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<meta name="ncbi_db" content="books" /><meta name="ncbi_pdid" content="book-part" /><meta name="ncbi_acc" content="NBK51967" /><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="/NBK51967/" /><meta name="ncbi_pagename" content="Probe Development Efforts to Identify Novel Antagonists of the Sphingosine 1-phosphate Receptor 4 (S1P4) - 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>Probe Development Efforts to Identify Novel Antagonists of the Sphingosine 1-phosphate Receptor 4 (S1P4) - 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="Probe Development Efforts to Identify Novel Antagonists of the Sphingosine 1-phosphate Receptor 4 (S1P4)" /><meta name="citation_publisher" content="National Center for Biotechnology Information (US)" /><meta name="citation_date" content="2010/12/16" /><meta name="citation_author" content="M Oldstone" /><meta name="citation_author" content="P Hodder" /><meta name="citation_author" content="M Crisp" /><meta name="citation_author" content="E Roberts" /><meta name="citation_author" content="M Guerrero" /><meta name="citation_author" content="M Urbano" /><meta name="citation_author" content="S Velaparthi" /><meta name="citation_author" content="J Zhao" /><meta name="citation_author" content="H Rosen" /><meta name="citation_author" content="MT Schaeffer" /><meta name="citation_author" content="S Brown" /><meta name="citation_author" content="J Ferguson" /><meta name="citation_pmid" content="21433398" /><meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK51967/" /><link rel="schema.DC" href="http://purl.org/DC/elements/1.0/" /><meta name="DC.Title" content="Probe Development Efforts to Identify Novel Antagonists of the Sphingosine 1-phosphate Receptor 4 (S1P4)" /><meta name="DC.Type" content="Text" /><meta name="DC.Publisher" content="National Center for Biotechnology Information (US)" /><meta name="DC.Contributor" content="M Oldstone" /><meta name="DC.Contributor" content="P Hodder" /><meta name="DC.Contributor" content="M Crisp" /><meta name="DC.Contributor" content="E Roberts" /><meta name="DC.Contributor" content="M Guerrero" /><meta name="DC.Contributor" content="M Urbano" /><meta name="DC.Contributor" content="S Velaparthi" /><meta name="DC.Contributor" content="J Zhao" /><meta name="DC.Contributor" content="H Rosen" /><meta name="DC.Contributor" content="MT Schaeffer" /><meta name="DC.Contributor" content="S Brown" /><meta name="DC.Contributor" content="J Ferguson" /><meta name="DC.Date" content="2010/12/16" /><meta name="DC.Identifier" content="https://www.ncbi.nlm.nih.gov/books/NBK51967/" /><meta name="description" content="Pandemic influenza represents a significant public health threat, due in part to immune cell-mediated lung tissue damage induced during viral infection. Sphingosine 1-phosphate (S1P), a bioactive phospholipid released by activated blood platelets, serves to influence endothelial integrity, lung epithelial integrity and lymphocyte recirculation through five related high affinity G-protein coupled receptors. Recent studies suggest that chemical activation of the S1P4 receptor subtype in the airways could be efficient at controlling the immunopathological response to viral infection. S1P4 is coupled to Gαi and Gαo G-proteins and activates ERK MAPK and PLC downstream pathways, indicating that selective antagonists of S1P4 may also serve as useful tools for understanding S1P4 biological function. The Scripps Research Institute Molecular Screening Center (SRIMSC), part of the Molecular Libraries Probe Production Centers Network (MLPCN), identified a selective S1P4 antagonist probe, ML131, by high-throughput screening employing cell-based assays, and SAR analysis of selected screening hits. Probe ML131 inhibits S1P4 receptor in a cell-based TANGO-format assay with an IC50 of 89 nM, is inactive against other members of the S1P receptor family with IC50s >25 µM against S1P1-S1P3 and S1P5 receptors, and is nontoxic to U2OS cells. Probe ML131 was profiled against a panel of potential receptors, transporters, and ion channels; the results suggest that probe ML131 is generally inactive against a broad array of off targets and does not likely exert unwanted effects. There are no S1P4 antagonist compounds currently available. Dose response assays against all five S1P receptors demonstrate that probe ML131 is the first submicromolar, completely selective S1P4 receptor antagonist to be identified." /><meta name="og:title" content="Probe Development Efforts to Identify Novel Antagonists of the Sphingosine 1-phosphate Receptor 4 (S1P4)" /><meta name="og:type" content="book" /><meta name="og:description" content="Pandemic influenza represents a significant public health threat, due in part to immune cell-mediated lung tissue damage induced during viral infection. Sphingosine 1-phosphate (S1P), a bioactive phospholipid released by activated blood platelets, serves to influence endothelial integrity, lung epithelial integrity and lymphocyte recirculation through five related high affinity G-protein coupled receptors. Recent studies suggest that chemical activation of the S1P4 receptor subtype in the airways could be efficient at controlling the immunopathological response to viral infection. S1P4 is coupled to Gαi and Gαo G-proteins and activates ERK MAPK and PLC downstream pathways, indicating that selective antagonists of S1P4 may also serve as useful tools for understanding S1P4 biological function. The Scripps Research Institute Molecular Screening Center (SRIMSC), part of the Molecular Libraries Probe Production Centers Network (MLPCN), identified a selective S1P4 antagonist probe, ML131, by high-throughput screening employing cell-based assays, and SAR analysis of selected screening hits. Probe ML131 inhibits S1P4 receptor in a cell-based TANGO-format assay with an IC50 of 89 nM, is inactive against other members of the S1P receptor family with IC50s >25 µM against S1P1-S1P3 and S1P5 receptors, and is nontoxic to U2OS cells. Probe ML131 was profiled against a panel of potential receptors, transporters, and ion channels; the results suggest that probe ML131 is generally inactive against a broad array of off targets and does not likely exert unwanted effects. There are no S1P4 antagonist compounds currently available. Dose response assays against all five S1P receptors demonstrate that probe ML131 is the first submicromolar, completely selective S1P4 receptor antagonist to be identified." /><meta name="og:url" content="https://www.ncbi.nlm.nih.gov/books/NBK51967/" /><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/ml131/" /><link rel="canonical" href="https://www.ncbi.nlm.nih.gov/books/NBK51967/" /><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="#__NBK51967_dtls__">Show details</a><div style="display:none" class="ui-widget" id="__NBK51967_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/ml132/" title="Previous page in this title">< Prev</a><a class="active page_link next" href="/books/n/mlprobe/ml130/" 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="_NBK51967_"><span class="title" itemprop="name">Probe Development Efforts to Identify Novel Antagonists of the Sphingosine 1-phosphate Receptor 4 (S1P4)</span></h1><p class="contrib-group"><span itemprop="author">M Oldstone</span>, <span itemprop="author">P Hodder</span>, <span itemprop="author">M Crisp</span>, <span itemprop="author">E Roberts</span>, <span itemprop="author">M Guerrero</span>, <span itemprop="author">M Urbano</span>, <span itemprop="author">S Velaparthi</span>, <span itemprop="author">J Zhao</span>, <span itemprop="author">H Rosen</span>, <span itemprop="author">MT Schaeffer</span>, <span itemprop="author">S Brown</span>, and <span itemprop="author">J Ferguson</span>.</p><a data-jig="ncbitoggler" href="#__NBK51967_ai__" style="border:0;text-decoration:none">Author Information and Affiliations</a><div style="display:none" class="ui-widget" id="__NBK51967_ai__"><p class="contrib-group"><h4>Authors</h4><span itemprop="author">M Oldstone</span>,<sup>*</sup> <span itemprop="author">P Hodder</span>,<sup>†</sup> <span itemprop="author">M Crisp</span>,<sup>†</sup> <span itemprop="author">E Roberts</span>,<sup>‡</sup> <span itemprop="author">M Guerrero</span>,<sup>‡</sup> <span itemprop="author">M Urbano</span>,<sup>‡</sup> <span itemprop="author">S Velaparthi</span>,<sup>‡</sup> <span itemprop="author">J Zhao</span>,<sup>‡</sup> <span itemprop="author">H Rosen</span>,<sup>**</sup> <span itemprop="author">MT Schaeffer</span>,<sup>**</sup> <span itemprop="author">S Brown</span>,<sup>**</sup> and <span itemprop="author">J Ferguson</span><sup>**</sup>.</p><h4>Affiliations</h4><div class="affiliation"><sup>*</sup>
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Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla CA 92037</div><div class="affiliation"><sup>†</sup>
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Department of Chemistry, The Scripps Research Institute, Jupiter, FL, 33458</div><div class="affiliation"><sup>‡</sup>
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Department of Chemistry, The Scripps Research Institute, La Jolla CA 92037</div><div class="affiliation"><sup>**</sup>
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Department of Chemical Physiology, The Scripps Research Institute Molecular Screening Center, The Scripps Research Institute, La Jolla, CA 92037</div></div><p class="small">Received: <span itemprop="datePublished">February 24, 2010</span>; Last Update: <span itemprop="dateModified">December 16, 2010</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>Pandemic influenza represents a significant public health threat, due in part to immune cell-mediated lung tissue damage induced during viral infection. Sphingosine 1-phosphate (S1P), a bioactive phospholipid released by activated blood platelets, serves to influence endothelial integrity, lung epithelial integrity and lymphocyte recirculation through five related high affinity G-protein coupled receptors. Recent studies suggest that chemical activation of the S1P4 receptor subtype in the airways could be efficient at controlling the immunopathological response to viral infection. S1P4 is coupled to Gαi and Gαo G-proteins and activates ERK MAPK and PLC downstream pathways, indicating that selective antagonists of S1P4 may also serve as useful tools for understanding S1P4 biological function. The Scripps Research Institute Molecular Screening Center (SRIMSC), part of the Molecular Libraries Probe Production Centers Network (MLPCN), identified a selective S1P4 antagonist probe, ML131, by high-throughput screening employing cell-based assays, and SAR analysis of selected screening hits. Probe ML131 inhibits S1P4 receptor in a cell-based TANGO-format assay with an IC50 of 89 nM, is inactive against other members of the S1P receptor family with IC50s >25 µM against S1P1-S1P3 and S1P5 receptors, and is nontoxic to U2OS cells. Probe ML131 was profiled against a panel of potential receptors, transporters, and ion channels; the results suggest that probe ML131 is generally inactive against a broad array of off targets and does not likely exert unwanted effects. There are no S1P4 antagonist compounds currently available. Dose response assays against all five S1P receptors demonstrate that probe ML131 is the first submicromolar, completely selective S1P4 receptor antagonist to be identified.</p></div><div class="h2"></div><p><b>Assigned Assay Grant #:</b> 1 U01 <a href="/nuccore/3401208" class="bk_tag" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=nuccore">AI074564</a></p><p><b>Screening Center Name & PI:</b> Scripps Research Institute Molecular Screening Center (SRIMSC), H Rosen</p><p><b>Chemistry Center Name & PI</b>: Scripps Research Institute Molecular Screening Center (SRIMSC), H Rosen</p><p><b>Assay Submitter & Institution:</b> Michael Oldstone, The Scripps Research Institute</p><p><b>PubChem Summary Bioassay Identifier (AID):</b>
