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<title>Identification of a Novel Agonist 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="Identification of a Novel Agonist 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" /><meta name="citation_author" content="Miguel Guerrero" /><meta name="citation_author" content="Mariangela Urbano" /><meta name="citation_author" content="Subash Velaparthi" /><meta name="citation_author" content="Marie-Therese Schaeffer" /><meta name="citation_author" content="Steven J Brown" /><meta name="citation_author" content="Melissa Crisp" /><meta name="citation_author" content="Jill Ferguson" /><meta name="citation_author" content="Peter Hodder" /><meta name="citation_author" content="Hugh Rosen" /><meta name="citation_author" content="Michael Oldstone" /><meta name="citation_author" content="Edward Roberts" /><meta name="citation_pmid" content="23762926" /><meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK143556/" /><link rel="schema.DC" href="http://purl.org/DC/elements/1.0/" /><meta name="DC.Title" content="Identification of a Novel Agonist 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="Miguel Guerrero" /><meta name="DC.Contributor" content="Mariangela Urbano" /><meta name="DC.Contributor" content="Subash Velaparthi" /><meta name="DC.Contributor" content="Marie-Therese Schaeffer" /><meta name="DC.Contributor" content="Steven J Brown" /><meta name="DC.Contributor" content="Melissa Crisp" /><meta name="DC.Contributor" content="Jill Ferguson" /><meta name="DC.Contributor" content="Peter Hodder" /><meta name="DC.Contributor" content="Hugh Rosen" /><meta name="DC.Contributor" content="Michael Oldstone" /><meta name="DC.Contributor" content="Edward Roberts" /><meta name="DC.Date" content="2010" /><meta name="DC.Identifier" content="https://www.ncbi.nlm.nih.gov/books/NBK143556/" /><meta name="description" content="Sphingosine 1-phosphate (S1P) is a bioactive phospholipid released by activated blood platelets that serves to influence heart rate, coronary artery caliber, endothelial integrity, lung epithelial integrity, and lymphocyte recirculation through five related high affinity G-protein coupled receptors. S1P4 receptor is coupled to Gαi and Gαo G proteins and activates extracellular signal-regulated kinases (ERK) mitogen-activated protein kinases (MAPK) and Phospholipase C (PLC) downstream pathways. Inhibition of lymphocyte recirculation by nonselective S1P receptor agonists produces clinical immunosuppression preventing transplant rejection, but is associated with transient bradycardia. Understanding the contribution of individual receptors has been limited by the unavailability of selective agonists or antagonists for the 5 receptor subtypes. The Scripps Research Institute Molecular Screening Center (SRIMSC), part of the Molecular Libraries Probe Production Centers Network (MLPCN), previously identified an S1P4 agonist probe, ML178, which has submicromolar potency and is completely selective against the five other S1P receptor family members; but has non-classic structure and flat medicinal chemistry. The SRIMSC reports here ML248, a novel compound with potent and selective S1P4 receptor agonist activity that is amenable to further medicinal chemistry optimization. ML248 was identified by high-throughput screening using a cell-based Tango™-format assay. ML248 activates S1P4 receptor with a half maximal effective concentration (EC50) of 37.7 nM–79.1 nM, and is inactive as an agonist against other members of the receptor family, with EC50s > 25 μM against S1P1, S1P2, and S1P3 receptors, and an EC50 of 2.1 μM against the S1P5 receptor. ML248 is inhibited by an S1P4 receptor-selective antagonist, inactive for agonism in the presence of ≥ 3.7 nM selective antagonist and is nontoxic to U2OS cells, with a CC50 of > 10 μM. ML248 was submitted to Ricerca Biosciences LLC., target profiling against a panel of receptors, transporters, or ion channels; the data suggest that ML248 is generally inactive against a broad array of off-targets and does not likely exert unwanted effects." /><meta name="og:title" content="Identification of a Novel Agonist of the Sphingosine 1-phosphate Receptor 4 (S1P4)" /><meta name="og:type" content="book" /><meta name="og:description" content="Sphingosine 1-phosphate (S1P) is a bioactive phospholipid released by activated blood platelets that serves to influence heart rate, coronary artery caliber, endothelial integrity, lung epithelial integrity, and lymphocyte recirculation through five related high affinity G-protein coupled receptors. S1P4 receptor is coupled to Gαi and Gαo G proteins and activates extracellular signal-regulated kinases (ERK) mitogen-activated protein kinases (MAPK) and Phospholipase C (PLC) downstream pathways. Inhibition of lymphocyte recirculation by nonselective S1P receptor agonists produces clinical immunosuppression preventing transplant rejection, but is associated with transient bradycardia. Understanding the contribution of individual receptors has been limited by the unavailability of selective agonists or antagonists for the 5 receptor subtypes. The Scripps Research Institute Molecular Screening Center (SRIMSC), part of the Molecular Libraries Probe Production Centers Network (MLPCN), previously identified an S1P4 agonist probe, ML178, which has submicromolar potency and is completely selective against the five other S1P receptor family members; but has non-classic structure and flat medicinal chemistry. The SRIMSC reports here ML248, a novel compound with potent and selective S1P4 receptor agonist activity that is amenable to further medicinal chemistry optimization. ML248 was identified by high-throughput screening using a cell-based Tango™-format assay. ML248 activates S1P4 receptor with a half maximal effective concentration (EC50) of 37.7 nM–79.1 nM, and is inactive as an agonist against other members of the receptor family, with EC50s > 25 μM against S1P1, S1P2, and S1P3 receptors, and an EC50 of 2.1 μM against the S1P5 receptor. ML248 is inhibited by an S1P4 receptor-selective antagonist, inactive for agonism in the presence of ≥ 3.7 nM selective antagonist and is nontoxic to U2OS cells, with a CC50 of > 10 μM. ML248 was submitted to Ricerca Biosciences LLC., target profiling against a panel of receptors, transporters, or ion channels; the data suggest that ML248 is generally inactive against a broad array of off-targets and does not likely exert unwanted effects." /><meta name="og:url" content="https://www.ncbi.nlm.nih.gov/books/NBK143556/" /><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/ml248/" /><link rel="canonical" href="https://www.ncbi.nlm.nih.gov/books/NBK143556/" /><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="#__NBK143556_dtls__">Show details</a><div style="display:none" class="ui-widget" id="__NBK143556_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/ml249/" title="Previous page in this title">< Prev</a><a class="active page_link next" href="/books/n/mlprobe/ml247/" 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="_NBK143556_"><span class="title" itemprop="name">Identification of a Novel Agonist of the Sphingosine 1-phosphate Receptor 4 (S1P4)</span></h1><p class="contrib-group"><span itemprop="author">Miguel Guerrero</span>, <span itemprop="author">Mariangela Urbano</span>, <span itemprop="author">Subash Velaparthi</span>, <span itemprop="author">Marie-Therese Schaeffer</span>, <span itemprop="author">Steven J Brown</span>, <span itemprop="author">Melissa Crisp</span>, <span itemprop="author">Jill Ferguson</span>, <span itemprop="author">Peter Hodder</span>, <span itemprop="author">Hugh Rosen</span>, <span itemprop="author">Michael Oldstone</span>, and <span itemprop="author">Edward Roberts</span>.</p><a data-jig="ncbitoggler" href="#__NBK143556_ai__" style="border:0;text-decoration:none">Author Information and Affiliations</a><div style="display:none" class="ui-widget" id="__NBK143556_ai__"><p class="contrib-group"><h4>Authors</h4><span itemprop="author">Miguel Guerrero</span>,<sup>*</sup> <span itemprop="author">Mariangela Urbano</span>,<sup>*</sup> <span itemprop="author">Subash Velaparthi</span>,<sup>*</sup> <span itemprop="author">Marie-Therese Schaeffer</span>,<sup>†</sup> <span itemprop="author">Steven J Brown</span>,<sup>†</sup> <span itemprop="author">Melissa Crisp</span>,<sup>‡</sup> <span itemprop="author">Jill Ferguson</span>,<sup>†</sup> <span itemprop="author">Peter Hodder</span>,<sup>‡</sup> <span itemprop="author">Hugh Rosen</span>,<sup>*</sup><sup>,1</sup> <span itemprop="author">Michael Oldstone</span>,<sup>§</sup> and <span itemprop="author">Edward Roberts</span><sup>*</sup>.</p><h4>Affiliations</h4><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 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 Immunology and Microbial Science, The Scripps Research Institute, La Jolla CA 92037</div><div class="affiliation"><sup>1</sup> Corresponding author:
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<span class="before-email-separator"></span><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="ude.sppircs@nesorh" class="oemail">ude.sppircs@nesorh</a></div></div></div><div class="jig-ncbiinpagenav body-content whole_rhythm" data-jigconfig="allHeadingLevels: ['h2'],smoothScroll: false" itemprop="text"><div id="_abs_rndgid_" itemprop="description"><p>Sphingosine 1-phosphate (S1P) is a bioactive phospholipid released by activated blood platelets that serves to influence heart rate, coronary artery caliber, endothelial integrity, lung epithelial integrity, and lymphocyte recirculation through five related high affinity G-protein coupled receptors. S1P4 receptor is coupled to Gαi and Gαo G proteins and activates extracellular signal-regulated kinases (ERK) mitogen-activated protein kinases (MAPK) and Phospholipase C (PLC) downstream pathways. Inhibition of lymphocyte recirculation by nonselective S1P receptor agonists produces clinical immunosuppression preventing transplant rejection, but is associated with transient bradycardia. Understanding the contribution of individual receptors has been limited by the unavailability of selective agonists or antagonists for the 5 receptor subtypes. The Scripps Research Institute Molecular Screening Center (SRIMSC), part of the Molecular Libraries Probe Production Centers Network (MLPCN), previously identified an S1P4 agonist probe, <a href="/pcsubstance/?term=ML178[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML178</a>, which has submicromolar potency and is completely selective against the five other S1P receptor family members; but has non-classic structure and flat medicinal chemistry. The SRIMSC reports here <a href="/pcsubstance/?term=ML248[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML248</a>, a novel compound with potent and selective S1P4 receptor agonist activity that is amenable to further medicinal chemistry optimization. <a href="/pcsubstance/?term=ML248[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML248</a> was identified by high-throughput screening using a cell-based Tango™-format assay. <a href="/pcsubstance/?term=ML248[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML248</a> activates S1P4 receptor with a half maximal effective concentration (EC50) of 37.7 nM–79.1 nM, and is inactive as an agonist against other members of the receptor family, with EC50s > 25 μM against S1P1, S1P2, and S1P3 receptors, and an EC50 of 2.1 μM against the S1P5 receptor. <a href="/pcsubstance/?term=ML248[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML248</a> is inhibited by an S1P4 receptor-selective antagonist, inactive for agonism in the presence of ≥ 3.7 nM selective antagonist and is nontoxic to U2OS cells, with a CC50 of > 10 μM. <a href="/pcsubstance/?term=ML248[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML248</a> was submitted to Ricerca Biosciences LLC., target profiling against a panel of receptors, transporters, or ion channels; the data suggest that <a href="/pcsubstance/?term=ML248[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML248</a> is generally inactive against a broad array of off-targets and does not likely exert unwanted effects.