B. Structure Verification and Purity: <sup>1</sup>H NMR, <sup>13</sup>C NMR, LCMS, and HRMS Data</h4><p><b>Proton and carbon NMR data for</b>
|
||
<a href="/pcsubstance/?term=ML336[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML336</a><b>/</b>
|
||
<a href="https://pubchem.ncbi.nlm.nih.gov/substance/144087340" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 144087340</a><b>/ CID 71301451:</b> Detailed analytical methods and instrumentation are described in <a href="#ml336.s10">Section 2.3, entitled “Probe Preparation”</a> under general experimental and analytical details. The numerical experimental proton and carbon data are represented below for <a href="https://pubchem.ncbi.nlm.nih.gov/substance/152199106" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 152199106</a>. The corresponding NMR data for <a href="https://pubchem.ncbi.nlm.nih.gov/substance/144087340" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 144087340</a> (a separate lot of <a href="/pcsubstance/?term=ML336[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML336</a>) was obtained and determined to match what is described for <a href="https://pubchem.ncbi.nlm.nih.gov/substance/152199106" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 152199106</a>. The experimental proton and carbon spectra are included for reference (<a class="figpopup" href="/books/NBK179829/figure/ml336.f12/?report=objectonly" target="object" rid-figpopup="figml336f12" rid-ob="figobml336f12">Appendix, Figures A6A</a> and <a class="figpopup" href="/books/NBK179829/figure/ml336.f13/?report=objectonly" target="object" rid-figpopup="figml336f13" rid-ob="figobml336f13">A6B</a>, respectively).</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml336f13" co-legend-rid="figlgndml336f13"><a href="/books/NBK179829/figure/ml336.f13/?report=objectonly" target="object" title="Figure A6B" class="img_link icnblk_img figpopup" rid-figpopup="figml336f13" rid-ob="figobml336f13"><img class="small-thumb" src="/books/NBK179829/bin/ml336f13.gif" src-large="/books/NBK179829/bin/ml336f13.jpg" alt="Figure A6B. 13CNMR spectrum for ML336, SID 152199106, CID 71301451." /></a><div class="icnblk_cntnt" id="figlgndml336f13"><h4 id="ml336.f13"><a href="/books/NBK179829/figure/ml336.f13/?report=objectonly" target="object" rid-ob="figobml336f13">Figure A6B</a></h4><p class="float-caption no_bottom_margin"><sup>13</sup>CNMR spectrum for ML336, SID 152199106, CID 71301451. </p></div></div><p><b>Proton NMR Data for</b>
|
||
<a href="/pcsubstance/?term=ML336[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML336</a><b>/</b>
|
||
<a href="https://pubchem.ncbi.nlm.nih.gov/substance/144087340" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 144087340</a><b>/ CID 7130145:</b><sup>1</sup>H NMR (500 MHz, CDCl<sub>3</sub>) δ 10.99 (s, 1H), 9.15 (d, <i>J</i> = 2.8 Hz, 1H), 8.15 (dd, <i>J</i> = 8.8, 2.8 Hz, 1H), 7.65 – 7.60 (m, 2H), 7.38 – 7.33 (m, 2H), 7.12 (tt, <i>J</i> = 7.3, 1.2 Hz, 1H), 6.80 (d, <i>J</i> = 8.8 Hz, 1H), 3.47 (t, <i>J</i> = 5.7 Hz, 2H), 3.28 (s, 3H), 3.13 (s, 2H), 2.69 (t, <i>J</i> = 5.7 Hz, 2H), 2.26 (s, 3H).</p><p><b>Carbon NMR Data for</b>
|
||
<a href="/pcsubstance/?term=ML336[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML336</a><b>/</b>
|
||
<a href="https://pubchem.ncbi.nlm.nih.gov/substance/144087340" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 144087340</a><b>/ CID 71301451:</b><sup>13</sup>C NMR (126 MHz, CDCl<sub>3</sub>) δ 162.63, 156.42, 153.97, 142.79, 138.19, 129.09, 127.64, 126.35, 126.16, 124.19, 123.75, 120.19, 55.12, 51.82, 49.66, 45.27, 36.88.</p><p><b>LCMS and HRMS Data for</b>
|
||
<a href="/pcsubstance/?term=ML336[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML336</a><b>,</b>
|
||
<a href="https://pubchem.ncbi.nlm.nih.gov/substance/144087340" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 144087340</a><b>/ CID 71301451:</b> Detailed analytical methods and instrumentation are described in <a href="#ml336.s10">section 2.3, entitled “Probe Preparation”</a> under general experimental and analytical details. The numerical experimental LCMS and HRMS data are represented as follows:</p><p>For <a href="https://pubchem.ncbi.nlm.nih.gov/substance/152199106" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 152199106</a>: LCMS retention time: 3.206 min. LCMS purity at 214 nm: 99.2%. HRMS <i>m/z</i> calculated for C<sub>19</sub>H<sub>22</sub>N<sub>5</sub>O<sub>3</sub> [M<sup>+</sup>+H] 368.1723, found 368.1718. The experimental HRMS and LCMS spectra are included for reference (<a class="figpopup" href="/books/NBK179829/figure/ml336.f14/?report=objectonly" target="object" rid-figpopup="figml336f14" rid-ob="figobml336f14">Appendix, Figure A6C</a> and <a class="figpopup" href="/books/NBK179829/figure/ml336.f15/?report=objectonly" target="object" rid-figpopup="figml336f15" rid-ob="figobml336f15">A6D</a>, respectively).</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml336f14" co-legend-rid="figlgndml336f14"><a href="/books/NBK179829/figure/ml336.f14/?report=objectonly" target="object" title="Figure A6C" class="img_link icnblk_img figpopup" rid-figpopup="figml336f14" rid-ob="figobml336f14"><img class="small-thumb" src="/books/NBK179829/bin/ml336f14.gif" src-large="/books/NBK179829/bin/ml336f14.jpg" alt="Figure A6C. High resolution MS for ML336, SID 152199106, CID 71301451." /></a><div class="icnblk_cntnt" id="figlgndml336f14"><h4 id="ml336.f14"><a href="/books/NBK179829/figure/ml336.f14/?report=objectonly" target="object" rid-ob="figobml336f14">Figure A6C</a></h4><p class="float-caption no_bottom_margin">High resolution MS for ML336, SID 152199106, CID 71301451. </p></div></div><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml336f15" co-legend-rid="figlgndml336f15"><a href="/books/NBK179829/figure/ml336.f15/?report=objectonly" target="object" title="Figure A6D" class="img_link icnblk_img figpopup" rid-figpopup="figml336f15" rid-ob="figobml336f15"><img class="small-thumb" src="/books/NBK179829/bin/ml336f15.gif" src-large="/books/NBK179829/bin/ml336f15.jpg" alt="Figure A6D. LCMS purity data at 214 nM for ML336, SID 152199106, CID 71301451." /></a><div class="icnblk_cntnt" id="figlgndml336f15"><h4 id="ml336.f15"><a href="/books/NBK179829/figure/ml336.f15/?report=objectonly" target="object" rid-ob="figobml336f15">Figure A6D</a></h4><p class="float-caption no_bottom_margin">LCMS purity data at 214 nM for ML336, SID 152199106, CID 71301451. </p></div></div><p>For <a href="https://pubchem.ncbi.nlm.nih.gov/substance/144087340" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 144087340</a>: LCMS retention time: 3.213 min. LCMS purity at 214 nm: 96.5%. HRMS: <i>m/z</i> calculated for C<sub>19</sub>H<sub>22</sub>N<sub>5</sub>O<sub>3</sub> [M<sup>+</sup>+H] 368.1723, found 368.1729. The experimental HRMS and LCMS spectra are included for reference (<a class="figpopup" href="/books/NBK179829/figure/ml336.f16/?report=objectonly" target="object" rid-figpopup="figml336f16" rid-ob="figobml336f16">Appendix, Figure A6E</a> and <a class="figpopup" href="/books/NBK179829/figure/ml336.f17/?report=objectonly" target="object" rid-figpopup="figml336f17" rid-ob="figobml336f17">A6F</a>, respectively).</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml336f16" co-legend-rid="figlgndml336f16"><a href="/books/NBK179829/figure/ml336.f16/?report=objectonly" target="object" title="Figure A6E" class="img_link icnblk_img figpopup" rid-figpopup="figml336f16" rid-ob="figobml336f16"><img class="small-thumb" src="/books/NBK179829/bin/ml336f16.gif" src-large="/books/NBK179829/bin/ml336f16.jpg" alt="Figure A6E. High resolution MS for ML336, SID 144087340, CID 71301451." /></a><div class="icnblk_cntnt" id="figlgndml336f16"><h4 id="ml336.f16"><a href="/books/NBK179829/figure/ml336.f16/?report=objectonly" target="object" rid-ob="figobml336f16">Figure A6E</a></h4><p class="float-caption no_bottom_margin">High resolution MS for ML336, SID 144087340, CID 71301451. </p></div></div><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml336f17" co-legend-rid="figlgndml336f17"><a href="/books/NBK179829/figure/ml336.f17/?report=objectonly" target="object" title="Figure A6F" class="img_link icnblk_img figpopup" rid-figpopup="figml336f17" rid-ob="figobml336f17"><img class="small-thumb" src="/books/NBK179829/bin/ml336f17.gif" src-large="/books/NBK179829/bin/ml336f17.jpg" alt="Figure A6F. LCMS purity data at 214 nM for ML336, SID 144087340, CID 71301451." /></a><div class="icnblk_cntnt" id="figlgndml336f17"><h4 id="ml336.f17"><a href="/books/NBK179829/figure/ml336.f17/?report=objectonly" target="object" rid-ob="figobml336f17">Figure A6F</a></h4><p class="float-caption no_bottom_margin">LCMS purity data at 214 nM for ML336, SID 144087340, CID 71301451. </p></div></div><p><b>If available from a vendor, please provide details.</b> This probe is not yet commercially available, as it was designed and prepared as an original analog for this project. A 20 mg sample of <a href="/pcsubstance/?term=ML336[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML336</a> synthesized at the KUSCC has been deposited in the MLSMR; however, the compound is also available free of charge from the KUSCC.<a class="bk_pop" href="#ml336.r1">1</a></p></div><div id="ml336.s8"><h4>C. Solubility</h4><p>Aqueous solubility<a class="bk_pop" href="#ml336.r2">2</a> was measured in phosphate buffered saline (PBS) at room temperature (23 °C). PBS by definition is 137 mM NaCl, 2.7 mM KCl, 10 mM sodium phosphate dibasic, 2 mM potassium phosphate monobasic and a pH of 7.4. Probe <a href="/pcsubstance/?term=ML336[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML336</a> (<a href="https://pubchem.ncbi.nlm.nih.gov/substance/144087340" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 144087340</a>, CID 71301451) was found to have a solubility measurement of 40.4 μg/mL, or 110.0 μM, under these conditions. Solubility in the TC-83 VEEV CPE assay medium, consisting of high glucose DMEM (Dulbecco's Modified Eagle's Medium) with 10% FBS and 1× Pen/Strep, was also assessed. Under these conditions, <a href="/pcsubstance/?term=ML336[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML336</a> solubility was lower than that found in PBS, with a value of 13.1 μg/mL, or 35.7 μM; however, this concentration is well above (∼ 1100-fold) the <a href="/pcsubstance/?term=ML336[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML336</a> IC<sub>50</sub> observed in this CPE assay.