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<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1853" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1853</a></p><div id="ml131.s1"><h2 id="_ml131_s1_">Probe Structure & Characteristics</h2><div id="ml131.fu1" class="figure"><div class="graphic"><img src="/books/NBK51967/bin/ml131fu1.jpg" alt="Image ml131fu1" /></div></div><div id="ml131.tu1" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK51967/table/ml131.tu1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml131.tu1_lrgtbl__"><table><thead><tr><th id="hd_h_ml131.tu1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">CID/ML#</th><th id="hd_h_ml131.tu1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Target Name</th><th id="hd_h_ml131.tu1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">IC50 (nM) [SID, AID]</th><th id="hd_h_ml131.tu1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Anti-target Name(s)</th><th id="hd_h_ml131.tu1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">IC50 (μM) [SID, AID]</th><th id="hd_h_ml131.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Fold Selective</th><th id="hd_h_ml131.tu1_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Secondary Assay(s) Name:<br />IC50 (µM) [SID, AID]</th></tr></thead><tbody><tr><td headers="hd_h_ml131.tu1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">CID-44607580/<a href="/pcsubstance/?term=ML131[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML131</a></td><td headers="hd_h_ml131.tu1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">S1P4</td><td headers="hd_h_ml131.tu1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">89 nM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/87357351" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-87357351</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2346" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-2346</a>]</td><td headers="hd_h_ml131.tu1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">S1P1</td><td headers="hd_h_ml131.tu1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">>25 µM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/87357351" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-87357351</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2351" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-2351</a>]</td><td headers="hd_h_ml131.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">>280</td><td headers="hd_h_ml131.tu1_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><b>S1P2 IC50:</b> >25 µM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/87357351" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-87357351</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2354" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-2354</a>]<br /><b>S1P3 IC50:</b> >25 µM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/87357351" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-87357351</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2349" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-2349</a>]<br /><b>S1P5 IC50:</b> >25 µM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/87357351" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-87357351</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2350" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-2350</a>]<br /><b>Cytotoxicity:</b> CC50 >20 µM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/87357351" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-87357351</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/489009" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-489009</a>]</td></tr></tbody></table></div></div></div><div id="ml131.s2"><h2 id="_ml131_s2_">Recommendations for scientific use of the probe</h2><p>These probes are useful for assays aiming to modulate S1P4 signaling without affecting S1P1, and will be invaluable in research efforts designed to determine the effects of S1P4 signaling. The probe reported herein represents a significant milestone that will allow experiments designed to improve understanding of the role of the S1P4 receptor in fundamental immune system processes, such as dendritic cell function, T-cell migration and other S1P-mediated biology. The S1P4 antagonist probe has already been provided under MTA to an expert in lymphocyte trafficking, Professor Antal Rot, of the MRC Centre for Immune Regulation, University of Birmingham.</p></div><div id="ml131.s3"><h2 id="_ml131_s3_">1. Introduction</h2><p>Pandemic influenza represents a significant public health threat, due in part to immune cell-mediated lung tissue damage induced during viral infection. Sphingosine 1-phosphate (S1P) is a bioactive phospholipid released by activated blood platelets and serves to influence endothelial integrity, lung epithelial integrity (<a class="bk_pop" href="#ml131.r1">1</a>, <a class="bk_pop" href="#ml131.r2">2</a>) and lymphocyte recirculation (<a class="bk_pop" href="#ml131.r3">3</a>–<a class="bk_pop" href="#ml131.r6">6</a>) through five related high affinity G-protein coupled receptors. Recently, modulation of S1P receptors locally in the lungs was shown to alter dendritic cell activation and accumulation in the mediastinal lymph nodes, resulting in blunted T-cell responses and control of immunopathological features of influenza virus infection (<a class="bk_pop" href="#ml131.r7">7</a>). Reports showing that S1P5 expression is very low in dendritic cells, while S1P4 is highly expressed (<a class="bk_pop" href="#ml131.r8">8</a>), suggest that chemical activation of the S1P4 receptor subtype in the airways could be efficient at controlling the immunopathological response to viral infection. S1P4 is coupled to Gαi and Gαo G-proteins and activates ERK MAPK and PLC downstream pathways (<a class="bk_pop" href="#ml131.r9">9</a>), indicating that selective antagonists of S1P4 may also serve as useful tools for understanding S1P4 biological function. There are no reports of selective inhibitor S1P4 antagonists in the literature and S1P4 antagonist compounds are not available. With the exception of the PubChem AIDs for this project, no S1P4 antagonist HTS efforts have been reported to date. Exhaustive search of the published literature—including U.S. and International Patent literature—has not identified any S1P4 antagonist compounds.</p></div><div id="ml131.s4"><h2 id="_ml131_s4_">2. Materials and Methods</h2><p>The following reagents were obtained from Invitrogen: Tango EDG6-bla U2OS cells (K1622), Tango EDG-1-bla U2OS cells (K1520), Tango EDG8 bla U2OS cells (K1518), GeneBLAzer FRET B/G Loading Kit (CCF4-AM) (K1025), LiveBLAzer (K1096), Freestyle Expression Medium (Assay media; 12338-018), McCoy’s 5A Medium (modified) (1X) (16600-082), Dulbecco’s Modified Eagle’s Media with phenol red (11965-092), Dulbecco’s Modified Eagle’s Media without phenol red (21063-029), Fetal Bovine Serum (26400-044), Fetal Bovine Serum, dialyzed (26400-036), NEAA (1114-050), Penicillin-Streptomycin-Neomycin antibiotic mix (15140-122), 100X Penicillin-Streptomycin-Neomycin mix (part 15640-055), Sodium Pyruvate (11360-070), PBS without calcium or magnesium (14190-136), HEPES (15630-080), Trypsin/EDTA (25300-054), Zeocin (R250-01), Hygromycin (10687-010), Geneticin (10131-027).</p><p>Fatty Acid Free BSA was obtained from Calbiochem (NC9734015). Probenecid was obtained from Sigma (part P8761). S1P was obtained from Avanti Polar Lipids (860492P) and Biomol (SL-140). U2OS cells were obtained from ATCC (HTB-96). 1536-well plates were obtained from Greiner (789072). 384-well plates were obtained from Greiner (788092) and Corning (3570). T175 tissue culture flasks (431080) were obtained from Corning. Charcoal/dextran treated fetal bovine serum was obtained from Hyclone (SH30068.03). Reagents for the Ricerca HitProfilingScreen + CYP450 were provided by Ricerca Biosciences, LLC.</p><div id="ml131.s5"><h3>2.1. Assays</h3><div id="ml131.s6"><h4>LC-MS/MS</h4><p>All analytical methods are in MRM mode where the parent ion is selected in Q1 of the mass spectrometer. The parent ion is fragmented and a characteristic fragment ion is monitored in Q3. MRM mass spectroscopy methods are particularly sensitive because additional time is spent monitoring the desired ions and not sweeping a large mass range. Methods will be rapidly set up using Automaton<sup>®</sup> (Applied Biosystems), where the compounds are listed with their name and mass in an Excel datasheet. Compounds are submitted in a 96-well plate to the HPLC autosampler and are slowly injected without a column present. A narrow range centered on the indicated mass is scanned to detect the parent ion. The software then evaluates a few pre-selected parameters to determine conditions that maximize the signal for the parent ion. The molecule is then fragmented in the collision cell of the mass spectrometer and fragments with m/z larger than 70 but smaller than the parent mass are determined. Three separate collision energies are evaluated to fragment the parent ion and the largest three ions are selected. Each of these three fragment ions is further optimized and the best fragment is chosen. The software then inserts the optimized masses and parameters into a template method and saves it with a unique name that indicates the individual compound being optimized. Spectra for the parent ion and the fragmentation pattern are saved and can be reviewed later.</p></div><div id="ml131.s7"><h4>Solubility</h4><p>The solubility of compounds was tested in phosphate buffered saline, pH 7.4. Compounds were inverted for 24 hours in test tubes containing 1–2 mg of compound with 1 mL of PBS. The samples were centrifuged and analyzed by HPLC (Agilent 1100 with diode-array detector). Peak area was compared to a standard of known concentration. In cases when the concentration was too low for UV analysis or when the compound did not possess a good chromophore, LC-MS/MS analysis was used.</p></div><div id="ml131.s8"><h4>Primary uHTS assay to identify S1P4 receptor antagonists (AIDs 1510)</h4><p><b>Assay Overview:</b> The purpose of this assay was to identify compounds that act as antagonists of S1P4. This assay uses Tango S1P4-BLA U2OS cells which contain the human Endothelial Differentiation Gene 6 (EDG6; S1P4) linked to a GAL4-VP16 transcription factor via a TEV protease site. The cells also express a beta-arrestin/TEV protease fusion protein and a beta-lactamase (BLA) reporter gene under the control of a UAS response element. Stimulation of the S1P4 receptor by agonist causes migration of the fusion protein to the GPCR, and through proteolysis liberates GAL4-VP16 from the receptor. The liberated VP16-GAL4 migrates to the nucleus, where it induces transcription of the BLA gene. BLA expression is monitored by measuring fluorescence resonance energy transfer (FRET) of a cleavable, fluorogenic, cell-permeable BLA substrate. As designed, test compounds that act as S1P4 antagonists will inhibit S1P4 activation and migration of the fusion protein, thus preventing proteolysis of GAL4-VP16 and BLA transcription, leading to no increase in well FRET. Compounds were tested in singlicate at a final nominal concentration of 5 µM.</p><p><b>Protocol Summary:</b> U2OS cells were cultured in T-175 square cm flasks at 37 degrees C and 95% relative humidity (RH). The growth media consisted of McCoy’s 5A Medium supplemented with 10% v/v dialyzed fetal bovine serum, 0.1 mM NEAA, 25 mM HEPES (pH 7.3), 1 mM sodium pyruvate, 100 U/mL penicillin-streptomycin-neomycin, 200 ug/mL Zeocin, 50 ug/mL Hygromycin, and 100 ug/mL Geneticin. Prior to the start of the assay, cells were suspended at a concentration of 250,000/mL in Assay Medium (Freestyle Expression Medium without supplements). The assay was started by dispensing 4 uL of cell suspension to each well, followed by overnight incubation at 37 degrees C in 5% CO<sub>2</sub> and 95% RH. The next day, 25 nL of test compound (5 µM final nominal concentration) in DMSO was added to sample wells, and DMSO alone (0.5 % final concentration) was added to control wells. Next, S1P prepared in 2% BSA (0.3 µM final nominal EC80 concentration) was added to the appropriate wells. Plates were then incubated at 37 degrees C in 5% CO<sub>2</sub> for 4 hours. After the incubation, 1 uL/well of the LiveBLAzer FRET substrate mixture, prepared according to the manufacturer’s protocol and containing 10 mM Probenicid, was added to all wells. After 2 hours of incubation at room temperature in the dark, plates were read on the EnVision plate reader (PerkinElmer Lifesciences, Turku, Finland) at an excitation wavelength of 405 nm and emission wavelengths of 460 nm and 535 nm. <b>Assay Cutoff:</b> compounds that exhibited greater than 29.02% inhibition of S1P4 receptor were considered active.</p></div><div id="ml131.s9"><h4>Confirmation uHTS assay to identify S1P4 receptor antagonists (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1524" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1524</a>)</h4><p><b>Assay Overview:</b> The purpose of this assay was to confirm activity of compounds identified as active in the uHTS primary screen (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1510" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1510</a>). This assay was as described above (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1510" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1510</a>). Compounds were tested in triplicate at a nominal concentration of 5 µM.</p><p><b>Protocol Summary:</b> The assay was performed as described above (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1510" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1510</a>). <b>Assay Cutoff:</b> compounds that exhibited greater than 29.02% inhibition of S1P4 receptor were considered active.