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<meta name="ncbi_db" content="books" /><meta name="ncbi_pdid" content="book-part" /><meta name="ncbi_acc" content="NBK280041" /><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="/NBK280041/" /><meta name="ncbi_pagename" content="Discovery of two small molecule inhibitors, ML387 and ML388, of human NAD+-dependent 15-hydroxyprostaglandin dehydrogenase - 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>Discovery of two small molecule inhibitors, ML387 and ML388, of human NAD+-dependent 15-hydroxyprostaglandin dehydrogenase - Probe Reports from the NIH Molecular Libraries Program - NCBI Bookshelf</title>
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<meta name="robots" content="INDEX,FOLLOW,NOARCHIVE" /><meta name="citation_inbook_title" content="Probe Reports from the NIH Molecular Libraries Program [Internet]" /><meta name="citation_title" content="Discovery of two small molecule inhibitors, ML387 and ML388, of human NAD+-dependent 15-hydroxyprostaglandin dehydrogenase" /><meta name="citation_publisher" content="National Center for Biotechnology Information (US)" /><meta name="citation_date" content="2015/02/11" /><meta name="citation_author" content="Damien Y. Duveau" /><meta name="citation_author" content="Adam Yasgar" /><meta name="citation_author" content="Xin Hu" /><meta name="citation_author" content="Jennifer Kouznetsova" /><meta name="citation_author" content="Kyle R. Brimacombe" /><meta name="citation_author" content="Edward H. Kerns" /><meta name="citation_author" content="Lesley Mathews" /><meta name="citation_author" content="Xiaohu Zhang" /><meta name="citation_author" content="Hongmao Sun" /><meta name="citation_author" content="Marc Ferrer" /><meta name="citation_author" content="Ajit Jadhav" /><meta name="citation_author" content="Anton Simeonov" /><meta name="citation_author" content="Craig J. Thomas" /><meta name="citation_author" content="David J. Maloney" /><meta name="citation_pmid" content="25834895" /><meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK280041/" /><link rel="schema.DC" href="http://purl.org/DC/elements/1.0/" /><meta name="DC.Title" content="Discovery of two small molecule inhibitors, ML387 and ML388, of human NAD+-dependent 15-hydroxyprostaglandin dehydrogenase" /><meta name="DC.Type" content="Text" /><meta name="DC.Publisher" content="National Center for Biotechnology Information (US)" /><meta name="DC.Contributor" content="Damien Y. Duveau" /><meta name="DC.Contributor" content="Adam Yasgar" /><meta name="DC.Contributor" content="Xin Hu" /><meta name="DC.Contributor" content="Jennifer Kouznetsova" /><meta name="DC.Contributor" content="Kyle R. Brimacombe" /><meta name="DC.Contributor" content="Edward H. Kerns" /><meta name="DC.Contributor" content="Lesley Mathews" /><meta name="DC.Contributor" content="Xiaohu Zhang" /><meta name="DC.Contributor" content="Hongmao Sun" /><meta name="DC.Contributor" content="Marc Ferrer" /><meta name="DC.Contributor" content="Ajit Jadhav" /><meta name="DC.Contributor" content="Anton Simeonov" /><meta name="DC.Contributor" content="Craig J. Thomas" /><meta name="DC.Contributor" content="David J. Maloney" /><meta name="DC.Date" content="2015/02/11" /><meta name="DC.Identifier" content="https://www.ncbi.nlm.nih.gov/books/NBK280041/" /><meta name="description" content="The central role of hydroxyprostaglandin dehydrogenase(HPGD) is to inactivate prostaglandins such as prostaglandin E2 (PGE2) or prostaglandin D2 (PGD2). These prostaglandins are involved in a number of processes, including inflammation, differentiation, and cellular signaling, which makes HPGD an important target for pharmacological intervention. High throughput screening of the Molecular Libraries Small Molecule Repository (MLSMR) led to the identification of three small molecule inhibitors of HPGD with differing mechanisms of action (ML147, ML148, and ML149). Herein, we report, the structure-activity relationship (SAR) studies, cell-based characterization and selectivity studies around the ML148 and ML149 scaffolds. These combined efforts led to the identification of two novel small molecule chemical probes (ML387 and ML388) with nanomolar inhibitory activity and excellent selectivity toward HPGD. Moreover, these compounds were shown to potently promote PGE2 production in both A549 lung cancer and LNCaP prostate cancer cells. As part of these studies, we also profiled top compounds for select in vitro ADME properties to facilitate future optimization efforts." /><meta name="og:title" content="Discovery of two small molecule inhibitors, ML387 and ML388, of human NAD+-dependent 15-hydroxyprostaglandin dehydrogenase" /><meta name="og:type" content="book" /><meta name="og:description" content="The central role of hydroxyprostaglandin dehydrogenase(HPGD) is to inactivate prostaglandins such as prostaglandin E2 (PGE2) or prostaglandin D2 (PGD2). These prostaglandins are involved in a number of processes, including inflammation, differentiation, and cellular signaling, which makes HPGD an important target for pharmacological intervention. High throughput screening of the Molecular Libraries Small Molecule Repository (MLSMR) led to the identification of three small molecule inhibitors of HPGD with differing mechanisms of action (ML147, ML148, and ML149). Herein, we report, the structure-activity relationship (SAR) studies, cell-based characterization and selectivity studies around the ML148 and ML149 scaffolds. These combined efforts led to the identification of two novel small molecule chemical probes (ML387 and ML388) with nanomolar inhibitory activity and excellent selectivity toward HPGD. Moreover, these compounds were shown to potently promote PGE2 production in both A549 lung cancer and LNCaP prostate cancer cells. As part of these studies, we also profiled top compounds for select in vitro ADME properties to facilitate future optimization efforts." /><meta name="og:url" content="https://www.ncbi.nlm.nih.gov/books/NBK280041/" /><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/ml388/" /><link rel="canonical" href="https://www.ncbi.nlm.nih.gov/books/NBK280041/" /><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="#__NBK280041_dtls__">Show details</a><div style="display:none" class="ui-widget" id="__NBK280041_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/ml382/" title="Previous page in this title">< Prev</a><a class="active page_link next" href="/books/n/mlprobe/ml389/" 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="_NBK280041_"><span class="title" itemprop="name">Discovery of two small molecule inhibitors, ML387 and ML388, of human NAD<sup>+</sup>-dependent 15-hydroxyprostaglandin dehydrogenase</span></h1><p class="contrib-group"><span itemprop="author">Damien Y. Duveau</span>, <span itemprop="author">Adam Yasgar</span>, <span itemprop="author">Xin Hu</span>, <span itemprop="author">Jennifer Kouznetsova</span>, <span itemprop="author">Kyle R. Brimacombe</span>, <span itemprop="author">Edward H. Kerns</span>, <span itemprop="author">Lesley Mathews</span>, <span itemprop="author">Xiaohu Zhang</span>, <span itemprop="author">Hongmao Sun</span>, <span itemprop="author">Marc Ferrer</span>, <span itemprop="author">Ajit Jadhav</span>, <span itemprop="author">Anton Simeonov</span>, <span itemprop="author">Craig J. Thomas</span>, and <span itemprop="author">David J. Maloney</span>.</p><a data-jig="ncbitoggler" href="#__NBK280041_ai__" style="border:0;text-decoration:none">Author Information and Affiliations</a><div style="display:none" class="ui-widget" id="__NBK280041_ai__"><p class="contrib-group"><h4>Authors</h4><span itemprop="author">Damien Y. Duveau</span>,<sup>1</sup> <span itemprop="author">Adam Yasgar</span>,<sup>1</sup> <span itemprop="author">Xin Hu</span>,<sup>1</sup> <span itemprop="author">Jennifer Kouznetsova</span>,<sup>1</sup> <span itemprop="author">Kyle R. Brimacombe</span>,<sup>1</sup> <span itemprop="author">Edward H. Kerns</span>,<sup>1</sup> <span itemprop="author">Lesley Mathews</span>,<sup>1</sup> <span itemprop="author">Xiaohu Zhang</span>,<sup>1</sup> <span itemprop="author">Hongmao Sun</span>,<sup>1</sup> <span itemprop="author">Marc Ferrer</span>,<sup>1</sup> <span itemprop="author">Ajit Jadhav</span>,<sup>1</sup> <span itemprop="author">Anton Simeonov</span>,<sup>1</sup> <span itemprop="author">Craig J. Thomas</span>,<sup>1</sup> and <span itemprop="author">David J. Maloney</span><sup>1</sup><sup>,*</sup>.</p><h4>Affiliations</h4><div class="affiliation"><sup>1</sup>
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NIH Chemical Genomics Center, National Center for Advancing Translational Sciences, 9800 Medical Center Drive, Rockville, MD, 20850</div><div class="affiliation">
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<sup>*</sup> To whom correspondence should be addressed: Email:
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<a href="mailto:dev@null" data-email="vog.hin.liam@dyenolam" class="oemail">vog.hin.liam@dyenolam</a></div></div><p class="small">Received: <span itemprop="datePublished">December 15, 2013</span>; Last Update: <span itemprop="dateModified">February 11, 2015</span>.</p></div><div class="jig-ncbiinpagenav body-content whole_rhythm" data-jigconfig="allHeadingLevels: ['h2'],smoothScroll: false" itemprop="text"><div id="_abs_rndgid_" itemprop="description"><p>The central role of hydroxyprostaglandin dehydrogenase(HPGD) is to inactivate prostaglandins such as prostaglandin E<sub>2</sub> (PGE<sub>2</sub>) or prostaglandin D<sub>2</sub> (PGD<sub>2</sub>). These prostaglandins are involved in a number of processes, including inflammation, differentiation, and cellular signaling, which makes HPGD an important target for pharmacological intervention. High throughput screening of the Molecular Libraries Small Molecule Repository (MLSMR) led to the identification of three small molecule inhibitors of HPGD with differing mechanisms of action (<a href="/pcsubstance/?term=ML147[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML147</a>, <a href="/pcsubstance/?term=ML148[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML148</a>, and <a href="/pcsubstance/?term=ML149[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML149</a>). Herein, we report, the structure-activity relationship (SAR) studies, cell-based characterization and selectivity studies around the <a href="/pcsubstance/?term=ML148[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML148</a> and <a href="/pcsubstance/?term=ML149[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML149</a> scaffolds. These combined efforts led to the identification of two novel small molecule chemical probes (<a href="/pcsubstance/?term=ML387[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML387</a> and <a href="/pcsubstance/?term=ML388[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML388</a>) with nanomolar inhibitory activity and excellent selectivity toward HPGD. Moreover, these compounds were shown to potently promote PGE<sub>2</sub> production in both A549 lung cancer and LNCaP prostate cancer cells. As part of these studies, we also profiled top compounds for select <i>in vitro</i> ADME properties to facilitate future optimization efforts.</p></div><div class="h2"></div><p><b>Assigned Assay Grant #:</b> <a href="/nuccore/1453778989" class="bk_tag" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=nuccore">MH084681</a></p><p><b>Date Submitted:</b> 04/11/2014</p><p><b>Screening Center Name & PI:</b> NIH Chemical Genomics Center (NCGC), NIH Center for Advancing Translation Sciences (NCATS)</p><p><b>Chemistry Center Name & PI:</b> NIH Chemical Genomics Center (NCGC), NIH Center for Advancing Translation Sciences (NCATS)</p><p><b>Assay Submitter & Institution:</b> Udo Oppermann, Structural Genomics Consortium, University of Oxford</p><p><b>PubChem Summary Bioassay Identifier (AID):</b>
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<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743196" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">743196</a></p><div id="ml388.s1"><h2 id="_ml388_s1_">Probe Structure & Characteristics</h2><div id="ml388.f1" class="figure"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Image%20ml388f1&p=BOOKS&id=280041_ml388f1.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/NBK280041/bin/ml388f1.jpg" alt="Image ml388f1" class="tileshop" title="Click on image to zoom" /></a></div></div><div id="ml388.t1" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK280041/table/ml388.t1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml388.t1_lrgtbl__"><table><thead><tr><th id="hd_h_ml388.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CID/ML#</th><th id="hd_h_ml388.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Target Name</th><th id="hd_h_ml388.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">IC<sub>50</sub>/EC<sub>50</sub> (nM)<br />[SID, AID]</th><th id="hd_h_ml388.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Anti-target Name(s)</th><th id="hd_h_ml388.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">IC<sub>50</sub>/EC<sub>50</sub> (μM)<br />[SID, AID]</th><th id="hd_h_ml388.t1_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Fold Selective</th><th id="hd_h_ml388.t1_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Secondary Assay(s) Name: IC<sub>50</sub>/EC<sub>50</sub> (μM) [SID, AID]</th></tr></thead><tbody><tr><td headers="hd_h_ml388.t1_1_1_1_1" rowspan="4" colspan="1" style="text-align:left;vertical-align:middle;"><b>CID 3474778/</b><a href="/pcsubstance/?term=ML387[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML387</a></td><td headers="hd_h_ml388.t1_1_1_1_2" rowspan="4" colspan="1" style="text-align:left;vertical-align:middle;">HPGD</td><td headers="hd_h_ml388.t1_1_1_1_3" rowspan="4" colspan="1" style="text-align:left;vertical-align:middle;">19 [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/162211043" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 162211043</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743171" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743171</a>]</td><td headers="hd_h_ml388.