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<script type="text/javascript" src="/corehtml/pmc/jatsreader/ptpmc_3.22/js/jr.boots.min.js"> </script><title>Probe Development Efforts for an Allosteric Agonist of the Sphingosine 1-phosphate Receptor 3 (S1P3) - Probe Reports from the NIH Molecular Libraries Program - NCBI Bookshelf</title>
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<meta name="citation_inbook_title" content="Probe Reports from the NIH Molecular Libraries Program [Internet]">
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<meta name="citation_title" content="Probe Development Efforts for an Allosteric Agonist of the Sphingosine 1-phosphate Receptor 3 (S1P3)">
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<meta name="citation_publisher" content="National Center for Biotechnology Information (US)">
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<meta name="citation_date" content="2013/02/25">
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<meta name="citation_author" content="Miguel Guerrero">
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<meta name="citation_author" content="Ramulu Poddutoori">
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<meta name="citation_author" content="Fernando Pinacho-Crisostomo">
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<meta name="citation_author" content="Marie-Therese Schaeffer">
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<meta name="citation_author" content="Steven J Brown">
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<meta name="citation_author" content="Timothy Spicer">
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<meta name="citation_author" content="Peter Chase">
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<meta name="citation_author" content="Jill Ferguson">
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<meta name="citation_author" content="Edward Roberts">
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<meta name="citation_author" content="Germana Sanna">
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<meta name="citation_author" content="Peter Hodder">
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<meta name="citation_author" content="Hugh Rosen">
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<meta name="citation_pmid" content="23658964">
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<meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK133421/">
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<link rel="schema.DC" href="http://purl.org/DC/elements/1.0/">
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<meta name="DC.Title" content="Probe Development Efforts for an Allosteric Agonist of the Sphingosine 1-phosphate Receptor 3 (S1P3)">
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<meta name="DC.Type" content="Text">
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<meta name="DC.Publisher" content="National Center for Biotechnology Information (US)">
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<meta name="DC.Contributor" content="Miguel Guerrero">
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<meta name="DC.Contributor" content="Ramulu Poddutoori">
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<meta name="DC.Contributor" content="Fernando Pinacho-Crisostomo">
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<meta name="DC.Contributor" content="Marie-Therese Schaeffer">
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<meta name="DC.Contributor" content="Steven J Brown">
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<meta name="DC.Contributor" content="Timothy Spicer">
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<meta name="DC.Contributor" content="Peter Chase">
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<meta name="DC.Contributor" content="Jill Ferguson">
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<meta name="DC.Contributor" content="Edward Roberts">
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<meta name="DC.Contributor" content="Germana Sanna">
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<meta name="DC.Contributor" content="Peter Hodder">
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<meta name="DC.Contributor" content="Hugh Rosen">
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<meta name="DC.Date" content="2013/02/25">
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<meta name="DC.Identifier" content="https://www.ncbi.nlm.nih.gov/books/NBK133421/">
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<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. S1P3 receptor couples promiscuously to Gi, Gq, and G12/13 proteins, and its tissue distribution is widespread. Bradycardia and hypertension are clearly associated with S1P3 activation and its expression patterns in cardiac tissue. S1P3 on dendritic cells has been identified as a major exacerbating factor for mortality during sepsis by playing a role in the critical linkage of inflammation and coagulation pathways downstream of the thrombin cascade. Understanding the contributions of the individual S1P receptors has been limited by the unavailability of selective modulators for the 5 receptor subtypes. In the pilot phase of the Molecular Libraries Probe Production Centers Network (MLPCN), The Scripps Research Institute Molecular Screening Center (SRIMSC) reported four low micromolar agonist probes for S1P3: ML003, ML004, ML005 and ML006. The current report describes the development of ML249, a submicromolar allosteric agonist probe for S1P3. ML249 resulted from high-throughput screening using a cell-based assay employing a Chinese Hamster Ovary (CHO) cell line stably transfected with human S1P3 receptor, nuclear