</p></div><div id="ml131.s10"><h4>Dose response uHTS assay to identify S1P4 receptor agonists (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1692" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1692</a>)</h4><p><b>Assay Overview:</b> The purpose of this assay was to determine dose response for compounds identified as active in the uHTS primary screen (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1510" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1510</a>) and that confirmed activity in the uHTS confirmation screen (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1524" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1524</a>). This assay was as described above (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1510" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1510</a>). Compounds were tested in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 50 µM.</p><p><b>Protocol Summary:</b> The assay was performed as described above (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1509" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1509</a>). <b>Assay Cutoff:</b> compounds with an IC50 equal to or less than 10 µM were considered active.</p></div><div id="ml131.s11"><h4>Counterscreen uHTS assay to identify S1P1 receptor antagonists (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1564" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1564</a>)</h4><p><b>Assay Overview:</b> The purpose of this assay was to determine whether compounds identified as active in the primary uHTS assay (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1510" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1510</a>), and that confirmed activity in the uHTS confirmation screen (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1524" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1524</a>), were nonselective antagonists due to inhibition of S1P1. This assay uses Tango S1P1-bla U2OS cells that express S1P1 (EDG1) linked to a GAL4-VP16 transcription factor via a TEV protease site. The cells also express a beta-arrestin/TEV protease fusion protein and a beta-lactamase (BLA) reporter gene under the control of a UAS response element. Stimulation of the S1P1 receptor by agonist causes migration of the fusion protein to the GPCR, and through proteolysis liberates GAL4-VP16 from the receptor. The liberated VP16-GAL4 migrates to the nucleus, where it induces transcription of the BLA gene. BLA expression is monitored by measuring fluorescence resonance energy transfer (FRET) of a cleavable, fluorogenic, cell-permeable BLA substrate. As designed, test compounds that act as S1P1 antagonists will inhibit S1P1 activation and migration of the fusion protein, thus preventing proteolysis of GAL4-VP16 and BLA transcription, leading to no increase in well FRET. Compounds were tested in triplicate at a final nominal concentration of 5 µM.</p><p><b>Protocol Summary:</b> Cells were cultured as described above (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1510" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1510</a>). Prior to the start of the assay, cells were suspended at a concentration of 250,000/mL in Assay Medium (Freestyle Expression Medium without supplements). The assay was started by dispensing 4 uL of cell suspension to each well, followed by overnight incubation at 37 degrees C in 5% CO<sub>2</sub> and 95% RH. The next day, 25 nL of test compound in DMSO (0.5 % final DMSO concentration), DMSO alone, or S1P (10 nM final nominal EC80 concentration) prepared in 2% BSA was added to the appropriate wells. Plates were then incubated at 37 degrees C in 5% CO<sub>2</sub> for 4 hours. After the incubation, 1 uL/well of the LiveBLAzer FRET substrate mixture, prepared according to the manufacturer’s protocol and containing 10 mM Probenicid, was added to all wells. After 2 hours of incubation at room temperature in the dark, plates were read on the EnVision plate reader (PerkinElmer Lifesciences, Turku, Finland) at an excitation wavelength of 405 nm and emission wavelengths of 460 nm and 535 nm. <b>Assay Cutoff:</b> compounds that exhibited greater than 40.0% inhibition of S1P1 receptor were considered active.</p></div><div id="ml131.s12"><h4>Counterscreen dose response uHTS assay to identify S1P1 receptor antagonists (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1821" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1821</a>)</h4><p><b>Assay Overview:</b> The purpose of this assay was to determine S1P1 antagonist dose response for compounds identified as active in the uHTS S1P1 receptor counterscreen assay (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1564" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1564</a>). The assay was as described above (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1564" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1564</a>), except that compounds were tested in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 50 µM.</p><p><b>Protocol Summary:</b> The assay was performed as described above (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1564" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1564</a>). <b>Assay Cutoff:</b> compounds with an IC50 equal to or less than 10 µM were considered active.</p></div><div id="ml131.s13"><h4>Late-stage dose response assay to identify S1P4 receptor agonists with purchased compounds (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2332" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-2332</a>)</h4><p><b>Assay Overview:</b> The purpose of this assay was to determine S1P4 antagonist dose response with purchased compounds. Compounds with S1P4 antagonist IC50 less than 1 µM in PubChem <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1692" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1692</a> were selected based on presence of a tractable chemical structure and absence of known activities towards other targets. This assay was as described above (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1510" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1510</a>). Compounds were tested in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 50 µM.</p><p><b>Protocol Summary:</b> Cells were cultured as described above (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1510" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1510</a>). Prior to the start of the assay, cells were suspended at a concentration of 1,000,000/mL in Assay Medium (Freestyle Expression Medium without supplements). The assay was started by dispensing 10 uL of cell suspension to each well in 384-well plates, followed by overnight incubation at 37 degrees C in 5% CO<sub>2</sub> and 95% RH. The next day, 50 nL of test compound in DMSO was added to sample wells, and DMSO alone (0.5 % final concentration) was added to control wells. Next, S1P prepared in 2% BSA (0.22 µM final nominal EC80 concentration) was added to the appropriate wells. Plates were then incubated at 37 degrees C in 5% CO<sub>2</sub> for 4 hours. After the incubation, 2.2 uL/well of the LiveBLAzer FRET substrate mixture, prepared according to the manufacturer’s protocol and containing 10 mM Probenicid, was added to all wells. After 2 hours of incubation at room temperature in the dark, plates were read on the EnVision plate reader (PerkinElmer Lifesciences, Turku, Finland) at an excitation wavelength of 405 nm and emission wavelengths of 460 nm and 535 nm. <b>Assay Cutoff:</b> compounds with an IC50 equal to or less than 10 µM were considered active.</p></div><div id="ml131.s14"><h4>Late-stage dose response assay to identify S1P4 receptor agonists with synthesized compounds (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2346" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-2346</a>)</h4><p><b>Assay Overview:</b> The purpose of this assay was to determine S1P4 antagonist dose response with synthesized compounds. This assay was as described above (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1510" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1510</a>). Compounds were tested in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 50 µM.</p><p><b>Protocol Summary:</b> The assay was performed as described above (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2332" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-2332</a>). <b>Assay Cutoff:</b> compounds with an IC50 equal to or less than 10 µM were considered active.</p></div><div id="ml131.s15"><h4>Late-stage counterscreen dose response assay to identify S1P1 receptor agonists (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2351" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-2351</a>)</h4><p><b>Assay Overview:</b> The purpose of this assay was to determine antagonist activity of powder compounds against S1P1 receptor. This assay was as described above (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1564" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1564</a>). Compounds were tested in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 25 µM.</p><p><b>Protocol Summary:</b> Cells were cultured as described above (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1510" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1510</a>). The assay was performed as described above (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2332" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-2332</a>). <b>Assay Cutoff:</b> compounds with an IC50 equal to or less than 10 µM were considered active.</p></div><div id="ml131.s16"><h4>Late-stage counterscreen dose response assay to identify S1P2 receptor agonists (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2354" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-2354</a>)</h4><p><b>Assay Overview:</b> The purpose of this assay was to determine antagonist activity of powder compounds against S1P2 receptor. A Chinese Hamster Ovary (CHO) cell line stably transfected with the human S1P2 receptor and a cAMP Response Element-beta lactamase (CRE-BLA) reporter construct was used to measure S1P2 antagonism. Under normal conditions, S1P2 has low basal activity and therefore cells express low BLA levels. In this assay, cells have been stimulated with the S1P agonist, which stimulates the S1P2 receptor, hence increasing BLA gene transcription. An antagonist is identified as a compound that prevents activation by exogenously added S1P. Therefore, in the presence of a potential S1P2 antagonist, transcription of the BLA gene will decrease. This decrease is monitored by measuring fluorescence resonance energy transfer (FRET) of a cleavable fluorogenic cell permeable BLA substrate. As designed, test compounds that act as S1P2 antagonists will inhibit BLA transcription and cleavage of the fluorogenic substrate, leading to no increase in well FRET. Compounds were tested in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 25 µM.</p><p><b>Protocol Summary:</b> Cells were cultured in T-175 square cm flasks at 37 degrees C and 95% RH. The growth media consisted of Dulbecco’s Modified Eagle’s Media supplemented with 10% v/v heat inactivated dialyzed fetal bovine serum, 0.1 mM NEAA, 1 mM Sodium Pyruvate, 25 mM HEPES, 5 mM L-Glutamine, 2 mg/mL Geneticin and 1X antibiotic mix (mix of penicillin, streptomycin and neomycin). Prior to assay, cells were suspended to a concentration of 1,250,000/mL in assay media, which consisted of phenol red-free Dulbecco’s Modified Eagle’s Media supplemented with 2% charcoal/dextran-treated fetal bovine serum, 0.1 mM NEAA, 1 mM Sodium Pyruvate, 25 mM HEPES, 5 mM L-Glutamine and 1X antibiotic mix (mix of penicillin, streptomycin and neomycin). The assay began by dispensing 4 uL of cell suspension to each test well of a 384-well plate (5000 cells/well) followed by incubation at 37 degrees C in 5% CO<sub>2</sub> for 16 hrs. To the appropriate wells were then added 26 nL of test compound in DMSO (final nominal concentration of 5 µM, final DMSO concentration of 0.5%) or DMSO only (for high control wells) followed directly afterwards by 1 uL of S1P in 2% BSA (final concentration of 750 nM, a concentration that resulted in 80% activity). The high control (EC80 challenge) and low control (100% antagonism) were added to the appropriate control wells and plates were incubated again at 37 degrees C in 5% CO<sub>2</sub> for 2 hrs. The fluorogenic LiveBLAzer substrate mixture with 10 mM Probenicid was prepared according to the manufacturer’s protocol and 1 uL of this mixture was then added to each well. After a further 2 hours of incubation at room temperature, plates were read on the EnVision plate reader (PerkinElmer Lifesciences, Turku, Finland) at an excitation wavelength of 405 nm and fluorescence emission wavelengths of 535 nm & 460 nm. <b>Assay Cutoff:</b> compounds with an IC50 equal to or less than 10 µM were considered active.