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">ALDH1A1</td><td headers="hd_h_ml388.t1_1_1_1_5" rowspan="4" colspan="1" style="text-align:left;vertical-align:top;">1358 [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/162211043" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 162211043</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743195" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743195</a>]<br />Inactive [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/162211043" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 162211043</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743169" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743169</a> & <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743192" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743192</a>]<br />Inactive [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/162211043" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 162211043</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743193" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743193</a>]<br />Inactive [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/162211043" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 162211043</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743187" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743187</a>]</td><td headers="hd_h_ml388.t1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">71</td><td headers="hd_h_ml388.t1_1_1_1_7" rowspan="4" colspan="1" style="text-align:left;vertical-align:top;">ALDH1A1 [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/162211043" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID162211043</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743195" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743195</a>]<br />LDHA, LDHB [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/162211043" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 162211043</a>,<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743169" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743169</a> & <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743192" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743192</a>]<br />rGAPDH [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/162211043" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 162211043</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743193" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743193</a>]<br />HSD17β4 [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/162211043" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 162211043</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743187" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743187</a>]</td></tr><tr><td headers="hd_h_ml388.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">LDHA, LDHB</td><td headers="hd_h_ml388.t1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">>1000</td></tr><tr><td headers="hd_h_ml388.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">rGAPDH</td><td headers="hd_h_ml388.t1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">>1000</td></tr><tr><td headers="hd_h_ml388.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">HSD17B4</td><td headers="hd_h_ml388.t1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">>1000</td></tr><tr><td headers="hd_h_ml388.t1_1_1_1_1 hd_h_ml388.t1_1_1_1_2 hd_h_ml388.t1_1_1_1_3 hd_h_ml388.t1_1_1_1_4 hd_h_ml388.t1_1_1_1_5 hd_h_ml388.t1_1_1_1_6 hd_h_ml388.t1_1_1_1_7" colspan="7" rowspan="1" style="text-align:center;vertical-align:bottom;">
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<span class="hr"></span></td></tr><tr><td headers="hd_h_ml388.t1_1_1_1_1" rowspan="4" colspan="1" style="text-align:left;vertical-align:middle;"><b>CID 71307851</b>/<a href="/pcsubstance/?term=ML388[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML388</a></td><td headers="hd_h_ml388.t1_1_1_1_2" rowspan="4" colspan="1" style="text-align:left;vertical-align:middle;">HPGD</td><td headers="hd_h_ml388.t1_1_1_1_3" rowspan="4" colspan="1" style="text-align:left;vertical-align:middle;">34 [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/162211017" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-162211017</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/substance/743171" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">743171</a>]</td><td headers="hd_h_ml388.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">ALDH1A1</td><td headers="hd_h_ml388.t1_1_1_1_5" rowspan="4" colspan="1" style="text-align:left;vertical-align:top;">2415[<a href="https://pubchem.ncbi.nlm.nih.gov/substance/162211017" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-162211017</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/substance/743195" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">743195</a>]<br />Inactive [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/162211017" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-162211017</a>,<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743169" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743169</a> & <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743192" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743192</a>]<br />Inactive [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/162211017" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-162211017</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743193" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743193</a>]<br />Inactive [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/162211017" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-162211017</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743187" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743187</a>]</td><td headers="hd_h_ml388.t1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">71</td><td headers="hd_h_ml388.t1_1_1_1_7" rowspan="4" colspan="1" style="text-align:left;vertical-align:top;">ALDH1A1 [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/162211017" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 162211017</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743195" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743195</a>]<br />LDHA, LDHB [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/162211017" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 162211017</a>,<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743169" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743169</a> & <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743192" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743192</a>]<br />rGAPDH [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/162211017" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 162211017</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743193" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743193</a>]<br />HSD17β4 [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/162211017" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 162211017</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743187" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743187</a>]</td></tr><tr><td headers="hd_h_ml388.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">LDHA, LDHB</td><td headers="hd_h_ml388.t1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">>1000</td></tr><tr><td headers="hd_h_ml388.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">GAPDH</td><td headers="hd_h_ml388.t1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">>1000</td></tr><tr><td headers="hd_h_ml388.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">HSD17B4</td><td headers="hd_h_ml388.t1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">>1000</td></tr></tbody></table></div></div></div><div id="ml388.s2"><h2 id="_ml388_s2_">1. Recommendations for scientific use of the probe</h2><p>Prostaglandins play central roles as signaling molecules, hence their levels are tightly regulated. One approach to study prostaglandins deregulation is through the modulation of their metabolism. HPGD is central to the metabolism of three key prostaglandins: PGE<sub>2</sub>, PGD<sub>2</sub>, and 15d-PGJ<sub>2</sub>. The two reported probes, <a href="/pcsubstance/?term=ML387[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML387</a> and <a href="/pcsubstance/?term=ML388[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML388</a>, are small molecules inhibitors of HPGD with nanomolar potency, excellent selectivity versus five other dehydrogenases, and improved ADME properties when compared to the original probes. Moreover, their efficacy was demonstrated at the cellular level through induction of PGE<sub>2</sub> production in A549 (lung cancer) cells and LNCaP prostate cancer cells. Given these attributes, <a href="/pcsubstance/?term=ML387[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML387</a> and <a href="/pcsubstance/?term=ML388[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML388</a> would be useful for studying the effect of pharmacological inhibition of HPGD in diseases such as cancer (e.g. cell migration, proliferation) and wound healing (prevention of excessive scar tissue).</p></div><div id="ml388.s3"><h2 id="_ml388_s3_">2. Materials and Methods</h2><div id="ml388.s4"><h3>2.1. Assays</h3><div id="ml388.s5"><h4>2.1.1. Cell lines and culture conditions</h4><p>The A549 cell line was obtained from ATCC. LNCaP cell line was generously donated by the lab of William Farrar at Frederick National Laboratories. Cells were cultured in RPMI 1640 medium supplanted with 10% dialyzed fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin. For the LNCaP cell line, arachidonic acid was added at 10 μM. The cultures were maintained in a 37 °C incubator with 5% CO<sub>2</sub> and under a humidified atmosphere.</p></div><div id="ml388.s6"><h4>2.1.2. HPGD qHTS Assay</h4><p>Inhibition of 15-PGDH (HPGD) activity was screened by utilizing prostaglandin as an electron donor and NAD<sup>+</sup> as an electron acceptor/cofactor. An increase in the fluorescence intensity due to conversion of NAD<sup>+</sup> to NADH was used to measure the enzyme activity. All compounds were screened using a qHTS approach, where compounds are assayed using at least seven concentrations to generate concentration-response curves for every compound. Briefly, qHTS uses an inter-plate dilution method where the first plate contains the highest concentration of a set of compounds in DMSO, while subsequent plates contain the same compounds in the same well locations, but at successive lower concentrations.</p><p>Three μL of enzyme (20 nM final concentration) were dispensed to 1536-well Greiner black solid bottom plates. Compounds (23 nL) were transferred via Kalypsys PinTool. The plates were incubated for 15 min at room temperature, and then 1 μL of substrate/cofactor solution (Substrate/cofactor solution: 1 mM NAD<sup>+</sup> and 30 μM prostaglandin (Sigma, prostaglandin E2, #P5640)) were added to start the reaction. The plates were immediately transferred to and read 4 times every 30 seconds on a ViewLux High-throughput CCD imager (Perkin-Elmer) using 340 nm excitation and 450 nm emission fluorescence protocol. The fluorescence intensity difference between the last and the first time points, normalized against controls, was used to compute reaction progress. [PubChem <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743171" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743171</a>]</p><div id="ml388.t2" class="table"><h3><span class="label">Table 1</span><span class="title">Stepwise protocol of the HPGD primary screen in 1536-well format</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK280041/table/ml388.t2/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml388.t2_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml388.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Sequence</th><th id="hd_h_ml388.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Parameter</th><th id="hd_h_ml388.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Value</th><th id="hd_h_ml388.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Description</th></tr></thead><tbody><tr><td headers="hd_h_ml388.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>1</b></td><td headers="hd_h_ml388.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Reagent</td><td headers="hd_h_ml388.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">3 μL</td><td headers="hd_h_ml388.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">20 nM HPGD</td></tr><tr><td headers="hd_h_ml388.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>2</b></td><td headers="hd_h_ml388.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Compound</td><td headers="hd_h_ml388.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">23 nL</td><td headers="hd_h_ml388.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Compound library and control</td></tr><tr><td headers="hd_h_ml388.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>3</b></td><td headers="hd_h_ml388.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Time</td><td headers="hd_h_ml388.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">15 min</td><td headers="hd_h_ml388.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Incubation with compounds at room temperature</td></tr><tr><td headers="hd_h_ml388.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>4</b></td><td headers="hd_h_ml388.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Reagent</td><td headers="hd_h_ml388.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">1 μL</td><td headers="hd_h_ml388.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">1 mM NAD<sup>+</sup> and 30 μM prostaglandin</td></tr><tr><td headers="hd_h_ml388.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>5</b></td><td headers="hd_h_ml388.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Detector</td><td headers="hd_h_ml388.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Fluorescence</td><td headers="hd_h_ml388.