</p></div><div id="ml336.s9"><h4>D. Stability</h4><p><b><u>Chemical Stability</u></b>: <a href="/pcsubstance/?term=ML336[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML336</a> was evaluated for susceptibility to nucleophilic addition and formation of conjugates by treatment with dithiothreitol (DTT). <a class="figpopup" href="/books/NBK179829/figure/ml336.f3/?report=objectonly" target="object" rid-figpopup="figml336f3" rid-ob="figobml336f3">Figure 1</a> represents the time course experiment with <a href="/pcsubstance/?term=ML336[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML336</a> under various conditions. <a href="/pcsubstance/?term=ML336[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML336</a> was dissolved at 10 μM in PBS at pH 7.4 (1% DMSO) and independently incubated at room temperature with no nucleophile present or 50 μM dithiothreitol (DTT). The test reactions were sampled every hour for eight hours and analyzed by LCMS. The analytical LCMS system utilized for the analysis was a Waters Acquity system with UV-detection and mass-detection (Waters LCT Premier). The analytical method conditions included a Waters Acquity HSS T3 C18 column (2.1 × 50mm, 1.8 μm) and elution with a linear gradient of 1% water to 100% CH<sub>3</sub>CN at 0.6 mL/min flow rate. Peaks on the 214 nm chromatographs were integrated using the Waters OpenLynx software. Absolute areas under the curve were compared at each time point to determine relative percent parent remaining. The masses of potential adducts and dimers of <a href="/pcsubstance/?term=ML336[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML336</a> were searched for in the final samples to determine if any detectable adduct formed or dimerization had occurred. In the case of <a href="/pcsubstance/?term=ML336[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML336</a>, no adducts were detected at any time point using LCMS detection. All samples were prepared in duplicate. Ethacrynic acid, a known Michael acceptor, was used as a positive control.<a class="bk_pop" href="#ml336.r3">3</a></p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml336f3" co-legend-rid="figlgndml336f3"><a href="/books/NBK179829/figure/ml336.f3/?report=objectonly" target="object" title="Figure 1" class="img_link icnblk_img figpopup" rid-figpopup="figml336f3" rid-ob="figobml336f3"><img class="small-thumb" src="/books/NBK179829/bin/ml336f3.gif" src-large="/books/NBK179829/bin/ml336f3.jpg" alt="Figure 1. Chemical stability of ML336 over 8 h in the presence of a 5-fold concentration of dithiothreitol." /></a><div class="icnblk_cntnt" id="figlgndml336f3"><h4 id="ml336.f3"><a href="/books/NBK179829/figure/ml336.f3/?report=objectonly" target="object" rid-ob="figobml336f3">Figure 1</a></h4><p class="float-caption no_bottom_margin">Chemical stability of ML336 over 8 h in the presence of a 5-fold concentration of dithiothreitol. </p></div></div><p><a class="figpopup" href="/books/NBK179829/table/ml336.t3/?report=objectonly" target="object" rid-figpopup="figml336t3" rid-ob="figobml336t3">Table 3</a> summarizes the percent remaining of <a href="/pcsubstance/?term=ML336[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML336</a> at the endpoints of each run in each experiment. The average amount of <a href="/pcsubstance/?term=ML336[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML336</a> remaining after 8h when no nucleophile was added was 93.9%, and when 5X DTT was added, nearly 99% of parent <a href="/pcsubstance/?term=ML336[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML336</a> remained after the same time period, indicating that <a href="/pcsubstance/?term=ML336[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML336</a> was not generally susceptible to degradation or adduct formation in the presence of a thiol-based nucleophile.<a class="bk_pop" href="#ml336.r3">3</a></p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml336t3"><a href="/books/NBK179829/table/ml336.t3/?report=objectonly" target="object" title="Table 3" class="img_link icnblk_img figpopup" rid-figpopup="figml336t3" rid-ob="figobml336t3"><img class="small-thumb" src="/books/NBK179829/table/ml336.t3/?report=thumb" src-large="/books/NBK179829/table/ml336.t3/?report=previmg" alt="Table 3. Percent of ML336 remaining at the conclusion of the experiment (8h)." /></a><div class="icnblk_cntnt"><h4 id="ml336.t3"><a href="/books/NBK179829/table/ml336.t3/?report=objectonly" target="object" rid-ob="figobml336t3">Table 3</a></h4><p class="float-caption no_bottom_margin">Percent of ML336 remaining at the conclusion of the experiment (8h). </p></div></div><p><b><u>Aqueous Stability</u></b>: <a class="figpopup" href="/books/NBK179829/figure/ml336.f4/?report=objectonly" target="object" rid-figpopup="figml336f4" rid-ob="figobml336f4">Figure 2</a> represents the time course experiment with <a href="/pcsubstance/?term=ML336[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML336</a> under various aqueous stability conditions. <a href="/pcsubstance/?term=ML336[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML336</a> was dissolved at 10 μM in PBS at pH 7.4 (1% DMSO) or dissolved at 10 μM in 50% PBS/50% acetonitrile at pH 7.4 (1% DMSO). The samples were evaluated over 14 timepoints (0,1, 2, 3, 4, 5, 6, 7, 8, 16, 24, 32, 40, and 48 h) and analyzed by LCMS. The analytical LCMS system utilized for the analysis was a Waters Acquity system with UV-detection and mass-detection (Waters LCT Premier). The analytical method conditions included a Waters Acquity HSS T3 C18 column (2.1 × 50mm, 1.8 μm) and elution with a linear gradient of 1% water to 100% CH<sub>3</sub>CN at 0.6 mL/min flow rate. Peaks on the 214 nm chromatographs were integrated using the Waters OpenLynx software. Absolute areas under the curve were compared at each time point to determine relative percent parent remaining.<a class="bk_pop" href="#ml336.r3">3</a></p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml336f4" co-legend-rid="figlgndml336f4"><a href="/books/NBK179829/figure/ml336.f4/?report=objectonly" target="object" title="Figure 2" class="img_link icnblk_img figpopup" rid-figpopup="figml336f4" rid-ob="figobml336f4"><img class="small-thumb" src="/books/NBK179829/bin/ml336f4.gif" src-large="/books/NBK179829/bin/ml336f4.jpg" alt="Figure 2. Aqueous stability of ML336 over 48 h in PBS or PBS/acetonitrile." /></a><div class="icnblk_cntnt" id="figlgndml336f4"><h4 id="ml336.f4"><a href="/books/NBK179829/figure/ml336.f4/?report=objectonly" target="object" rid-ob="figobml336f4">Figure 2</a></h4><p class="float-caption no_bottom_margin">Aqueous stability of ML336 over 48 h in PBS or PBS/acetonitrile. </p></div></div><p>In PBS, <a href="/pcsubstance/?term=ML336[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML336</a> showed and average 82.93% remaining after 48h; however, no other mass peaks were observed. In order to account for precipitation/solubility effects, <a href="/pcsubstance/?term=ML336[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML336</a> was also evaluated in 50/50 PBS:acetonitrile and was shown to have an average of 95.89% remaining after 48 h (<a class="figpopup" href="/books/NBK179829/table/ml336.t4/?report=objectonly" target="object" rid-figpopup="figml336t4" rid-ob="figobml336t4">Table 4</a>).<a class="bk_pop" href="#ml336.r3">3</a></p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml336t4"><a href="/books/NBK179829/table/ml336.t4/?report=objectonly" target="object" title="Table 4" class="img_link icnblk_img figpopup" rid-figpopup="figml336t4" rid-ob="figobml336t4"><img class="small-thumb" src="/books/NBK179829/table/ml336.t4/?report=thumb" src-large="/books/NBK179829/table/ml336.t4/?report=previmg" alt="Table 4. Percent of ML336 remaining at the conclusion of the experiment (48h)." /></a><div class="icnblk_cntnt"><h4 id="ml336.t4"><a href="/books/NBK179829/table/ml336.t4/?report=objectonly" target="object" rid-ob="figobml336t4">Table 4</a></h4><p class="float-caption no_bottom_margin">Percent of ML336 remaining at the conclusion of the experiment (48h). </p></div></div></div></div><div id="ml336.s10"><h3>2.3. Probe Preparation</h3><p><b>General experimental and analytical details:</b><sup>1</sup>H and <sup>13</sup>C NMR spectra were recorded on a Bruker AM 400 spectrometer (operating at 400 and 101 MHz respectively) or a Bruker AVIII spectrometer (operating at 500 and 126 MHz respectively) in CDCl<sub>3</sub> with 0.03% TMS as an internal standard or DMSO-<i>d<sub>6</sub></i>. The chemical shifts (δ) reported are given in parts per million (ppm) and the coupling constants (<i>J</i>) are in Hertz (Hz). The spin multiplicities are reported as s = singlet, bs = broad singlet, d = doublet, t = triplet, q = quartet, dd = doublet of doublet and m = multiplet. The LCMS analysis was performed on an Agilent 1200 RRL chromatograph with photodiode array UV detection and an Agilent 6224 TOF mass spectrometer. The chromatographic method utilized the following parameters: a Waters Acquity BEH C-18 2.1 × 50mm, 1.7 um column; UV detection wavelength = 214 nm; flow rate = 0.4ml/min; gradient = 5 - 100% acetonitrile over 3 minutes with a hold of 0.8 minutes at 100% acetonitrile; the aqueous mobile phase contained 0.15% ammonium hydroxide (v/v). The mass spectrometer utilized the following parameters: an Agilent multimode source which simultaneously acquires ESI+/APCI+; a reference mass solution consisting of purine and hexakis(1H, 1H, 3H-tetrafluoropropoxy) phosphazine; and a make-up solvent of 90:10:0.1 MeOH:Water:Formic Acid which was introduced to the LC flow prior to the source to assist ionization. Melting points were determined on a Stanford Research Systems OptiMelt apparatus.</p><p>Probe <a href="/pcsubstance/?term=ML336[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML336</a> was prepared by the route depicted in <a class="figpopup" href="/books/NBK179829/figure/ml336.f5/?report=objectonly" target="object" rid-figpopup="figml336f5" rid-ob="figobml336f5">Figure 3</a>. Anthranilic acid <b>1</b> was treated with 2-chloroacetyl chloride in the presence of triethylamine to afford 2-(chloromethyl)benzoxazinone <b>2</b>. Dehydrative amidation was carried out by treating <b>2</b> with aniline and POCl<sub>3</sub>.<a class="bk_pop" href="#ml336.r4">4</a> The resulting 2-(chloromethyl)phenylquinazolinone <b>3</b> was aminated with mono-BOC-protected <i>tert</i>-butyl methyl(2-(methylamino)ethyl)carbamate to provide <b>4</b> which was subsequently deprotected with TFA to afford <a href="/pcsubstance/?term=ML336[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML336</a> in 11% overall yield over four steps.<a class="bk_pop" href="#ml336.r5">5</a> This process has been used to prepare <a href="/pcsubstance/?term=ML336[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML336</a> on a 260 mg scale.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml336f5" co-legend-rid="figlgndml336f5"><a href="/books/NBK179829/figure/ml336.f5/?report=objectonly" target="object" title="Figure 3" class="img_link icnblk_img figpopup" rid-figpopup="figml336f5" rid-ob="figobml336f5"><img class="small-thumb" src="/books/NBK179829/bin/ml336f5.gif" src-large="/books/NBK179829/bin/ml336f5.jpg" alt="Figure 3. Synthetic scheme for preparation of ML336." /></a><div class="icnblk_cntnt" id="figlgndml336f5"><h4 id="ml336.f5"><a href="/books/NBK179829/figure/ml336.f5/?report=objectonly" target="object" rid-ob="figobml336f5">Figure 3</a></h4><p class="float-caption no_bottom_margin">Synthetic scheme for preparation of ML336. </p></div></div><p>Detailed protocols used for the assembly of <a href="/pcsubstance/?term=ML336[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML336</a> are as follows:</p><div id="ml336.f6" class="figure"><div class="graphic"><img src="/books/NBK179829/bin/ml336f6.jpg" alt="Image ml336f6" /></div></div><p><b>2-(chloromethyl)-6-nitro-4H-benzo[d][1,3]oxazin-4-one.