</p></div><div id="ml131.s17"><h4>Late-stage counterscreen dose response assay to identify S1P3 receptor agonists (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2349" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-2349</a>)</h4><p><b>Assay Overview:</b> The purpose of this assay was to determine antagonist activity of powder compounds against S1P3 receptor. In this assay, a CHO cell line containing human S1P3 and the beta-lactamase (BLA) reporter-gene under control of the nuclear factor of activated T-cells (NFAT) promoter was used to measure S1P3 antagonism by test compound. Stimulation of S1P3 by S1P induces transcription of NFAT-BLA via a G-alpha16 protein coupled signaling cascade, and an increase in BLA activity. BLA activity is measured using a fluorescent BLA substrate. As designed, a compound that acts as a S1P3 antagonist will decrease NFAT-BLA transcription and decrease well fluorescence. Compounds were tested in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 25 µM.</p><p><b>Protocol Summary:</b> Cells were cultured in T-175 square cm flasks at 37 degrees C and 95% relative humidity (RH). The growth media consisted of Dulbecco’s Modified Eagle’s Media (DMEM) containing 10% v/v heat inactivated bovine growth serum, 0.1 mM NEAA, 1 mM sodium pyruvate, 25 mM HEPES, 5 mM L-Glutamine, 2 mg/mL Geneticin, 0.2 mg/mL Hygromycin B, and 1x penicillin-streptomycin-neomycin. Prior to the start of the assay, cells were suspended at a concentration of 1,250,000/mL in phenol red-free DMEM supplemented as above, except with 0.5% charcoal/dextran-treated fetal bovine serum and no antibiotics. The assay was started by dispensing 4 uL of cell suspension to each well followed by overnight incubation at 37 degrees C in 5% CO<sub>2</sub> and 95% RH. The next day, 25 nL of test compound (5 µM final nominal concentration) in DMSO was added to sample wells, and DMSO alone (0.5 % final concentration) was added to High Control wells. Next, S1P prepared in 1% BSA (0.8 µM final nominal concentration, corresponding to the EC80 of S1P) was added to the appropriate wells. After 4 hours of incubation, 1 uL/well of the GeneBLAzer fluorescent substrate mixture, prepared according to the manufacturer’s protocol and containing 10 mM Probenicid, was added to all wells. The plates were then incubated for 2 hours at room temperature. Plates were read on the EnVision plate reader (PerkinElmer Lifesciences, Turku, Finland) at an excitation wavelength of 405 nm and emission wavelengths of 535 nm and 460 nm. <b>Assay Cutoff:</b> compounds with an IC50 equal to or less than 10 µM were considered active.</p></div><div id="ml131.s18"><h4>Late-stage counterscreen dose response assay to identify S1P5 receptor agonists (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2350" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-2350</a>)</h4><p><b>Assay Overview:</b> The purpose of this assay was to determine antagonist activity of powder compounds against S1P5 receptor. This assay uses Tango S1P5-BLA U2OS cells which contain the human Endothelial Differentiation Gene 8 (EDG8; S1P5) linked to a GAL4-VP16 transcription factor via a TEV protease site. The cells also express a beta-arrestin/TEV protease fusion protein and a beta-lactamase (BLA) reporter gene under the control of a UAS response element. Stimulation of the S1P5 receptor by agonist causes migration of the fusion protein to the GPCR, and through proteolysis liberates GAL4-VP16 from the receptor. The liberated VP16-GAL4 migrates to the nucleus, where it induces transcription of the BLA gene. BLA expression is monitored by measuring fluorescence resonance energy transfer (FRET) of a cleavable, fluorogenic, cell-permeable BLA substrate. As designed, test compounds that act as S1P5 antagonists will inhibit S1P5 activation and migration of the fusion protein, thus preventing proteolysis of GAL4-VP16 and BLA transcription, leading to no increase in well FRET. Compounds were tested in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 25 µM.</p><p><b>Protocol Summary:</b> Cells were cultured as described above (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1510" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1510</a>). The assay was performed as described above (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2332" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-2332</a>). <b>Assay Cutoff:</b> compounds with an IC50 equal to or less than 10 µM were considered active.</p></div><div id="ml131.s19"><h4>Luminescence-based cell-based dose response assay to determine cytotoxicity of antagonist compounds (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/489009" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-489009</a>)</h4><p><b>Assay Overview:</b> The purpose of this assay was to determine cytotoxicity of a powder sample of probe compound <a href="/pcsubstance/?term=ML131[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML131</a>. In this assay, U2OS cells are incubated with test compound, followed by determination of cell viability. The assay utilizes the CellTiter-Glo luminescent reagent to measure intracellular ATP in viable cells. Luciferase present in the reagent catalyzes the oxidation of beetle luciferin to oxyluciferin and light in the presence of cellular ATP. Well luminescence is directly proportional to ATP levels and cell viability. As designed, compounds that reduce cell viability will reduce ATP levels, luciferin oxidation and light production, resulting in decreased well luminescence. Compound <a href="/pcsubstance/?term=ML131[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML131</a> was tested in six replicates in a 7-point 1:3 dilution series starting at a nominal test concentration of 20 µM.</p><p><b>Prococol Summary:</b> This assay was started by dispensing 20 uL U2OS cells (200,000 cells/well) in McCoy’s 5A medium plus 10% FBS, penicillin 100 U/ml and streptomycin 100 ug/ml into the wells of a 384-well plate. Eight 1:3 serial dilutions of compound (100 µM in growth media) were made. 5 uL of diluted compound or media were added to wells, giving final compound concentrations of 0–20 µM. The plate was spun for 1 minute at 1000 rpm, then incubated at 37 degrees C in a humidified incubator for 6 hours, then equilibrated to room temperature for 30 minutes. 25 uL CellTitre-Glo reagent was added to each well, followed by incubation of the plate in the dark for 10 minutes. Well luminescence was measured on the Envision plate reader. <b>Assay Cutoff:</b> Compounds with a CC50 value of less than 10 µM were considered active (cytotoxic). Compounds with a CC50 value greater than 10 µM were considered inactive (non-cytotoxic).</p></div><div id="ml131.s20"><h4>Ricerca HitProfilingScreen + CYP450 Counterscreen assay (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/489017" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-489017</a>)</h4><p><b>Assay Overview:</b> The purpose of this panel of binding assays performed by Ricerca Biosciences, LLC, was to identify a subset of potential receptors, transporters, ion channels, etc. for which probe <a href="/pcsubstance/?term=ML131[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML131</a> displays affinity.</p><p><b>Protocol Summary:</b> Assays for CYP450, 1A2; CYP450, 2C19; CYP450, 2C9; CYP450, 2D6; and CYP450, 3A4 were enzyme assays using human recombinant insect Sf9 cells with 5 µM 3-cyano-7-ethoxycoumarin as substrate (except for CYP450, 3A4, which used 50 µM 7-benzyloxy-4-(trifluoromethyl)-coumarin as substrate). Detection was based on spectrofluorimetric quantitation of the enzymatic product produced. Assays for the other targets were radioligand binding assays. <b>Assay Cutoff:</b> A response of at least 50% inhibition or stimulation was considered “active”. Negative inhibition represents a stimulation of binding.</p></div></div><div id="ml131.s21"><h3>2.2. Probe Chemical Characterization</h3><div id="ml131.fu2" class="figure"><div class="graphic"><img src="/books/NBK51967/bin/ml131fu2.jpg" alt="Image ml131fu2" /></div></div><p>The probe structure was verified by NMR (proton and carbon) and LC-MS (<a class="figpopup" href="/books/NBK51967/figure/ml131.f1/?report=objectonly" target="object" rid-figpopup="figml131f1" rid-ob="figobml131f1">Figure 1</a>).</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml131f1" co-legend-rid="figlgndml131f1"><a href="/books/NBK51967/figure/ml131.f1/?report=objectonly" target="object" title="Figure 1" class="img_link icnblk_img figpopup" rid-figpopup="figml131f1" rid-ob="figobml131f1"><img class="small-thumb" src="/books/NBK51967/bin/ml131f1.gif" src-large="/books/NBK51967/bin/ml131f1.jpg" alt="Figure 1. LC-MS results for probe ML131." /></a><div class="icnblk_cntnt" id="figlgndml131f1"><h4 id="ml131.f1"><a href="/books/NBK51967/figure/ml131.f1/?report=objectonly" target="object" rid-ob="figobml131f1">Figure 1</a></h4><p class="float-caption no_bottom_margin">LC-MS results for probe ML131. </p></div></div><p><sup>1</sup>H NMR (300 MHz, CDCl<sub>3</sub>): δ8.43 (s, 1H), 8.00 (d, <i>J</i> = 2.50 Hz, 1H), 7.36 (d, <i>J</i> = 8.52 Hz, 1H), 7.25 (m, 2H), 7.22-7.17 (m, 2H), 7.11 (m, 2H), 2.62 (q, <i>J</i> = 7.56 Hz, 4H), 1.16 (t, <i>J</i> = 7.12 Hz, 6H); <sup>13</sup>C NMR (75 MHz, CDCl<sub>3</sub>) δ 157.12, 150.66, 147.50, 142.06, 133.29, 132.20, 129.63, 129.12, 128.34, 126.46, 116.77, 113.95, 25.10, 14.63.</p><p>Solubility in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM sodium phosphate dibasic, 2 mM potassium phosphate monobasic, pH 7.4) at room temperature (23 degrees C) was determined to be 0.036 µM by dilution of 10 mM stock solution in DMSO into PBS. Solubility was too low to determine stability or Michael Acceptor reactivity. Properties for probe <a href="/pcsubstance/?term=ML131[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML131</a> are listed in <a class="figpopup" href="/books/NBK51967/table/ml131.t1/?report=objectonly" target="object" rid-figpopup="figml131t1" rid-ob="figobml131t1">Table 1</a>.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml131t1"><a href="/books/NBK51967/table/ml131.t1/?report=objectonly" target="object" title="Table 1" class="img_link icnblk_img figpopup" rid-figpopup="figml131t1" rid-ob="figobml131t1"><img class="small-thumb" src="/books/NBK51967/table/ml131.t1/?report=thumb" src-large="/books/NBK51967/table/ml131.t1/?report=previmg" alt="Table 1. Probe Properties for ML131." /></a><div class="icnblk_cntnt"><h4 id="ml131.t1"><a href="/books/NBK51967/table/ml131.t1/?report=objectonly" target="object" rid-ob="figobml131t1">Table 1</a></h4><p class="float-caption no_bottom_margin">Probe Properties for ML131. </p></div></div><p>The following compounds have been submitted to the SMR collection. Compound numbers refer to the <b><a class="figpopup" href="/books/NBK51967/table/ml131.t3/?report=objectonly" target="object" rid-figpopup="figml131t3" rid-ob="figobml131t3">SAR Table</a></b>.</p><div id="ml131.tu2" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK51967/table/ml131.tu2/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml131.tu2_lrgtbl__"><table><thead><tr><th id="hd_h_ml131.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Designation</th><th id="hd_h_ml131.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Compound Number</th><th id="hd_h_ml131.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CID</th><th id="hd_h_ml131.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SID</th><th id="hd_h_ml131.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SRID</th><th id="hd_h_ml131.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS</th></tr></thead><tbody><tr><td headers="hd_h_ml131.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Probe</td><td headers="hd_h_ml131.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b>1</b></td><td headers="hd_h_ml131.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">44607580</td><td headers="hd_h_ml131.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87357351" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87357351</a></td><td headers="hd_h_ml131.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-02000000248-1</td><td headers="hd_h_ml131.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS002699003</td></tr><tr><td headers="hd_h_ml131.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Analog 1</td><td headers="hd_h_ml131.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b>2</b></td><td headers="hd_h_ml131.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">44607581</td><td headers="hd_h_ml131.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87357343" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87357343</a></td><td headers="hd_h_ml131.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-02000000240-1</td><td headers="hd_h_ml131.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS002698999</td></tr><tr><td headers="hd_h_ml131.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Analog 2</td><td headers="hd_h_ml131.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b>3</b></td><td headers="hd_h_ml131.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1007755</td><td headers="hd_h_ml131.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87357344" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87357344</a></td><td headers="hd_h_ml131.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-02000000241-1</td><td headers="hd_h_ml131.