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Read 4 times, 30 sec interval (ViewLux; ex 340, em 450)</td></tr></tbody></table></div></div></div><div id="ml388.s7"><h4>2.1.3. Prostaglandin E<sub>2</sub> (PGE<sub>2</sub>) HTRF Cell-based 384-well Assay: A549 or LNCaP Cell Line</h4><p>A549 cells, LNCaP cells or media were dispensed in 384-well plates (ProxiPlate-384 Plus, PerkinElmer, Shelton, CT) at 5,000 cells/9.9 μL/well using a CappAero384 multichannel pipette (Odense, Denmark). Separately on the plate, 10 μL of PGE<sub>2</sub> calibration standards at a starting assay concentration of 5,000 pg/mL (1:2 dilution, 12-points, n = 1, prepared in media, Cisbio, Bedford, MA) were dispensed. Compounds (100 nL, final concentration range from 41.9 nM to 100 μM) were transferred using an ATS Acoustic Liquid Dispenser (EDC Biosystems, Fremont, CA). After being mixed using an automated liquid handler (CyBio, Jena, Germany), samples were incubated (37 °C, 5% CO<sub>2,</sub> under a humidified atmosphere) for 16 hours. PGE<sub>2</sub> labeled conjugates were added to each well, first 5 μL of PGE<sub>2</sub>-<i>d<sub>2</sub></i>, followed by 5 μL of anti-PGE<sub>2</sub>-cryptate, centrifuged (1,500 rpm, 15 sec) and incubated (RT, protected from light) for 5 hours. Time-resolved fluorescence intensity (320 nm excitation, 615 and 665 nm emission) was measured using an Envision plate reader (PerkinElmer). Data was expressed as the ratio of 665 nm/615 nm emissions. Concentration (pg/mL) of samples were calculated from a 4-parameter logistic calibration curves of the PGE<sub>2</sub> standards. [PubChem <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743172" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743172</a>]</p><div id="ml388.t3" class="table"><h3><span class="label">Table 2</span><span class="title">Stepwise protocol of the PGE<sub>2</sub> HTRF assay using LNCaP cells in 384-well format</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK280041/table/ml388.t3/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml388.t3_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml388.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Sequence</th><th id="hd_h_ml388.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Parameter</th><th id="hd_h_ml388.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Value</th><th id="hd_h_ml388.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Description</th></tr></thead><tbody><tr><td headers="hd_h_ml388.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>1</b></td><td headers="hd_h_ml388.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Reagent</td><td headers="hd_h_ml388.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">9.9 μL</td><td headers="hd_h_ml388.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">5,000 cells/well (A549, LNCaP, or media)</td></tr><tr><td headers="hd_h_ml388.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>2</b></td><td headers="hd_h_ml388.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Compound</td><td headers="hd_h_ml388.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">100 nL</td><td headers="hd_h_ml388.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Compound addition through acoustic liquid dispenser (final concentration ranges 41.9 nM t0 100 μM)</td></tr><tr><td headers="hd_h_ml388.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>3</b></td><td headers="hd_h_ml388.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Time</td><td headers="hd_h_ml388.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">16 hr</td><td headers="hd_h_ml388.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Upon mixing, incubation at 37 ºC, 5% CO<sub>2</sub></td></tr><tr><td headers="hd_h_ml388.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>4</b></td><td headers="hd_h_ml388.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Reagent</td><td headers="hd_h_ml388.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">5 μL</td><td headers="hd_h_ml388.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">PGE<sub>2</sub>-<i>d</i><sub>2</sub></td></tr><tr><td headers="hd_h_ml388.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>5</b></td><td headers="hd_h_ml388.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Reagent</td><td headers="hd_h_ml388.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">5 μL</td><td headers="hd_h_ml388.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">anti-PGE<sub>2</sub>-cryptate</td></tr><tr><td headers="hd_h_ml388.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>6</b></td><td headers="hd_h_ml388.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Time</td><td headers="hd_h_ml388.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">15 sec</td><td headers="hd_h_ml388.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Centrifuged at 1,500 RPM</td></tr><tr><td headers="hd_h_ml388.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>7</b></td><td headers="hd_h_ml388.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Time</td><td headers="hd_h_ml388.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">5 hr</td><td headers="hd_h_ml388.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Incubation at room temperature, protected from light</td></tr><tr><td headers="hd_h_ml388.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>8</b></td><td headers="hd_h_ml388.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Detector</td><td headers="hd_h_ml388.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">HTRF</td><td headers="hd_h_ml388.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Envision (320 nm, 615 and 665 nm emission</td></tr></tbody></table></div></div></div><div id="ml388.s8"><h4>2.1.4. Prostaglandin E<sub>2</sub> (PGE<sub>2</sub>) HTRF Cell-based 1,536-well Assay: A549 Cell Line</h4><p>A549 cells or media were dispensed onto a 1,536-well solid-bottom T/C white plate (Greiner Bio One, Monroe, NC) at 2,500 cells/5 μL/well using a Thermo Scientific Multidrop Combi (Thermo Fisher Scientific Inc., Waltham, MA). Separately on the plate, 5 μL of PGE<sub>2</sub> calibration standards at a starting assay concentration of 5,000 pg/mL (1:2 dilution, 12-points, n = 2, prepared in media, Cisbio, Bedford, MA) were manually dispensed, followed by compounds (23 nL, final concentration range from 775 pM to 45.8 μM) and positive control titration (CID- 3243760, final concentration range 1.4 nM to 45.8 μM) transferred using a Kalypsys Pin-tool dispenser (Wako Automation, San Diego, CA). Plates were incubated (37 °C, 5% CO<sub>2</sub>, under a humidified atmosphere) at 4, 8, 12, 16, and 20 hours. PGE<sub>2</sub> labeled conjugates were added to each well, first 2.5 μL of PGE<sub>2</sub>-d<sub>2</sub> (or buffer), followed by 2.5 μL of anti-PGE<sub>2</sub>-cryptate, centrifuged (1,000 rpm, 15 sec) and incubated (RT, protected from light) for 5 hours. Time-resolved fluorescence intensity (320 nm excitation, 615 and 665 nm emission) was measured using an Envision plate reader (PerkinElmer). Data was expressed as the ratio of 665 nm/615 nm emissions. Concentration (pg/mL) of samples were calculated from a 4-parameter logistic calibration curves of the PGE<sub>2</sub> standards. [PubChem <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743167" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743167</a>]</p><div id="ml388.t4" class="table"><h3><span class="label">Table 3</span><span class="title">Stepwise protocol of the PGE<sub>2</sub> HTRF assay using A549 cells in 1536-well format</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK280041/table/ml388.t4/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml388.t4_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml388.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Sequence</th><th id="hd_h_ml388.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Parameter</th><th id="hd_h_ml388.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Value</th><th id="hd_h_ml388.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Description</th></tr></thead><tbody><tr><td headers="hd_h_ml388.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>1</b></td><td headers="hd_h_ml388.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Reagent</td><td headers="hd_h_ml388.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">5 μL</td><td headers="hd_h_ml388.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">2,500 cells/well (A549 or media)</td></tr><tr><td headers="hd_h_ml388.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>2</b></td><td headers="hd_h_ml388.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Compound</td><td headers="hd_h_ml388.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">23 nL</td><td headers="hd_h_ml388.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Compounds and controls</td></tr><tr><td headers="hd_h_ml388.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>3</b></td><td headers="hd_h_ml388.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Time</td><td headers="hd_h_ml388.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Varied</td><td headers="hd_h_ml388.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Incubation at 37 ºC, 5% CO<sub>2</sub>, 4, 8, 12, and 16 hours</td></tr><tr><td headers="hd_h_ml388.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>4</b></td><td headers="hd_h_ml388.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Reagent</td><td headers="hd_h_ml388.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">2.5 μL</td><td headers="hd_h_ml388.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">PGE<sub>2</sub>-<i>d</i><sub>2</sub></td></tr><tr><td headers="hd_h_ml388.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>5</b></td><td headers="hd_h_ml388.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Reagent</td><td headers="hd_h_ml388.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">2.5 μL</td><td headers="hd_h_ml388.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">anti-PGE<sub>2</sub>-cryptate</td></tr><tr><td headers="hd_h_ml388.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>6</b></td><td headers="hd_h_ml388.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Time</td><td headers="hd_h_ml388.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">15 sec</td><td headers="hd_h_ml388.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Centrifuged at 1,500 RPM</td></tr><tr><td headers="hd_h_ml388.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>7</b></td><td headers="hd_h_ml388.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Time</td><td headers="hd_h_ml388.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">5 hr</td><td headers="hd_h_ml388.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Incubation at room temperature, protected from light</td></tr><tr><td headers="hd_h_ml388.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>8</b></td><td headers="hd_h_ml388.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Detector</td><td headers="hd_h_ml388.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">HTRF</td><td headers="hd_h_ml388.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Envision (320 nm, 615 and 665 nm emission)</td></tr></tbody></table></div></div></div><div id="ml388.s9"><h4>2.1.5. HSD17β4 Counterscreen</h4><p>The mitochondrial enzyme 17β-hydroxysteroid dehydrogenase 1 (HSD17β4 catalyzes the NAD<sup>+</sup>-dependent oxidation of a variety of substrates including acyl-CoAs, androgens and neurosteroids, as well as bile acids. HSD17β4 activity was screened by utilizing an electron donor (estradiol) and NAD<sup>+</sup> as an electron acceptor/cofactor. An increase in the fluorescence intensity due to conversion of NAD<sup>+</sup> to NADH was used to measure the enzyme activity.</p><p>Briefly, 3 μL of enzyme (final concentration 100 nM) or buffer were dispensed into a 1,536-well solid-bottom black plate (Greiner Bio One, Monroe, NC) followed by pin-tool transfer (23 nL) of compound (final concentration range 324 pM to 57.2 μM) and control (CID-24867619, final concentration range 5.23 nM to 11.4 μM). Samples were incubated (RT, protected from light) for 15 minutes followed by a 1 μL substrate addition of NAD<sup>+</sup> and Estradiol (final concentrations of 1 mM and 50 μM, respectively, in 15% DMSO). Plates were centrifuged at 1,000 rpm for 15 seconds, then read immediately on a ViewLux High-throughput CCD imager (Perkin-Elmer) equipped with standard UV fluorescence optics (340 nm excitation, 450 nm emission), incubated for 1 hour (RT, protected from light) followed by a second read. Data were normalized (Read 2<sub>1 hour</sub> – Read 1<sub>0 hour</sub>) against no-enzyme (positive) and enzyme-containing (neutral) wells. A stepwise description of the 1536-well assay is shown in <a class="figpopup" href="/books/NBK280041/table/ml388.t5/?report=objectonly" target="object" rid-figpopup="figml388t5" rid-ob="figobml388t5">Table 4</a>.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml388t5"><a href="/books/NBK280041/table/ml388.t5/?report=objectonly" target="object" title="Table 4" class="img_link icnblk_img figpopup" rid-figpopup="figml388t5" rid-ob="figobml388t5"><img class="small-thumb" src="/books/NBK280041/table/ml388.t5/?report=thumb" src-large="/books/NBK280041/table/ml388.t5/?report=previmg" alt="Table 4. Stepwise protocol of the HSD17β4 counterscreen in 1536-well format." /></a><div class="icnblk_cntnt"><h4 id="ml388.t5"><a href="/books/NBK280041/table/ml388.t5/?report=objectonly" target="object" rid-ob="figobml388t5">Table 4</a></h4><p class="float-caption no_bottom_margin">Stepwise protocol of the HSD17β4 counterscreen in 1536-well format. </p></div></div></div><div id="ml388.s10"><h4>2.1.6. ALDH1A1 Counterscreen</h4><p>Aldehyde dehydrogenase 1 family, member A1 (ALDH1A1) catalyzes the NAD<sup>+</sup> dependent oxidation of a variety of endogenous and exogenous aldehydes to the corresponding carboxylic acids. The enzyme is the critical step in the metabolic activation of retinoic acid, which plays an essential role as a nuclear receptor ligand. Furthermore, the precursor, retinaldehyde, has recently been shown to play a fundamental role in adipogenesis and obesity, which makes inhibitor development a possible target in metabolic diseases. ALDH1A1 shares some homology with HPGD, and it served as a suitable anti-target of HPGD to determine chemical probe selectivity. The assay system is similar to the HPGD primary screening protocol. Inhibition of ALDH1A1 activity was screened by utilizing propionaldehyde as an electron donor and NAD<sup>+</sup> as an electron acceptor/cofactor. An increase in the fluorescence intensity due to conversion of NAD<sup>+</sup> to NADH was used to measure the enzyme activity.