</b> Following a previously reported procedure,<a class="bk_pop" href="#ml336.r4">4</a> 2-amino-5-nitro-benzoic acid (6.718 g, 36.9 mmol, 1 eq) was placed under nitrogen and dissolved in CH<sub>2</sub>Cl<sub>2</sub> (100 mL). After addition of triethylamine (5.9 mL, 42.3 mmol, 1.15 eq), the mixture was lowered to 0 °C in an ice bath and a solution of chloroacetyl chloride (3.2 mL, 40.2 mmol, 1.1 eq) in CH<sub>2</sub>Cl<sub>2</sub> (50 mL) was slowly added. The reaction mixture was stirred at 0 °C for 1 hour, then at rt for 1 additional hour. The solvent was removed <i>in vacuo</i> and water was added to the remaining solid. The solid was filtered and rinsed with water (3 × 20 mL), followed by 5% Et<sub>2</sub>O/Hexanes (3 × 30 mL) to give 2-(chloromethyl)-6-nitro-4H-benzo[d][1,3]oxazin-4-one (8.87 g, 100%) along with residual triethylamine as a pale, yellow solid. The product was used in the following step without further purification. <sup>1</sup>H NMR (400 MHz, acetone-<i>d</i><sub>6</sub>) δ 8.96 – 8.91 (m, 2H), 8.48 (dd, <i>J</i> = 9.3, 2.8 Hz, 1H), 4.44 (s, 2H).</p><div id="ml336.f7" class="figure"><div class="graphic"><img src="/books/NBK179829/bin/ml336f7.jpg" alt="Image ml336f7" /></div></div><p><b>2-(chloromethyl)-6-nitro-3-phenylquinazolin-4(3H)-one.</b> To a microwave vial was added 2-(chloromethyl)-6-nitro-4H-benzo[d][1,3]oxazin-4-one (1.47 g, 6.12 mmol, 1 eq) under Ar, and the solid was dissolved in acetonitrile (13 mL). Phosphorus oxychloride (1.15 mL, 12.34 mmol, 2 eq) was added, followed by the addition of solution of aniline (0.73 mL, 8.00 mmol, 1.3 eq) in acetonitrile (4 mL). The mixture was heated in a MW reactor at 150 °C for 15 min. The reaction mixture was transferred to a larger flask and slowly quenched with saturated aq. NaHCO<sub>3</sub> (20 mL). The precipitate was filtered and rinsed with water (3 × 20 mL) to give 2-(chloromethyl)-6-nitro-3-phenylquinazolin-4(3H)-one (1.24 g, 64%) as a burnt-orange solid. <sup>1</sup>H NMR (400 MHz, CDCl<sub>3</sub>) δ 9.13 (d, <i>J</i> = 2.6 Hz, 1H), 8.59 (dd, <i>J</i> = 8.9, 2.6 Hz, 1H), 7.91 (d, <i>J</i> = 9.0 Hz, 1H), 7.65 – 7.58 (m, 3H), 7.39 – 7.35 (m, 2H), 4.28 (s, 2H).</p><div id="ml336.f8" class="figure"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Image%20ml336f8&p=BOOKS&id=179829_ml336f8.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/NBK179829/bin/ml336f8.jpg" alt="Image ml336f8" class="tileshop" title="Click on image to zoom" /></a></div></div><p><b><i>tert</i>-butyl methyl(2-(methyl((6-nitro-4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)methyl)amino)ethyl)carbamate.</b> Following a previously reported procedure,<a class="bk_pop" href="#ml336.r5">5</a> 2-(chloromethyl)-6-nitro-3-phenylquinazolin-4(3H)-one (1.40 g, 4.43 mmol, 1 eq) was dissolved in acetonitrile (18 mL). Potassium carbonate (1.226 g, 8.87 mmol, 2 eq), tert-butyl methyl(2-(methylamino)ethyl)carbamate (ChemBridge, 1.18 g, 6.25 mmol, 1.4 eq), and potassium iodide (0.280 g, 1.687 mmol, 0.4 eq) were added sequentially, and the mixture was heated in a MW reactor at 80 °C for 5 min. The crude reaction mixture was adsorbed onto Celite®, and the product was purified (2×) by flash chromatography (CombiFlash, 40 g silica, 0-10% MeOH/CH<sub>2</sub>Cl<sub>2</sub>, followed by CombiFlash, 80 g silica, 0-80% EtOAc/Hexanes) to give <i>tert</i>-butyl methyl(2-(methyl((6-nitro-4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)methyl)amino)ethyl)carbamate (0.72 g, 35%) as a pale orange solid. <sup>1</sup>H NMR (400 MHz, CDCl<sub>3</sub>) δ 9.13 (d, <i>J</i> = 2.6 Hz, 1H), 8.56 (dd, <i>J</i> = 9.0, 2.7 Hz, 1H), 7.89 (d, <i>J</i> = 9.0 Hz, 1H), 7.60 - 7.52 (m, 3H), 7.32 – 7.27 (m, 2H), 3.36 (s, 2H), 3.22 - 3.06 (m, 2H), 2.75 (br s, 3H), 2.48 (br s, 2H), 2.20 (s, 3H), 1.39 (br s, 9H).</p><div id="ml336.f9" class="figure"><div class="graphic"><img src="/books/NBK179829/bin/ml336f9.jpg" alt="Image ml336f9" /></div></div><p><b>PROBE</b>
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<a href="/pcsubstance/?term=ML336[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML336</a><b>: (<i>E</i>)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-5-nitro-N-phenylbenzamide.</b> To a stirred solution of <i>tert</i>-butyl methyl(2-(methyl((6-nitro-4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)methyl)amino)ethyl)carbamate (678 mg, 1.45 mmol) in CH<sub>2</sub>Cl<sub>2</sub> (20 mL), TFA (9.5 mL, 124 mmol) was slowly added, and the resulting mixture was stirred at rt for 45 min. Water (40 mL) and CH<sub>2</sub>Cl<sub>2</sub> (40 mL) were then added, and the mixture was adjusted to pH 10 using saturated aq. Na<sub>2</sub>CO<sub>3</sub> (40 mL). The organic phase was separated and the aqueous layer was extracted with CH<sub>2</sub>Cl<sub>2</sub> (2 × 40 mL). The combined organic phase was concentrated and purified by flash chromatography (CombiFlash, 40 g silica, 0-5% MeOH/ CH<sub>2</sub>Cl<sub>2</sub>) to give (<i>E</i>)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-5-nitro-N-phenylbenzamide (265 mg, 49%) as a pale yellow solid. <sup>1</sup>H NMR (500 MHz, CDCl<sub>3</sub>) δ 10.99 (s, 1H), 9.15 (d, <i>J</i> = 2.8 Hz, 1H), 8.15 (dd, <i>J</i> = 8.8, 2.8 Hz, 1H), 7.65 – 7.60 (m, 2H), 7.38 – 7.33 (m, 2H), 7.12 (tt, <i>J</i> = 7.3, 1.2 Hz, 1H), 6.80 (d, <i>J</i> = 8.8 Hz, 1H), 3.47 (t, <i>J</i> = 5.7 Hz, 2H), 3.28 (s, 3H), 3.13 (s, 2H), 2.69 (t, <i>J</i> = 5.7 Hz, 2H), 2.26 (s, 3H). <sup>13</sup>C NMR (126 MHz, CDCl<sub>3</sub>) δ 162.63, 156.42, 153.97, 142.79, 138.19, 129.09, 127.64, 126.35, 126.16, 124.19, 123.75, 120.19, 55.12, 51.82, 49.66, 45.27, 36.88. LCMS retention time: 3.206 min, purity at 214 nm = 99.2%. HRMS <i>m/z</i> calculated for C<sub>19</sub>H<sub>22</sub>N<sub>5</sub>O<sub>3</sub> [M<sup>+</sup>+H] 368.1723, found 368.1718. Pale yellow needles, mp 168-173 °C (recrystallized from CH<sub>2</sub>Cl<sub>2</sub>).</p></div></div><div id="ml336.s11"><h2 id="_ml336_s11_">3. Results</h2><div id="ml336.s12"><h3>3.1. Dose Response Curves for Probe</h3><div id="ml336.f10" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%203.%20Each%20data%20point%20represents%20the%20mean%20of%20%25%20cell%20viability%20from%20triplicate%20runs.&p=BOOKS&id=179829_ml336f10.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/NBK179829/bin/ml336f10.jpg" alt="Figure 3. Each data point represents the mean of % cell viability from triplicate runs." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 3</span><span class="title">Each data point represents the mean of % cell viability from triplicate runs</span></h3><div class="caption"><p>Dose response curve and IC<sub>50</sub> were generated using the Four Parameter Logistic Model or Sigmoidal Dose-Response model. A. Fourteen point CPE assay dose response curve for ML336 using TC-83 VEEV strain. B. CPE assay dose response curves for ML336 using wild type Trinidad donkey VEEV strain (triangles), V3526 VEEV strain (circles), and Chikungunya virus (squares)</p></div></div></div><div id="ml336.s13"><h3>3.2. Cellular Activity</h3><p>All of the assays utilized in this project were cell-based (see <a class="figpopup" href="/books/NBK179829/table/ml336.t2/?report=objectonly" target="object" rid-figpopup="figml336t2" rid-ob="figobml336t2">Table 2</a>, <a href="#ml336.s4">section 2.1</a>). The probe and many analogs show potent inhibition of a VEEV-induced cytopathic effect and dramatic attenuation of viral turnover at sub- to low micromolar concentrations in cellular titer reduction assays. Cytotoxic effects were not observed for the series. As the mechanism of action appears to target the viral nsP2, <a href="/pcsubstance/?term=ML336[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML336</a> and its analogs must penetrate host cells to engage cytoplasmic targets involved in viral replication.</p></div><div id="ml336.s14"><h3>3.3. Profiling Assays</h3><p><b>Profiling against host targets.</b> Probe <a href="/pcsubstance/?term=ML336[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML336</a> was submitted to Eurofins PanLabs (formerly Ricerca Biosciences) to evaluate it in radioligand binding assays against a panel of 67 GPCRs, ion channels and transporters at a single concentration of 10 μM, each in duplicate. <a href="/pcsubstance/?term=ML336[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML336</a> inhibited the human norepinephrine transporter with 91% inhibition at 10 μM, and an IC<sub>50</sub> determination has been ordered and will be reported in due course. With the exception of this one target in the panel, inhibition was not observed above 36%. Norepinephrine inhibition (or reuptake inhibitors) is associated with treatment of depression, attention deficit hyperactivity disorder (ADHD), as well as other brain-related disorders. Given that <a href="/pcsubstance/?term=ML336[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML336</a> is blood-brain barrier penetrant and has potential in the development as a PET imaging agent, the activity against the norepinephrine transporter (and notable lack of inhibition on dopamine and seratonin transporters) may indicate other therapeutic significance. For the full report, see <a href="#ml336.ack">Appendix</a>.</p><p><b>NCI-60 panel.</b> Probe <a href="/pcsubstance/?term=ML336[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML336</a> was submitted to the NCI-60 panel for anti-cancer profiling. The NCI looks for one of two scenarios when evaluating a test compound at a single dose: (A) at least 8 cell lines with a growth percent of 40 or less (at least 60% inhibition), or (b) very specific and maybe strong inhibition of only a few cell lines. At a single dose of <a href="/pcsubstance/?term=ML336[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML336</a> at 10 μM concentration, neither of these criteria was met to warrant confirmation in the standard, follow-up dose response assay. For the full report, see <a href="#ml336.ack">Appendix</a>.</p></div></div><div id="ml336.s15"><h2 id="_ml336_s15_">4. Discussion</h2><div id="ml336.s16"><h3>4.1. Comparison to existing art and how the new probe is an improvement</h3><p><b>Prior Art Overview:</b> A number of compounds were identified from patent and literature sources with reported VEEV inhibitory activity (<a class="figpopup" href="/books/NBK179829/figure/ml336.f11/?report=objectonly" target="object" rid-figpopup="figml336f11" rid-ob="figobml336f11">Figure 4</a>, <a class="figpopup" href="/books/NBK179829/table/ml336.t5/?report=objectonly" target="object" rid-figpopup="figml336t5" rid-ob="figobml336t5">Table 5</a>); however, closer inspection of the data and/or testing in the CPE assay and considering their dependency on host mediated mechanisms of action revealed these compounds were severely limited and not particularly relevant given the remarkable results seen with <a href="/pcsubstance/?term=ML336[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML336</a>.