</p></div><div class="h2"></div><p><b>Assigned Assay Grant #:</b> 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, Scripps Research Institute Molecular Screening Center, H. Rosen, W. Roush</p><p><b>Chemistry Center Name & PI:</b> Scripps Research Institute Molecular Screening Center, Scripps Research Institute Molecular Screening Center, H. Rosen, W. Roush</p><p><b>Assay Submitter & Institution:</b> Michael B. Oldstone MD, Professor, 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/1801" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1801</a></p><div id="ml248.s1"><h2 id="_ml248_s1_">Probe Structure & Characteristics</h2><div id="ml248.fu1" class="figure bk_fig"><div class="graphic"><img src="/books/NBK143556/bin/ml248fu1.jpg" alt="ML248." /></div><h3><span class="title">ML248</span></h3></div><div id="ml248.tu1" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK143556/table/ml248.tu1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml248.tu1_lrgtbl__"><table><thead><tr><th id="hd_h_ml248.tu1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">CID/ML#</th><th id="hd_h_ml248.tu1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Target Name</th><th id="hd_h_ml248.tu1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">EC50 (nM) [SID, AID]</th><th id="hd_h_ml248.tu1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Anti-target Name</th><th id="hd_h_ml248.tu1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">EC50 (μM) [SID, AID]</th><th id="hd_h_ml248.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Fold Selective</th><th id="hd_h_ml248.tu1_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Secondary Assay(s) Name: EC50 (μM) [SID, AID]</th></tr></thead><tbody><tr><td headers="hd_h_ml248.tu1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CID 49835928/<a href="/pcsubstance/?term=ML248[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML248</a></td><td headers="hd_h_ml248.tu1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">S1P4</td><td headers="hd_h_ml248.tu1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">37.7–79.1 nM[<a href="https://pubchem.ncbi.nlm.nih.gov/substance/103911214" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 103911214</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504877" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504877</a>]</td><td headers="hd_h_ml248.tu1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">S1P1</td><td headers="hd_h_ml248.tu1_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>25 μM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/103911214" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 103911214</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504870" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504870</a>]</td><td headers="hd_h_ml248.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>316–663</td><td headers="hd_h_ml248.tu1_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;"><b>S1P1 AG Counterscreen:</b> >25 μM<br />[<a href="https://pubchem.ncbi.nlm.nih.gov/substance/103911214" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 103911214</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504870" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504870</a>]<br /><b>S1P2 AG Counterscreen:</b> >25 μM<br />[<a href="https://pubchem.ncbi.nlm.nih.gov/substance/103911214" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 103911214</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504873" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504873</a>]<br /><b>S1P3 AG Counterscreen:</b> >25 μM<br />[<a href="https://pubchem.ncbi.nlm.nih.gov/substance/103911214" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 103911214</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504879" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504879</a>]<br /><b>S1P5 AG Counterscreen:</b> 2.1 μM<br />[<a href="https://pubchem.ncbi.nlm.nih.gov/substance/103911214" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 103911214</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504867" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504867</a>]<br /><b>Cytotoxicity</b>: CC50>10 μM<br />[<a href="https://pubchem.ncbi.nlm.nih.gov/substance/103911214" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 103911214</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504875" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504875</a>]<br /><b>Inhibition by antagonist control:</b> EC50 15.4 nM<br />[<a href="https://pubchem.ncbi.nlm.nih.gov/substance/103911214" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 103911214</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504871" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504871</a>]<br /><b>Inhibition by 0.4 nM antagonist:</b> EC50 40 nM<br />[<a href="https://pubchem.ncbi.nlm.nih.gov/substance/103911214" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 103911214</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504918" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504918</a>]<br /><b>Inhibition by 1.2 nM antagonist:</b> EC50 173 nM<br />[<a href="https://pubchem.ncbi.nlm.nih.gov/substance/103911214" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 103911214</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504917" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504917</a>]<br /><b>Inhibition by 3.7 nM antagonist:</b> EC50>3.3 μM<br />[<a href="https://pubchem.ncbi.nlm.nih.gov/substance/103911214" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 103911214</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504924" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504924</a>]<br /><b>Inhibition by 11 nM antagonist:</b> EC50>3.3 μM<br />[<a href="https://pubchem.ncbi.nlm.nih.gov/substance/103911214" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 103911214</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504919" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504919</a>]<br /><b>Inhibition by 33 nM antagonist:</b> EC50>3.3 μM<br />[<a href="https://pubchem.ncbi.nlm.nih.gov/substance/103911214" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 103911214</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504921" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504921</a>]<br /><b>Inhibition by 100 nM antagonist:</b> EC50>3.3 μM<br />[<a href="https://pubchem.ncbi.nlm.nih.gov/substance/103911214" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 103911214</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504923" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504923</a>]<br /><b>Ricerca profiling:</b><br />[<a href="https://pubchem.ncbi.nlm.nih.gov/substance/103911214" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 103911214</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/540332" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 540332</a>]</td></tr></tbody></table></div></div></div><div id="ml248.s2"><h2 id="_ml248_s2_">Recommendations for Scientific Use of the Probe</h2><p>Sphingosine 1-phosphate (S1P), a bioactive phospholipid released by activated blood platelets, influences multiple physiological systems, including endothelial integrity, lung epithelial integrity (<a class="bk_pop" href="#ml248.r1">1</a>–<a class="bk_pop" href="#ml248.r2">2</a>), and lymphocyte recirculation (<a class="bk_pop" href="#ml248.r3">3</a>–<a class="bk_pop" href="#ml248.r7">7</a>) through five related high affinity G-protein coupled receptors (<a class="bk_pop" href="#ml248.r4">4</a>). Understanding the contributions of individual S1P receptors to these physiological processes has been limited by the unavailability of selective agonists or antagonists for the 5 receptor subtypes. Following influenza infection, an excessive immune response on the part of the host can be detrimental by causing damage to tissues. 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="#ml248.r8">8</a>). The elucidation of the exact S1P receptors to be modulated for inhibition of pulmonary immune response is limited by the availability of specific probes that are water-soluble and suitable for local delivery in the airways. Reports showing that S1P5 receptor expression is very low in dendritic cells but that S1P4 receptor is highly expressed (<a class="bk_pop" href="#ml248.r9">9</a>), suggest that chemical activation of the S1P4 receptor subtype in the airways could be efficient at controlling the immunopathological response to viral infection. The availability of a selective S1P4 receptor antagonist may potentially provide a therapeutic tool for ensuring a balanced host immune response to influenza infection, in addition to serving as a useful tool for understanding S1P4 receptor biological function. <a href="/pcsubstance/?term=ML248[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML248</a> has a structure that is amenable to further medicinal chemistry optimization. This probe should be useful for extended optimization, characterization, and assays in animal models of influenza.</p></div><div id="ml248.s3"><h2 id="_ml248_s3_">1. Introduction</h2><p>Sphingosine 1-phosphate (S1P) is a bioactive phospholipid released by activated blood platelets that serves to influence heart rate (<a class="bk_pop" href="#ml248.r4">4</a>, <a class="bk_pop" href="#ml248.r6">6</a>), coronary artery caliber, endothelial integrity, lung epithelial integrity (<a class="bk_pop" href="#ml248.r1">1</a>–<a class="bk_pop" href="#ml248.r2">2</a>), and lymphocyte recirculation (<a class="bk_pop" href="#ml248.r3">3</a>–<a class="bk_pop" href="#ml248.r7">7</a>) through five related high affinity G-protein coupled receptors (<a class="bk_pop" href="#ml248.r4">4</a>). S1P4 receptor, the subject of this report, is coupled to Gαi and Gαo G proteins and activates ERK MAPK and PLC downstream pathways (<a class="bk_pop" href="#ml248.r10">10</a>). Inhibition of lymphocyte recirculation by nonselective S1P receptor agonists produces clinical immunosuppression preventing transplant rejection, but is associated with transient bradycardia. In addition, it has been suggested that S1P4 expression and function may play a role in stimulating the ERK 1/2 pathway in a breast cancer cell line (<a class="bk_pop" href="#ml248.r11">11</a>). Understanding the contribution of individual receptors has been limited by the unavailability of selective agonists or antagonists for the 5 receptor subtypes.