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS002699000</td></tr><tr><td headers="hd_h_ml131.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Analog 3</td><td headers="hd_h_ml131.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b>4</b></td><td headers="hd_h_ml131.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">44607576</td><td headers="hd_h_ml131.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87357347" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87357347</a></td><td headers="hd_h_ml131.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-02000000244-1</td><td headers="hd_h_ml131.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS002699001</td></tr><tr><td headers="hd_h_ml131.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Analog 4</td><td headers="hd_h_ml131.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b>5</b></td><td headers="hd_h_ml131.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">44607573</td><td headers="hd_h_ml131.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87357341" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87357341</a></td><td headers="hd_h_ml131.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-02000000238-1</td><td headers="hd_h_ml131.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS002699002</td></tr><tr><td headers="hd_h_ml131.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Analog 5</td><td headers="hd_h_ml131.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b>6</b></td><td headers="hd_h_ml131.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">44607577</td><td headers="hd_h_ml131.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87357349" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87357349</a></td><td headers="hd_h_ml131.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-02000000246-1</td><td headers="hd_h_ml131.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS002699004</td></tr></tbody></table></div></div></div><div id="ml131.s22"><h3>2.3. Probe Preparation</h3><div id="ml131.f2" class="figure bk_fig"><div class="graphic"><img src="/books/NBK51967/bin/ml131f2.jpg" alt="Figure 2. Synthesis scheme for ML131." /></div><h3><span class="label">Figure 2</span><span class="title">Synthesis scheme for <a href="/pcsubstance/?term=ML131[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML131</a></span></h3></div><p>A solution of 5-(2,5-dichlorophenyl)furan-2-carbonyl chloride (0.070 g, 0.25 mmol) in dichloromethane (4 mL) was treated with 2,6-diethylaniline (0.037 g, 0.25 mmol) and N,N-diisopropylethylamine (0.1 mL). The mixture was stirred at ambient temperature for 6 hours. The mixture was diluted with dichloromethane (5 mL) and washed with 5% NaHCO3 solution (5 mL), water (5 mL) and brine (5 mL). The organic phase was dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography (25% EtOAc/Hexane) to afford 5-(2,5-dichlorophenyl)-N-(2,6-diethylphenyl)furan-2-carboxamide (0.078 g, 81%) as a white solid. MS (EI) m/z 388, 400 (M+).</p></div></div><div id="ml131.s23"><h2 id="_ml131_s23_">3. Results</h2><p>Probe <a href="/pcsubstance/?term=ML131[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML131</a> satisfies the goals for the probe characteristics identified in the CPDP: 1) probes should selectively inhibit S1P4 in cell-based TANGO-format assays, and 2) probes should not inhibit S1P1, S1P2, S1P3 and S1P5 in cell-based assays. Probe <a href="/pcsubstance/?term=ML131[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML131</a>:</p><ul><li class="half_rhythm"><div>Inhibits S1P4 receptor in a cell-based TANGO-format assay with an IC50 of 89 nM (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2346" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-2346</a>).</div></li><li class="half_rhythm"><div>Does not inhibit other members of the receptor family in cell-based assays, with IC50s > 25 µM against S1P1 receptor (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2351" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-2351</a>), S1P2 receptor (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2354" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-2354</a>), S1P3 receptor (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2349" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-2349</a>), and S1P5 receptor (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2350" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-2350</a>).</div></li></ul><p>In addition, <a href="/pcsubstance/?term=ML131[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML131</a> is nontoxic to U202 cells with a CC50 of > 20 µM (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/489009" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-489009</a>).</p><div id="ml131.s24"><h3>3.1. Summary of Screening Results</h3><p>In the uHTS primary assay (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1510" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1510</a>), 218K compounds were screened at 5 µM. A total of 569 compounds (0.26%) were active, passing the set threshold of 29.02% S1P4 receptor antagonism. For the uHTS confirmation assay, (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1524" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1524</a>), 569 active compounds were retested in triplicate, and 366 compounds (71.07%) were confirmed as active. In a uHTS counterscreen assay against the S1P1 receptor (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1564" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1564</a>), 179 of these compounds were found to be inactive against the S1P1 receptor. Of the top 128 confirmed S1P4 hits, 100 were available from the NIH small molecule repository. These 100 compounds were tested in uHTS dose response assay for S1P4 receptor antagonism (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1692" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1692</a>) and for S1P1 receptor antagonism (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1821" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1821</a>), and 51 were found to be selective for S1P4 receptor—having an IC50 of less than 10 µM for S1P4 receptor and an IC50 of greater than 10 µM for S1P1 receptor.</p><div id="ml131.f3" class="figure bk_fig"><div class="graphic"><img src="/books/NBK51967/bin/ml131f3.jpg" alt="Figure 3. S1P4 antagonist HTS overview." /></div><h3><span class="label">Figure 3</span><span class="title">S1P4 antagonist HTS overview</span></h3></div><p>The four most potent hit compounds confirmed following the HTS screening effort, representing four unique chemical scaffolds, were purchased as powders and profiled against all of the S1P receptor family. Results of these assays are summarized in <a class="figpopup" href="/books/NBK51967/table/ml131.t2/?report=objectonly" target="object" rid-figpopup="figml131t2" rid-ob="figobml131t2">Table 2</a>. Based on potency towards S1P4, selectivity, and chemical tractability, <a href="https://pubchem.ncbi.nlm.nih.gov/substance/87241457" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-87241457</a> was chosen as the hit compound. Analogs were synthesized and tested in dose response assays against S1P4. In collaboration with the assay provider’s lab, selected compounds were profiled against the remaining members of the S1P family, S1P1-3 and S1P5; the results are listed in <a class="figpopup" href="/books/NBK51967/table/ml131.t3/?report=objectonly" target="object" rid-figpopup="figml131t3" rid-ob="figobml131t3">Table 3</a>. These efforts led to the identification of the probe compound <a href="https://pubchem.ncbi.nlm.nih.gov/substance/87357351" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-87357351</a> belonging to the 5-phenylfuran-2-arylcarboxamide scaffold. The probe and analogs span an IC50 range of 78 nM to 2 μM, while having no significant antagonist activity toward the other members of the S1P family at 25 µM concentrations. The probe compound is one of the most potent S1P4 antagonists. The original screening hit <a href="https://pubchem.ncbi.nlm.nih.gov/substance/87241457" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-87241457</a> can be conceptualized as two components, A and B. In region <b>A</b> (<a class="figpopup" href="/books/NBK51967/table/ml131.t3/?report=objectonly" target="object" rid-figpopup="figml131t3" rid-ob="figobml131t3">Table 3</a>), both chlorines 2 and 5 are fundamental for the binding, as evidenced by the fact that the appropriate monochlorines <b>10</b> and <b>16</b> were less potent than <a href="https://pubchem.ncbi.nlm.nih.gov/substance/87357351" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-87357351</a>. Replacement of 2,5-dichlorine in region <b>A</b> by polar substituents such as fluorine and small alkoxy groups (<b>5</b>, <b>17</b>, <b>18</b>) was detrimental to the potency. Replacement of the 2,5 dichlorophenyl moiety by a tiophene bioisotere (<b>2</b>) was successful. In the arylcarboxamide region (region <b>B</b>), replacement of ethyl by methyl, methoxy or isopropyl moieties at the 2, 6 positions produced minor impact on the potency (<b>2, 4</b>, <b>6</b>). Shifting or removing a methyl group in position 2 (<b>7</b>, <b>8</b> compared to <b>2</b>), as well as addition of substituents (<b>14</b>) in the phenyl ring is permitted without significant loss of activity.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml131t2"><a href="/books/NBK51967/table/ml131.t2/?report=objectonly" target="object" title="Table 2" class="img_link icnblk_img figpopup" rid-figpopup="figml131t2" rid-ob="figobml131t2"><img class="small-thumb" src="/books/NBK51967/table/ml131.t2/?report=thumb" src-large="/books/NBK51967/table/ml131.t2/?report=previmg" alt="Table 2. Potency and selectivity of lead scaffolds from HTS screen." /></a><div class="icnblk_cntnt"><h4 id="ml131.t2"><a href="/books/NBK51967/table/ml131.t2/?report=objectonly" target="object" rid-ob="figobml131t2">Table 2</a></h4><p class="float-caption no_bottom_margin">Potency and selectivity of lead scaffolds from HTS screen. </p></div></div></div><div id="ml131.s25"><h3>3.2. Dose Response Curves for Probe</h3><p>Probe <a href="/pcsubstance/?term=ML131[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML131</a> is a potent, dose-dependent S1P4 antagonist, displaying a dose-dependent inhibition with a calculated IC50 of 89 nM for S1P4 receptor (<a class="figpopup" href="/books/NBK51967/figure/ml131.f4/?report=objectonly" target="object" rid-figpopup="figml131f4" rid-ob="figobml131f4">Figure 4</a>).</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml131f4" co-legend-rid="figlgndml131f4"><a href="/books/NBK51967/figure/ml131.f4/?report=objectonly" target="object" title="Figure 4" class="img_link icnblk_img figpopup" rid-figpopup="figml131f4" rid-ob="figobml131f4"><img class="small-thumb" src="/books/NBK51967/bin/ml131f4.gif" src-large="/books/NBK51967/bin/ml131f4.jpg" alt="Figure 4. Dose response curve for probe ML131 assayed against the S1P4 receptor." /></a><div class="icnblk_cntnt" id="figlgndml131f4"><h4 id="ml131.f4"><a href="/books/NBK51967/figure/ml131.f4/?report=objectonly" target="object" rid-ob="figobml131f4">Figure 4</a></h4><p class="float-caption no_bottom_margin">Dose response curve for probe ML131 assayed against the S1P4 receptor. </p></div></div></div><div id="ml131.s26"><h3>3.3. Scaffold/Moiety Chemical Liabilities</h3><p>No scaffold or moiety chemical liabilities are observed with probe <a href="/pcsubstance/?term=ML131[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML131</a>.</p></div><div id="ml131.s27"><h3>3.4. SAR Table</h3><p>The original screening hit <a href="https://pubchem.ncbi.nlm.nih.gov/substance/87241457" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-87241457</a> can be conceptualized as two components, A and B. <a class="figpopup" href="/books/NBK51967/table/ml131.t3/?report=objectonly" target="object" rid-figpopup="figml131t3" rid-ob="figobml131t3">Table 3</a> shows structures of compounds used for probe optimization.