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml336f11" co-legend-rid="figlgndml336f11"><a href="/books/NBK179829/figure/ml336.f11/?report=objectonly" target="object" title="Figure 4" class="img_link icnblk_img figpopup" rid-figpopup="figml336f11" rid-ob="figobml336f11"><img class="small-thumb" src="/books/NBK179829/bin/ml336f11.gif" src-large="/books/NBK179829/bin/ml336f11.jpg" alt="Figure 4. Structures for which VEEV activity is reported." /></a><div class="icnblk_cntnt" id="figlgndml336f11"><h4 id="ml336.f11"><a href="/books/NBK179829/figure/ml336.f11/?report=objectonly" target="object" rid-ob="figobml336f11">Figure 4</a></h4><p class="float-caption no_bottom_margin">Structures for which VEEV activity is reported. </p></div></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml336t5"><a href="/books/NBK179829/table/ml336.t5/?report=objectonly" target="object" title="Table 5" class="img_link icnblk_img figpopup" rid-figpopup="figml336t5" rid-ob="figobml336t5"><img class="small-thumb" src="/books/NBK179829/table/ml336.t5/?report=thumb" src-large="/books/NBK179829/table/ml336.t5/?report=previmg" alt="Table 5. Data and liabilities of compounds with reported VEEV activity." /></a><div class="icnblk_cntnt"><h4 id="ml336.t5"><a href="/books/NBK179829/table/ml336.t5/?report=objectonly" target="object" rid-ob="figobml336t5">Table 5</a></h4><p class="float-caption no_bottom_margin">Data and liabilities of compounds with reported VEEV activity. </p></div></div><p>The reported compounds are either weakly inhibitory against VEEV and/or do not act selectively on viral components. The interaction of these compounds via host mechanisms can lead to toxicity and was an undesirable characteristic for a new probe. Ribavirin (<b>5</b>) and carbodine (<b>6</b>) are nucleoside anti-metabolite prodrugs with notable clinical toxicities and are not specific in their mechanism of action to viral targets. For example, ribavirin has a broad <i>in vitro</i> inhibitory activity against RNA viruses. The activities include 1) depleting the cellular GTP pool (IMPDH inhibitor), 2) increasing mutations in the viral genome, and 3) inhibiting the GTP capping enzyme. Direct, antiviral activity for ribavirin<a class="bk_pop" href="#ml336.r6">6</a> against Sindbis virus (another alphavirus) has been disclosed, but inhibitory activity for VEEV has not yet been reported. Our internal assessment of ribavirin against VEEV in a CPE assay showed an IC<sub>50</sub> of 126 μM. Carbodine (<b>6</b>) has been shown to have <i>in vitro</i> activity, but has associated toxicity and is only moderately efficacious <i>in vivo</i>.<a class="bk_pop" href="#ml336.r7">7</a> Urea VX-497 (<b>7</b>) is a potent, reversible uncompetitive IMP dehydrogenase (IMPDH) inhibitor with modest VEEV activity.<a class="bk_pop" href="#ml336.r8">8</a> IMPDH catalyzes an essential step in the de novo biosynthesis of guanine nucleotides, and as such, VX-497 is known to target cellular processes.</p><p>The quinazolinone structure (<b>8</b>) had a reported VEEV CPE IC<sub>50</sub> of 16.7 μM, and importantly, it is the most closely related structurally to selected chemotypes for our investigation.<a class="bk_pop" href="#ml336.r9">9</a> The KU SCC synthesized the compound for assessment in our internal CPE assay; however, we found the IC<sub>50</sub> for this compound to be > 25 μM. Completed SAR studies for our quinazolinone series leading to <a href="/pcsubstance/?term=ML336[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML336</a>
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<i>do not</i> support that quinazolinone (<b>8</b>) should be active for VEEV.</p><p>Thienylpyrrole (<b>9</b>) is reported to inhibit WEEV viral replication with an IC<sub>50</sub> = 9.3 μM.<a class="bk_pop" href="#ml336.r10">10</a> VEEV inhibition is referred to (< 100 μM) for the compound class but is not explicitly reported for any one compound. To dispel ambiguity, the KU SCC synthesized this lead compound and assessed it in our internal CPE assay, corroborating the finding with a VEEV IC<sub>50</sub> > 25 μM.</p><p>Antioxidant 80 (<b>10</b>) is a broad spectrum antiviral agent<a class="bk_pop" href="#ml336.r11">11</a> due to its antioxidant activity, and it has notable CYP450 (2C9, 2C19) inhibition to the tune of about 3 μM.<a class="bk_pop" href="#ml336.r12">12</a> This compound was purchased and purified prior to submitting to the CPE assay, which resulted in a VEEV IC<sub>50</sub> of > 50 μM. Didemnin (<b>11</b>) was reported in Pubchem<a class="bk_pop" href="#ml336.r13">13</a> as an active compound for a VEEV screen; however, it is a large cyclic peptide and does not represent prior art against which <a href="/pcsubstance/?term=ML336[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML336</a>, a small molecule, should be compared. Lastly, BIOder (<b>12</b>) is a recently disclosed compound<a class="bk_pop" href="#ml336.r14">14</a> (April 2012) that was reported during the course of our own project investigation and should not be considered prior art according to NIH; however, it is worth noting that BIOder inhibited VEEV via the host protein GSK-3β and was only partially efficacious in protecting mice from succumbing to VEEV infection. Moreover, BIOder has significant structural liabilities such as the presence of a diazodiimine and the electrophilic character of the dimeric core that are a cause for concern.</p><p>In summary, many of the compounds with disclosed anti-VEEV activity are weakly potent against VEEV, and when four selected compounds were assessed in an internal TC-83 CPE assay, we found them to have CPE IC<sub>50</sub> values > 25 μM. Of the small molecule prior art compounds disclosed, quinazolinone compound <b>8</b> (CID 13182904) and thienylpyrrole <b>9</b> (CID 3240671) do not have described mechanisms of action and they have TC-83 CPE IC<sub>50</sub> values > 25 μM. The depsipeptide didemnin <b>11</b> (CID 44287859) also has an unknown mechanism of action, but is not a small molecule against which <a href="/pcsubstance/?term=ML336[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML336</a> can be compared. Moreover, didemnin is exceedingly cytotoxic which led to it being dropped from clinical evaluation.<a class="bk_pop" href="#ml336.r15">15</a> The remaining compounds (carbodine <b>6</b>, VX-497 <b>7</b>, and recently disclosed BIOder <b>12</b>) have mechanisms of action that involve host cellular targets. <a href="/pcsubstance/?term=ML336[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML336</a> is a first-in-class probe that represents a significant improvement over the prior art landscape in terms of CPE nanomolar potency (∼ 16-fold more potent than BIOder), lack of notable cytotoxicity (> 50 μM in Vero 76 cells), ability to reduce VEEV viral titer by more than 7.2 log at 1 μM, and according to mechanistic data, <a href="/pcsubstance/?term=ML336[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML336</a> appears to inhibit VEEV replication by means of viral nsP2 interference and not host-mediated targets.</p></div></div><div id="ml336.s17"><h2 id="_ml336_s17_">5. References</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="ml336.r1">Contact the KU SCC for more information: <a href="http://www.scc.ku.edu/contact.shtml" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">http://www<wbr style="display:inline-block"></wbr>.scc.ku.edu/contact.shtml</a></div></dd><dt>2.</dt><dd><div class="bk_ref" id="ml336.r2">Data provided by Layton Smith's lab, Arianna Mangravita-Novo, Sanford Burnham Institute</div></dd><dt>3.</dt><dd><div class="bk_ref" id="ml336.r3">Data provided by University of Kansas Analytical Chemistry Core, Specialized Chhemistry Center, Patrick Porubsky</div></dd><dt>4.</dt><dd><div class="bk_ref" id="ml336.r4">Compounds and compositions that cause non-apoptotic cell death and uses thereof. B. Stockwell US20080299076 A1.</div></dd><dt>5.</dt><dd><div class="bk_ref" id="ml336.r5">Acharyulu PVR, Dubey PK, Prasada Reddy PVV, Suresh T. Synthesis of Novel New 2-(2-(4-((3,4-Dihydro-4-oxo-3-aryl quinazolin-2-yl)methyl)piperazin-1-yl)acetoyloxy)-2-phenyl Acetic Acid Esters. <span><span class="ref-journal">Synthetic Communications. </span>2009;<span class="ref-vol">3</span>:3217–3231.</span></div></dd><dt>6.</dt><dd><div class="bk_ref" id="ml336.r6">Malinoski F, Stollar V. Inhibition of sindbis virus replication by ribavirin: Influence of cultural conditions and of the host cell phenotype. <span><span class="ref-journal">Antiviral Research. </span>1981;<span class="ref-vol">1</span>:287–299.</span></div></dd><dt>7.</dt><dd><div class="bk_ref" id="ml336.r7">Julander JG, Bowen RA, Rao JR, Day C, Shafer K, Smee DF, Morrey JD, Chu C. Treatment of Venezuelan equine encephalitis virus infection with (-)-carbodine. <span><span class="ref-journal">Antiviral Research. </span>2008;<span class="ref-vol">80</span>:309–315.</span> [<a href="/pmc/articles/PMC2612642/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2612642</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/18675850" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18675850</span></a>]</div></dd><dt>8.</dt><dd><div class="bk_ref" id="ml336.r8">Markland W, McQuaid TJ, Jain J, Kwong AD. Broad-spectrum antiviral activity of the IMP dehydrogenase inhibitor VX-497: a comparison with ribavirin and demonstration of antiviral additivity with alpha interferon. <span><span class="ref-journal">Antimicrobial Agents and Chemotherapy. </span>2000;<span class="ref-vol">44</span>:859–866.</span> [<a href="/pmc/articles/PMC89783/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC89783</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/10722482" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 10722482</span></a>]</div></dd><dt>9.</dt><dd><div class="bk_ref" id="ml336.r9">Selvam P, et al. Novel 3-sulphonamido-quinazolin-4(3H)-one derivatives: microwave assisted synthesis and evaluation of antiviral activitiesagainst respiratory and biodefense viruses. <span><span class="ref-journal">Antivial Research and Chemotherapy. </span>2007;<span class="ref-vol">18</span>:301–305.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/18046963" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18046963</span></a>]</div></dd><dt>10.</dt><dd><div class="bk_ref" id="ml336.r10">Peng W, Peltier DC, Lasen MJ, Kirchcoff PD, Larsen SD, Neubig RR, Miller DJ. Identification of Thieno[3,2-<em>b</em>]Pyrrole Derivatives as Novel Small Molecule Inhibitors of Neurotropic Alphaviruses. <span><span class="ref-journal">J Infectious Diseases. </span>2009;<span class="ref-vol">199</span>:950–957.</span> [<a href="/pmc/articles/PMC2788236/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2788236</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/19239364" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19239364</span></a>]</div></dd><dt>11.</dt><dd><div class="bk_ref" id="ml336.r11">Panchal RG, Reid SP, Tran JP, Bergeron AA, Wells J, Kota KP, Aman J, Bavari S. Identification of an antioxidant small-molecule with broad-spectrum antiviral activity. <span><span class="ref-journal">Antiviral Research. </span>2012;<span class="ref-vol">93</span>:23–29.