</p><p>We have recently published a proof-of-concept paper showing that modulation of S1P receptors locally in the lungs during influenza infection altered dendritic cell (DC) activation and accumulation in the mediastinal lymph nodes, resulting in blunted T-cell response and allowing control of immunopathological features of the infection (<a class="bk_pop" href="#ml248.r8">8</a>). Upon influenza virus infection, pulmonary DC become activated and migrate towards mediastinal lymph nodes to initiate the T-cell response. Influenza virus-specific T cells are rapidly induced and proliferate in mediastinal lymph node, then migrate to the infected sites, including the lungs, where they cause damage. While airway delivery of the broad S1P receptor agonist AFD-R, with activity on S1P1, S1P3, S1P4, and S1P5 receptors, but not S1P2 receptor, efficiently inhibited DC activation after influenza infection; specific chemical activation of the S1P1 receptor did not show any inhibitory effect. Moreover, we recently observed that the inhibitory effect of AFD-R was not diminished in S1P3-null mice. This series of experiments therefore rules out the involvement of S1P1, S1P2, and S1P3 receptors in the inhibition of the DC response to influenza virus infection. Based on the fact that S1P5 expression is very low in DC while S1P4 receptor was shown to be highly expressed (<a class="bk_pop" href="#ml248.r9">9</a>), we have hypothesized that chemical activation of S1P4 receptor in the airways could be effective at controlling the immunopathological response to viral infections. S1P4 receptor knockout mice exhibit limited phenotypes, but demonstrate that the receptor is important in megakaryocyte differentiation and platelet formation (<a class="bk_pop" href="#ml248.r12">12</a>). Pandemic influenza represents a significant public health threat, with much of the morbidity and mortality associated with outbreaks such as H5N1 or the 1918 flu reflecting excessive immune damage to the lungs (<a class="bk_pop" href="#ml248.r13">13</a>). Modulating this pathology could represent a promising novel therapeutic approach for influenza.</p><p>Several reports of compounds with S1P4 receptor agonist activity have been published (for a review see (<a class="bk_pop" href="#ml248.r14">14</a>)). A patent report from Azzaoui et al. (Novartis) claims a selective compound (compound <b>1</b> in <a class="figpopup" href="/books/NBK143556/table/ml248.t1/?report=objectonly" target="object" rid-figpopup="figml248t1" rid-ob="figobml248t1">Table 1</a>) (<a class="bk_pop" href="#ml248.r15">15</a>). Oddly, these compounds were later reported by Novartis as partial agonists of the N-methyl-D-aspartate (NMDA) receptor complex with no mention of S1P4 receptor activity (<a class="bk_pop" href="#ml248.r16">16</a>). Clemens et al. report the synthesis of enantiomeric agonist compounds (compounds <b>2a</b> and <b>2b</b> in <a class="figpopup" href="/books/NBK143556/table/ml248.t1/?report=objectonly" target="object" rid-figpopup="figml248t1" rid-ob="figobml248t1">Table 1</a>) that incorporate a benzimidazole ring system that also have low nanomolar partial agonist activity on S1P5 receptor and is thus not selective for S1P4 (<a class="bk_pop" href="#ml248.r17">17</a>). The constrained azacyclic FTY720 analogue reported by Hanessian (compound <b>3</b> in <a class="figpopup" href="/books/NBK143556/table/ml248.t1/?report=objectonly" target="object" rid-figpopup="figml248t1" rid-ob="figobml248t1">Table 1</a>) is a mixed S1P4 and S1P5 receptor agonist with single digit potencies in a calcium flux assay and thus is also not selective for S1P4 (<a class="bk_pop" href="#ml248.r18">18</a>). Notably, all three of these compounds have zwitterionic headgroup mimetics of S1P and are unlike to provide selectivity and specificity required for elucidating specific S1P4 receptor-mediated biological function. A Japanese patent has been filed for a benzothiophene derivative as an S1P4 regulator (<a class="bk_pop" href="#ml248.r19">19</a>), but no further information is available about this compound. In summary, previously reported S1P4 receptor agonist compounds are unavailable, poorly characterized, and/or not selective. The Scripps Research Institute Molecular Screening Center (SRIMSC), part of the Molecular Libraries Probe Production Centers Network (MLPCN), previously identified a selective S1P4 agonist probe, <a href="/pcsubstance/?term=ML178[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML178</a> (compound <b>4</b> in <a class="figpopup" href="/books/NBK143556/table/ml248.t1/?report=objectonly" target="object" rid-figpopup="figml248t1" rid-ob="figobml248t1">Table 1</a>), a 46 nM agonist that has >1000-fold selectivity for the other members of the S1P receptor family (<a class="bk_pop" href="#ml248.r20">20</a>). Given that prior art compounds other than <a href="/pcsubstance/?term=ML178[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML178</a> are not available and do not have the desired potency and selectivity for a lead optimization for <i>in vivo</i> studies, we embarked on a search for a novel probe which can serve for lead optimization for <i>in vivo</i> studies.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml248t1"><a href="/books/NBK143556/table/ml248.t1/?report=objectonly" target="object" title="Table 1" class="img_link icnblk_img figpopup" rid-figpopup="figml248t1" rid-ob="figobml248t1"><img class="small-thumb" src="/books/NBK143556/table/ml248.t1/?report=thumb" src-large="/books/NBK143556/table/ml248.t1/?report=previmg" alt="Table 1. Published S1P4 receptor agonists." /></a><div class="icnblk_cntnt"><h4 id="ml248.t1"><a href="/books/NBK143556/table/ml248.t1/?report=objectonly" target="object" rid-ob="figobml248t1">Table 1</a></h4><p class="float-caption no_bottom_margin">Published S1P4 receptor agonists. </p></div></div><p><a href="/pcsubstance/?term=ML178[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML178</a> has non-classic structure and flat medicinal chemistry. A structure that is more amenable to optimization was desired in order to pursue extended studies in animal models of influenza. In this report, we describe the identification and optimization of a novel compound with potent and selective S1P4 receptor agonist activity. This compound is amenable to further medicinal chemistry optimization. Extended optimization, characterization, and assays in animal models of influenza are planned.</p></div><div id="ml248.s4"><h2 id="_ml248_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 (part K1520), Tango™ EDG8-bla U2OS cells (K1518), GeneBLAzer FRET B/G Loading Kit (CCF4-AM) (part K1025), LiveBLAzer (K1096), Freestyle Expression Medium (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, dialyzed (26400-036), NEAA (1114-050), Penicillin-Streptomycin-Neomycin antibiotic mix (15140-122), 100X Penicillin-Streptomycin-Neomycin mix (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), L-Glutamine (25030-081).</p><p>Probenecid was obtained from Sigma (P8761). S1P was obtained from Avanti Polar Lipids (860492P). Fatty Acid Free BSA was obtained from Calbiochem (NC9734015). 1536-well plates and 384-well plates were obtained from Greiner (789072 and 788092, respectively). T175 tissue culture flasks were obtained from Corning (431080). Charcoal/dextran treated fetal bovine serum (SH30068.03) and Bovine Growth Serum (SH30541.03) were obtained from Hyclone. U2OS cells were obtained from ATCC (HTB-96). Cell Titer-Glo was obtained from Promega (G7572). Reagents for the Ricerca HitProfilingScreen + CYP450 were provided by Ricerca Biosciences, LLC.</p><div id="ml248.s5"><h3>2.1. Assays</h3><div id="ml248.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="ml248.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="ml248.s8"><h4>Stability</h4><p>Demonstration of stability in PBS was conducted under conditions likely to be experienced in a laboratory setting. The compound was dissolved in 1 mL of PBS at a concentration of 10 μM, unless its maximum solubility was insufficient to achieve this concentration. Low solubility compounds were tested between ten and fifty percent of their solubility limit. The solution was immediately aliquoted into seven standard polypropylene microcentrifuge tubes which were stored at ambient temperature in a block microcentrifuge tube holder. Individual tubes were frozen at −80°C at 0, 1, 2, 4, 8, 24, and 48 hours. The frozen samples were thawed in a room temperature and an equal volume of acetonitrile was added prior to determination of concentration by LC-MS/MS.</p></div><div id="ml248.s9"><h4>Determination of glutathione reactivity</h4><p>One μL of a 10 mM compound stock solution was added to 1 mL of a freshly prepared solution of 100 μM reduced glutathione. Final compound concentration is 10 μM unless solubility limited. The solution was allowed to incubate at 37°C for two hours prior to being directly analyzed for glutathione adduct formation. LC-MS/MS analysis of GSH adducts was performed on an API 4000 Q-TrapTM mass spectrometer equipped with a Turboionspray source (Applied Biosystems, Foster City, CA). Two methodologies were utilized—a negative precursor ion (PI) scan of m/z 272, corresponding to GSH fragmenting at the thioether bond, and a neutral loss scan of −129 AMU to detect GSH adducts. This triggered positive ion enhanced resolution and enhanced product ion scans (<a class="bk_pop" href="#ml248.r21">21</a>–<a class="bk_pop" href="#ml248.r22">22</a>).</p></div><div id="ml248.s10"><h4>Spectroscopy analyses</h4><p><sup>1</sup>H NMR, heteronuclear multiple bond correlation (<sup>1</sup>H,<sup>13</sup>C-HMBC), and nuclear Overhauser effect spectroscopy (<sup>1</sup>H,<sup>1</sup>H-NOESY) experiments were performed on spectrometer Bruker DRX-600 equipped with a 5 mm DCH cryoprobe. <sup>13</sup>C NMR spectrum was acquired on Bruker DRX-500. Chemical shifts are given as <i>δ</i> values (ppm) using trimethylsilane as the internal standard.</p></div><div id="ml248.s11"><h4>Primary uHTS assay to identify S1P4 receptor agonists (AID 1509)</h4><p><b>Assay Overview:</b> The purpose of this assay was to identify compounds that act as agonists of the S1P4 receptor. 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 receptor agonists will activate S1P4 receptor and increase 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 sq cm flasks at 37°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 μg/mL zeocin, 50 μg/mL hygromycin, 100 μg/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 μL of cell suspension to each well, followed by overnight incubation at 37°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 low control wells. S1P prepared in 2% fatty-acid free BSA was added to the high control wells to a final concentration of 5 μM. Plates were then incubated at 37 in 5% CO<sub>2</sub> for 4 hrs. After the incubation, 1 μL of the LiveBLAzer FRET substrate mixture, prepared according to the manufacturer's protocol and containing 10 mM probenecid, 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 10.98% agonism for S1P4 receptor were considered active.</p></div><div id="ml248.s12"><h4>Confirmation uHTS assay to identify S1P4 receptor agonists (AID 1523)</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/1509" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1509</a>). This assay was run 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>). Compounds were tested in triplicate at a nominal concentration of 5 μM.</p><p><b>Protocol Summary:</b> The assay was performed as previously described (<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 that exhibited greater than 10.98% agonism for S1P4 receptor were considered active.</p></div><div id="ml248.s13"><h4>Dose response uHTS assay to identify S1P4 receptor agonists (AID 1686)</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/1509" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1509</a>) and that confirmed activity in the uHTS confirmation screen (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1523" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1523</a>). This assay was run as described previously (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1509" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1509</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 (<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 EC50 equal to or less than 10 μM were considered active.