</p><div id="ml131.t3" class="table"><h3><span class="label">Table 3</span><span class="title">SAR Table for S1P4 receptor antagonist probe optimization</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK51967/table/ml131.t3/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml131.t3_lrgtbl__"><table class="no_margin"><thead><tr><th id="hd_h_ml131.t3_1_1_1_1" colspan="5" rowspan="1" style="text-align:center;vertical-align:middle;">SAR Analysis for S1P4 ANT</th><th id="hd_h_ml131.t3_1_1_1_2" colspan="2" rowspan="1" style="text-align:center;vertical-align:middle;"><div class="graphic"><img src="/books/NBK51967/bin/ml131fu7.jpg" alt="Image ml131fu7.jpg" /></div></th><th id="hd_h_ml131.t3_1_1_1_3" colspan="5" rowspan="1" style="text-align:center;vertical-align:middle;">IC50 89 nM</th><th id="hd_h_ml131.t3_1_1_1_4" rowspan="2" colspan="1" headers="hd_h_ml131.t3_1_1_1_4" style="text-align:center;vertical-align:middle;">S1P1 IC50/S1P4 IC50 Fold Selectivity</th></tr><tr><th headers="hd_h_ml131.t3_1_1_1_1" id="hd_h_ml131.t3_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Entry</th><th headers="hd_h_ml131.t3_1_1_1_1" id="hd_h_ml131.t3_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">CID</th><th headers="hd_h_ml131.t3_1_1_1_1" id="hd_h_ml131.t3_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">SID</th><th headers="hd_h_ml131.t3_1_1_1_1" id="hd_h_ml131.t3_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Center Internal Number</th><th headers="hd_h_ml131.t3_1_1_1_1" id="hd_h_ml131.t3_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">P/S<sup><a class="bk_pop" href="#ml131.tfn1">*</a></sup></th><th headers="hd_h_ml131.t3_1_1_1_2" id="hd_h_ml131.t3_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">A</th><th headers="hd_h_ml131.t3_1_1_1_2" id="hd_h_ml131.t3_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">B</th><th headers="hd_h_ml131.t3_1_1_1_3" id="hd_h_ml131.t3_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">S1P4 IC50 (nM)</th><th headers="hd_h_ml131.t3_1_1_1_3" id="hd_h_ml131.t3_1_1_2_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">S1P1 IC50 (µM)</th><th headers="hd_h_ml131.t3_1_1_1_3" id="hd_h_ml131.t3_1_1_2_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">S1P2 IC50 (µM)</th><th headers="hd_h_ml131.t3_1_1_1_3" id="hd_h_ml131.t3_1_1_2_11" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">S1P3 IC50 (µM)</th><th headers="hd_h_ml131.t3_1_1_1_3" id="hd_h_ml131.t3_1_1_2_12" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">S1P5 IC50 (µM)</th></tr></thead><tbody><tr><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"><b>1 (Probe)</b></td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">44607580</td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">87357351</td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-02000000248-1</td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">S</td><td headers="hd_h_ml131.t3_1_1_1_2 hd_h_ml131.t3_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><div class="graphic"><img src="/books/NBK51967/bin/ml131fu8.jpg" alt="Image ml131fu8.jpg" /></div></td><td headers="hd_h_ml131.t3_1_1_1_2 hd_h_ml131.t3_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><div class="graphic"><img src="/books/NBK51967/bin/ml131fu9.jpg" alt="Image ml131fu9.jpg" /></div></td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">89</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>25</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>25</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_11" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>25</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_12" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>25</td><td headers="hd_h_ml131.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>280</td></tr><tr><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"><b>2 (Analog 1)</b></td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">44607581</td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">87357343</td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-02000000240-1</td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">S</td><td headers="hd_h_ml131.t3_1_1_1_2 hd_h_ml131.t3_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><div class="graphic"><img src="/books/NBK51967/bin/ml131fu10.jpg" alt="Image ml131fu10.jpg" /></div></td><td headers="hd_h_ml131.t3_1_1_1_2 hd_h_ml131.t3_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><div class="graphic"><img src="/books/NBK51967/bin/ml131fu11.jpg" alt="Image ml131fu11.jpg" /></div></td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">286</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>25</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>25</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_11" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>25</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_12" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>25</td><td headers="hd_h_ml131.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>87</td></tr><tr><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"><b>3 (Analog 2)</b></td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1007755</td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">87357344</td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-02000000241-1</td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">S</td><td headers="hd_h_ml131.t3_1_1_1_2 hd_h_ml131.t3_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><div class="graphic"><img src="/books/NBK51967/bin/ml131fu12.jpg" alt="Image ml131fu12.jpg" /></div></td><td headers="hd_h_ml131.t3_1_1_1_2 hd_h_ml131.t3_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><div class="graphic"><img src="/books/NBK51967/bin/ml131fu13.jpg" alt="Image ml131fu13.jpg" /></div></td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">182</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>25</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>25</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_11" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>25</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_12" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>25</td><td headers="hd_h_ml131.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>137</td></tr><tr><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"><b>4 (Analog 3)</b></td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">44607576</td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">87357347</td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-02000000244-1</td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">S</td><td headers="hd_h_ml131.t3_1_1_1_2 hd_h_ml131.t3_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><div class="graphic"><img src="/books/NBK51967/bin/ml131fu14.jpg" alt="Image ml131fu14.jpg" /></div></td><td headers="hd_h_ml131.t3_1_1_1_2 hd_h_ml131.t3_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><div class="graphic"><img src="/books/NBK51967/bin/ml131fu15.jpg" alt="Image ml131fu15.jpg" /></div></td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">417</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>25</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>25</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_11" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">21</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_12" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">10</td><td headers="hd_h_ml131.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>60</td></tr><tr><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"><b>5 (Analog 4)</b></td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">44607573</td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">87357341</td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-02000000238-1</td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">S</td><td headers="hd_h_ml131.t3_1_1_1_2 hd_h_ml131.t3_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><div class="graphic"><img src="/books/NBK51967/bin/ml131fu16.jpg" alt="Image ml131fu16.jpg" /></div></td><td headers="hd_h_ml131.t3_1_1_1_2 hd_h_ml131.t3_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><div class="graphic"><img src="/books/NBK51967/bin/ml131fu17.jpg" alt="Image ml131fu17.jpg" /></div></td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">308</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>25</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>25</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_11" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>25</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_12" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>25</td><td headers="hd_h_ml131.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>81</td></tr><tr><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"><b>6 (Analog 5)</b></td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">44607577</td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">87357349</td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-02000000246-1</td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">S</td><td headers="hd_h_ml131.t3_1_1_1_2 hd_h_ml131.t3_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><div class="graphic"><img src="/books/NBK51967/bin/ml131fu18.jpg" alt="Image ml131fu18.jpg" /></div></td><td headers="hd_h_ml131.t3_1_1_1_2 hd_h_ml131.t3_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><div class="graphic"><img src="/books/NBK51967/bin/ml131fu19.jpg" alt="Image ml131fu19.jpg" /></div></td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">105</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>25</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>25</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_11" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>25</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_12" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">13</td><td headers="hd_h_ml131.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>238</td></tr><tr><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"><b>7</b></td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1114196</td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">87357345</td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-02000000242-1</td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">S</td><td headers="hd_h_ml131.t3_1_1_1_2 hd_h_ml131.t3_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><div class="graphic"><img src="/books/NBK51967/bin/ml131fu20.jpg" alt="Image ml131fu20.jpg" /></div></td><td headers="hd_h_ml131.t3_1_1_1_2 hd_h_ml131.t3_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><div class="graphic"><img src="/books/NBK51967/bin/ml131fu21.jpg" alt="Image ml131fu21.jpg" /></div></td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">165</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>25</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>25</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_11" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>25</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_12" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>25</td><td headers="hd_h_ml131.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>151</td></tr><tr><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"><b>8</b></td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">779010</td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">87357346</td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-02000000243-1</td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">S</td><td headers="hd_h_ml131.t3_1_1_1_2 hd_h_ml131.t3_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><div class="graphic"><img src="/books/NBK51967/bin/ml131fu22.jpg" alt="Image ml131fu22.jpg" /></div></td><td headers="hd_h_ml131.t3_1_1_1_2 hd_h_ml131.t3_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><div class="graphic"><img src="/books/NBK51967/bin/ml131fu23.jpg" alt="Image ml131fu23.jpg" /></div></td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">393</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>25</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>25</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_11" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>25</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_12" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>25</td><td headers="hd_h_ml131.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>63</td></tr><tr><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"><b>9</b></td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">44607575</td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">87357348</td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-02000000245-1</td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">S</td><td headers="hd_h_ml131.t3_1_1_1_2 hd_h_ml131.t3_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><div class="graphic"><img src="/books/NBK51967/bin/ml131fu24.jpg" alt="Image ml131fu24.jpg" /></div></td><td headers="hd_h_ml131.t3_1_1_1_2 hd_h_ml131.t3_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><div class="graphic"><img src="/books/NBK51967/bin/ml131fu25.jpg" alt="Image ml131fu25.jpg" /></div></td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">163</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">9.3</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">4.8</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_11" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">9.9</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_12" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">11.2</td><td headers="hd_h_ml131.