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/22027648" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 22027648</span></a>]</div></dd><dt>12.</dt><dd><div class="bk_ref" id="ml336.r12">PubChem Bioassay data <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/883" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubchem">AID883</a> and <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/899" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubchem">AID899</a></div></dd><dt>13.</dt><dd><div class="bk_ref" id="ml336.r13">PubChem Bioassay data <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/396870" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubchem">AID396870</a></div></dd><dt>14.</dt><dd><div class="bk_ref" id="ml336.r14">Kehn-Hall K, et al. Modulation of GSK-3beta activity in Venezuelan equine encephalitis virus infection. <span><span class="ref-journal">PLoS One. </span>2012;<span class="ref-vol">7</span>:e34761.</span> [<a href="/pmc/articles/PMC3319612/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3319612</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/22496857" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 22496857</span></a>]</div></dd><dt>15.</dt><dd><div class="bk_ref" id="ml336.r15">Nuijen B, Bouma M, Manada C, Jimeno JM, Schellens JHM, Bult A, Beijnen JH. <span><span class="ref-journal">Anti-Cancer Drugs. </span>2000;<span class="ref-vol">11</span>:793.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/11142687" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 11142687</span></a>]</div></dd></dl></div><div id="ml336.ack"><h2 id="_ml336_ack_">Appendix</h2><div id="ml336.s19"><h3>Quality control <sup>1</sup>H-NMR and <sup>13</sup>C-NMR spectra for ML336</h3><div id="ml336.f12" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%20A6A.%201H%20NMR%20spectrum%20for%20ML336%2C%20SID%20152199106%2C%20CID%2071301451.&p=BOOKS&id=179829_ml336f12.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/NBK179829/bin/ml336f12.jpg" alt="Figure A6A. 1H NMR spectrum for ML336, SID 152199106, CID 71301451." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure A6A</span><span class="title"><sup>1</sup>H NMR spectrum for ML336, SID 152199106, CID 71301451</span></h3></div></div><div id="ml336.s20"><h3>Quality control LCMS Purity and Mass ID data: HRMS and HPLC for ML336</h3><div id="ml336.s21"><h4>Assay Description</h4><div id="ml336.s22"><h5>HTS Primary and Confirmatory Assay Using TC-83</h5><div id="ml336.s30"><h5>Protocol</h5><p>Cell Culture: Vero 76 cells obtained from ATCC (CRL-1587) were cultured and maintained in MEM-E (Invitrogen, 10370-088) with 10% Hi-FBS (Invitrogen 16000), 1% Penicillin/Streptomycin/L-glutamine (Invitrogen 10378-024) and 1% HEPES (Invitrogen 15630-080). The cells are maintained at 37 °C, 5.0% CO<sub>2</sub> to 100% confluence being passaged 1:4 every 3-4 days. For cell plating, cells were detached from flask bottom by using Trypsin-EDTA solution and then re-suspended in a growth media. Cells were passaged no more than ten times after being thawed.</p><p>VEEV culture: VEEV TC-83 was used for screening. The VEEV stock was prepared in Vero76 cells using an initial stock obtained from Dr. Chung.</p><p>Compound Dosing/Plating: The positive control was MPA at 10 μM final well concentration. The compounds were diluted in complete growth medium to 6× concentrated dosing solution which was dispensed into 384-well black clear-bottom tissue culture treated plates (5 μL volume).</p><p>Single Dose Compound Preparation: The MLSMR library was plated at 25 μM single dose concentration.</p><p>Dose Response Compound Preparation: The compounds were tested in a dose response format using a 1:2 serial dilution with the highest concentrations starting at 25 μM and extending to .05 μM over a 10-plate 1:2 serial dilution pattern. DMSO and compounds were diluted in assay media to 6× and 5μL was dispensed to assay plates. The final DMSO in the assay for all screening concentrations was 0.25%.</p><p>Virus Addition: VEEV stock was diluted in the culture media to 6.44 pfu/mL. (MOI 4e-5)</p><p>VEEV and Cell Plating: 3,000 cells/well alone or with VEEV virus at the previously indicated dilution(180,000 cells/ml) were plated in 25 uL using a Matrix WellMate. All additions were done using a Matrix WellMate housed in a class II Biosafety Cabinet within the BSL-2 laboratory. The plates were incubated in an actively humidified incubator with 5.0% CO<sub>2</sub> at 37 °C for 72h and 95% humidity.</p><p>Endpoint Read: The assay plates were equilibrated to room temperature for 30 minutes and then an equal volume of CellTiter-Glo reagent (Promega Inc.) was added to each well. Plates were incubated for 10 min at room temperature and luminescence was measured using a Perkin Elmer Envision multi-label reader.</p><p><b>Time of Addition Assay:</b> Vero 76 cells were seeded into a 6-well plate in a volume of 2 mL/well and incubated for overnight at 37 °C with 5% CO<sub>2</sub> and high humidity. The next day, cell culture media from the 6-well plates was aspirated and the cells were infected with 5 MOI TC-83 VEEV. After the infection, cell culture media was replaced (1.5 mL per well of complete culture media, 0-hr post infection). At the time of addition, 1.5 mL of cell culture media containing a test compound was added to designated wells. Sixteen hours post infection, culture media was harvested and the progeny viruses were titrated. The virus titers of the progeny virus were measured with a “microplaque” method employing methyl cellulose as a semi-solid substance in a 96-well format. The harvested virus were diluted by 10-fold serial dilutions in DMEM supplemented with 5% FBS using the epMotion™ (Eppendorf Inc.) liquid handler.</p></div></div></div></div><div id="ml336.s23"><h3>Eurofins Panlabs Target Profiling Report</h3><div id="ml336.s24"><h4>Summary</h4><p><b>STUDY OBJECTIVE:</b> To evaluate, in Radioligand Binding assays, the activity of compound CID60156253 (UK-13, PT# 1167309).</p><p><b>METHODS:</b> Methods employed in this study have been adapted from the scientific literature to maximize reliability and reproducibility. Reference standards were run as an integral part of each assay to ensure the validity of the results obtained. Assays were performed under conditions described in the accompanying <a href="#ml336.s3">“Methods” section</a> of this report. Where presented, IC<sub>50</sub> values were determined by a non-linear, least squares regression analysis using MathIQ™ (ID Business Solutions Ltd., UK). Where inhibition constants (K<sub>i</sub>) are presented, the K<sub>i</sub> values were calculated using the equation of Cheng and Prusoff (Cheng, Y., Prusoff, W.H., Biochem. Pharmacol. 22:3099-3108, 1973) using the observed IC<sub>50</sub> of the tested compound, the concentration of radioligand employed in the assay, and the historical values for the K<sub>D</sub> of the ligand (obtained experimentally at <b>Eurofins Panlabs, Inc.</b>). Where presented, the Hill coefficient (n<sub>H</sub>), defining the slope of the competitive binding curve, was calculated using MathIQ™. Hill coefficients significantly different than 1.0, may suggest that the binding displacement does not follow the laws of mass action with a single binding site. Where IC<sub>50</sub>, K<sub>i</sub>, and/or n<sub>H</sub> data are presented without Standard Error of the Mean (SEM), data are insufficient to be quantitative, and the values presented (K<sub>i</sub>, IC<sub>50</sub>, n<sub>H</sub>) should be interpreted with caution.</p><p><b>RESULTS:</b> A summary of results meeting the significance criteria is presented in the following sections. Complete results are presented under the section labeled “Experimental Results”. Individual responses, if requested, are presented in the section labeled “Individual Responses”.</p><p><b>SUMMARY/CONCLUSION:</b> Significant results are displayed in the following table(s) in rank order of potency for estimated IC<sub>50</sub> and/or K<sub>i</sub> values.</p></div><div id="ml336.s25"><h4>Summary of Significant Results</h4><p>Biochemical assay results are presented as the percent inhibition of specific binding or activity throughout the report. All other results are expressed in terms of that assay's quantitation method.</p><ul><li class="half_rhythm"><div>For primary assays, only the lowest concentration with a significant response judged by the assays' criteria, is shown in this summary.</div></li><li class="half_rhythm"><div>Where applicable, either the secondary assay results with the lowest dose/concentration meeting the significance criteria or, if inactive, the highest dose/concentration that did not meet the significance criteria is shown.</div></li><li class="half_rhythm"><div>Unless otherwise requested, primary screening in duplicate with quantitative data (e.g., IC<sub>50</sub> ± SEM, K<sub>i</sub> ± SEM and n<sub>H</sub>) are shown where applicable for individual requested assays. In screening packages, primary screening in duplicate with semi-quantitative data (e.g., estimated IC<sub>50</sub>, K<sub>i</sub> and n<sub>H</sub>) are shown where applicable (concentration range of 4 log units); available secondary functional assays are carried out (30 mM) and MEC or MIC determined only if active in primary assays >50% at 1 log unit below initial test concentration. Significant responses (≥ 50% inhibition or stimulation for Biochemical assays) were noted in the primary assays listed in <a class="figpopup" href="/books/NBK179829/table/ml336.t12/?report=objectonly" target="object" rid-figpopup="figml336t12" rid-ob="figobml336t12">Table 6</a>:</div></li></ul><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml336t12"><a href="/books/NBK179829/table/ml336.t12/?report=objectonly" target="object" title="Table 6" class="img_link icnblk_img figpopup" rid-figpopup="figml336t12" rid-ob="figobml336t12"><img class="small-thumb" src="/books/NBK179829/table/ml336.t12/?report=thumb" src-large="/books/NBK179829/table/ml336.t12/?report=previmg" alt="Table 6. Results of Eurofins Profiling with ML336, Compound: CID60156253." /></a><div class="icnblk_cntnt"><h4 id="ml336.t12"><a href="/books/NBK179829/table/ml336.t12/?report=objectonly" target="object" rid-ob="figobml336t12">Table 6</a></h4><p class="float-caption no_bottom_margin">Results of Eurofins Profiling with ML336, Compound: CID60156253. </p></div></div><div id="ml336.t11" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK179829/table/ml336.t11/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml336.t11_lrgtbl__"><table><thead><tr><th id="hd_h_ml336.t11_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Catalog #</th><th id="hd_h_ml336.t11_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Assay Name</th><th id="hd_h_ml336.t11_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Species</th><th id="hd_h_ml336.t11_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Concentration</th><th id="hd_h_ml336.t11_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">% Inhibition</th></tr></thead><tbody><tr><td headers="hd_h_ml336.t11_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">204410</td><td headers="hd_h_ml336.t11_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Norepinephrine Transporter (NET)</td><td headers="hd_h_ml336.t11_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">human</td><td headers="hd_h_ml336.t11_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">10 μM</td><td headers="hd_h_ml336.t11_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">91</td></tr></tbody></table></div></div><div id="ml336.t13" class="table"><h3><span class="label">Table 7</span><span class="title">Results of Eurofins Profiling with ML336, Compound: CID60156253 continued</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK179829/table/ml336.t13/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml336.t13_lrgtbl__"><table class="no_margin"><thead><tr><th id="hd_h_ml336.t13_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Cat #</th><th id="hd_h_ml336.t13_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Assay name</th><th id="hd_h_ml336.t13_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Batch</th><th id="hd_h_ml336.t13_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Species</th><th id="hd_h_ml336.t13_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Rep.