</p><p>Briefly, 3 μL of enzyme (final concentration 50 nM) or buffer were dispensed into a 1,536-well solid-bottom black plate (Greiner Bio One, Monroe, NC) followed by pin-tool transfer (23 nL) of compound (final concentration range 324 pM to 57.2 μM) and control (Bay 11-7085 (CID-5353432), final concentration range 1.31 nM to 2.86 μM). Samples were incubated (RT, protected from light) for 15 minutes followed by a 1 μL substrate addition of NAD<sup>+</sup> and Propionaldehyde (final concentrations of 1 mM and 80 μM, respectively). Plates were centrifuged at 1,000 rpm for 15 seconds, then read in kinetic mode on a ViewLux High-throughput CCD imager (Perkin-Elmer) equipped with standard UV fluorescence optics (340 nm excitation, 450 nm emission) for 10 minutes at 5 minute intervals. Data were normalized (Read 2<sub>10 min</sub> – Read 1<sub>0 min</sub>) against no-enzyme (positive) and enzyme-containing (neutral) wells. A stepwise description of the 1536-well assay is shown in <a class="figpopup" href="/books/NBK280041/table/ml388.t6/?report=objectonly" target="object" rid-figpopup="figml388t6" rid-ob="figobml388t6">Table 5</a>.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml388t6"><a href="/books/NBK280041/table/ml388.t6/?report=objectonly" target="object" title="Table 5" class="img_link icnblk_img figpopup" rid-figpopup="figml388t6" rid-ob="figobml388t6"><img class="small-thumb" src="/books/NBK280041/table/ml388.t6/?report=thumb" src-large="/books/NBK280041/table/ml388.t6/?report=previmg" alt="Table 5. Stepwise protocol of the LDHA counterscreen in 1536-well format." /></a><div class="icnblk_cntnt"><h4 id="ml388.t6"><a href="/books/NBK280041/table/ml388.t6/?report=objectonly" target="object" rid-ob="figobml388t6">Table 5</a></h4><p class="float-caption no_bottom_margin">Stepwise protocol of the LDHA counterscreen in 1536-well format. </p></div></div></div><div id="ml388.s11"><h4>2.1.7. LDHA Counterscreen</h4><p>LDHA converts pyruvate to lactate while regenerating NAD<sup>+</sup> from NADH. LDHA enzymatic activity is then coupled to the activity of diaphorase, which reversibly catalyzes the reaction of NAD<sup>+</sup> to NADH while simultaneously reducing resazurin to the fluorescent resorufin. This change in fluorescence signal (excitation 544 nm; emission, 590 nm) was monitored using the ViewLux CCD imager (Perkin Elmer). Data were normalized against ‘no enzyme’ control wells and ‘no compound’ control wells.</p><p>The enzymatic reaction was performed in buffer consisting of 200 mM Tris-HCl pH 7.4, containing 0.01% Tween 20, and the assay was run in black solid-bottom Greiner plates. A stepwise description of the 1536-well assay is shown in <a class="figpopup" href="/books/NBK280041/table/ml388.t7/?report=objectonly" target="object" rid-figpopup="figml388t7" rid-ob="figobml388t7">Table 6</a>.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml388t7"><a href="/books/NBK280041/table/ml388.t7/?report=objectonly" target="object" title="Table 6" class="img_link icnblk_img figpopup" rid-figpopup="figml388t7" rid-ob="figobml388t7"><img class="small-thumb" src="/books/NBK280041/table/ml388.t7/?report=thumb" src-large="/books/NBK280041/table/ml388.t7/?report=previmg" alt="Table 6. Stepwise protocol of the LDHA counterscreen in 1536-well format." /></a><div class="icnblk_cntnt"><h4 id="ml388.t7"><a href="/books/NBK280041/table/ml388.t7/?report=objectonly" target="object" rid-ob="figobml388t7">Table 6</a></h4><p class="float-caption no_bottom_margin">Stepwise protocol of the LDHA counterscreen in 1536-well format. </p></div></div></div><div id="ml388.s12"><h4>2.1.8. LDHB Counterscreen</h4><p>LDHB converts pyruvate to lactate while regenerating NAD<sup>+</sup> from NADH. LDHB enzymatic activity is then coupled to the activity of diaphorase, which reversibly catalyzes the reaction of NAD<sup>+</sup> to NADH while simultaneously reducing resazurin to the fluorescent resorufin. This change in fluorescence signal (excitation 544 nm; emission, 590 nm) was monitored using the ViewLux CCD imager (Perkin Elmer). Data were normalized against ‘no enzyme’ control wells and ‘no compound’ control wells.</p><p>The enzymatic reaction was performed in buffer consisting of 200 mM Tris-HCl pH 7.4, containing 0.01% Tween 20, and the assay was run in black solid-bottom Greiner plates. A stepwise description of the 1536-well assay is shown in <a class="figpopup" href="/books/NBK280041/table/ml388.t8/?report=objectonly" target="object" rid-figpopup="figml388t8" rid-ob="figobml388t8">Table 7</a>.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml388t8"><a href="/books/NBK280041/table/ml388.t8/?report=objectonly" target="object" title="Table 7" class="img_link icnblk_img figpopup" rid-figpopup="figml388t8" rid-ob="figobml388t8"><img class="small-thumb" src="/books/NBK280041/table/ml388.t8/?report=thumb" src-large="/books/NBK280041/table/ml388.t8/?report=previmg" alt="Table 7. Stepwise protocol of the LDHA counterscreen in 1536-well format." /></a><div class="icnblk_cntnt"><h4 id="ml388.t8"><a href="/books/NBK280041/table/ml388.t8/?report=objectonly" target="object" rid-ob="figobml388t8">Table 7</a></h4><p class="float-caption no_bottom_margin">Stepwise protocol of the LDHA counterscreen in 1536-well format. </p></div></div></div><div id="ml388.s13"><h4>2.1.9. Rabbit GAPDH Counterscreen</h4><p>The counterscreen for non-specific dehydrogenase activity was conducted using a rabbit glyceraldehyde-3-phosphate dehydrogenase (rGAPDH; Sigma Aldrich, St. Louis, MO) coupled assay system. In this assay, the rGAPDH-catalyzed conversion of NAD<sup>+</sup> to NADH is coupled downstream to an additional enzyme, diaphorase (Sigma Aldrich, St. Louis, MO), which reduces the non-fluorescent molecule, resazurin, to a red fluorescent product, resorufin, in an NADH-dependent fashion. Thus, rGAPDH activity leads to an increase in fluorescence at 598 nm, and inhibitors of the reaction lead to a decrease in resorufin production and a subsequent decrease in fluorescence. The change in fluorescence between 0 and 30 minutes was measured, and data were normalized to control columns representing maximum signal (all components) and background (no rGAPDH). The buffer consisted of 100mM triethanolamine, pH 7.6, 5mM MgCl<sub>2</sub> and 0.01% Tween-20, and the assay was run in black, solid-bottom, non-tissue culture-treated Greiner plates. A stepwise description of the 1536-well assay is shown in <a class="figpopup" href="/books/NBK280041/table/ml388.t9/?report=objectonly" target="object" rid-figpopup="figml388t9" rid-ob="figobml388t9">Table 8</a>.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml388t9"><a href="/books/NBK280041/table/ml388.t9/?report=objectonly" target="object" title="Table 8" class="img_link icnblk_img figpopup" rid-figpopup="figml388t9" rid-ob="figobml388t9"><img class="small-thumb" src="/books/NBK280041/table/ml388.t9/?report=thumb" src-large="/books/NBK280041/table/ml388.t9/?report=previmg" alt="Table 8. Stepwise protocol of the rGAPDH counterscreen in 1536-well format." /></a><div class="icnblk_cntnt"><h4 id="ml388.t9"><a href="/books/NBK280041/table/ml388.t9/?report=objectonly" target="object" rid-ob="figobml388t9">Table 8</a></h4><p class="float-caption no_bottom_margin">Stepwise protocol of the rGAPDH counterscreen in 1536-well format. </p></div></div></div><div id="ml388.s14"><h4>2.1.10. NF-κB-<i>bla</i> ME180 Cell Line and Culture Conditions</h4><p>The CellSensor™ NF-κB-<i>bla</i> ME180 cell line (NF-κB-<i>bla</i> cells) were obtained from Invitrogen (Carlsbad, CA). Cells were cultured in High Glucose GlutaMAX™ DMEM medium supplemented with 10% dialyzed fetal bovine serum, 0.1 mM non-essential amino acids, 25 mM HEPES (pH 7.3), 100 U/mL penicillin and 100 μg/mL streptomycin, and 5 μg/mL of blasticidin. The cultures were maintained in a 37 °C incubator with 5% CO<sub>2</sub> and under a humidified atmosphere.</p></div><div id="ml388.s15"><h4>2.1.11. NF-κB β-Lactamase Reporter Gene Assay (NF-κB <i>bla</i> assay) and Cell Viability Assay</h4><p>The NF-κB-bla cells were suspended in OPTI-MEM medium supplemented with 0.5% dialyzed fetal bovine serum, 0.1 mM nonessential amino acids, 25 mM sodium pyruvate, 100 U/mL penicillin, and 100 μg/mL streptomycin. Cells were dispensed at 2,000 cells/5 μL/well using a Thermo Scientific Multidrop Combi (Thermo Fisher Scientific Inc., Waltham, MA) and incubated (37 °C, 5% CO<sub>2,</sub> under a humidified atmosphere) overnight. Compounds (23 nL, final concentration range from 647 pM to 38.2 μM) and positive control titration (MG132, final concentration range 17.5 nM to 38.2 μM) were transferred to a 1,536-well clear-bottom T/C black plate (Greiner Bio One) using a Kalypsys Pin-tool dispenser (Wako Automation, San Diego, CA). Cells were incubated (37 °C) 15 minutes, followed by addition of either 1 μL assay buffer with or without tumor necrosis factor alpha (TNF-α, final concentration 1 ng/mL). The plates were incubated for 4, 8, 12, 18, and 24 hours at 37 °C, then 1 μL of LiveBLAzer™ B/G FRET substrate (Invitrogen, CA) detection mix was added and the plates incubated at room temperature for 2 hours. Fluorescence intensity (405 nm excitation, 460 and 530 nm emission) was measured using an Envision plate reader (Perkin Elmer, Shelton, CT). Data was expressed as the ratio of 460nm/530 nm emissions. At the completion of the read, each plate received a 2.5 μL dispense of CellTiter-Glo (Promega, Madison, WI), then after a 10 minute incubation (RT) samples were analyzed for luminescence intensity using a ViewLux High-throughput CCD imager (Perkin-Elmer) equipped with clear filters.</p><div id="ml388.t10" class="table"><h3><span class="label">Table 9</span><span class="title">Stepwise protocol for the NF-κB Lactamase Reporter Gene Assay</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK280041/table/ml388.t10/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml388.t10_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml388.t10_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Sequence</th><th id="hd_h_ml388.t10_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Parameter</th><th id="hd_h_ml388.t10_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Value</th><th id="hd_h_ml388.t10_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Description</th></tr></thead><tbody><tr><td headers="hd_h_ml388.t10_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>1</b></td><td headers="hd_h_ml388.t10_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Compound</td><td headers="hd_h_ml388.t10_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">23 nL</td><td headers="hd_h_ml388.t10_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Compound Library and MG132</td></tr><tr><td headers="hd_h_ml388.t10_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>2</b></td><td headers="hd_h_ml388.t10_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Reagent</td><td headers="hd_h_ml388.t10_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">5 μL</td><td headers="hd_h_ml388.t10_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">2000cells/well Cell Sensor™ NF- κB-<i>bla</i> ME180</td></tr><tr><td headers="hd_h_ml388.t10_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>3</b></td><td headers="hd_h_ml388.t10_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Time</td><td headers="hd_h_ml388.t10_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">15 min</td><td headers="hd_h_ml388.t10_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Incubation at 37 ºC</td></tr><tr><td headers="hd_h_ml388.t10_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>4</b></td><td headers="hd_h_ml388.t10_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Reagent</td><td headers="hd_h_ml388.t10_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">1 μL</td><td headers="hd_h_ml388.t10_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">TNF-α (final conc. 1 ng/mL) or assay buffer</td></tr><tr><td headers="hd_h_ml388.t10_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>5</b></td><td headers="hd_h_ml388.t10_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Time</td><td headers="hd_h_ml388.t10_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">5 hr</td><td headers="hd_h_ml388.t10_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Incubation at 37 ºC</td></tr><tr><td headers="hd_h_ml388.t10_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>6</b></td><td headers="hd_h_ml388.t10_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Reagent</td><td headers="hd_h_ml388.t10_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">1 μL</td><td headers="hd_h_ml388.t10_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">LiveBLAzer™ B/G FRET substrate</td></tr><tr><td headers="hd_h_ml388.t10_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>7</b></td><td headers="hd_h_ml388.t10_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Time</td><td headers="hd_h_ml388.t10_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">2 hr</td><td headers="hd_h_ml388.t10_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Room temperature incubation</td></tr><tr><td headers="hd_h_ml388.t10_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>8</b></td><td headers="hd_h_ml388.t10_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Detector</td><td headers="hd_h_ml388.t10_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Fluorescence</td><td headers="hd_h_ml388.t10_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Envision (460 nm Ex, 530 nm Em)</td></tr><tr><td headers="hd_h_ml388.t10_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>9</b></td><td headers="hd_h_ml388.t10_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Reagent</td><td headers="hd_h_ml388.t10_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">2.5 μL</td><td headers="hd_h_ml388.t10_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CellTiter-Glo</td></tr><tr><td headers="hd_h_ml388.t10_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>10</b></td><td headers="hd_h_ml388.t10_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Time</td><td headers="hd_h_ml388.t10_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">15 sec</td><td headers="hd_h_ml388.t10_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Centrifuge at 1,500 RPM</td></tr><tr><td headers="hd_h_ml388.t10_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>11</b></td><td headers="hd_h_ml388.t10_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Time</td><td headers="hd_h_ml388.t10_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">10 min</td><td headers="hd_h_ml388.t10_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Incubate at room temperature</td></tr><tr><td headers="hd_h_ml388.t10_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>12</b></td><td headers="hd_h_ml388.t10_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Detector</td><td headers="hd_h_ml388.t10_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Luminescence</td><td headers="hd_h_ml388.t10_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">ViewLux (clear filters)</td></tr></tbody></table></div></div></div></div><div id="ml388.s16"><h3>2.2. Probe Chemical Characterization</h3><div id="ml388.s17"><h4>2.2.1. ML387 (Probe 1)</h4><div id="ml388.f2" class="figure"><div class="graphic"><img src="/books/NBK280041/bin/ml388f2.jpg" alt="Image ml388f2" /></div></div><div id="ml388.s18"><h5>(1-(4-methoxyphenyl)-1H-benzo[d]imidazol-5-yl)(piperidin-1-yl)methanone, TFA (ML387)</h5><p>LC-MS rt: 4.15 min; <sup>1</sup>H NMR (400 MHz, DMSO-<i>d</i><sub>6</sub>) δ 8.81 – 8.70 (m, 1H), 7.77 (s, 1H), 7.66 – 7.60 (m, 2H), 7.58 (d, <i>J</i> = 8.5 Hz, 1H), 7.37 (d, <i>J</i> = 8.6 Hz, 1H), 7.23 – 7.15 (m, 2H), 3.86 (s, 3H), 3.65 – 3.35 (m, 4H), 1.68 – 1.58 (m, 2H), 1.58 – 1.44 (m, 4H); <sup>13</sup>C NMR (100 MHz, DMSO-<i>d<sub>6</sub></i>) δ 168.9, 159.2, 144.4, 133.6, 131.5, 128.0, 125.8, 123.0, 117.4, 115.2, 111.0, 55.6, and 24.1; HRMS (ESI) <i>m/z</i> 336.1703 (M + H)<sup>+</sup> (C<sub>20</sub>H<sub>22</sub>N<sub>3</sub>O<sub>2</sub> requires 336.1707).