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">57</td></tr><tr><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"><b>10</b></td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">878124</td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">87357342</td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-02000000239-1</td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">S</td><td headers="hd_h_ml131.t3_1_1_1_2 hd_h_ml131.t3_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><div class="graphic"><img src="/books/NBK51967/bin/ml131fu26.jpg" alt="Image ml131fu26.jpg" /></div></td><td headers="hd_h_ml131.t3_1_1_1_2 hd_h_ml131.t3_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><div class="graphic"><img src="/books/NBK51967/bin/ml131fu27.jpg" alt="Image ml131fu27.jpg" /></div></td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">497</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>25</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>25</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_11" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>25</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_12" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>25</td><td headers="hd_h_ml131.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>50</td></tr><tr><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"><b>11</b></td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">44607574</td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">87357352</td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-02000000249-1</td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">S</td><td headers="hd_h_ml131.t3_1_1_1_2 hd_h_ml131.t3_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><div class="graphic"><img src="/books/NBK51967/bin/ml131fu28.jpg" alt="Image ml131fu28.jpg" /></div></td><td headers="hd_h_ml131.t3_1_1_1_2 hd_h_ml131.t3_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><div class="graphic"><img src="/books/NBK51967/bin/ml131fu29.jpg" alt="Image ml131fu29.jpg" /></div></td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">253</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>25</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>25</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_11" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>25</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_12" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>25</td><td headers="hd_h_ml131.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>99</td></tr><tr><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"><b>12</b></td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1363299</td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">87457330</td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-01000669308-3</td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">S</td><td headers="hd_h_ml131.t3_1_1_1_2 hd_h_ml131.t3_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><div class="graphic"><img src="/books/NBK51967/bin/ml131fu30.jpg" alt="Image ml131fu30.jpg" /></div></td><td headers="hd_h_ml131.t3_1_1_1_2 hd_h_ml131.t3_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><div class="graphic"><img src="/books/NBK51967/bin/ml131fu31.jpg" alt="Image ml131fu31.jpg" /></div></td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2030</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">N.D.<sup><a class="bk_pop" href="#ml131.tfn2">†</a></sup></td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">N.D.<sup><a class="bk_pop" href="#ml131.tfn2">†</a></sup></td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_11" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">N.D.<sup><a class="bk_pop" href="#ml131.tfn2">†</a></sup></td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_12" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">N.D.<sup><a class="bk_pop" href="#ml131.tfn2">†</a></sup></td><td headers="hd_h_ml131.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">N.D.<sup><a class="bk_pop" href="#ml131.tfn2">†</a></sup></td></tr><tr><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"><b>13</b></td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">4492589</td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">87457331</td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-02000000251-1</td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">S</td><td headers="hd_h_ml131.t3_1_1_1_2 hd_h_ml131.t3_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><div class="graphic"><img src="/books/NBK51967/bin/ml131fu32.jpg" alt="Image ml131fu32.jpg" /></div></td><td headers="hd_h_ml131.t3_1_1_1_2 hd_h_ml131.t3_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><div class="graphic"><img src="/books/NBK51967/bin/ml131fu33.jpg" alt="Image ml131fu33.jpg" /></div></td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">78</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>25</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">7.9</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_11" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">19.5</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_12" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>25</td><td headers="hd_h_ml131.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>320</td></tr><tr><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"><b>14</b></td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">44607582</td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">87357350</td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-02000000247-1</td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">S</td><td headers="hd_h_ml131.t3_1_1_1_2 hd_h_ml131.t3_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><div class="graphic"><img src="/books/NBK51967/bin/ml131fu34.jpg" alt="Image ml131fu34.jpg" /></div></td><td headers="hd_h_ml131.t3_1_1_1_2 hd_h_ml131.t3_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><div class="graphic"><img src="/books/NBK51967/bin/ml131fu35.jpg" alt="Image ml131fu35.jpg" /></div></td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">302</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>25</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">13.8</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_11" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">15.8</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_12" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>25</td><td headers="hd_h_ml131.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>82</td></tr><tr><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"><b>15</b></td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">44607579</td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">87357353</td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-02000000250-1</td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">S</td><td headers="hd_h_ml131.t3_1_1_1_2 hd_h_ml131.t3_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><div class="graphic"><img src="/books/NBK51967/bin/ml131fu36.jpg" alt="Image ml131fu36.jpg" /></div></td><td headers="hd_h_ml131.t3_1_1_1_2 hd_h_ml131.t3_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><div class="graphic"><img src="/books/NBK51967/bin/ml131fu37.jpg" alt="Image ml131fu37.jpg" /></div></td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">169</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>25</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">9.5</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_11" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>25</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_12" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>25</td><td headers="hd_h_ml131.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>145</td></tr><tr><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"><b>16</b></td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">7275219</td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">87357338</td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-02000000235-1</td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">S</td><td headers="hd_h_ml131.t3_1_1_1_2 hd_h_ml131.t3_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><div class="graphic"><img src="/books/NBK51967/bin/ml131fu38.jpg" alt="Image ml131fu38.jpg" /></div></td><td headers="hd_h_ml131.t3_1_1_1_2 hd_h_ml131.t3_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><div class="graphic"><img src="/books/NBK51967/bin/ml131fu39.jpg" alt="Image ml131fu39.jpg" /></div></td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">269</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>25</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>25</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_11" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>25</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_12" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>25</td><td headers="hd_h_ml131.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>92</td></tr><tr><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"><b>17</b></td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">44607572</td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">87357339</td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-02000000236-1</td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">S</td><td headers="hd_h_ml131.t3_1_1_1_2 hd_h_ml131.t3_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><div class="graphic"><img src="/books/NBK51967/bin/ml131fu40.jpg" alt="Image ml131fu40.jpg" /></div></td><td headers="hd_h_ml131.t3_1_1_1_2 hd_h_ml131.t3_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><div class="graphic"><img src="/books/NBK51967/bin/ml131fu41.jpg" alt="Image ml131fu41.jpg" /></div></td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">513</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>25</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>25</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_11" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>25</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_12" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>25</td><td headers="hd_h_ml131.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>55</td></tr><tr><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"><b>18</b></td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">44607578</td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">87357340</td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-02000000237-1</td><td headers="hd_h_ml131.t3_1_1_1_1 hd_h_ml131.t3_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">S</td><td headers="hd_h_ml131.t3_1_1_1_2 hd_h_ml131.t3_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><div class="graphic"><img src="/books/NBK51967/bin/ml131fu42.jpg" alt="Image ml131fu42.jpg" /></div></td><td headers="hd_h_ml131.t3_1_1_1_2 hd_h_ml131.t3_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><div class="graphic"><img src="/books/NBK51967/bin/ml131fu43.jpg" alt="Image ml131fu43.jpg" /></div></td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">414</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>25</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>25</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_11" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>25</td><td headers="hd_h_ml131.t3_1_1_1_3 hd_h_ml131.t3_1_1_2_12" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">19.2</td><td headers="hd_h_ml131.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>60</td></tr></tbody></table></div><div><div><dl class="temp-labeled-list small"><dt>*</dt><dd><div id="ml131.tfn1"><p class="no_margin">P = Purchased; S = Synthesized</p></div></dd><dt>†</dt><dd><div id="ml131.tfn2"><p class="no_margin">N.D. = Not Determined; this compound was not tested in counterscreen assays because its S1P4 ANT IC50 was unacceptably high.</p></div></dd></dl></div></div></div></div><div id="ml131.s28"><h3>3.5. Cellular Activity</h3><p>All the assays employed in the development of probe <a href="/pcsubstance/?term=ML131[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML131</a> were cell-based assays.</p><p><a href="/pcsubstance/?term=ML131[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML131</a> was evaluated for cell toxicity (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/489009" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-489009</a>) and its CC50 was determined to be >20 µM. This result is expected since cytotoxic compounds would disrupt signaling. The ligand binding domain of S1P4 receptor is extracellular and an antagonist is not required to cross the plasma membrane.</p></div><div id="ml131.s29"><h3>3.6. Profiling Assays</h3><p>To date, the lead hit (CID 1363299) has been tested in 420 other bioassays deposited in PubChem, and has shown activity in only 18 of those assays, five of which are for the S1P4 receptor antagonist project. The other 13 assays give a hit rate of 3.1%, indicating that this series is not generally active across a broad range of cell-based and non-cell based assays.