</th><th id="hd_h_ml336.t13_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Conc.</th><th id="hd_h_ml336.t13_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">% inhibition</th></tr></thead><tbody><tr><td headers="hd_h_ml336.t13_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">241000</td><td headers="hd_h_ml336.t13_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Imidazoline I2, Central</td><td headers="hd_h_ml336.t13_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">326329</td><td headers="hd_h_ml336.t13_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">rat</td><td headers="hd_h_ml336.t13_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2</td><td headers="hd_h_ml336.t13_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">10 μM</td><td headers="hd_h_ml336.t13_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">14</td></tr><tr><td headers="hd_h_ml336.t13_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">243520</td><td headers="hd_h_ml336.t13_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Interleukin IL-1</td><td headers="hd_h_ml336.t13_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">326355</td><td headers="hd_h_ml336.t13_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">hum</td><td headers="hd_h_ml336.t13_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2</td><td headers="hd_h_ml336.t13_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">10 μM</td><td headers="hd_h_ml336.t13_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2</td></tr><tr><td headers="hd_h_ml336.t13_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">250460</td><td headers="hd_h_ml336.t13_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Leukotriene, Cysteinyl CysLT1</td><td headers="hd_h_ml336.t13_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">326336</td><td headers="hd_h_ml336.t13_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">hum</td><td headers="hd_h_ml336.t13_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2</td><td headers="hd_h_ml336.t13_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">10 μM</td><td headers="hd_h_ml336.t13_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">11</td></tr><tr><td headers="hd_h_ml336.t13_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">251600</td><td headers="hd_h_ml336.t13_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Melatonin MT1</td><td headers="hd_h_ml336.t13_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">326356</td><td headers="hd_h_ml336.t13_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">hum</td><td headers="hd_h_ml336.t13_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2</td><td headers="hd_h_ml336.t13_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">10 μM</td><td headers="hd_h_ml336.t13_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">18</td></tr><tr><td headers="hd_h_ml336.t13_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">252610</td><td headers="hd_h_ml336.t13_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Muscarinic M1</td><td headers="hd_h_ml336.t13_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">326408</td><td headers="hd_h_ml336.t13_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">hum</td><td headers="hd_h_ml336.t13_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2</td><td headers="hd_h_ml336.t13_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">10 μM</td><td headers="hd_h_ml336.t13_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1</td></tr><tr><td headers="hd_h_ml336.t13_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">252710</td><td headers="hd_h_ml336.t13_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Muscarinic M2</td><td headers="hd_h_ml336.t13_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">326409</td><td headers="hd_h_ml336.t13_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">hum</td><td headers="hd_h_ml336.t13_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2</td><td headers="hd_h_ml336.t13_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">10 μM</td><td headers="hd_h_ml336.t13_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2</td></tr><tr><td headers="hd_h_ml336.t13_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">252810</td><td headers="hd_h_ml336.t13_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Muscarinic M3</td><td headers="hd_h_ml336.t13_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">326348</td><td headers="hd_h_ml336.t13_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">hum</td><td headers="hd_h_ml336.t13_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2</td><td headers="hd_h_ml336.t13_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">10 μM</td><td headers="hd_h_ml336.t13_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td></tr><tr><td headers="hd_h_ml336.t13_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">257010</td><td headers="hd_h_ml336.t13_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Neuropeptide Y Y1</td><td headers="hd_h_ml336.t13_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">326330</td><td headers="hd_h_ml336.t13_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">hum</td><td headers="hd_h_ml336.t13_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2</td><td headers="hd_h_ml336.t13_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">10 μM</td><td headers="hd_h_ml336.t13_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">26</td></tr><tr><td headers="hd_h_ml336.t13_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">257110</td><td headers="hd_h_ml336.t13_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Neuropeptide Y Y2</td><td headers="hd_h_ml336.t13_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">326331</td><td headers="hd_h_ml336.t13_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">hum</td><td headers="hd_h_ml336.t13_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2</td><td headers="hd_h_ml336.t13_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">10 μM</td><td headers="hd_h_ml336.t13_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">-6</td></tr><tr><td headers="hd_h_ml336.t13_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">258590</td><td headers="hd_h_ml336.t13_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Nicotinic Acetylcholine</td><td headers="hd_h_ml336.t13_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">326298</td><td headers="hd_h_ml336.t13_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">hum</td><td headers="hd_h_ml336.t13_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2</td><td headers="hd_h_ml336.t13_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">10 μM</td><td headers="hd_h_ml336.t13_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">11</td></tr><tr><td headers="hd_h_ml336.t13_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">258700</td><td headers="hd_h_ml336.t13_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Nicotinic Acetylcholine α1, Bungarotoxin</td><td headers="hd_h_ml336.t13_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">326300</td><td headers="hd_h_ml336.t13_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">hum</td><td headers="hd_h_ml336.t13_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2</td><td headers="hd_h_ml336.t13_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">10 μM</td><td headers="hd_h_ml336.t13_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1</td></tr><tr><td headers="hd_h_ml336.t13_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">260130</td><td headers="hd_h_ml336.t13_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Opiate δ1 (OP1, DOP)</td><td headers="hd_h_ml336.t13_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">326357</td><td headers="hd_h_ml336.t13_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">hum</td><td headers="hd_h_ml336.t13_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2</td><td headers="hd_h_ml336.t13_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">10 μM</td><td headers="hd_h_ml336.t13_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">-2</td></tr><tr><td headers="hd_h_ml336.t13_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">260210</td><td headers="hd_h_ml336.t13_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Opiate κ(OP2, KOP)</td><td headers="hd_h_ml336.t13_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">326371</td><td headers="hd_h_ml336.t13_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">hum</td><td headers="hd_h_ml336.t13_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2</td><td headers="hd_h_ml336.t13_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">10 μM</td><td headers="hd_h_ml336.t13_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">10</td></tr><tr><td headers="hd_h_ml336.t13_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">260410</td><td headers="hd_h_ml336.t13_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Opiate μ(OP3, MOP)</td><td headers="hd_h_ml336.t13_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">326372</td><td headers="hd_h_ml336.t13_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">hum</td><td headers="hd_h_ml336.t13_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2</td><td headers="hd_h_ml336.t13_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">10 μM</td><td headers="hd_h_ml336.t13_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">25</td></tr><tr><td headers="hd_h_ml336.t13_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">264500</td><td headers="hd_h_ml336.t13_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Phorbol Ester</td><td headers="hd_h_ml336.t13_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">326373</td><td