</p><div id="ml388.f3" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%201.%201H%20and13C%20NMR%20spectra%20of%20ML387.&p=BOOKS&id=280041_ml388f3.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/NBK280041/bin/ml388f3.jpg" alt="Figure 1. 1H and13C NMR spectra of ML387." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 1</span><span class="title"><sup>1</sup>H and<sup>13</sup>C NMR spectra of <b>ML387</b></span></h3></div><div id="ml388.f4" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%202.%20LC-MS%20of%20ML387.&p=BOOKS&id=280041_ml388f4.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/NBK280041/bin/ml388f4.jpg" alt="Figure 2. LC-MS of ML387." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 2</span><span class="title">LC-MS of <b>ML387</b></span></h3></div></div><div id="ml388.s19"><h5>Probe analogs</h5><div id="ml388.f5" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%203.%20Structures%20of%20the%20five%20analogs%20of%20ML387%20that%20have%20been%20submitted%20to%20the%20MLSMR.&p=BOOKS&id=280041_ml388f5.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/NBK280041/bin/ml388f5.jpg" alt="Figure 3. Structures of the five analogs of ML387 that have been submitted to the MLSMR." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 3</span><span class="title">Structures of the five analogs of <b>ML387</b> that have been submitted to the MLSMR</span></h3><div id="ml388.t11" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK280041/table/ml388.t11/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml388.t11_lrgtbl__"><table><thead><tr><th id="hd_h_ml388.t11_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Internal ID</th><th id="hd_h_ml388.t11_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS ID</th><th id="hd_h_ml388.t11_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">SID</th><th id="hd_h_ml388.t11_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CID</th><th id="hd_h_ml388.t11_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">ML #</th><th id="hd_h_ml388.t11_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Type</th><th id="hd_h_ml388.t11_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Source</th></tr></thead><tbody><tr><td headers="hd_h_ml388.t11_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">NCGC00262228</td><td headers="hd_h_ml388.t11_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">MLS005565827</td><td headers="hd_h_ml388.t11_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/162211043" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">162211043</a></td><td headers="hd_h_ml388.t11_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">3474778</td><td headers="hd_h_ml388.t11_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="/pcsubstance/?term=ML387[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML387</a></td><td headers="hd_h_ml388.t11_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Probe</td><td headers="hd_h_ml388.t11_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">NCGC</td></tr><tr><td headers="hd_h_ml388.t11_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">NCGC00262223</td><td headers="hd_h_ml388.t11_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">MLS004857446</td><td headers="hd_h_ml388.t11_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/162211012" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">162211012</a></td><td headers="hd_h_ml388.t11_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">71307827</td><td headers="hd_h_ml388.t11_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"></td><td headers="hd_h_ml388.t11_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Analog</td><td headers="hd_h_ml388.t11_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">NCGC</td></tr><tr><td headers="hd_h_ml388.t11_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">NCGC00262277</td><td headers="hd_h_ml388.t11_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">MLS004857445</td><td headers="hd_h_ml388.t11_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/162210983" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">162210983</a></td><td headers="hd_h_ml388.t11_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">71307865</td><td headers="hd_h_ml388.t11_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"></td><td headers="hd_h_ml388.t11_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Analog</td><td headers="hd_h_ml388.t11_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">NCGC</td></tr><tr><td headers="hd_h_ml388.t11_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">NCGC00262222</td><td headers="hd_h_ml388.t11_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">MLS004857448</td><td headers="hd_h_ml388.t11_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/162210969" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">162210969</a></td><td headers="hd_h_ml388.t11_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">71307836</td><td headers="hd_h_ml388.t11_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"></td><td headers="hd_h_ml388.t11_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Analog</td><td headers="hd_h_ml388.t11_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">NCGC</td></tr><tr><td headers="hd_h_ml388.t11_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">NCGC00262238</td><td headers="hd_h_ml388.t11_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">MLS004857449</td><td headers="hd_h_ml388.t11_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/162211008" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">162211008</a></td><td headers="hd_h_ml388.t11_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">71307876</td><td headers="hd_h_ml388.t11_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"></td><td headers="hd_h_ml388.t11_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Analog</td><td headers="hd_h_ml388.t11_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">NCGC</td></tr><tr><td headers="hd_h_ml388.t11_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">NCGC00262242</td><td headers="hd_h_ml388.t11_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">MLS004857450</td><td headers="hd_h_ml388.t11_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/162211025" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">162211025</a></td><td headers="hd_h_ml388.t11_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">71307812</td><td headers="hd_h_ml388.t11_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"></td><td headers="hd_h_ml388.t11_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Analog</td><td headers="hd_h_ml388.t11_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">NCGC</td></tr></tbody></table></div></div></div><div id="ml388.f6" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%204.%20Stability%20of%20ML387%20over%20time%20in%20aqueous%20solution%20at%20room%20temperature%2C%20as%20measured%20by%20the%20area%20under%20the%20curve%20(AUC)%20of%20the%20probe%20HPLC%20signal%20observed%20at%20254%20nm.&p=BOOKS&id=280041_ml388f6.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/NBK280041/bin/ml388f6.jpg" alt="Figure 4. Stability of ML387 over time in aqueous solution at room temperature, as measured by the area under the curve (AUC) of the probe HPLC signal observed at 254 nm." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 4</span><span class="title">Stability of <b>ML387</b> over time in aqueous solution at room temperature, as measured by the area under the curve (AUC) of the probe HPLC signal observed at 254 nm</span></h3><div class="caption"><p>PBS: phosphate buffer saline, pH 7.4; HPGD: assay buffer; GSH: 10 mM GSH in PBS buffer.</p></div></div></div></div><div id="ml388.s20"><h4>2.2.2. ML388 (Probe 2)</h4><div id="ml388.f7" class="figure"><div class="graphic"><img src="/books/NBK280041/bin/ml388f7.jpg" alt="Image ml388f7" /></div></div><div id="ml388.s21"><h5>3-(2,5-dimethyl-1-(p-tolyl)-1H-pyrrol-3-yl)-6,7,8,9-tetrahydro-5H-[1,2,4]triazolo[4,3-a]azepine, TFA (ML388)</h5><p>LC-MS rt: 4.75 min; <sup>1</sup>H NMR (400 MHz, DMSO-<i>d</i><sub>6</sub>) δ 7.39 (d, <i>J</i> = 8.0 Hz, 2H), 7.26 (d, <i>J</i> = 8.2 Hz, 2H), 6.19 (s, 1H), 4.30 – 4.06 (m, 2H), 3.16 – 3.01 (m, 2H), 2.41 (s, 3H), 2.05 (s, 3H), 2.02 (s, 3H), 1.90 – 1.78 (m, 4H), 1.75 (m, 2H); <sup>13</sup>C NMR (100 MHz, DMSO-<i>d<sub>6</sub></i>) δ 157.0, 150.0, 138.4, 134.2, 131.4, 130.2, 130.1, 127.6, 106.5, 69.8, 46.5, 29.3, 26.7, 25.4, 23.9, 20.7, 12.5, and 11.8; HRMS (ESI) <i>m/z</i> 321.2076 (M + H)<sup>+</sup> (C<sub>20</sub>H<sub>25</sub>N<sub>4</sub> requires 321.2074).</p><div id="ml388.f8" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%205.%201H%20and13C%20spectra%20of%20ML388.&p=BOOKS&id=280041_ml388f8.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/NBK280041/bin/ml388f8.jpg" alt="Figure 5. 1H and13C spectra of ML388." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 5</span><span class="title"><sup>1</sup>H and<sup>13</sup>C spectra of <b>ML388</b></span></h3></div><div id="ml388.f9" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%206.%20LC-MS%20of%20ML388.&p=BOOKS&id=280041_ml388f9.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/NBK280041/bin/ml388f9.jpg" alt="Figure 6. LC-MS of ML388." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 6</span><span class="title">LC-MS of <b>ML388</b></span></h3></div></div><div id="ml388.s22"><h5>Probe analogs</h5><div id="ml388.f10" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%207.%20Structures%20of%20the%20five%20analogs%20of%20ML388%20that%20have%20been%20submitted%20to%20the%20MLSMR.&p=BOOKS&id=280041_ml388f10.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/NBK280041/bin/ml388f10.jpg" alt="Figure 7. Structures of the five analogs of ML388 that have been submitted to the MLSMR." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 7</span><span class="title">Structures of the five analogs of <b>ML388</b> that have been submitted to the MLSMR</span></h3><div id="ml388.t12" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK280041/table/ml388.t12/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml388.t12_lrgtbl__"><table><thead><tr><th id="hd_h_ml388.t12_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Internal ID</th><th id="hd_h_ml388.t12_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS ID</th><th id="hd_h_ml388.t12_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">SID</th><th id="hd_h_ml388.t12_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CID</th><th id="hd_h_ml388.t12_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">ML #</th><th id="hd_h_ml388.t12_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Type</th><th id="hd_h_ml388.t12_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Source</th></tr></thead><tbody><tr><td headers="hd_h_ml388.t12_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">NCGC00249661</td><td headers="hd_h_ml388.t12_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">MLS005565826</td><td headers="hd_h_ml388.t12_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/162211017" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">162211017</a></td><td headers="hd_h_ml388.t12_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">71307851</td><td headers="hd_h_ml388.t12_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="/pcsubstance/?term=ML388[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML388</a></td><td headers="hd_h_ml388.t12_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Probe</td><td headers="hd_h_ml388.t12_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">NCGC</td></tr><tr><td headers="hd_h_ml388.t12_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">NCGC00249658</td><td headers="hd_h_ml388.t12_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">MLS004857440</td><td headers="hd_h_ml388.t12_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/162211019" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">162211019</a></td><td headers="hd_h_ml388.t12_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">71307849</td><td headers="hd_h_ml388.t12_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"></td><td headers="hd_h_ml388.t12_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Analog</td><td headers="hd_h_ml388.t12_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">NCGC</td></tr><tr><td headers="hd_h_ml388.t12_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">NCGC00249646</td><td headers="hd_h_ml388.t12_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">MLS004857441</td><td headers="hd_h_ml388.t12_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/162211016" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">162211016</a></td><td headers="hd_h_ml388.t12_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">71307856</td><td headers="hd_h_ml388.t12_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"></td><td headers="hd_h_ml388.t12_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Analog</td><td headers="hd_h_ml388.t12_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">NCGC</td></tr><tr><td