</p><p>Compound <a href="/pcsubstance/?term=ML131[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML131</a> was submitted to Ricerca Biosciences, LLC for HitProfilingScreen + CYP450 (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/489017" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-489017</a>). The purpose of this panel of binding assays was to identify any potential receptors, transporters, or ion channels for which compound <a href="/pcsubstance/?term=ML131[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML131</a> displays affinity. Out of 35 targets tested, only one resulted in ≥50% inhibition or stimulation of activity; <a href="/pcsubstance/?term=ML131[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML131</a> inhibited CYP450 (2C9) by 51%. These data suggest that compound <b><a href="/pcsubstance/?term=ML131[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML131</a></b> is generally inactive against a broad array of off targets and does not likely exert unwanted effects.</p></div></div><div id="ml131.s30"><h2 id="_ml131_s30_">4. Discussion</h2><p>Probe <a href="/pcsubstance/?term=ML131[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML131</a> inhibits S1P4 receptor in a cell-based TANGO-format assay (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2346" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-2346</a>) with an IC50 of 89 nM; and is inactive against other members of the receptor family, with IC50s >25 µM against S1P1 receptor (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2351" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-2351</a>), S1P2 receptor (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2354" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-2354</a>), S1P3 receptor (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2349" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-2349</a>), and S1P5 receptor (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2350" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-2350</a>), and is nontoxic to U2OS cells (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/489009" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-489009</a>), with a CC50 of >20 µM.</p><div id="ml131.s31"><h3>4.1. Comparison to existing art and how the new probe is an improvement</h3><p>There are no S1P4 antagonist compounds currently available. Dose response assays against all five S1P receptors demonstrate that <a href="/pcsubstance/?term=ML131[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML131</a> is the first submicromolar, completely selective S1P4 receptor antagonist to be identified. This probe will be invaluable in research efforts designed to determine the effects of S1P4 signaling.</p></div><div id="ml131.s32"><h3>4.2. Mechanism of Action Studies</h3><p>The S1P5 receptor counterscreen assay is in the same TANGO format as the S1P4 receptor assay used for the primary screening based upon arrestin recruitment to activated receptor. From this we infer that the probe compound directly interacts and activates the S1P4 receptor. Work is ongoing in the assay provider’s lab with this probe to elucidate the signaling pathway activated in native, S1P4 receptor-expressing cells.</p></div><div id="ml131.s33"><h3>4.3. Planned Future Studies</h3><p>Medicinal chemistry to enhance solubility and potency are planned. In the extended probe development period, our aim will be to develop these further using traditional MedChem techniques and scaffold hopping methods to produce an increase in potency while maintaining selectivity. The pharmacokinetic (PK) properties of the lead compounds will also be optimized using a series of <i>in vitro</i> and <i>in vivo</i> (mouse) PK studies to identify a compound that is suitable for use in animal studies. The most promising compounds will be tested for selectivity against the panel of off-target proteins including GPCRs and ion channels.</p><p>Compounds with improved solubility and potency will be useful for mechanism of action studies planned by Dr. Oldstone, including the role of S1P4 receptor in dendritic cell migration, production of pro-inflammatory cytokines, and the role of S1P4 receptor in the response to influenza in mice.</p></div></div><div id="ml131.s34"><h2 id="_ml131_s34_">5. References</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="ml131.r1">Marsolais D, Hahm B, Walsh K, Edelmann K, MacGavern D, Hatta Y, Kawaoka Y, Rosen Ha, OM A critical role for the sphingosine analog AAL-R in dampening the cytokine response during influenza virus infection. <span><span class="ref-journal">Proc. Natl. Acad. Sci. </span>2009;<span class="ref-vol">106</span>(5):1560–5.</span> [<a href="/pmc/articles/PMC2635800/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2635800</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/19164548" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19164548</span></a>]</div></dd><dt>2.</dt><dd><div class="bk_ref" id="ml131.r2">Marsolais D, Rosen H. Chemical modulators of sphingosine-1-phosphate receptors as barrier-oriented therapeutic molecules. <span><span class="ref-journal">Nat. Rev. Drug Disc. </span>2009;<span class="ref-vol">8</span>(4):297–307.</span> [<a href="/pmc/articles/PMC4455967/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC4455967</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/19300460" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19300460</span></a>]</div></dd><dt>3.</dt><dd><div class="bk_ref" id="ml131.r3">Sanna MG, Liao J, Jo E, Alfonso C, Ahn MY, Peterson MS, Webb B, Lefebvre S, Chun J, Gray N, Rosen H. Sphingosine 1-phosphate (S1P) receptor subtypes S1P1 and S1P3, respectively, regulate lymphocyte recirculation and heart rate. <span><span class="ref-journal">J Biol Chem. </span>2004;<span class="ref-vol">279</span>(14):13839–48.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/14732717" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 14732717</span></a>]</div></dd><dt>4.</dt><dd><div class="bk_ref" id="ml131.r4">Wei SH, Rosen H, Matheu MP, Sanna MG, Wang SK, Jo E, Wong CH, Parker I, Cahalan MD. Sphingosine 1-phosphate type 1 receptor agonism inhibits transendothelial migration of medullary T cells to lymphatic sinuses. <span><span class="ref-journal">Nat Immunol. </span>2005;<span class="ref-vol">6</span>(12):1228–35.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/16273098" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 16273098</span></a>]</div></dd><dt>5.</dt><dd><div class="bk_ref" id="ml131.r5">Alfonso C, McHeyzer-Williams MG, Rosen H. CD69 down-modulation and inhibition of thymic egress by short- and long-term selective chemical agonism of sphingosine 1-phosphate receptors. <span><span class="ref-journal">Eur J Immunol. </span>2006;<span class="ref-vol">36</span>(1):149–59.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/16342326" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 16342326</span></a>]</div></dd><dt>6.</dt><dd><div class="bk_ref" id="ml131.r6">Jo E, Sanna MG, Gonzalez-Cabrera PJ, Thangada S, Tigyi G, Osborne DA, Hla T, Parrill AL, Rosen H. S1P1-selective in vivo-active agonists from high-throughput screening: off-the-shelf chemical probes of receptor interactions, signaling, and fate. <span><span class="ref-journal">Chem Biol. </span>2005;<span class="ref-vol">12</span>(6):703–15.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/15975516" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 15975516</span></a>]</div></dd><dt>7.</dt><dd><div class="bk_ref" id="ml131.r7">Marsolais D, Hahm B, Edelmann KH, Walsh KB, Guerrero M, Hatta Y, Kawaoka Y, Roberts E, Oldstone MB, Rosen H. Local not systemic modulation of dendritic cell S1P receptors in lung blunts virus-specific immune responses to influenza. <span><span class="ref-journal">Mol Pharmacol. </span>2008;<span class="ref-vol">74</span>(3):896–903.</span> [<a href="/pmc/articles/PMC2574812/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2574812</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/18577684" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18577684</span></a>]</div></dd><dt>8.</dt><dd><div class="bk_ref" id="ml131.r8">Maeda Y, Matsuyuki H, Shimano K, Kataoka H, Sugahara K, Chiba K. Migration of CD4 T cells and dendritic cells toward sphingosine 1-phosphate (S1P) is mediated by different receptor subtypes: S1P regulates the functions of murine mature dendritic cells via S1P receptor type 3. <span><span class="ref-journal">J Immunol. </span>2007;<span class="ref-vol">178</span>(6):3437–46.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/17339438" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17339438</span></a>]</div></dd><dt>9.</dt><dd><div class="bk_ref" id="ml131.r9">Toman RE, Spiegel S. Lysophospholipid receptors in the nervous system. <span><span class="ref-journal">Neurochem Res. </span>2002;<span class="ref-vol">27</span>(7-8):619–27.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/12374197" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 12374197</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/NBK51967/?report=reader">PubReader</a></li><li><a href="/books/NBK51967/?report=printable">Print View</a></li><li><a data-jig="ncbidialog" href="#_ncbi_dlg_citbx_NBK51967" data-jigconfig="width:400,modal:true">Cite this Page</a><div id="_ncbi_dlg_citbx_NBK51967" style="display:none" title="Cite this Page"><div class="bk_tt">Oldstone M, Hodder P, Crisp M, et al. Probe Development Efforts to Identify Novel Antagonists of the Sphingosine 1-phosphate Receptor 4 (S1P4) 2010 Feb 24 [Updated 2010 Dec 16]. 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><li><a href="/books/NBK51967/pdf/Bookshelf_NBK51967.pdf">PDF version of this page</a> (588K)</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="#ml131.s1" ref="log$=inpage&link_id=inpage">Probe Structure & Characteristics</a></li><li><a href="#ml131.s2" ref="log$=inpage&link_id=inpage">Recommendations for scientific use of the probe</a></li><li><a href="#ml131.s3" ref="log$=inpage&link_id=inpage">Introduction</a></li><li><a href="#ml131.s4" ref="log$=inpage&link_id=inpage">Materials and Methods</a></li><li><a href="#ml131.s23" ref="log$=inpage&link_id=inpage">Results</a></li><li><a href="#ml131.s30" ref="log$=inpage&link_id=inpage">Discussion</a></li><li><a href="#ml131.s34" ref="log$=inpage&link_id=inpage">References</a></li></ul></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Related information</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="discovery_db_links" id="Shutter"></a></div><div class="portlet_content"><ul><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pmc&DbFrom=books&Cmd=Link&LinkName=books_pmc_refs&IdsFromResult=2412246" ref="log$=recordlinks">PMC</a><div class="brieflinkpop offscreen_noflow">PubMed Central citations</div></li><li class="brieflinkpopper"><a class="brieflinkpopperctrl" 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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/22834032" ref="ordinalpos=1&linkpos=1&log$=relatedreviews&logdbfrom=pubmed"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> Probe Development Efforts to Identify Novel Agonists of the Sphingosine 1-phosphate Receptor 4 (S1P4).</a><span class="source">[Probe Reports from the NIH Mol...]</span><div class="brieflinkpop offscreen_noflow"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> Probe Development Efforts to Identify Novel Agonists of the Sphingosine 1-phosphate Receptor 4 (S1P4).<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Guerrero M, Urbano M, Velaparthi S, Zhao J, Schaeffer MT, Brown SJ, Crisp M, Ferguson J, Roberts E, Oldstone M, et al. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Probe Reports from the NIH Molecular Libraries Program. 2010</em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/23762926" ref="ordinalpos=1&linkpos=2&log$=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> Identification of a Novel Agonist of the Sphingosine 1-phosphate Receptor 4 (S1P4).</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> Identification of a Novel Agonist of the Sphingosine 1-phosphate Receptor 4 (S1P4).<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Guerrero M, Urbano M, Velaparthi S, Schaeffer MT, Brown SJ, Crisp M, Ferguson J, Hodder P, Rosen H, Oldstone M, et al. </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/23658964" ref="ordinalpos=1&linkpos=3&log$=relatedreviews&logdbfrom=pubmed"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> Probe 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ref="ordinalpos=1&linkpos=5&log$=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> Optimization and characterization of an opioid kappa receptor (OPRK1) antagonist.</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> Optimization and characterization of an opioid kappa receptor (OPRK1) antagonist.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Guerrero M, Urbano M, Brown SJ, Cayanan C, Ferguson J, Cameron M, Devi LA, Roberts E, Rosen H. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Probe Reports 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