headers="hd_h_ml336.t13_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">mouse</td><td headers="hd_h_ml336.t13_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2</td><td headers="hd_h_ml336.t13_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">10 μM</td><td headers="hd_h_ml336.t13_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0</td></tr><tr><td headers="hd_h_ml336.t13_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">265010</td><td headers="hd_h_ml336.t13_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Platelet Activating Factor (PAF)</td><td headers="hd_h_ml336.t13_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">326383</td><td headers="hd_h_ml336.t13_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">hum</td><td headers="hd_h_ml336.t13_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2</td><td headers="hd_h_ml336.t13_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">10 μM</td><td headers="hd_h_ml336.t13_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">7</td></tr><tr><td headers="hd_h_ml336.t13_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">265600</td><td headers="hd_h_ml336.t13_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Potassium Channel [KATP]</td><td headers="hd_h_ml336.t13_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">326384</td><td headers="hd_h_ml336.t13_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">ham</td><td headers="hd_h_ml336.t13_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2</td><td headers="hd_h_ml336.t13_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">10 μM</td><td headers="hd_h_ml336.t13_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">5</td></tr><tr><td headers="hd_h_ml336.t13_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">265900</td><td headers="hd_h_ml336.t13_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Potassium Channel hERG</td><td headers="hd_h_ml336.t13_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">326385</td><td headers="hd_h_ml336.t13_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">hum</td><td headers="hd_h_ml336.t13_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2</td><td headers="hd_h_ml336.t13_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">10 μM</td><td headers="hd_h_ml336.t13_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">30</td></tr><tr><td headers="hd_h_ml336.t13_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">268420</td><td headers="hd_h_ml336.t13_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Prostanoid EP4</td><td headers="hd_h_ml336.t13_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">326343</td><td headers="hd_h_ml336.t13_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">hum</td><td headers="hd_h_ml336.t13_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2</td><td headers="hd_h_ml336.t13_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">10 μM</td><td headers="hd_h_ml336.t13_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">5</td></tr><tr><td headers="hd_h_ml336.t13_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">268700</td><td headers="hd_h_ml336.t13_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Purinergic P2X</td><td headers="hd_h_ml336.t13_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">326287</td><td headers="hd_h_ml336.t13_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">rabbit</td><td headers="hd_h_ml336.t13_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2</td><td headers="hd_h_ml336.t13_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">10 μM</td><td headers="hd_h_ml336.t13_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">14</td></tr><tr><td headers="hd_h_ml336.t13_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">268810</td><td headers="hd_h_ml336.t13_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Purinergic P2Y</td><td headers="hd_h_ml336.t13_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">326288</td><td headers="hd_h_ml336.t13_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">rat</td><td headers="hd_h_ml336.t13_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2</td><td headers="hd_h_ml336.t13_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">10 μM</td><td headers="hd_h_ml336.t13_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">6</td></tr><tr><td headers="hd_h_ml336.t13_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">270000</td><td headers="hd_h_ml336.t13_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Rolipram</td><td headers="hd_h_ml336.t13_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">326374</td><td headers="hd_h_ml336.t13_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">rat</td><td headers="hd_h_ml336.t13_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2</td><td headers="hd_h_ml336.t13_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">10 μM</td><td headers="hd_h_ml336.t13_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">35</td></tr><tr><td headers="hd_h_ml336.t13_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">271110</td><td headers="hd_h_ml336.t13_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Serotonin (5-Hydroxytryptamine) 5-HT1A</td><td headers="hd_h_ml336.t13_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">326289</td><td headers="hd_h_ml336.t13_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">hum</td><td headers="hd_h_ml336.t13_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2</td><td headers="hd_h_ml336.t13_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">10 μM</td><td headers="hd_h_ml336.t13_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2</td></tr><tr><td headers="hd_h_ml336.t13_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">271700</td><td headers="hd_h_ml336.t13_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Serotonin (5-Hydroxytryptamine) 5-HT2B</td><td headers="hd_h_ml336.t13_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">326386</td><td headers="hd_h_ml336.t13_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">hum</td><td headers="hd_h_ml336.t13_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2</td><td headers="hd_h_ml336.t13_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">10 μM</td><td headers="hd_h_ml336.t13_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">10</td></tr><tr><td headers="hd_h_ml336.t13_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">271910</td><td headers="hd_h_ml336.t13_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Serotonin (5-Hydroxytryptamine) 5-HT3</td><td headers="hd_h_ml336.t13_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">326387</td><td headers="hd_h_ml336.t13_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">hum</td><td headers="hd_h_ml336.t13_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2</td><td headers="hd_h_ml336.t13_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">10 μM</td><td headers="hd_h_ml336.t13_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2</td></tr><tr><td headers="hd_h_ml336.t13_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">278110</td><td headers="hd_h_ml336.t13_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Sigma σ1</td><td headers="hd_h_ml336.t13_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">326284</td><td headers="hd_h_ml336.t13_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">hum</td><td headers="hd_h_ml336.t13_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2</td><td headers="hd_h_ml336.t13_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">10 μM</td><td headers="hd_h_ml336.t13_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">7</td></tr><tr><td headers="hd_h_ml336.t13_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">279510</td><td headers="hd_h_ml336.t13_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Sodium Channel, Site 2</td><td headers="hd_h_ml336.t13_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">326318</td><td headers="hd_h_ml336.t13_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">rat</td><td headers="hd_h_ml336.t13_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2</td><td headers="hd_h_ml336.t13_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">10 μM</td><td headers="hd_h_ml336.t13_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">9</td></tr><tr><td headers="hd_h_ml336.t13_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">255520</td><td headers="hd_h_ml336.t13_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Tachykinin NK1</td><td headers="hd_h_ml336.t13_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">326382</td><td headers="hd_h_ml336.t13_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">hum</td><td headers="hd_h_ml336.t13_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2</td><td headers="hd_h_ml336.t13_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">10 μM</td><td headers="hd_h_ml336.t13_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">8</td></tr><tr><td headers="hd_h_ml336.t13_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">285900</td><td headers="hd_h_ml336.t13_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Thyroid Hormone</td><td headers="hd_h_ml336.t13_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">326388</td><td headers="hd_h_ml336.t13_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">rat</td><td headers="hd_h_ml336.t13_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2</td><td headers="hd_h_ml336.t13_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">10 μM</td><td headers="hd_h_ml336.t13_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2</td></tr><tr><td headers="hd_h_ml336.t13_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">220320</td><td headers="hd_h_ml336.t13_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Transporter, Dopamine (DAT)</td><td headers="hd_h_ml336.t13_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">326376</td><td headers="hd_h_ml336.t13_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">hum</td><td headers="hd_h_ml336.t13_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2</td><td headers="hd_h_ml336.t13_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">10 μM</td><td headers="hd_h_ml336.t13_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">11</td></tr><tr><td headers="hd_h_ml336.t13_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">226400</td><td headers="hd_h_ml336.t13_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Transporter, GABA</td><td headers="hd_h_ml336.t13_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">326281</td><td headers="hd_h_ml336.t13_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">rat</td><td headers="hd_h_ml336.t13_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2</td><td headers="hd_h_ml336.t13_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">10 μM</td><td headers="hd_h_ml336.t13_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0</td></tr><tr><td headers="hd_h_ml336.t13_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">204410</td><td headers="hd_h_ml336.t13_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Transporter, Norepinephrine (NET)</td><td headers="hd_h_ml336.t13_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">326375</td><td headers="hd_h_ml336.t13_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">hum</td><td headers="hd_h_ml336.t13_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2</td><td headers="hd_h_ml336.t13_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">10 μM</td><td headers="hd_h_ml336.t13_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">91</td></tr><tr><td headers="hd_h_ml336.t13_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">274030</td><td headers="hd_h_ml336.t13_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Transporter, Serotonin (5-Hydroxytryptamine)</td><td headers="hd_h_ml336.t13_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">326377</td><td headers="hd_h_ml336.t13_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">hum</td><td headers="hd_h_ml336.t13_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2</td><td headers="hd_h_ml336.t13_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">10 μM</td><td headers="hd_h_ml336.t13_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">-6</td></tr></tbody></table></div><div><div><dl class="temp-labeled-list small"><dt></dt><dd><div id="ml336.tfn8"><p class="no_margin">Note: Items meeting criteria for significance (≥50% stimulation or inhibition) are highlighted.