factor of activated T-cell-beta lactamase (NFAT-BLA) reporter construct, and the Gα16 pathway coupling protein, followed by medicinal chemistry efforts. ML249 activates S1P3 receptor with an EC50 of 72.3 nM–132 nM, and is inactive as an agonist against other members of the receptor family S1P1 (EC50 >10 μM), S1P2 (EC50 >50 μM), S1P4 (EC50 >50 μM), and S1P5 (EC50 >25 μM). Evidence is presented showing that ML249 is an allosteric agonist; it does not compete with physiological ligand S1P for binding to S1P3. In addition, ML249 is nontoxic to U2OS cells, with a CC50 >10 μM. ML249 was submitted to Ricerca Biosciences, LLC for target profiling against a panel of receptors, transporters, and ion channels; the data suggest that compound ML249 is generally inactive against a broad array of off-targets and does not likely exert unwanted effects. ML249 is the first submicromolar, completely selective S1P3 receptor agonist to be identified, and as an allosteric agonist promises to facilitate determination of key receptor interactions that would not otherwise be possible.">
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<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. S1P3 receptor couples promiscuously to Gi, Gq, and G12/13 proteins, and its tissue distribution is widespread. Bradycardia and hypertension are clearly associated with S1P3 activation and its expression patterns in cardiac tissue. S1P3 on dendritic cells has been identified as a major exacerbating factor for mortality during sepsis by playing a role in the critical linkage of inflammation and coagulation pathways downstream of the thrombin cascade. Understanding the contributions of the individual S1P receptors has been limited by the unavailability of selective modulators for the 5 receptor subtypes. In the pilot phase of the Molecular Libraries Probe Production Centers Network (MLPCN), The Scripps Research Institute Molecular Screening Center (SRIMSC) reported four low micromolar agonist probes for S1P3: ML003, ML004, ML005 and ML006. The current report describes the development of ML249, a submicromolar allosteric agonist probe for S1P3. ML249 resulted from high-throughput screening using a cell-based assay employing a Chinese Hamster Ovary (CHO) cell line stably transfected with human S1P3 receptor, nuclear factor of activated T-cell-beta lactamase (NFAT-BLA) reporter construct, and the Gα16 pathway coupling protein, followed by medicinal chemistry efforts. ML249 activates S1P3 receptor with an EC50 of 72.3 nM–132 nM, and is inactive as an agonist against other members of the receptor family S1P1 (EC50 >10 μM), S1P2 (EC50 >50 μM), S1P4 (EC50 >50 μM), and S1P5 (EC50 >25 μM). Evidence is presented showing that ML249 is an allosteric agonist; it does not compete with physiological ligand S1P for binding to S1P3. In addition, ML249 is nontoxic to U2OS cells, with a CC50 >10 μM. ML249 was submitted to Ricerca Biosciences, LLC for target profiling against a panel of receptors, transporters, and ion channels; the data suggest that compound ML249 is generally inactive against a broad array of off-targets and does not likely exert unwanted effects. ML249 is the first submicromolar, completely selective S1P3 receptor agonist to be identified, and as an allosteric agonist promises to facilitate determination of key receptor interactions that would not otherwise be possible.">
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match">◀</a><button id="jr-fip-matches">no matches yet</button><a id="jr-fip-next" class="wsprkl btn" title="Jump to next match">▶</a></nav></nav></div><div id="jr-epub-interstitial" class="hidden"></div><div id="jr-content"><article data-type="main"><div class="main-content lit-style" itemscope="itemscope" itemtype="http://schema.org/CreativeWork"><div class="meta-content fm-sec"><div class="fm-sec"><h1 id="_NBK133421_"><span class="title" itemprop="name">Probe Development Efforts for an Allosteric Agonist of the Sphingosine
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1-phosphate Receptor 3 (S1P3)</span></h1><p class="contribs">Guerrero M, Poddutoori R, Pinacho-Crisostomo F, et al.</p><p class="fm-aai"><a href="#_NBK133421_pubdet_">Publication Details</a></p></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
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platelets that serves to influence heart rate, coronary artery caliber, endothelial
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integrity, lung epithelial integrity and lymphocyte recirculation through five
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related high affinity G-protein coupled receptors. S1P3 receptor couples
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promiscuously to Gi, Gq, and G12/13 proteins, and its tissue distribution is
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widespread. Bradycardia and hypertension are clearly associated with S1P3 activation
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and its expression patterns in cardiac tissue. S1P3 on dendritic cells has been
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identified as a major exacerbating factor for mortality during sepsis by playing a
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role in the critical linkage of inflammation and coagulation pathways downstream of
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the thrombin cascade. Understanding the contributions of the individual S1P
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receptors has been limited by the unavailability of selective modulators for the 5
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receptor subtypes. In the pilot phase of the Molecular Libraries Probe Production