headers="hd_h_ml388.t12_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">NCGC00249647</td><td headers="hd_h_ml388.t12_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">MLS004857442</td><td headers="hd_h_ml388.t12_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/162210974" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">162210974</a></td><td headers="hd_h_ml388.t12_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">71307853</td><td headers="hd_h_ml388.t12_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"></td><td headers="hd_h_ml388.t12_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Analog</td><td headers="hd_h_ml388.t12_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">NCGC</td></tr><tr><td headers="hd_h_ml388.t12_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">NCGC00249642</td><td headers="hd_h_ml388.t12_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">MLS004857443</td><td headers="hd_h_ml388.t12_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/162210963" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">162210963</a></td><td headers="hd_h_ml388.t12_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">71307824</td><td headers="hd_h_ml388.t12_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"></td><td headers="hd_h_ml388.t12_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Analog</td><td headers="hd_h_ml388.t12_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">NCGC</td></tr><tr><td headers="hd_h_ml388.t12_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">NCGC00249643</td><td headers="hd_h_ml388.t12_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">MLS004857444</td><td headers="hd_h_ml388.t12_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/162210962" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">162210962</a></td><td headers="hd_h_ml388.t12_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">71307846</td><td headers="hd_h_ml388.t12_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"></td><td headers="hd_h_ml388.t12_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Analog</td><td headers="hd_h_ml388.t12_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">NCGC</td></tr></tbody></table></div></div></div><div id="ml388.f11" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%208.%20Stability%20of%20ML388%20over%20time%20in%20aqueous%20solution%20at%20room%20temperature%2C%20as%20measured%20by%20the%20area%20under%20the%20curve%20(AUC)%20of%20the%20probe%20HPLC%20signal%20observed%20at%20254%20nm.&p=BOOKS&id=280041_ml388f11.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/NBK280041/bin/ml388f11.jpg" alt="Figure 8. Stability of ML388 over time in aqueous solution at room temperature, as measured by the area under the curve (AUC) of the probe HPLC signal observed at 254 nm." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 8</span><span class="title">Stability of <b>ML388</b> over time in aqueous solution at room temperature, as measured by the area under the curve (AUC) of the probe HPLC signal observed at 254 nm</span></h3><div class="caption"><p>PBS: phosphate buffer saline, pH 7.4; HPGD: assay buffer; GSH: 10 mM GSH in PBS buffer.</p></div></div></div></div></div><div id="ml388.s23"><h3>2.3. Probe Preparation</h3><div id="ml388.s24"><h4>2.3.1. ML387 (Probe 1)</h4><div id="ml388.f12" class="figure"><div class="graphic"><img src="/books/NBK280041/bin/ml388f12.jpg" alt="Image ml388f12" /></div></div><div id="ml388.s25"><h5>(4-fluoro-3-nitrophenyl)(piperidin-1-yl)methanone</h5><p>A mixture containing 4-fluoro-3-nitrobenzoic acid (2.00 g, 10.80 mmol), and oxalyl chloride (4.75 mL, 54.0 mmol) in DCM (54mL) was carefully treated with DMF (0.084 mL, 1.080 mmol). The reaction mixture was stirred at 23 °C for 3 hr. The reaction mixture was concentrated under diminished pressure. The obtained residue was taken up in 10 mL of toluene, and the mixture was concentrated under diminished pressure. This process was repeated to yield crude 4-fluoro-3-nitrobenzoyl chloride as a clear, dark yellow syrup.</p><p>A solution containing 4-fluoro-3-nitrobenzoyl chloride (2.20 g, 10.8 mmol) in DCM (27 mL) was treated with piperidine hydrochloride (1.71 g, 14.0 mmol), and with diisopropylethylamine (2.10 mL, 11.9 mmol) at 23 °C for 69 hr. The reaction mixture was diluted with 25 mL of DCM. The organic layer was washed with two 25 mL portions of 0.50 M aqueous HCl. The product was extracted with two 25 mL portions of DCM. The combined organic layer was dried (MgSO<sub>4</sub>), and was then concentrated under diminished pressure. The obtained clear pale yellow oil was applied to a silica gel column (50 g); eluting with 80:20 → 20:80 Hex-EtOAc afforded (4-fluoro-3-nitrophenyl)(piperidin-1-yl)methanone as a clear pale yellow syrup: yield 1.72 g (63 % over two steps); <sup>1</sup>H NMR (400 MHz, CDCl<sub>3</sub>) δ 8.11 (dd, <i>J</i> = 7.0, 2.2 Hz, 1H), 7.69 (ddd, <i>J</i> = 8.5, 4.2, 2.2 Hz, 1H), 7.34 (dd, <i>J</i> = 10.4, 8.5 Hz, 1H), 3.70 (brs, 2H), 3.36 (brs, 2H), 1.63 (m, 6H).</p><div id="ml388.f13" class="figure"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Image%20ml388f13&p=BOOKS&id=280041_ml388f13.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/NBK280041/bin/ml388f13.jpg" alt="Image ml388f13" class="tileshop" title="Click on image to zoom" /></a></div></div></div><div id="ml388.s26"><h5>(1-(4-methoxyphenyl)-1H-benzo[d]imidazol-5-yl)(piperidin-1-yl)methanone, TFA (ML387)</h5><p>A mixture containing (4-fluoro-3-nitrophenyl)(piperidin-1-yl)methanone (200 mg, 0.793 mmol), 4-methoxyaniline (98.0 mg, 0.793 mmol), and DIPEA (140 μL, 0.800 mmol) in NMP (1.60 mL) was heated in the microwave reactor at 200 °C for 3 hr. The reaction mixture was partitioned between 40 mL of water and 20 mL of EtOAc. The layers were separated, then the product was extracted with three 20 mL portions of EtOAc. The combined organic layer was washed with 20 mL of water, then with 20 mL of brine, was then dried (Na<sub>2</sub>SO<sub>4</sub>), and was then concentrated under diminished pressure. The obtained brown oil was applied to a silica gel column (100 g); eluting with 80:20 → 20:80 Hex-EtOAc afforded (4-((4-methoxyphenyl)amino)-3-nitrophenyl)(piperidin-1-yl)methanone as a red syrup; yield: 141 mg (50%).</p><p>To a stirred suspension containing(4-((4-methoxyphenyl)amino)-3-nitrophenyl)(piperidin-1-yl)methanone (0.141 g, 0.397 mmol) and Raney nickel (slurry in water) (50 μL) in methanol (7.90 mL) was added hydrazine (0.137 mL, 4.36 mmol). The reaction mixture was stirred at 23 °C for 18 hr. Some Raney nickel (slurry in water) (50 μL) and hydrazine (0.137 mL, 4.36 mmol) were added. Stirring was continued at 23 °C for 30 min. Then, some celite was added, followed by 20 mL of MeOH. The suspension was filtered, and the collected filtrate was concentrated to afford (3-amino-4-((4-methoxyphenyl)amino)phenyl)(piperidin-1-yl)methanone as a brown syrup; yield: 0.125 g (97 % yield). The crude aniline was used in the next step without further purification.</p><p>A mixture containing (3-amino-4-((4-methoxyphenyl)amino)phenyl)(piperidin-1-yl)methanone (0.125 g, 0.384 mmol), triethyl orthoformate (0.338 mL, 1.921 mmol) and <i>p</i>-toluenesulfonic acid monohydrate (3.65 mg, 0.019 mmol) in THF (1.90 mL) was heated in the microwave reactor at 90 °C for 1.0 hr. The reaction mixture was allowed to cool down to 23 °C, and was then diluted with 2 mL of MeOH. The resulting solution was purified by reversed-phase HPLC to afford (1-(4-methoxyphenyl)-1H-benzo[d]imidazol-5-yl)(piperidin-1-yl)methanone, TFA; yield: 76.3 mg (44%).</p></div></div><div id="ml388.s27"><h4>2.3.2. ML388 (Probe 2)</h4><div id="ml388.f14" class="figure"><div class="graphic"><img src="/books/NBK280041/bin/ml388f14.jpg" alt="Image ml388f14" /></div></div><div id="ml388.s28"><h5>2,5-dimethyl-1H-pyrrole-3-carbohydrazide</h5><p>A solution containing 2,5-dimethyl-1H-pyrrole-3-carboxylic acid (2.00 g, 14.37 mmol) in THF (29 mL) was treated with CDI (2.56 g, 15.81 mmol) at 23 °C for 3.5 hr. The resulting brown/orange suspension was then treated with hydrazine monohydrate (2.1 mL, 43.1 mmol) at 23 °C for 18 hr. Within 1 hr, the precipitate had dissolved and a clear orange solution was obtained. The solvent was evaporated under diminished pressure; the resulting mixture was applied to a silica gel column (50 g); eluting with 95:5 → 75:25 DCM-MeOH led to a mixture containing the product and imidazole. The obtained solid was recrystallized from EtOAc to afford 2,5-dimethyl-1H-pyrrole-3-carbohydrazide as a pale yellow crystalline solid: yield 1.43 g (65.0 %); <sup>1</sup>H NMR (400 MHz, DMSO-<i>d<sub>6</sub></i>) δ 10.62 (s, 1H), 8.65 (s, 1H), 6.08 – 5.97 (m, 1H), 4.13 (s, 2H), 2.35 (s, 3H), 2.07 (s, 3H).</p><div id="ml388.f15" class="figure"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Image%20ml388f15&p=BOOKS&id=280041_ml388f15.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/NBK280041/bin/ml388f15.jpg" alt="Image ml388f15" class="tileshop" title="Click on image to zoom" /></a></div></div></div><div id="ml388.s29"><h5>3-(2,5-dimethyl-1H-pyrrol-3-yl)-6,7,8,9-tetrahydro-5H-[1,2,4]triazolo[4,3-a]azepine</h5><p>A suspension containing 2,5-dimethyl-1H-pyrrole-3-carbohydrazide (2.00 g, 13.1 mmol) and 7-methoxy-3,4,5,6-tetrahydro-2H-azepine (2.80 mL, 19.6 mmol) in chlorobenzene (15 mL) was heated in a capped reaction vial in the microwave oven at 180 °C for 45 min. The reaction mixture was allowed to cool down to room temperature and was then concentrated under diminished pressure. The residue was applied to a silica gel column (100 g); eluting with 95:5 → 75:25 DCM-MeOH (methanol containing 10% NH<sub>4</sub>OH) afforded 3-(2,5-dimethyl-1H-pyrrol-3-yl)-6,7,8,9-tetrahydro-5H-[1,2,4]triazolo[4,3-a]azepine as a white solid; yield 2.14 g (71%); LC-MS rt (min): 2.58 min; <sup>1</sup>H NMR (400 MHz, CDCl<sub>3</sub>) δ 9.02 (s, 1H), 5.77 (d, <i>J</i> = 2.6 Hz, 1H), 4.03 - 3.96 (m, 2H), 3.05 - 2.95 (m, 2H), 2.23 (d, <i>J</i> = 1.5 Hz, 6H), 1.88 (q, <i>J</i> = 6.0 Hz, 2H), 1.75 (dt, <i>J</i> = 10.0, 5.1 Hz, 4H).</p></div><div id="ml388.s30"><h5>3-(2,5-dimethyl-1-(p-tolyl)-1H-pyrrol-3-yl)-6,7,8,9-tetrahydro-5H-[1,2,4]triazolo[4,3-a]azepine, TFA (ML388)</h5><div id="ml388.f16" class="figure"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Image%20ml388f16&p=BOOKS&id=280041_ml388f16.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/NBK280041/bin/ml388f16.jpg" alt="Image ml388f16" class="tileshop" title="Click on image to zoom" /></a></div></div><p>In a sealed vial, a mixture containing 3-(2,5-dimethyl-1H-pyrrol-3-yl)-6,7,8,9-tetrahydro-5H-[1,2,4]triazolo[4,3-a]azepine (150 mg, 0.651 mmol), <i>p</i>-toluyl iodide (170 mg, 0.782 mmol), cesium carbonate (318 mg, 0.977 mmol), copper(I) oxide (9.32 mg, 0.065 mmol), 4,7-dimethoxy-1,10-phenanthroline (L) (31.3 mg, 0.130 mmol), and poly(ethylene glycol) (150 mg, approx. 100 mg/mmol of pyrrole) in NMP (1.30 mL) was stirred at 100 °C for 18 hr. The reaction mixture was diluted with 3 mL of acetonitrile, and was then filtered. The collected clear brown solution was concentrated under diminished pressure and the resulting residue was purified by reversed phase HPLC to afford 3-(2,5-dimethyl-1-(p-tolyl)-1H-pyrrol-3-yl)-6,7,8,9-tetrahydro-5H-[1,2,4]triazolo[4,3-a]azepine, TFA; yield: 38.3 mg (14%).</p></div></div></div></div><div id="ml388.s31"><h2 id="_ml388_s31_">3. Results</h2><div id="ml388.s32"><h3>3.1. Dose Response Curves for Probes ML387 and ML388</h3><div id="ml388.f17" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%209.%20Dose-dependent%20inhibition%20of%20HPGD%20by%20ML387%20and%20ML388%20in%20the%20qHTS%20assay%20(see%20section%202.2.2).&p=BOOKS&id=280041_ml388f17.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/NBK280041/bin/ml388f17.jpg" alt="Figure 9. Dose-dependent inhibition of HPGD by ML387 and ML388 in the qHTS assay (see section 2.2.2)." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 9</span><span class="title">Dose-dependent inhibition of HPGD by <b>ML387</b> and <b>ML388</b> in the qHTS assay (see <a href="#ml388.s20">section 2.2.2</a>)</span></h3></div></div><div id="ml388.s33"><h3>3.2. Cellular Activity</h3><div id="ml388.t13" class="table"><h3><span class="label">Table 10</span><span class="title">PGE<sub>2</sub> produced in A549 cells and LNCaP cells</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK280041/table/ml388.t13/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml388.t13_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml388.t13_1_1_1_1" rowspan="2" colspan="1" headers="hd_h_ml388.t13_1_1_1_1" style="text-align:center;vertical-align:middle;">Entry</th><th id="hd_h_ml388.t13_1_1_1_2" rowspan="2" colspan="1" headers="hd_h_ml388.t13_1_1_1_2" style="text-align:center;vertical-align:middle;">CID</th><th id="hd_h_ml388.t13_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">HPGD Assay</th><th id="hd_h_ml388.t13_1_1_1_4" colspan="2" rowspan="1" style="text-align:center;vertical-align:middle;">Prostagladin E<sub>2</sub> (PGE<sub>2</sub>) Assay in A549 cells</th><th id="hd_h_ml388.t13_1_1_1_5" colspan="2" rowspan="1" style="text-align:center;vertical-align:middle;">Prostagladin E<sub>2</sub> (PGE<sub>2</sub>) Assay in LNCaP cells</th></tr><tr><th headers="hd_h_ml388.t13_1_1_1_3" id="hd_h_ml388.t13_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">IC<sub>50</sub> (nM)</th><th headers="hd_h_ml388.t13_1_1_1_4" id="hd_h_ml388.t13_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">AC<sub>50</sub> (μM)</th><th headers="hd_h_ml388.t13_1_1_1_4" id="hd_h_ml388.t13_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">% Induction</th><th headers="hd_h_ml388.t13_1_1_1_5" id="hd_h_ml388.t13_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">AC<sub>50</sub> (μM)</th><th headers="hd_h_ml388.t13_1_1_1_5" id="hd_h_ml388.t13_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">% Induction</th></tr></thead><tbody><tr><td headers="hd_h_ml388.t13_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="/pcsubstance/?term=ML149[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML149</a>