</p></div></dd><dt>*</dt><dd><div id="ml336.tfn9"><p class="no_margin">Batch: Represents compounds tested concurrently in the same assay(s).</p></div></dd><dt></dt><dd><div id="ml336.tfn10"><p class="no_margin">ham=Hamster; hum=Human;</p></div></dd></dl></div></div></div><div id="ml336.s26"><h5>NCI-60 Panel Profile</h5><div id="ml336.t14" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Table%208.%20Single%20dose%20profile%20of%20ML336%20at%2010%20micromolar%20concentration.&p=BOOKS&id=179829_ml336f18.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/NBK179829/bin/ml336f18.jpg" alt="Table 8. Single dose profile of ML336 at 10 micromolar concentration." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Table 8</span><span class="title">Single dose profile of ML336 at 10 micromolar concentration</span></h3></div></div></div></div></div><div style="display:none"><div style="display:none" id="figml336f12"><img alt="Image ml336f12" src-large="/books/NBK179829/bin/ml336f12.jpg" /></div></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/NBK179829/?report=reader">PubReader</a></li><li><a href="/books/NBK179829/?report=printable">Print View</a></li><li><a data-jig="ncbidialog" href="#_ncbi_dlg_citbx_NBK179829" data-jigconfig="width:400,modal:true">Cite this Page</a><div id="_ncbi_dlg_citbx_NBK179829" style="display:none" title="Cite this Page"><div class="bk_tt">Chung D, Schroeder CE, Sotsky J, et al. ML336: Development of Quinazolinone-Based Inhibitors Against Venezuelan Equine Encephalitis Virus (VEEV) 2012 Dec 17 [Updated 2013 Nov 7]. 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="#ml336.s1" ref="log$=inpage&link_id=inpage">Probe Structure & Characteristics</a></li><li><a href="#ml336.s2" ref="log$=inpage&link_id=inpage">Recommendations for Scientific Use of the Probe</a></li><li><a href="#ml336.s3" ref="log$=inpage&link_id=inpage">Materials and Methods</a></li><li><a href="#ml336.s11" ref="log$=inpage&link_id=inpage">Results</a></li><li><a href="#ml336.s15" ref="log$=inpage&link_id=inpage">Discussion</a></li><li><a href="#ml336.s17" ref="log$=inpage&link_id=inpage">References</a></li><li><a href="#ml336.ack" ref="log$=inpage&link_id=inpage">Appendix</a></li></ul></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Related information</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="discovery_db_links" id="Shutter"></a></div><div class="portlet_content"><ul><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pmc&DbFrom=books&Cmd=Link&LinkName=books_pmc_refs&IdsFromResult=3069982" ref="log$=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=3069982" ref="log$=recordlinks">PubChem BioAssay for Chemical Probe</a><div class="brieflinkpop offscreen_noflow">PubChem BioAssay records reporting screening data for the development of the chemical probe(s) described in this book chapter</div></li><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pcsubstance&DbFrom=books&Cmd=Link&LinkName=books_pcsubstance&IdsFromResult=3069982" ref="log$=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=3069982" ref="log$=recordlinks">PubMed</a><div class="brieflinkpop offscreen_noflow">Links to PubMed</div></li></ul></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Similar articles in PubMed</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="PBooksDiscovery_RA" id="Shutter"></a></div><div class="portlet_content"><ul><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/31816348" ref="ordinalpos=1&linkpos=1&log$=relatedarticles&logdbfrom=pubmed">Benzamidine ML336 inhibits plus and minus strand RNA synthesis of Venezuelan equine encephalitis virus without affecting host RNA production.</a><span class="source">[Antiviral Res. 2020]</span><div class="brieflinkpop offscreen_noflow">Benzamidine ML336 inhibits plus and minus strand RNA synthesis of Venezuelan equine encephalitis virus without affecting host RNA production.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Skidmore AM, Adcock RS, Jonsson CB, Golden JE, Chung DH. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Antiviral Res. 2020 Feb; 174:104674. Epub 2019 Dec 6.</em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/27852852" ref="ordinalpos=1&linkpos=2&log$=relatedarticles&logdbfrom=pubmed">Ablation of Programmed -1 Ribosomal Frameshifting in Venezuelan Equine Encephalitis Virus Results in Attenuated Neuropathogenicity.</a><span class="source">[J Virol. 2017]</span><div class="brieflinkpop offscreen_noflow">Ablation of Programmed -1 Ribosomal Frameshifting in Venezuelan Equine Encephalitis Virus Results in Attenuated Neuropathogenicity.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Kendra JA, de la Fuente C, Brahms A, Woodson C, Bell TM, Chen B, Khan YA, Jacobs JL, Kehn-Hall K, Dinman JD. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">J Virol. 2017 Feb 1; 91(3). Epub 2017 Jan 18.</em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/32878897" ref="ordinalpos=1&linkpos=3&log$=relatedarticles&logdbfrom=pubmed">Emergence and Magnitude of ML336 Resistance in Venezuelan Equine Encephalitis Virus Depend on the Microenvironment.</a><span class="source">[J Virol. 2020]</span><div class="brieflinkpop offscreen_noflow">Emergence and Magnitude of ML336 Resistance in Venezuelan Equine Encephalitis Virus Depend on the Microenvironment.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Lee J, Parvathareddy J, Yang D, Bansal S, O'Connell K, Golden JE, Jonsson CB. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">J Virol. 2020 Oct 27; 94(22). Epub 2020 Oct 27.</em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/32503232" ref="ordinalpos=1&linkpos=4&log$=relatedreviews&logdbfrom=pubmed"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> Vaccine Advances against Venezuelan, Eastern, and Western Equine Encephalitis Viruses.</a><span class="source">[Vaccines (Basel). 2020]</span><div class="brieflinkpop offscreen_noflow"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> Vaccine Advances against Venezuelan, Eastern, and Western Equine Encephalitis Viruses.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Stromberg ZR, Fischer W, Bradfute SB, Kubicek-Sutherland JZ, Hraber P. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Vaccines (Basel). 2020 Jun 3; 8(2). Epub 2020 Jun 3.</em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/20028250" ref="ordinalpos=1&linkpos=5&log$=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> Alphavirus antiviral drug development: scientific gap analysis and prospective research areas.</a><span class="source">[Biosecur Bioterror. 2009]</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> Alphavirus antiviral drug development: scientific gap analysis and prospective research areas.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Reichert E, Clase A, Bacetty A, Larsen J. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Biosecur Bioterror. 2009 Dec; 7(4):413-27. </em></div></div></li></ul><a class="seemore" href="/sites/entrez?db=pubmed&cmd=link&linkname=pubmed_pubmed_reviews&uid=24479198" ref="ordinalpos=1&log$=relatedreviews_seeall&logdbfrom=pubmed">See reviews...</a><a class="seemore" href="/sites/entrez?db=pubmed&cmd=link&linkname=pubmed_pubmed&uid=24479198" ref="ordinalpos=1&log$=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$=activity&linkpos=1" href="/portal/utils/pageresolver.fcgi?recordid=67d66b5667c23b31e0b1005f">ML336: Development of Quinazolinone-Based Inhibitors Against Venezuelan Equine E...</a><div class="ralinkpop offscreen_noflow">ML336: Development of Quinazolinone-Based Inhibitors Against Venezuelan Equine Encephalitis Virus (VEEV) - Probe Reports from the NIH Molecular Libraries Program<div class="brieflinkpopdesc"></div></div><div class="tertiary"></div></li><li class="ra_rcd ralinkpopper two_line"><a class="htb ralinkpopperctrl" ref="log$=activity&linkpos=2" href="/portal/utils/pageresolver.fcgi?recordid=67d66b5567c23b31e0b0fe20">Development of a Second Generation mGlu3 NAM Probe - Probe Reports from the NIH ...</a><div class="ralinkpop offscreen_noflow">Development of a Second Generation mGlu3 NAM Probe - 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$=activity&linkpos=3" href="/portal/utils/pageresolver.fcgi?recordid=67d66b542f30673f7bf5f941">Elucidation of the physiology of non-replicating, drug tolerant Mycobacterium tu...</a><div class="ralinkpop offscreen_noflow">Elucidation of the physiology of non-replicating, drug tolerant Mycobacterium tuberculosis with the aid of the small molecule probe ML338 - Probe Reports from the NIH Molecular Libraries Program<div class="brieflinkpopdesc"></div></div><div class="tertiary"></div></li><li class="ra_rcd ralinkpopper two_line"><a class="htb ralinkpopperctrl" ref="log$=activity&linkpos=4" href="/portal/utils/pageresolver.fcgi?recordid=67d66b53cde49f3df7da2797">Probing the CXCR6/CXCL16 Axis: Targeting Prevention of Prostate Cancer Metastasi...</a><div class="ralinkpop offscreen_noflow">Probing the CXCR6/CXCL16 Axis: Targeting Prevention of Prostate Cancer Metastasis - 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=67d66b5284f3725e592b0ef0">Small-molecule antagonists of Gli function - Probe Reports from the NIH Molecula...</a><div class="ralinkpop offscreen_noflow">Small-molecule antagonists of Gli function - 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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<a href="https://www.google.com/maps/place/8600+Rockville+Pike,+Bethesda,+MD+20894/@38.9959508,-77.101021,17z/data=!3m1!4b1!4m5!3m4!1s0x89b7c95e25765ddb:0x19156f88b27635b8!8m2!3d38.9959508!4d-77.0988323" class="text-white" target="_blank" rel="noopener noreferrer">8600 Rockville Pike<br />
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