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Centers Network (MLPCN), The Scripps Research Institute Molecular Screening Center
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(SRIMSC) reported four low micromolar agonist probes for S1P3: <a href="/pcsubstance/?term=ML003[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML003</a>, <a href="/pcsubstance/?term=ML004[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML004</a>, <a href="/pcsubstance/?term=ML005[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML005</a> and <a href="/pcsubstance/?term=ML006[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML006</a>. The current report describes the development of <a href="/pcsubstance/?term=ML249[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML249</a>, a submicromolar allosteric agonist probe for S1P3. <a href="/pcsubstance/?term=ML249[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML249</a> resulted from high-throughput screening using a cell-based
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assay employing a Chinese Hamster Ovary (CHO) cell line stably transfected with
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human S1P3 receptor, nuclear factor of activated T-cell-beta lactamase (NFAT-BLA)
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reporter construct, and the Gα16 pathway coupling protein, followed by
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medicinal chemistry efforts. <a href="/pcsubstance/?term=ML249[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML249</a> activates S1P3
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receptor with an EC50 of 72.3 nM–132 nM, and is inactive as an agonist
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against other members of the receptor family S1P1 (EC50 >10 μM), S1P2
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(EC50 >50 μM), S1P4 (EC50 >50 μM), and S1P5 (EC50
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>25 μM). Evidence is presented showing that <a href="/pcsubstance/?term=ML249[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML249</a> is an allosteric agonist; it does not compete with
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physiological ligand S1P for binding to S1P3. In addition, <a href="/pcsubstance/?term=ML249[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML249</a> is nontoxic to U2OS cells, with a CC50 >10 μM.
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<a href="/pcsubstance/?term=ML249[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML249</a> was submitted to Ricerca Biosciences, LLC for target profiling
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against a panel of receptors, transporters, and ion channels; the data suggest that
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compound <a href="/pcsubstance/?term=ML249[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML249</a> is generally inactive against a broad
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array of off-targets and does not likely exert unwanted effects. <a href="/pcsubstance/?term=ML249[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML249</a> is the first submicromolar, completely selective S1P3 receptor
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agonist to be identified, and as an allosteric agonist promises to facilitate
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determination of key receptor interactions that would not otherwise be possible.</p></div><div class="h2"></div><p><b>Assigned Assay Grant #:</b> 1R03MH076534-01</p><p><b>Screening Center Name & PI:</b> Scripps Research Institute Molecular
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Screening Center, Scripps Research Institute Molecular Screening Center, H. Rosen, W.
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Roush</p><p><b>Chemistry Center Name & PI:</b> Scripps Research Institute Molecular
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Screening Center, Scripps Research Institute Molecular Screening Center, H. Rosen, W.
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Roush</p><p><b>Assay Submitter & Institution:</b> Germana Sanna, The Scripps Research
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Institute</p><p><b>PubChem Summary Bioassay Identifier (AID):</b>
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<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/540309" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">540309</a></p><div id="ml249.s1"><h2 id="_ml249_s1_">Probe Structure & Characteristics</h2><div id="ml249.fu1" class="figure bk_fig"><div class="graphic"><img src="/books/NBK133421/bin/ml249fu1.jpg" alt="ML249." /></div><h3><span class="title"><a href="/pcsubstance/?term=ML249[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML249</a></span></h3></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml249tu1"><a href="/books/NBK133421/table/ml249.tu1/?report=objectonly" target="object" title="Table" class="img_link icnblk_img figpopup" rid-figpopup="figml249tu1" rid-ob="figobml249tu1"><img class="small-thumb" src="/books/NBK133421/table/ml249.tu1/?report=thumb" src-large="/books/NBK133421/table/ml249.tu1/?report=previmg" alt="Image " /></a><div class="icnblk_cntnt"><h4 id="ml249.tu1"><a href="/books/NBK133421/table/ml249.tu1/?report=objectonly" target="object" rid-ob="figobml249tu1">Table</a></h4></div></div></div><div id="ml249.s2"><h2 id="_ml249_s2_">1. Recommendations for Scientific Use of the Probe</h2><p>Sphingosine 1-phosphate (S1P) is a lysophospholipid signaling molecule that regulates
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important biological functions in both intracellular (<a class="bibr" href="#ml249.r1" rid="ml249.r1">1</a>) and extracellular compartments (<a class="bibr" href="#ml249.r2" rid="ml249.r2">2</a>), including a wide variety of physiological responses