</p></div><div id="ml248.s14"><h4>Counterscreen uHTS assay to identify S1P1 receptor agonists (AID 1563)</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/1509" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1509</a>), and that confirmed activity in the uHTS confirmation screen (PubChem <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1523" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1523</a>), were nonselective agonists due to activation of the S1P1 receptor. This assay uses Tango™ S1P1-bla U2OS cells which 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 receptor agonists will activate S1P1 receptor and increase 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 previously described (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1509" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1509</a>). Prior to the start of the assay, cells were suspended at a concentration of 625,000/mL in Assay Medium (Freestyle Expression Medium without supplements). The assay was started by dispensing 4 μL of cell suspension to each well, followed by overnight incubation at 37°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 (low control), or S1P (high control; 5 μM final concentration) prepared in 2% fatty-acid free BSA, was added to the appropriate wells. Plates were then incubated at 37°C in 5% CO<sub>2</sub> for 4 hours. After the incubation, 1 μL/well of the LiveBLAzer FRET substrate mixture, prepared according to the manufacturer's protocol and containing 10 mM probenecid, 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 20.14% agonism for S1P1 receptor were considered active.</p></div><div id="ml248.s15"><h4>Counterscreen dose response uHTS assay to identify S1P1 receptor agonists (AID 1701)</h4><p><b>Assay Overview:</b> The purpose of this assay was to determine S1P1 agonist dose response for compounds identified as active in the uHTS S1P1 receptor counterscreen assay (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1563" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1563</a>). The assay was performed as described (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1563" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1563</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 previously (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1563" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1563</a>). <b>Assay Cutoff:</b> compounds with an EC50 equal to or less than 10 μM were considered active.</p></div><div id="ml248.s16"><h4>Late-stage dose response assay to identify S1P4 receptor agonists with synthesized compounds (AID 504877)</h4><p><b>Assay Overview:</b> The purpose of this assay was to determine S1P4 receptor agonist dose response with synthesized compounds. This assay was run as described(<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1509" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1509</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> U2OS cells were cultured as described (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1509" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1509</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 μL of cell suspension to each well, followed by overnight incubation at 37°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 (low control), or S1P (high control; 10 nM final nominal EC80 concentration) prepared in 2% BSA was added to the appropriate wells. Plates were then incubated at 37°C in 5% CO<sub>2</sub> for 4 hours. After the incubation, 1 μL/well of the LiveBLAzer FRET substrate mixture, prepared according to the manufacturer's protocol and containing 10 mM probenecid, 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 EC50 equal to or less than 10 μM were considered active.</p></div><div id="ml248.s17"><h4>Late-stage counterscreen dose response assay to identify S1P1 receptor agonists (AID 504870)</h4><p><b>Assay Overview:</b> The purpose of this assay was to determine whether synthesized compounds identified as active in the previous assay <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504877" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504877</a> were nonselective agonists as assayed by activation of the S1P1 receptor. The assay was performed as described previously(<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1563" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1563</a>), except that 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> U2OS cells were cultured as described previously(<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1509" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1509</a>). Prior to the start of the assay, cells were suspended at a concentration of 275,000/mL in Assay Medium (Freestyle Expression Medium without supplements). The assay was started by dispensing 10 μL of cell suspension to each well of a 384-well plate (10,000 cells/well), followed by overnight incubation at 37°C in 5% CO<sub>2</sub> and 95% RH. The next day, 50 nL of test compound in DMSO (0.5% final DMSO concentration), DMSO alone (low control), or S1P (high control; 40 nM final nominal EC80 concentration) prepared in 2% BSA was added to the appropriate wells. Plates were then incubated at 37°C in 5% CO<sub>2</sub> for 4 hours. After the incubation, 2.2 μL/well of the LiveBLAzer FRET substrate mixture, prepared according to the manufacturer's protocol and containing 10 mM probenecid, 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 EC50 equal to or less than 10 μM were considered active.</p></div><div id="ml248.s18"><h4>Late-stage counterscreen dose response assay to identify S1P2 receptor agonists (AID 504873)</h4><p><b>Assay Overview:</b> The purpose of this assay was to determine whether synthesized compounds identified as active in the previous assay <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504877" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504877</a> were nonselective agonists as assayed by activation of the S1P2 receptor. A 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 agonism. Under normal conditions, S1P2 has low basal activity and therefore cells express low BLA levels. Stimulation of the S1P2 receptor by agonist increases BLA gene transcription. This increase 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 receptor agonists will activate S1P2 receptor and increase 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> CHE cells were cultured in T-175 sq cm flasks at 37°C and 95% RH. The growth media consisted of Dulbecco's Modified Eagle's Media (DMEM) 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 was initiated by dispensing 10 μL of cell suspension to each test well of a 384 well plate (6,000 cells/well) followed by incubation at 37°C in 5% CO<sub>2</sub> for 16 hrs. To the appropriate wells were then added 50 nL of test compound in DMSO (final nominal concentration of 50 μM, final DMSO concentration of 0.5%) or DMSO only (low control). Next, 1 μL of S1P in 2% BSA (final concentration of 370 nM, i.e. a concentration that resulted in 80% activity) was added to the high control wells and plates were incubated again at 37°C in 5% CO<sub>2</sub> for 2 hrs. The fluorogenic LiveBLAzer substrate mixture with 10 mM probenecid was prepared according to the manufacturer's protocol and 2.2 microliter 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 EC50 equal to or less than 10 μM were considered active.</p></div><div id="ml248.s19"><h4>Late-stage counterscreen dose response assay to identify S1P3 receptor agonists (AID 504879)</h4><p><b>Assay Overview:</b> The purpose of this assay was to determine whether synthesized compounds identified as active in the previous assay <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504877" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504877</a> were nonselective agonists as assayed by activation of the S1P3 receptor. In this assay, a CHO cell line containing human S1P3 receptor and the beta-lactamase (BLA) reporter gene under control of the nuclear factor of activated T-cells (NFAT) promoter was used to measure S1P3 receptor agonism by test compound. Stimulation of S1P3 receptor 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 receptor agonist will increase NFAT-BLA transcription and increase 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> CHO cells were cultured were cultured as described previously (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504873" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504873</a>). 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 10 μL of cell suspension to each well of a 384 well plate (8,000 cells/well), followed by overnight incubation at 37°C in 5% CO<sub>2</sub> and 95% RH. The next day, 50 nL of test compound (50 μM final nominal concentration) in DMSO was added to sample wells, and DMSO alone (0.5 % final concentration) was added to low control wells. Next, S1P prepared in 2% BSA (0.7 μM final nominal concentration, corresponding to the EC80 of S1P) was added to the high control wells. After 4 hours of incubation, 2.2 μL/well of the GeneBLAzer fluorescent substrate mixture, prepared according to the manufacturer's protocol and containing 10 mM probenecid, 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 EC50 equal to or less than 10 μM were considered active.</p></div><div id="ml248.s20"><h4>Late-stage counterscreen dose response assay to identify S1P5 receptor agonists (AID 504867)</h4><p><b>Assay Overview:</b> The purpose of this assay was to determine whether synthesized compounds identified as active in the previous assay <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504877" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504877</a> were nonselective agonists as assayed by activation of the 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 receptor agonists will stimulate migration of the fusion protein, thus increasing proteolysis of GAL4-VP16 and BLA transcription, leading to an 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> U2OS cells were cultured as described previously (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1509" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1509</a>). Prior to the start of the assay, cells were suspended at a concentration of 275,000/mL in Assay Medium (Freestyle Expression Medium without supplements). The assay was started by dispensing 10 μL of cell suspension to each well of a 384 well plate (10,000 cells/well), followed by overnight incubation at 37°C in 5% CO<sub>2</sub> and 95% RH. The next day, 50 nL of test compound in DMSO (0.5% final DMSO concentration), DMSO alone (low control), or S1P (high control; 1.5 nM final nominal EC80 concentration) prepared in 2% BSA was added to the appropriate wells. Plates were then incubated at 37°C in 5% CO<sub>2</sub> for 4 hours. After the incubation, 2.2 μL/well of the LiveBLAzer FRET substrate mixture, prepared according to the manufacturer's protocol and containing 10 mM probenecid, 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 EC50 equal to or less than 10 μM were considered active.</p></div><div id="ml248.s21"><h4>Late-stage analysis of cytotoxicity (AID 504875)</h4><p><b>Assay Overview:</b> The purpose of this assay was to determine cytotoxicity of a synthesized compound identified as active in the previous assay <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504877" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504877</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. Compounds were tested in quadruplicate in a 7-point 1:3 dilution series starting at a nominal test concentration of 20 μM.