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<b>(1)</b></td><td headers="hd_h_ml388.t13_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2331284</td><td headers="hd_h_ml388.t13_1_1_1_3 hd_h_ml388.t13_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">54</td><td headers="hd_h_ml388.t13_1_1_1_4 hd_h_ml388.t13_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.743</td><td headers="hd_h_ml388.t13_1_1_1_4 hd_h_ml388.t13_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">106</td><td headers="hd_h_ml388.t13_1_1_1_5 hd_h_ml388.t13_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2.797</td><td headers="hd_h_ml388.t13_1_1_1_5 hd_h_ml388.t13_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">73</td></tr><tr><td headers="hd_h_ml388.t13_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b>2</b></td><td headers="hd_h_ml388.t13_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">71307587</td><td headers="hd_h_ml388.t13_1_1_1_3 hd_h_ml388.t13_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">22</td><td headers="hd_h_ml388.t13_1_1_1_4 hd_h_ml388.t13_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.296</td><td headers="hd_h_ml388.t13_1_1_1_4 hd_h_ml388.t13_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">110</td><td headers="hd_h_ml388.t13_1_1_1_5 hd_h_ml388.t13_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.352</td><td headers="hd_h_ml388.t13_1_1_1_5 hd_h_ml388.t13_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">105</td></tr><tr><td headers="hd_h_ml388.t13_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b>3</b></td><td headers="hd_h_ml388.t13_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">71307858</td><td headers="hd_h_ml388.t13_1_1_1_3 hd_h_ml388.t13_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">34</td><td headers="hd_h_ml388.t13_1_1_1_4 hd_h_ml388.t13_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.590</td><td headers="hd_h_ml388.t13_1_1_1_4 hd_h_ml388.t13_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">114</td><td headers="hd_h_ml388.t13_1_1_1_5 hd_h_ml388.t13_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.558</td><td headers="hd_h_ml388.t13_1_1_1_5 hd_h_ml388.t13_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">68</td></tr><tr><td headers="hd_h_ml388.t13_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b>7</b></td><td headers="hd_h_ml388.t13_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">71307838</td><td headers="hd_h_ml388.t13_1_1_1_3 hd_h_ml388.t13_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">48</td><td headers="hd_h_ml388.t13_1_1_1_4 hd_h_ml388.t13_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2.093</td><td headers="hd_h_ml388.t13_1_1_1_4 hd_h_ml388.t13_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">145</td><td headers="hd_h_ml388.t13_1_1_1_5 hd_h_ml388.t13_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2.222</td><td headers="hd_h_ml388.t13_1_1_1_5 hd_h_ml388.t13_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">44</td></tr><tr><td headers="hd_h_ml388.t13_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="/pcsubstance/?term=ML388[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML388</a>
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<b>(9)</b></td><td headers="hd_h_ml388.t13_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">71307851</td><td headers="hd_h_ml388.t13_1_1_1_3 hd_h_ml388.t13_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">34</td><td headers="hd_h_ml388.t13_1_1_1_4 hd_h_ml388.t13_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.166</td><td headers="hd_h_ml388.t13_1_1_1_4 hd_h_ml388.t13_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">107</td><td headers="hd_h_ml388.t13_1_1_1_5 hd_h_ml388.t13_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.703</td><td headers="hd_h_ml388.t13_1_1_1_5 hd_h_ml388.t13_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">71</td></tr><tr><td headers="hd_h_ml388.t13_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b>10</b></td><td headers="hd_h_ml388.t13_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">71307843</td><td headers="hd_h_ml388.t13_1_1_1_3 hd_h_ml388.t13_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">76</td><td headers="hd_h_ml388.t13_1_1_1_4 hd_h_ml388.t13_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2.635</td><td headers="hd_h_ml388.t13_1_1_1_4 hd_h_ml388.t13_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">350</td><td headers="hd_h_ml388.t13_1_1_1_5 hd_h_ml388.t13_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1.765</td><td headers="hd_h_ml388.t13_1_1_1_5 hd_h_ml388.t13_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">63</td></tr><tr><td headers="hd_h_ml388.t13_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="/pcsubstance/?term=ML148[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML148</a>
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<b>(27)</b></td><td headers="hd_h_ml388.t13_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3243760</td><td headers="hd_h_ml388.t13_1_1_1_3 hd_h_ml388.t13_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">19</td><td headers="hd_h_ml388.t13_1_1_1_4 hd_h_ml388.t13_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.469</td><td headers="hd_h_ml388.t13_1_1_1_4 hd_h_ml388.t13_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">129</td><td headers="hd_h_ml388.t13_1_1_1_5 hd_h_ml388.t13_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.884</td><td headers="hd_h_ml388.t13_1_1_1_5 hd_h_ml388.t13_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">62</td></tr><tr><td headers="hd_h_ml388.t13_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b>31</b></td><td headers="hd_h_ml388.t13_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">71307871</td><td headers="hd_h_ml388.t13_1_1_1_3 hd_h_ml388.t13_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">68</td><td headers="hd_h_ml388.t13_1_1_1_4 hd_h_ml388.t13_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.743</td><td headers="hd_h_ml388.t13_1_1_1_4 hd_h_ml388.t13_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">127</td><td headers="hd_h_ml388.t13_1_1_1_5 hd_h_ml388.t13_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">4.433</td><td headers="hd_h_ml388.t13_1_1_1_5 hd_h_ml388.t13_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">53</td></tr><tr><td headers="hd_h_ml388.t13_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b>32</b></td><td headers="hd_h_ml388.t13_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">71307813</td><td headers="hd_h_ml388.t13_1_1_1_3 hd_h_ml388.t13_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">68</td><td headers="hd_h_ml388.t13_1_1_1_4 hd_h_ml388.t13_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1.321</td><td headers="hd_h_ml388.t13_1_1_1_4 hd_h_ml388.t13_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">121</td><td headers="hd_h_ml388.t13_1_1_1_5 hd_h_ml388.t13_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1.765</td><td headers="hd_h_ml388.t13_1_1_1_5 hd_h_ml388.t13_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">62</td></tr><tr><td headers="hd_h_ml388.t13_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b>44</b></td><td headers="hd_h_ml388.t13_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">71307865</td><td headers="hd_h_ml388.t13_1_1_1_3 hd_h_ml388.t13_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">12</td><td headers="hd_h_ml388.t13_1_1_1_4 hd_h_ml388.t13_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.372</td><td headers="hd_h_ml388.t13_1_1_1_4 hd_h_ml388.t13_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">170</td><td headers="hd_h_ml388.t13_1_1_1_5 hd_h_ml388.t13_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.222</td><td headers="hd_h_ml388.t13_1_1_1_5 hd_h_ml388.t13_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">85</td></tr><tr><td headers="hd_h_ml388.t13_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b>46</b></td><td headers="hd_h_ml388.t13_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">71307863</td><td headers="hd_h_ml388.t13_1_1_1_3 hd_h_ml388.t13_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">48</td><td headers="hd_h_ml388.t13_1_1_1_4 hd_h_ml388.t13_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2.635</td><td headers="hd_h_ml388.t13_1_1_1_4 hd_h_ml388.t13_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">173</td><td headers="hd_h_ml388.t13_1_1_1_5 hd_h_ml388.t13_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2.797</td><td headers="hd_h_ml388.t13_1_1_1_5 hd_h_ml388.t13_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">70</td></tr><tr><td headers="hd_h_ml388.t13_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="/pcsubstance/?term=ML387[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML387</a>
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<b>(51)</b></td><td headers="hd_h_ml388.t13_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3474778</td><td headers="hd_h_ml388.t13_1_1_1_3 hd_h_ml388.t13_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">19</td><td headers="hd_h_ml388.t13_1_1_1_4 hd_h_ml388.t13_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.662</td><td headers="hd_h_ml388.t13_1_1_1_4 hd_h_ml388.t13_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">174</td><td headers="hd_h_ml388.t13_1_1_1_5 hd_h_ml388.t13_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.884</td><td headers="hd_h_ml388.t13_1_1_1_5 hd_h_ml388.t13_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">69</td></tr></tbody></table></div></div></div><div id="ml388.s34"><h3>3.3. Profiling Assays</h3><div id="ml388.s35"><h4>3.3.1. <i>In-vitro</i> ADME profiles of ML387 and ML388</h4><div id="ml388.t14" class="table"><h3><span class="label">Table 11</span><span class="title"><i>In vitro</i> ADME profiles of <b>ML387</b> and of <b>ML388</b></span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK280041/table/ml388.t14/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml388.t14_lrgtbl__"><table class="no_margin"><thead><tr><th id="hd_h_ml388.t14_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Compound</th><th id="hd_h_ml388.t14_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">PAMPA<sup><a class="bk_pop" href="#ml388.tfn1">a</a></sup>Permeability at pH 7.4</th><th id="hd_h_ml388.t14_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Aqueous Kinetic Solubility in PBS Buffer pH 7.4</th><th id="hd_h_ml388.t14_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Rat Liver Microsomal Stability<sup><a class="bk_pop" href="#ml388.tfn2">b</a></sup>T<sub>1/2</sub></th></tr></thead><tbody><tr><td headers="hd_h_ml388.t14_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="/pcsubstance/?term=ML387[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML387</a></td><td headers="hd_h_ml388.t14_1_1_1_2" rowspan="1" colspan="1" style="background-color:#FFC000;text-align:center;vertical-align:middle;">624 × 10<sup>-6</sup> cm/s</td><td headers="hd_h_ml388.t14_1_1_1_3" rowspan="1" colspan="1" style="background-color:#00B050;text-align:center;vertical-align:middle;">> 50 μM</td><td headers="hd_h_ml388.t14_1_1_1_4" rowspan="1" colspan="1" style="background-color:#00B050;text-align:center;vertical-align:middle;">23 min</td></tr><tr><td headers="hd_h_ml388.t14_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="/pcsubstance/?term=ML388[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML388</a></td><td headers="hd_h_ml388.t14_1_1_1_2" rowspan="1" colspan="1" style="background-color:#00B050;text-align:center;vertical-align:middle;">1330 × 10<sup>-6</sup> cm/s</td><td headers="hd_h_ml388.t14_1_1_1_3" rowspan="1" colspan="1" style="background-color:#00B050;text-align:center;vertical-align:middle;">28 μM</td><td headers="hd_h_ml388.t14_1_1_1_4" rowspan="1" colspan="1" style="background-color:#FF0000;text-align:center;vertical-align:middle;">1.8 min</td></tr></tbody></table></div><div><div><dl class="temp-labeled-list small"><dt>a</dt><dd><div id="ml388.tfn1"><p class="no_margin">PAMPA: Parallel Artificial Membrane Permeability Assay; the assay measures the passive transport of the probes through a lipid bilayer.</p></div></dd><dt>b</dt><dd><div id="ml388.tfn2"><p class="no_margin">represents the stability in the presence of NADPH. The probes showed no degradation without NADPH over a 1 h period.</p></div></dd></dl></div></div></div></div><div id="ml388.s36"><h4>3.3.1. Selectivity profiles of ML387 and ML388</h4><div id="ml388.t15" class="table"><h3><span class="label">Table 12</span><span class="title">Inhibitory activities of <b>ML387</b> and of <b>ML388</b> and related analogs versus a panel of dehydrogenases</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK280041/table/ml388.t15/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml388.t15_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml388.t15_1_1_1_1" rowspan="2" colspan="1" headers="hd_h_ml388.t15_1_1_1_1" style="text-align:center;vertical-align:middle;">Compound</th><th id="hd_h_ml388.t15_1_1_1_2" rowspan="2" colspan="1" headers="hd_h_ml388.t15_1_1_1_2" style="text-align:center;vertical-align:middle;">CID</th><th id="hd_h_ml388.t15_1_1_1_3" colspan="6" rowspan="1" style="text-align:center;vertical-align:middle;">IC<sub>50</sub> (nM)</th></tr><tr><th headers="hd_h_ml388.t15_1_1_1_3" id="hd_h_ml388.t15_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">HPGD</th><th headers="hd_h_ml388.t15_1_1_1_3" id="hd_h_ml388.t15_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">ALDH1A1</th><th headers="hd_h_ml388.t15_1_1_1_3" id="hd_h_ml388.t15_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">LDHA</th><th headers="hd_h_ml388.t15_1_1_1_3" id="hd_h_ml388.t15_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">LDHB</th><th headers="hd_h_ml388.t15_1_1_1_3" id="hd_h_ml388.t15_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">rGAPDH</th><th headers="hd_h_ml388.t15_1_1_1_3" id="hd_h_ml388.t15_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">HSD17β4</th></tr></thead><tbody><tr><td headers="hd_h_ml388.t15_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>2</b></td><td headers="hd_h_ml388.t15_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">71307587</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">22</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">607</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">inactive</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">inactive</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">inactive</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">inactive</td></tr><tr><td headers="hd_h_ml388.t15_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>3</b></td><td headers="hd_h_ml388.t15_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">71307858</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">34</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">4818</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">inactive</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">inactive</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">inactive</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">inactive</td></tr><tr><td headers="hd_h_ml388.t15_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>7</b></td><td headers="hd_h_ml388.t15_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">71307838</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">48</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">1524</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">inactive</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">inactive</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">inactive</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">inactive</td></tr><tr><td headers="hd_h_ml388.t15_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="/pcsubstance/?term=ML388[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML388</a>