</p><p><b>Protocol Summary:</b> U2OS cells were cultured as described previously (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1509" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1509</a>). This assay was started by dispensing U2OS cells in McCoy’s 5A medium plus 10% FBS, penicillin 100 U/ml and streptomycin 100 μg/ml (20 μL; 4,000 cells/well) into the wells of a 384-well plate. Eight 1:3 serial dilutions of compound (100 μM in growth media) were made. 5 μL of diluted compound or media were added to wells, giving final compound concentrations of 0–20 μM. The plate was incubated at 37°C in a humidified incubator for 24 hours, and then equilibrated to room temperature for 30 minutes. 25 μL 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 equal to or less than 10 μM were considered active (cytotoxic).</p></div><div id="ml248.s22"><h4>Late-stage inhibition by S1P4-selective antagonist (AIDs 504871, 504918, 504917, 504924, 504919, 504921, 504923)</h4><p><b>Assay Overview:</b> The purpose of this assay was to determine whether a test compound with S1P4 agonist activity is inhibited by an S1P4-selective antagonist. 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. The presence of an S1P4-selective antagonist compound in the assay inhibits S1P4 activation and migration of the fusion protein, thus preventing proteolysis of GAL4-VP16 and BLA transcription, leading to no increase in well FRET. In the presence of a test agonist compound, the S1P4 receptor will be stimulated, leading to an increase in well FRET. Compound was tested in triplicate using a 14-point, 1:3 dilution series starting at a nominal concentration of 3.3 μM.</p><p><b>Protocol Summary:</b> U2OS cells were cultured 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>). 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 μL of cell suspension to each well, followed by overnight incubation at 37°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) or S1P (10 nM final nominal EC80 concentration prepared in 2% BSA) was added to the appropriate control wells. Next, S1P4-selective antagonist <a href="https://pubchem.ncbi.nlm.nih.gov/substance/117682935" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 117682935</a> was added to the appropriate wells to result in a final concentration of 0.4 nM, 1.2 nM, 3.7 nM, 11 nM, 33 nM, or 100 nM. Plates were then incubated at 37°C in 5% CO<sub>2</sub> for 4 hours. After the incubation, 2.2 μL/well of the LiveBLAzer FRET substrate mixture, prepared according to the manufacturer's protocol and containing 10 mM probenecid, 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 EC50 equal to or less than 10 μM were considered active.</p></div><div id="ml248.s23"><h4>Counterscreen panel assay for S1P4 agonists: Ricerca HitProfilingScreen + CYP450 (AID 540332)</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 the S1P4 agonist compound CID 49835928 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 spectrofluorometric 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><p><a class="figpopup" href="/books/NBK143556/table/ml248.t2/?report=objectonly" target="object" rid-figpopup="figml248t2" rid-ob="figobml248t2">Table 2</a> gives an overview of the PubChem assays associated with the S1P4 agonist project and indicates which are associated with the current probe development effort for <a href="/pcsubstance/?term=ML248[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML248</a>.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml248t2"><a href="/books/NBK143556/table/ml248.t2/?report=objectonly" target="object" title="Table 2" class="img_link icnblk_img figpopup" rid-figpopup="figml248t2" rid-ob="figobml248t2"><img class="small-thumb" src="/books/NBK143556/table/ml248.t2/?report=thumb" src-large="/books/NBK143556/table/ml248.t2/?report=previmg" alt="Table 2. Overview of PubChem assays for S1P4 agonist project." /></a><div class="icnblk_cntnt"><h4 id="ml248.t2"><a href="/books/NBK143556/table/ml248.t2/?report=objectonly" target="object" rid-ob="figobml248t2">Table 2</a></h4><p class="float-caption no_bottom_margin">Overview of PubChem assays for S1P4 agonist project. </p></div></div></div></div><div id="ml248.s24"><h3>2.2. Probe Chemical Characterization</h3><div id="ml248.fu2" class="figure bk_fig"><div class="graphic"><img src="/books/NBK143556/bin/ml248fu6.jpg" alt="CID 49835928 SID 103911214 ML248." /></div><h3><span class="title">CID 49835928<br />SID 103911214<br />ML248</span></h3></div><p>The probe’s structure and purity (> 95%) were verified by <sup>1</sup>H NMR, <sup>13</sup>C NMR, heteronuclear multiple bond correlation (<sup>1</sup>H,<sup>13</sup>C-HMBC), nuclear Overhauser effect spectroscopy (<sup>1</sup>H,<sup>1</sup>H-NOESY) and LC-MS.</p><p><sup><i><u>1</u></i></sup><i><u>H NMR (600 MHz, CDCl<sub>3</sub>)</u>: δ</i> 7.68 (s, 1H), 7.22 (q, <i>J</i> = 6 Hz, 1H), 7.04-7.02 (m, 2H), 6.32 (s, 1H), 3.68 (t, <i>J</i> = 6 Hz, 2H), 3.57 (t, <i>J</i> = 6 Hz, 2H), 3.42 (s, 3H), 3.29 (s, 3H), 2.12 (s, 3H), 2.03 (s, 3H). <sup>13</sup>C NMR (125 MHz, CDCl<sub>3</sub>): <i>δ</i> 167.82, 163.12 (dd, <i>J</i> = 250.9, 10.8 Hz), 158.75 (dd, <i>J</i> = 252.7, 12.4 Hz), 151.93, 135.19, 131.77, 131.44 (d, <i>J</i> = 9.8 Hz), 124.08, 122.1 (dd, <i>J</i> = 13.1, 4 Hz), 116.70, 115.19, 112.55 (dd, <i>J</i> = 21.9, 4 Hz), 106.26, 105.78 (dd, <i>J</i> = 26.0, 23.7 Hz), 72.98, 59.46, 52.97, 29.65, 12.74, 11.12. MS (EI) <i>m/z</i> 406 (M<sup>+</sup>).</p><p><sup><i><u>1</u></i></sup><i><u>H,</u></i>
|
||
<sup><i><u>13</u></i></sup><i><u>C-HMBC (CDCl<sub>3</sub>)</u>:</i> the α-protons of the 2-(2-methoxyethyl)imino substituent only coupled with carbon C2 of the thiazolidin-4-one scaffold. Protons of the methyl group at N3 coupled with carbons C4 and C2.</p><p><sup><i><u>1</u></i></sup><i><u>H,</u></i>
|
||
<sup><i><u>1</u></i></sup><i><u>H-NOESY (CDCl<sub>3</sub>)</u>:</i> No NOE effects (<a class="figpopup" href="/books/NBK143556/figure/ml248.f1/?report=objectonly" target="object" rid-figpopup="figml248f1" rid-ob="figobml248f1">Figure 1</a>) were observed between the α-protons of the 2-(2-methoxyethyl)imino and the 3-methyl substituent of the thiazolidin-4-one core; the proton of the exocyclic olefinic bond at position 5 gave rise to a cross-peak only with the 2-methyl group of the pyrrol-3-yl ring.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml248f1" co-legend-rid="figlgndml248f1"><a href="/books/NBK143556/figure/ml248.f1/?report=objectonly" target="object" title="Figure 1" class="img_link icnblk_img figpopup" rid-figpopup="figml248f1" rid-ob="figobml248f1"><img class="small-thumb" src="/books/NBK143556/bin/ml248f1.gif" src-large="/books/NBK143556/bin/ml248f1.jpg" alt="Figure 1. Potential nuclear Overhauser effects in ML248." /></a><div class="icnblk_cntnt" id="figlgndml248f1"><h4 id="ml248.f1"><a href="/books/NBK143556/figure/ml248.f1/?report=objectonly" target="object" rid-ob="figobml248f1">Figure 1</a></h4><p class="float-caption no_bottom_margin">Potential nuclear Overhauser effects in ML248. </p></div></div><p><i><u>LC-MS results</u>:</i> LC-MS results for <a href="/pcsubstance/?term=ML248[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML248</a> are shown in <a class="figpopup" href="/books/NBK143556/figure/ml248.f2/?report=objectonly" target="object" rid-figpopup="figml248f2" rid-ob="figobml248f2">Figure 2</a>.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml248f2" co-legend-rid="figlgndml248f2"><a href="/books/NBK143556/figure/ml248.f2/?report=objectonly" target="object" title="Figure 2" class="img_link icnblk_img figpopup" rid-figpopup="figml248f2" rid-ob="figobml248f2"><img class="small-thumb" src="/books/NBK143556/bin/ml248f2.gif" src-large="/books/NBK143556/bin/ml248f2.jpg" alt="Figure 2. LC-MS results for ML248." /></a><div class="icnblk_cntnt" id="figlgndml248f2"><h4 id="ml248.f2"><a href="/books/NBK143556/figure/ml248.f2/?report=objectonly" target="object" rid-ob="figobml248f2">Figure 2</a></h4><p class="float-caption no_bottom_margin">LC-MS results for ML248. </p></div></div><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 °C) was determined to be 1.11 μM as determined by dilution of 10 mM stock solution in DMSO into PBS. The probe has a half-life of 32 hours in PBS at room temperature (37% compound remaining at 48 hours) (<a class="figpopup" href="/books/NBK143556/figure/ml248.f3/?report=objectonly" target="object" rid-figpopup="figml248f3" rid-ob="figobml248f3">Figure 3</a>).</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml248f3" co-legend-rid="figlgndml248f3"><a href="/books/NBK143556/figure/ml248.f3/?report=objectonly" target="object" title="Figure 3" class="img_link icnblk_img figpopup" rid-figpopup="figml248f3" rid-ob="figobml248f3"><img class="small-thumb" src="/books/NBK143556/bin/ml248f3.gif" src-large="/books/NBK143556/bin/ml248f3.jpg" alt="Figure 3. Stability of probe ML248 in PBS." /></a><div class="icnblk_cntnt" id="figlgndml248f3"><h4 id="ml248.f3"><a href="/books/NBK143556/figure/ml248.f3/?report=objectonly" target="object" rid-ob="figobml248f3">Figure 3</a></h4><p class="float-caption no_bottom_margin">Stability of probe ML248 in PBS. </p></div></div><p>No Michael acceptor adducts were observed when a sample of the probe was incubated with 100 μM glutathione and analyzed by LC-MS.</p><p>The following compounds (<a class="figpopup" href="/books/NBK143556/table/ml248.t3/?report=objectonly" target="object" rid-figpopup="figml248t3" rid-ob="figobml248t3">Table 3</a>) have been submitted to the SMR collection. Compound numbers refer to <a class="figpopup" href="/books/NBK143556/table/ml248.t5/?report=objectonly" target="object" rid-figpopup="figml248t5" rid-ob="figobml248t5">Table 5</a>.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml248t3"><a href="/books/NBK143556/table/ml248.t3/?report=objectonly" target="object" title="Table 3" class="img_link icnblk_img figpopup" rid-figpopup="figml248t3" rid-ob="figobml248t3"><img class="small-thumb" src="/books/NBK143556/table/ml248.t3/?report=thumb" src-large="/books/NBK143556/table/ml248.t3/?report=previmg" alt="Table 3. Compounds submitted to the MLSMR." /></a><div class="icnblk_cntnt"><h4 id="ml248.t3"><a href="/books/NBK143556/table/ml248.t3/?report=objectonly" target="object" rid-ob="figobml248t3">Table 3</a></h4><p class="float-caption no_bottom_margin">Compounds submitted to the MLSMR. </p></div></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml248t5"><a href="/books/NBK143556/table/ml248.t5/?report=objectonly" target="object" title="Table 5" class="img_link icnblk_img figpopup" rid-figpopup="figml248t5" rid-ob="figobml248t5"><img class="small-thumb" src="/books/NBK143556/table/ml248.t5/?report=thumb" src-large="/books/NBK143556/table/ml248.t5/?report=previmg" alt="Table 5. SAR Table for S1P4 receptor agonist probe optimization." /></a><div class="icnblk_cntnt"><h4 id="ml248.t5"><a href="/books/NBK143556/table/ml248.t5/?report=objectonly" target="object" rid-ob="figobml248t5">Table 5</a></h4><p class="float-caption no_bottom_margin">SAR Table for S1P4 receptor agonist probe optimization. </p></div></div></div><div id="ml248.s25"><h3>2.3. Probe Preparation</h3><div id="ml248.f4" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%204.%20Synthesis%20scheme%20for%20ML248%20(CYM50308).&p=BOOKS&id=143556_ml248f4.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img src="/books/NBK143556/bin/ml248f4.jpg" alt="Figure 4. Synthesis scheme for ML248 (CYM50308)." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 4</span><span class="title">Synthesis scheme for ML248 (CYM50308)</span></h3></div><p>2-methoxyethylamine <b>2</b> was added dropwise to a solution of methyl isothiocyanate <b>1</b> in CH<sub>2</sub>Cl<sub>2</sub> at 0 °C. The reaction mixture was stirred at room temperature for two hours. The solvent was evaporated under reduced pressure. The residue was purified by CC (2% MeOH/ CH<sub>2</sub>Cl<sub>2</sub>) to provide <b>3</b> (90%) [MS (EI) m/z 149 (M+)].</p><p>Ethyl chloroacetate <b>4</b> was added to a stirred suspension of <b>3</b> and NaOAc in EtOH. The reaction mixture was heated at 60°C overnight. The reaction mixture was evaporated under reduced pressure and purified by CC (20% EtOAc/Hexane) to provide <b>5</b> (85%) [MS (EI) m/z 189 (M+)].</p><p>Sulfamic acid was added to a mixture of hexane-2,5-dione <b>6</b> and 2,4-difluoroaniline <b>7</b>. The mixture was stirred at room temperature for three hours, and successively purified by CC (1% EtOAc/Hexane) to afford <b>8</b> (90%) [MS (EI) m/z 208 (M+)].</p><p>A solution of <b>8</b> in DMF was added dropwise to a mixture of phosphorus oxychloride in DMF at 0°C. The reaction mixture was heated at 60°C for three hours. The mixture was cooled to room temperature, poured on crushed ice, followed by the addition of 2 M aqueous solution NaOH. The resulting precipitate was filtered and washed with 2 M aqueous solution NaOH to yield 9 (95%) [MS (EI) m/z 236 (M+)].</p><p>Piperidine was added to a suspension of <b>5</b> and <b>9</b> in EtOH. The mixture was heated at 60°C overnight. EtOH was evaporated and the resulting residue purified by CC (15% EtOAc/Hexane) to afford <a href="/pcsubstance/?term=ML248[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML248</a> (<a href="/protein/994210350/?report=GenPept" class="bk_tag" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=genpept">CYM50308</a>) (60%) [MS (EI) m/z 406 (M+)].</p></div></div><div id="ml248.s26"><h2 id="_ml248_s26_">3. Results</h2><p>S1P4 is natively coupled to G<sub>i</sub> or G<sub>12/13</sub> (<a class="bk_pop" href="#ml248.r23">23</a>). The Tango™ assays are based on beta-arrestin recruitment, a response that occurs subsequent to G-protein activation and G protein-coupled receptor kinase-directed phosphorylation of intracellular residues on the GPCR. The Tango™ beta-lactamase reporter technology is well described in the literature and has been used in high-throughput screening campaigns for a range of target classes, including GPCRs (<a class="bk_pop" href="#ml248.r24">24</a>–<a class="bk_pop" href="#ml248.r26">26</a>). Numerous assays based on beta-arrestin recruitment have been used for GPCR screening (e.g. PubChem <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2764" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 2764</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/493098" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 493098</a>, and <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504459" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504459</a>).</p><p><a href="/pcsubstance/?term=ML248[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML248</a>: 1) activates S1P4 receptor (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504877" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504877</a>) with an EC50 of 37.7 nM–79.1 nM, 2) is inactive as an agonist against other members of the receptor family, with EC50s > 25 μM against S1P1 (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504870" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504870</a>), S1P2 (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504873" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504873</a>), and S1P3 receptors (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504879" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504879</a>), and an EC50 of 2.1 μM against the S1P5 receptor (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504867" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504867</a>), 3) is nontoxic to U2OS cells (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504875" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504875</a>), with a CC50 of > 10 μM, and 4) is inhibited by an S1P4 receptor-selective antagonist, inactive for agonism in the presence of ≥ 3.7 nM selective antagonist (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504871" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504871</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504918" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504918</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504917" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504917</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504924" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504924</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504919" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504919</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504921" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504921</a>, and <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504923" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504923</a>). <a href="/pcsubstance/?term=ML248[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML248</a> was submitted to Ricerca Biosciences, LLC target profiling against a panel of receptors, transporters, or ion channels; the data suggest that <a href="/pcsubstance/?term=ML248[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML248</a> is generally inactive against a broad array of off-targets and does not likely exert unwanted effects.</p><div id="ml248.s27"><h3>3.1. Summary of Screening Results</h3><p>The uHTS screening strategy for this project is shown in <a class="figpopup" href="/books/NBK143556/figure/ml248.f5/?report=objectonly" target="object" rid-figpopup="figml248f5" rid-ob="figobml248f5">Figure 5</a>. In the uHTS primary assay (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1509" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1509</a>), 218K compounds were screened at 5 μM. A total of 770 compounds (0.35%) were active, passing the set threshold of 10.98% S1P4 receptor agonism. For the uHTS confirmation assay, (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1523" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1523</a>), 737 active compounds were retested in triplicate, and 53 compounds (7.19%) were confirmed as active. In a uHTS counterscreen assay against the S1P1 receptor (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1563" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1563</a>), 28 of these compounds were found to be inactive against S1P1 receptor. Out of the 53 compounds confirmed as active in the confirmation assay (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1523" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1523</a>), 46 were available from the NIH small molecule repository. These 46 compounds were tested in uHTS dose response assays for S1P4 receptor agonism (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1686" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1686</a>) and for S1P1 receptor agonism (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1563" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1563</a>), and 11 were found to be selective for S1P4 receptor—having an EC50 of less than 10 μM for S1P4 receptor and an EC50 of greater than 10 μM for S1P1 receptor. S1P4 receptor agonists considered for probe optimization are shown in <a class="figpopup" href="/books/NBK143556/table/ml248.t4/?report=objectonly" target="object" rid-figpopup="figml248t4" rid-ob="figobml248t4">Table 4</a>.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml248f5" co-legend-rid="figlgndml248f5"><a href="/books/NBK143556/figure/ml248.f5/?report=objectonly" target="object" title="Figure 5" class="img_link icnblk_img figpopup" rid-figpopup="figml248f5" rid-ob="figobml248f5"><img class="small-thumb" src="/books/NBK143556/bin/ml248f5.gif" src-large="/books/NBK143556/bin/ml248f5.jpg" alt="Figure 5. S1P4 agonist HTS overview." /></a><div class="icnblk_cntnt" id="figlgndml248f5"><h4 id="ml248.f5"><a href="/books/NBK143556/figure/ml248.f5/?report=objectonly" target="object" rid-ob="figobml248f5">Figure 5</a></h4><p class="float-caption no_bottom_margin">S1P4 agonist HTS overview. </p></div></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml248t4"><a href="/books/NBK143556/table/ml248.t4/?report=objectonly" target="object" title="Table 4" class="img_link icnblk_img figpopup" rid-figpopup="figml248t4" rid-ob="figobml248t4"><img class="small-thumb" src="/books/NBK143556/table/ml248.t4/?report=thumb" src-large="/books/NBK143556/table/ml248.t4/?report=previmg" alt="Table 4. Selected S1P4 receptor agonists from the primary screening campaign." /></a><div class="icnblk_cntnt"><h4 id="ml248.t4"><a href="/books/NBK143556/table/ml248.t4/?report=objectonly" target="object" rid-ob="figobml248t4">Table 4</a></h4><p class="float-caption no_bottom_margin">Selected S1P4 receptor agonists from the primary screening campaign. </p></div></div><p>Lead molecule <a href="https://pubchem.ncbi.nlm.nih.gov/substance/22409359" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 22409359</a> provided the scaffold for development of <a href="/pcsubstance/?term=ML178[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML178</a>. This report focuses on the lead compound <a href="https://pubchem.ncbi.nlm.nih.gov/substance/17406856" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 17406856</a>.</p></div><div id="ml248.s28"><h3>3.2. Dose Response Curve for Probe</h3><p><a class="figpopup" href="/books/NBK143556/figure/ml248.f6/?report=objectonly" target="object" rid-figpopup="figml248f6" rid-ob="figobml248f6">Figure 6</a> shows the dose-response curve for binding of <a href="/pcsubstance/?term=ML248[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML248</a> to Tango™ S1P4-BLA U2OS cells which contain the human S1P4 gene. <a href="/pcsubstance/?term=ML248[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML248</a> acts as a full agonist; the level of activity achieved by increasing the dose of <a href="/pcsubstance/?term=ML248[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML248</a> is equivalent to the level of activity elicited by the physiologic ligand S1P (control).</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml248f6" co-legend-rid="figlgndml248f6"><a href="/books/NBK143556/figure/ml248.f6/?report=objectonly" target="object" title="Figure 6" class="img_link icnblk_img figpopup" rid-figpopup="figml248f6" rid-ob="figobml248f6"><img class="small-thumb" src="/books/NBK143556/bin/ml248f6.gif" src-large="/books/NBK143556/bin/ml248f6.jpg" alt="Figure 6. Dose response curve for ML248." /></a><div class="icnblk_cntnt" id="figlgndml248f6"><h4 id="ml248.f6"><a href="/books/NBK143556/figure/ml248.f6/?report=objectonly" target="object" rid-ob="figobml248f6">Figure 6</a></h4><p class="float-caption no_bottom_margin">Dose response curve for ML248. </p></div></div></div><div id="ml248.s29"><h3>3.3. Scaffold/Moiety Chemical Liabilities</h3><p>No reactive functional groups are observed in the probe molecule, which is very stable with a half-life of 32 hours.</p></div><div id="ml248.s30"><h3>3.4. SAR Table</h3><p>The original screening hit CID 1776080 can be conceptualized as three components consisting of regions A, B, and C. <a class="figpopup" href="/books/NBK143556/table/ml248.t5/?report=objectonly" target="object" rid-figpopup="figml248t5" rid-ob="figobml248t5">Table 5</a> shows structures of compounds used for probe optimization.</p><p>Rational structure-activity relationship (SAR) studies were carried out at the indicated regions (A, B, and C) of the original HTS hit (<b>1</b>).