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<b>(9)</b></td><td headers="hd_h_ml388.t15_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">71307851</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">34</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">2415</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">inactive</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">inactive</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">inactive</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">inactive</td></tr><tr><td headers="hd_h_ml388.t15_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>10</b></td><td headers="hd_h_ml388.t15_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">71307843</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">76</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">6806</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">inactive</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">inactive</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">inactive</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">inactive</td></tr><tr><td headers="hd_h_ml388.t15_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>31</b></td><td headers="hd_h_ml388.t15_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">71307871</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">68</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">1918</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">inactive</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">inactive</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">inactive</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">inactive</td></tr><tr><td headers="hd_h_ml388.t15_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>32</b></td><td headers="hd_h_ml388.t15_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">71307813</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">68</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">6806</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">inactive</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">inactive</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">inactive</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">inactive</td></tr><tr><td headers="hd_h_ml388.t15_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>44</b></td><td headers="hd_h_ml388.t15_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">71307865</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">12</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">1918</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">inactive</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">inactive</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">inactive</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">inactive</td></tr><tr><td headers="hd_h_ml388.t15_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>46</b></td><td headers="hd_h_ml388.t15_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">71307863</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">48</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">2415</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">inactive</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">inactive</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">inactive</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">inactive</td></tr><tr><td headers="hd_h_ml388.t15_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="/pcsubstance/?term=ML387[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML387</a>
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<b>(51)</b></td><td headers="hd_h_ml388.t15_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">3474778</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">19</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">1358</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">inactive</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">inactive</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">inactive</td><td headers="hd_h_ml388.t15_1_1_1_3 hd_h_ml388.t15_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">inactive</td></tr></tbody></table></div></div></div></div></div><div id="ml388.s37"><h2 id="_ml388_s37_">4. Discussion</h2><div id="ml388.s38"><h3>4.1. Comparison to existing art and how the new probe is an improvement</h3><p><a href="/pcsubstance/?term=ML387[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML387</a> and <a href="/pcsubstance/?term=ML388[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML388</a> are two inhibitors of HPGD with enzymatic activities in the nanomolar range; IC<sub>50</sub> = 19 nM for <a href="/pcsubstance/?term=ML387[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML387</a> and IC<sub>50</sub> = 34 nM for <a href="/pcsubstance/?term=ML388[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML388</a>. In that respect, they are a slight improvement to CT-8, which has a reported IC<sub>50</sub> value of 51 nM.<a class="bk_pop" href="#ml388.r1">1</a> To the best of our knowledge, the selectivity of CT-8 towards HGPD has not been reported, and the thiazolidinedione chemotype is known to be highly promiscuous. Our two probes displayed excellent selectivity toward five other dehydrogenases (<a class="figpopup" href="/books/NBK280041/table/ml388.t15/?report=objectonly" target="object" rid-figpopup="figml388t15" rid-ob="figobml388t15">Table 12</a>). Furthermore, while the ADME properties of <a href="/pcsubstance/?term=ML387[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML387</a> and <a href="/pcsubstance/?term=ML388[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML388</a> were assessed, the ADME properties of CT-8 have not been reported. Finally, we demonstrated that HPGD inhibition using <a href="/pcsubstance/?term=ML387[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML387</a> and <a href="/pcsubstance/?term=ML388[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML388</a> potentiated PGE<sub>2</sub> production in two cell lines in a dose-dependent fashion. In order to measure PGE<sub>2</sub> production levels, Wu <i>et al</i>. treated A549 cells with a high concentration of CT-8 (5 μM).<a class="bk_pop" href="#ml388.r2">2</a> Our assay provided the measurement of PGE<sub>2</sub> production in A549 and LNCaP cells in a dose-dependent fashion, leading to the measurement of a half-maximum activity concentration. Finally, the PGE<sub>2</sub> percentage induction was calculated to account for the difference of prostaglandin production, as compared to its basal levels.</p></div></div><div id="ml388.s39"><h2 id="_ml388_s39_">5. References</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="ml388.r1">Cho H, Tai H-H. <span><span class="ref-journal">Prostaglandins Leukot. Essent. Fatty Acids. </span>2002;<span class="ref-vol">67</span>:461.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/12468268" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 12468268</span></a>]</div></dd><dt>2.</dt><dd><div class="bk_ref" id="ml388.r2">Wu Y, Karna S, Choi CH, Tong M, Tai H-H, Na DH, Jang CH, Cho H. <span><span class="ref-journal">J. Med. Chem. </span>2011;<span class="ref-vol">54</span>:5260.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/21650226" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 21650226</span></a>]</div></dd></dl></div><div id="bk_toc_contnr"></div></div></div>
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<div xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance"></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Views</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="PDF_download" id="Shutter"></a></div><div class="portlet_content"><ul xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="simple-list"><li><a href="/books/NBK280041/?report=reader">PubReader</a></li><li><a href="/books/NBK280041/?report=printable">Print View</a></li><li><a data-jig="ncbidialog" href="#_ncbi_dlg_citbx_NBK280041" data-jigconfig="width:400,modal:true">Cite this Page</a><div id="_ncbi_dlg_citbx_NBK280041" style="display:none" title="Cite this Page"><div class="bk_tt">Duveau DY, Yasgar A, Hu X, et al. Discovery of two small molecule inhibitors, ML387 and ML388, of human NAD+-dependent 15-hydroxyprostaglandin dehydrogenase. 2013 Dec 15 [Updated 2015 Feb 11]. 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="#ml388.s1" ref="log$=inpage&link_id=inpage">Probe Structure & Characteristics</a></li><li><a href="#ml388.s2" ref="log$=inpage&link_id=inpage">Recommendations for scientific use of the probe</a></li><li><a href="#ml388.s3" ref="log$=inpage&link_id=inpage">Materials and Methods</a></li><li><a href="#ml388.s31" ref="log$=inpage&link_id=inpage">Results</a></li><li><a href="#ml388.s37" ref="log$=inpage&link_id=inpage">Discussion</a></li><li><a href="#ml388.s39" ref="log$=inpage&link_id=inpage">References</a></li></ul></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Related information</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="discovery_db_links" id="Shutter"></a></div><div class="portlet_content"><ul><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pcsubstance&DbFrom=books&Cmd=Link&LinkName=books_pcsubstance&IdsFromResult=3412276" ref="log$=recordlinks">PubChem Substance</a><div class="brieflinkpop offscreen_noflow">Related PubChem Substances</div></li><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pubmed&DbFrom=books&Cmd=Link&LinkName=books_pubmed_refs&IdsFromResult=3412276" ref="log$=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/21735612" ref="ordinalpos=1&linkpos=1&log$=relatedreviews&logdbfrom=pubmed"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> Potent and selective inhibitors of NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (HPGD).</a><span class="source">[Probe Reports from the NIH Mol...]</span><div class="brieflinkpop offscreen_noflow"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> Potent and selective inhibitors of NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (HPGD).<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Jadhav A, Niesen FH, Schultz L, Oppermann U, Maloney DJ, Simeonov A. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Probe Reports from the NIH Molecular Libraries Program. 2010</em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/24360556" ref="ordinalpos=1&linkpos=2&log$=relatedarticles&logdbfrom=pubmed">Structure-activity relationship studies and biological characterization of human NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase inhibitors.</a><span class="source">[Bioorg Med Chem Lett. 2014]</span><div class="brieflinkpop offscreen_noflow">Structure-activity relationship studies and biological characterization of human NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase inhibitors.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Duveau DY, Yasgar A, Wang Y, Hu X, Kouznetsova J, Brimacombe KR, Jadhav A, Simeonov A, Thomas CJ, Maloney DJ. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Bioorg Med Chem Lett. 2014 Jan 15; 24(2):630-5. Epub 2013 Dec 4.</em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/26068950" ref="ordinalpos=1&linkpos=3&log$=relatedarticles&logdbfrom=pubmed">miR-620 promotes tumor radioresistance by targeting 15-hydroxyprostaglandin dehydrogenase (HPGD).</a><span class="source">[Oncotarget. 2015]</span><div class="brieflinkpop offscreen_noflow">miR-620 promotes tumor radioresistance by targeting 15-hydroxyprostaglandin dehydrogenase (HPGD).<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Huang X, Taeb S, Jahangiri S, Korpela E, Cadonic I, Yu N, Krylov SN, Fokas E, Boutros PC, Liu SK. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Oncotarget. 2015 Sep 8; 6(26):22439-51. </em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/15908215" ref="ordinalpos=1&linkpos=4&log$=relatedarticles&logdbfrom=pubmed">Understanding human 15-hydroxyprostaglandin dehydrogenase binding with NAD+ and PGE2 by homology modeling, docking and molecular dynamics simulation.</a><span class="source">[Bioorg Med Chem. 2005]</span><div class="brieflinkpop offscreen_noflow">Understanding human 15-hydroxyprostaglandin dehydrogenase binding with NAD+ and PGE2 by homology modeling, docking and molecular dynamics simulation.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Hamza A, Cho H, Tai HH, Zhan CG. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Bioorg Med Chem. 2005 Jul 15; 13(14):4544-51. </em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/25968070" 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> Close teamwork between Nrf2 and peroxiredoxins 1 and 6 for the regulation of prostaglandin D2 and E2 production in macrophages in acute inflammation.</a><span class="source">[Free Radic Biol Med. 2015]</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> Close teamwork between Nrf2 and peroxiredoxins 1 and 6 for the regulation of prostaglandin D2 and E2 production in macrophages in acute inflammation.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Ishii T. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Free Radic Biol Med. 2015 Nov; 88(Pt B):189-198. 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