</p><p><i><u>SAR of regions A and C</u>:</i> Removal of the methyl group from position 3 of the thiazolidin-4-one nucleus (<b>3</b>) led to substantial decrease of potency. Moreover, complete loss of activity was observed for the 3-unsubstituted 2-phenylimino analog <b>2</b>. Compounds containing bulky alkylic groups at both positions 2 and 3, including but not limited (structures not shown) to the di-<i>iso</i>propyl derivative <b>5</b> were devoid of potency. By contrast, 3-fold improved activity compared to <b>1</b> was found in the presence of ethyl groups at the same positions (<b>4</b>). The thiazolidine-2,4-dione analog <b>7</b> was devoid of potency. Complete loss of activity was found for the conformationally restricted analog <b>6</b> that contains the 2-alkylimino functionality locked into the <i>E</i>-geometry, thus suggesting that the <i>Z</i>-configuration at position 2 may be a binding requirement.</p><p>When the <i>ortho</i>-fluorine of <b>1</b> was replaced with either bromine or a methyl group (<b>14</b>, <b>13</b>), no activity was observed. Among the di-substituted phenyl rings, the 2-methoxy-5-methyl phenyl analog <b>9</b> was found to be inactive, whereas the 4-chloro-2-fluorophenyl analogs <b>12</b> and <b>13</b> were respectively 2-fold more potent compared to <b>1</b>, and less potent than <b>4</b>. Moreover, the 2,4-difluorophenyl analogs <b>10</b> and <b>11</b> were 4-fold and 2.4-fold more potent than <b>1</b> and <b>4</b>, respectively, while the unsubstituted phenyl derivative <b>24</b> showed only 2–3-fold lower potency than <b>4</b>. Interestingly, the pyridinyl analog <b>23</b> showed comparable activity with respect to <b>4</b>, whereas 21-fold reduced activity compared to <b>4</b> was found for the 3-fluoropyridinyl derivative <b>22</b>. Elongation of the hydrophobic coil (A) by insertion of a methylene at the pyrrole nitrogen of <b>1</b> and <b>4</b> was tolerated as observed for the unsubstituted benzyl derivatives (<b>16</b>, <b>17</b>), and 2,4-difluorobenzyl (<b>20</b>) and 2-methoxybenzyl (<b>19)</b> analogs. However, the 2-fluorobenzyl 2,3-diethylthiazolidin-4-one derivative <b>18</b> was devoid of activity. Furthermore, the introduction of a methyl group at the benzylic carbon (<b>21</b>), negatively affected the potency, probably as a result of a steric clash within the binding site or due to conformational changes in the molecule.</p><p>Notably, the 2-(2-methoxyethyl)imino 2,4-difluorophenyl analog <b>31</b> was 4- and 30-fold more potent than <b>10</b> and the regioisomer <b>30</b>, respectively. Lack of activity was found for the di-substituted 3-(2-methoxyethyl)-2-(2-methoxyethyl)imino derivative <b>29</b>. These findings suggested that larger polar groups may be better tolerated at position 2 of the thizolidin-4-one head.</p><p><i><u>SAR of region B</u>:</i> 2,5-unsubstituted pyrrol-3-yl analogs (<b>25</b>, <b>26</b>, <b>27</b>, <b>28</b>) represent a sampling of compounds synthesized to explore the effects of modifications at region B. However, complete loss of potency was observed in all cases.</p><p><i><u>SAR and selectivity</u>:</i> The most active compounds were tested for selectivity against S1P1, S1P2, S1P3, and S1P5 receptors (<a class="figpopup" href="/books/NBK143556/table/ml248.t6/?report=objectonly" target="object" rid-figpopup="figml248t6" rid-ob="figobml248t6">Table 6</a>). With the exception of <b>4</b>, <b>10</b> and <b>31</b>, all compounds showed low selectivity (< 10-fold) against the antitarget S1P1. Generally, the tested compounds were highly selective against S1P2 and S1P3, producing no response at concentrations up to 25 μM. However, micromolar activity for S1P2 was observed for the unsubstituted benzyl derivatives <b>16</b> and <b>17. 17</b> and the 2-methoxybenzyl analog <b>19</b> were the only compounds with micromolar S1P3 activity. Moreover, with the exception of compounds <b>4</b>, <b>10</b> and <b>31</b>, all compounds showed low to high nanomolar activity versus S1P5. Remarkably, <b>31</b> elicited an exquisite selectivity profile against S1P5, displaying 26.5–55.7-fold selectivity with no appreciable activity for S1P1, S1P2, and S1P3 at concentrations up to 25 μM. <b>31</b> was therefore selected as the probe (<a href="/pcsubstance/?term=ML248[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML248</a>).</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml248t6"><a href="/books/NBK143556/table/ml248.t6/?report=objectonly" target="object" title="Table 6" class="img_link icnblk_img figpopup" rid-figpopup="figml248t6" rid-ob="figobml248t6"><img class="small-thumb" src="/books/NBK143556/table/ml248.t6/?report=thumb" src-large="/books/NBK143556/table/ml248.t6/?report=previmg" alt="Table 6. Selectivity of selected S1P4 receptor agonist compounds against S1P1, S1P2, S1P3, and S1P5." /></a><div class="icnblk_cntnt"><h4 id="ml248.t6"><a href="/books/NBK143556/table/ml248.t6/?report=objectonly" target="object" rid-ob="figobml248t6">Table 6</a></h4><p class="float-caption no_bottom_margin">Selectivity of selected S1P4 receptor agonist compounds against S1P1, S1P2, S1P3, and S1P5. Compound numbers in column 1 refer to the entry numbers in Table 5. </p></div></div><p>Data for molecular weight and calculated parameters logD, polar surface area, hydrogen bond acceptor and hydrogen bond donor for <a href="/pcsubstance/?term=ML248[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML248</a> and analogs 1–5 are shown in <a class="figpopup" href="/books/NBK143556/table/ml248.t7/?report=objectonly" target="object" rid-figpopup="figml248t7" rid-ob="figobml248t7">Table 7</a>.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml248t7"><a href="/books/NBK143556/table/ml248.t7/?report=objectonly" target="object" title="Table 7" class="img_link icnblk_img figpopup" rid-figpopup="figml248t7" rid-ob="figobml248t7"><img class="small-thumb" src="/books/NBK143556/table/ml248.t7/?report=thumb" src-large="/books/NBK143556/table/ml248.t7/?report=previmg" alt="Table 7. Molecular weight and physiochemical properties for ML248 and analogs 1–5." /></a><div class="icnblk_cntnt"><h4 id="ml248.t7"><a href="/books/NBK143556/table/ml248.t7/?report=objectonly" target="object" rid-ob="figobml248t7">Table 7</a></h4><p class="float-caption no_bottom_margin">Molecular weight and physiochemical properties for ML248 and analogs 1–5. </p></div></div></div><div id="ml248.s31"><h3>3.5. Cellular Activity</h3><p><a href="/pcsubstance/?term=ML248[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML248</a> has been evaluated in a series of cell-based assays; the primary screen and S1P1, S1P5, and cytotoxicity counterscreens are U2OS-based assays. The S1P3 and S1P2 counterscreens are CHO-based assays.</p></div><div id="ml248.s32"><h3>3.6. Profiling Assays</h3><p>To date, the lead hit (CID 1776080) has been tested in 486 other bioassays deposited in PubChem, and has shown activity in 37 of those assays, five of which are for the S1P4 receptor agonist project. The other 32 assays give a hit rate of 6.6%, 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=ML248[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML248</a> was submitted to Ricerca Biosciences, LLC for HitProfilingScreen + CYP450 (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/540332" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 540332</a>). The purpose of this panel of binding assays was to identify potential receptors, transporters, or ion channels for which compound <a href="/pcsubstance/?term=ML248[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML248</a> displays affinity. Out of 35 targets tested, eight (CYP450 3A4, norepinephrine transporter NET, dihydropyridine calcium channel L-type, cannabinoid CB<sub>1</sub>, histamine H<sub>1</sub>, opiate μ, serotonin 5-HT<sub>2B</sub>, and sodium channel site 2) resulted in modest inhibition of activity, and one (nicotinic acetylcholine) resulted in modest activation (a range of 52% inhibition to 71% inhibition) when tested at 30 μM (<a class="figpopup" href="/books/NBK143556/table/ml248.t8/?report=objectonly" target="object" rid-figpopup="figml248t8" rid-ob="figobml248t8">Table 8</a>). If these compounds have normal dose response curves, the IC50s will clearly be in the double digit micromolar range. <a href="/pcsubstance/?term=ML248[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML248</a> has an EC50 of ~50 nM, and with an estimate of IC50 of 10 uM for the Ricerca activities listed in <a class="figpopup" href="/books/NBK143556/table/ml248.t8/?report=objectonly" target="object" rid-figpopup="figml248t8" rid-ob="figobml248t8">Table 8</a>, <a href="/pcsubstance/?term=ML248[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML248</a> is 200 fold selective towards S1P4 as compared to the off-target Ricerca activities. These data suggest that compound <a href="/pcsubstance/?term=ML248[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML248</a> is generally inactive against a broad array of off targets and does not likely exert unwanted effects.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml248t8"><a href="/books/NBK143556/table/ml248.t8/?report=objectonly" target="object" title="Table 8" class="img_link icnblk_img figpopup" rid-figpopup="figml248t8" rid-ob="figobml248t8"><img class="small-thumb" src="/books/NBK143556/table/ml248.t8/?report=thumb" src-large="/books/NBK143556/table/ml248.t8/?report=previmg" alt="Table 8. Targets that exhibited ≥ 50% inhibiton in Ricerca screening." /></a><div class="icnblk_cntnt"><h4 id="ml248.t8"><a href="/books/NBK143556/table/ml248.t8/?report=objectonly" target="object" rid-ob="figobml248t8">Table 8</a></h4><p class="float-caption no_bottom_margin">Targets that exhibited ≥ 50% inhibiton in Ricerca screening. </p></div></div></div></div><div id="ml248.s33"><h2 id="_ml248_s33_">4. Discussion</h2><p><a href="/pcsubstance/?term=ML248[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML248</a> activates S1P4 receptor in a cell-based Tango™-format assay (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504877" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504877</a>) with an EC50 of 37.7 nM–79.1 nM; is inactive as an agonist against other members of the receptor family, with EC50s > 25 μM against S1P1 (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504870" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504870</a>), S1P2 (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504873" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504873</a>), and S1P3 receptors (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504879" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504879</a>), and an EC50 of 2.1 μM against the S1P5 receptor (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504867" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504867</a>); is nontoxic to U2OS cells (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504875" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504875</a>), with a CC50 of > 10 μM; and is inhibited by an S1P4 receptor-selective antagonist, inactive for agonism in the presence of ≥3.7 nM selective antagonist (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504871" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504871</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504918" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504918</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504917" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504917</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504924" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504924</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504919" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504919</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504921" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504921</a>, and <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504923" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504923</a>). In all of the S1P receptor assays, a control dose response of S1P is run and results are normalized to S1P as the reference compound. The S1P EC50 in the S1P4-Tango™ assay is 3 nM. The Tango™ assay is a well characterized reporter readout of β-arrestin recruitment (<a class="bk_pop" href="#ml248.r27">27</a>), which is not able to distinguish partial agonists from full agonists (<a class="bk_pop" href="#ml248.r26">26</a>).</p><p>The employment of engineered cell lines in early discovery is well established. The potency of known drugs and tool compounds is the best way to benchmark the utility of a readout in predicting “native cell” pharmacology. In this particular case, S1P4 biology is poorly understood and S1P4 selective agonists are not available. Indeed, this provides significant rationale for the development of S1P family selective tool compounds, such as selective S1P4 agonists. A relevant example is the S1P1 agonist, RCP1063, currently in Phase I clinical trials. This molecule was developed from a screening hit found with a reporter assay (<a class="bk_pop" href="#ml248.r28">28</a>). Significant effort was required to develop the compound characteristics (potency, selectivity, solubility, stability ect) that enabled <i>in vivo</i> experiments. Discovery of these novel pharmacophores with S1P4 selectivity is important in that they provide starting points for chemical optimization of properties required for investigating S1P4 biology <i>in vitro</i> and <i>in vivo</i>.</p><div id="ml248.s34"><h3>4.1. Comparison to Existing Art and How the New Probe is an Improvement</h3><p>Other than <a href="/pcsubstance/?term=ML178[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML178</a>, previously reported S1P4 receptor agonist compounds are unavailable, poorly characterized, and not selective. <a href="/pcsubstance/?term=ML178[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML178</a> was the first submicromolar, completely selective S1P4 receptor agonist to be identified (<a class="bk_pop" href="#ml248.r20">20</a>); however it has non-classic structure and flat medicinal chemistry. <a href="/pcsubstance/?term=ML248[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML248</a> has comparable potency and selectivity to <a href="/pcsubstance/?term=ML178[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML178</a>, but it is more amenable to optimization by medicinal chemistry efforts. Although the improvement is potency is modest, <a href="/pcsubstance/?term=ML248[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML248</a> is a completely orthogonal chemotype to other S1P4 agonists and provides a novel starting point for chemical optimization efforts. We have recently published extended descriptions of these two lead series (<a class="bk_pop" href="#ml248.r29">29</a>–<a class="bk_pop" href="#ml248.r30">30</a>).</p></div><div id="ml248.s35"><h3>4.2. Mechanism of Action</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. Experiments are ongoing with the probe in other assay formats to further examine its mechanism of action and the signaling pathways activated by S1P4. In collaboration with Dr. Oldstone’s laboratory, experiments are being conduction to elucidate the role of S1P4 in animal models of influenza and autoimmune diseases.</p></div><div id="ml248.s36"><h3>4.3. Planned Future Studies</h3><p>The SAR data obtained thus far indicate that in this project we have identified a molecular scaffold with good physicochemical properties and reasonable potency and selectivity against S1P receptor family members. In the Extended Characterization phase of this investigation (“Optimization of S1P4 Ligands for use in Inflammatory Disorders”), medicinal chemistry to enhance solubility and potency are planned. Our aim will be to develop these further using traditional MedChem techniques and scaffold hopping methods to produce at least a tenfold increase in potency while maintaining selectivity. In particular, elements that may be undesirable <i>in vivo</i> such as brominated pyridines will be replaced with suitable bio-isosteres. The most promising compounds will be tested to selectivity against the panel of off target proteins including GPCRs, transporters and ion channels. The pharmacokinetic (PK) properties of 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. The ultimate goal of these extended probe optimization studies of <a href="/pcsubstance/?term=ML248[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML248</a> is to generate an optimized candidate with appropriate solubility, metabolic stability, mouse PK properties, and overall physical properties to enable the compound to be used in animal models of influenza and autoimmune diseases. 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 effect of S1P4-specific agonist compounds on the response to influenza in mice. Employing this strategy, we have recently shown that S1P1-specific modulators directly suppress cytokine amplification, inflammation, and enhance survival following infection of mice with human pathogenic influenza virus (<a class="bk_pop" href="#ml248.r31">31</a>)</p></div></div><div id="ml248.s37"><h2 id="_ml248_s37_">5. References</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="ml248.r1">Marsolais D, Hahm B, Walsh KB, Edelmann KH, McGavern D, Hatta Y, Kawaoka Y, Rosen H, Oldstone MB. 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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/NBK143556/?report=reader">PubReader</a></li><li><a href="/books/NBK143556/?report=printable">Print View</a></li><li><a data-jig="ncbidialog" href="#_ncbi_dlg_citbx_NBK143556" data-jigconfig="width:400,modal:true">Cite this Page</a><div id="_ncbi_dlg_citbx_NBK143556" style="display:none" title="Cite this Page"><div class="bk_tt">Guerrero M, Urbano M, Velaparthi S, et al. Identification of a Novel Agonist of the Sphingosine 1-phosphate Receptor 4 (S1P4) 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/NBK143556/pdf/Bookshelf_NBK143556.pdf">PDF version of this page</a> (1.0M)</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="#ml248.s1" ref="log$=inpage&link_id=inpage">Probe Structure & Characteristics</a></li><li><a href="#ml248.s2" ref="log$=inpage&link_id=inpage">Recommendations for Scientific Use of the Probe</a></li><li><a href="#ml248.s3" ref="log$=inpage&link_id=inpage">Introduction</a></li><li><a href="#ml248.s4" ref="log$=inpage&link_id=inpage">Materials and Methods</a></li><li><a href="#ml248.s26" ref="log$=inpage&link_id=inpage">Results</a></li><li><a href="#ml248.s33" ref="log$=inpage&link_id=inpage">Discussion</a></li><li><a href="#ml248.s37" ref="log$=inpage&link_id=inpage">References</a></li></ul></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Related information</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="discovery_db_links" id="Shutter"></a></div><div class="portlet_content"><ul><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pmc&DbFrom=books&Cmd=Link&LinkName=books_pmc_refs&IdsFromResult=3036516" ref="log$=recordlinks">PMC</a><div class="brieflinkpop offscreen_noflow">PubMed Central citations</div></li><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pcassay&DbFrom=books&Cmd=Link&LinkName=books_pcassay_probe&IdsFromResult=3036516" ref="log$=recordlinks">PubChem BioAssay for Chemical Probe</a><div class="brieflinkpop offscreen_noflow">PubChem BioAssay records reporting screening data for the development of the chemical probe(s) described in this book chapter</div></li><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pcsubstance&DbFrom=books&Cmd=Link&LinkName=books_pcsubstance&IdsFromResult=3036516" ref="log$=recordlinks">PubChem Substance</a><div class="brieflinkpop offscreen_noflow">Related PubChem Substances</div></li><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pubmed&DbFrom=books&Cmd=Link&LinkName=books_pubmed_refs&IdsFromResult=3036516" ref="log$=recordlinks">PubMed</a><div class="brieflinkpop offscreen_noflow">Links to PubMed</div></li></ul></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Similar articles in PubMed</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="PBooksDiscovery_RA" id="Shutter"></a></div><div class="portlet_content"><ul><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/22834032" ref="ordinalpos=1&linkpos=1&log$=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/23658964" ref="ordinalpos=1&linkpos=2&log$=relatedreviews&logdbfrom=pubmed"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> Probe Development Efforts for an Allosteric Agonist of the Sphingosine 1-phosphate Receptor 3 (S1P3).</a><span class="source">[Probe Reports from the NIH Mol...]</span><div class="brieflinkpop offscreen_noflow"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> Probe Development Efforts for an Allosteric Agonist of the Sphingosine 1-phosphate Receptor 3 (S1P3).<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Guerrero M, Poddutoori R, Pinacho-Crisostomo F, Schaeffer MT, Brown SJ, Spicer T, Chase P, Ferguson J, Roberts E, Sanna G, et al. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Probe Reports from the NIH Molecular Libraries Program. 2010</em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/21433398" ref="ordinalpos=1&linkpos=3&log$=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 Development Efforts to Identify Novel Antagonists 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 Antagonists 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">Oldstone M, Hodder P, Crisp M, Roberts E, Guerrero M, Urbano M, Velaparthi S, Zhao J, Rosen H, Schaeffer MT, 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 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Epub 2004 Jan 19.</em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/24479196" ref="ordinalpos=1&linkpos=5&log$=relatedreviews&logdbfrom=pubmed"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> Optimization and characterization of an opioid kappa receptor (OPRK1) antagonist.</a><span class="source">[Probe Reports from the NIH Mol...]</span><div class="brieflinkpop offscreen_noflow"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> Optimization and characterization of an opioid kappa receptor (OPRK1) antagonist.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Guerrero M, Urbano M, Brown SJ, Cayanan C, Ferguson J, Cameron M, Devi LA, Roberts E, Rosen H. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Probe Reports from the NIH Molecular Libraries Program. 2010</em></div></div></li></ul><a class="seemore" href="/sites/entrez?db=pubmed&cmd=link&linkname=pubmed_pubmed_reviews&uid=23762926" ref="ordinalpos=1&log$=relatedreviews_seeall&logdbfrom=pubmed">See reviews...</a><a class="seemore" href="/sites/entrez?db=pubmed&cmd=link&linkname=pubmed_pubmed&uid=23762926" ref="ordinalpos=1&log$=relatedarticles_seeall&logdbfrom=pubmed">See all...</a></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Recent Activity</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="recent_activity" id="Shutter"></a></div><div class="portlet_content"><div xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" id="HTDisplay" class=""><div class="action"><a href="javascript:historyDisplayState('ClearHT')">Clear</a><a href="javascript:historyDisplayState('HTOff')" class="HTOn">Turn Off</a><a href="javascript:historyDisplayState('HTOn')" class="HTOff">Turn On</a></div><ul id="activity"><li class="ra_rcd ralinkpopper two_line"><a class="htb ralinkpopperctrl" ref="log$=activity&linkpos=1" href="/portal/utils/pageresolver.fcgi?recordid=67d66ba22f30673f7bf71993">Identification of a Novel Agonist of the Sphingosine 1-phosphate Receptor 4 (S1P...</a><div class="ralinkpop offscreen_noflow">Identification of a Novel Agonist of the Sphingosine 1-phosphate Receptor 4 (S1P4) - 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