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such as heart rate (<a class="bibr" href="#ml249.r3" rid="ml249.r3">3</a>–<a class="bibr" href="#ml249.r4" rid="ml249.r4">4</a>), coronary artery caliber, endothelial
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integrity, and lymphocyte recirculation (<a class="bibr" href="#ml249.r4" rid="ml249.r4">4</a>–<a class="bibr" href="#ml249.r7" rid="ml249.r7">7</a>). These
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responses are mediated through high-affinity interactions with five members of the
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endothelial differentiation gene (EDG) family of plasma membrane-localized
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G-protein-coupled receptors (GPCRs), the sphingosine lipid receptors, S1P1–5
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(<a class="bibr" href="#ml249.r8" rid="ml249.r8">8</a>–<a class="bibr" href="#ml249.r10" rid="ml249.r10">10</a>). Understanding the contributions of individual S1P
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receptors to these physiological processes has been limited by the unavailability of
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selective modulators for the 5 receptor subtypes. Most S1P-mediated responses on
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endothelial cells occur via the S1P1 receptor alone or in combination with the S1P3
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receptor. S1P-mediated migration, angiogenesis, and adherens junction formation all
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require both the Gi-mediated activity of the S1P1 receptor and the
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Gq/G12,13-mediated activity of the S1P3 receptor (<a class="bibr" href="#ml249.r11" rid="ml249.r11">11</a>–<a class="bibr" href="#ml249.r13" rid="ml249.r13">13</a>). Bradycardia and hypertension are clearly associated with S1P3
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activation and its expression patterns in cardiac tissue (<a class="bibr" href="#ml249.r3" rid="ml249.r3">3</a>, <a class="bibr" href="#ml249.r14" rid="ml249.r14">14</a>). S1P3
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on dendritic cells has been identified as a major exacerbating factor for mortality
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during sepsis by playing a role in the critical linkage of inflammation and
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coagulation pathways downstream of the thrombin cascade (<a class="bibr" href="#ml249.r15" rid="ml249.r15">15</a>).</p><p><a href="/pcsubstance/?term=ML249[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML249</a> is an allosteric agonist; it does not compete with the
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physiologic ligand S1P for binding to S1P3. Key residues that constitute the
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hydrophobic binding pocket of the S1P3 receptor have been identified (<a class="bibr" href="#ml249.r16" rid="ml249.r16">16</a>). An allosteric modulator can
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facilitate determination of key receptor interactions that would not otherwise be
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possible. These types of compounds derived from Molecular Libraries Program Small
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Molecule Repository (MLSMR) hits have been crucial for determination of the crystal
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structure of the S1P1 receptor (unpublished results). Allosteric modulators may also
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offer advantages over classic orthosteric ligands as therapeutic agents, including
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the potential for greater GPCR-subtype selectivity and lower side effect potential
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(<a class="bibr" href="#ml249.r17" rid="ml249.r17">17</a>–<a class="bibr" href="#ml249.r20" rid="ml249.r20">20</a>). We expect the potent and selective S1P3 agonist
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described here to be useful in dissecting the complexities of S1P-mediated
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physiological processes in which S1P3 is involved, including bradycardia and
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hypertension. Further, we expect that as an allosteric agonist it will be a useful
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tool for dissecting receptor structure and function.</p></div><div id="ml249.s3"><h2 id="_ml249_s3_">2. Materials and Methods</h2><p>The following reagents were obtained from Invitrogen: Tango™ EDG6-bla U2OS
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cells (K1622), Tango™ EDG-1-BLA U2OS cells (part K1520), Tango™
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EDG8-bla U2OS cells (K1518), GeneBLAzer FRET B/G Loading Kit (CCF4-AM) (part K1025),
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LiveBLAzer (K1096), GeneBLAzer (part K1085), Freestyle Expression Medium
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(12338-018), McCoy's 5A Medium (modified) (1X) (16600-082), Dulbecco's
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Modified Eagle's Media with phenol red (11965-092), Dulbecco's
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Modified Eagle's Media without phenol red (21063-029), Fetal Bovine Serum,
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dialyzed (26400-036), NEAA (1114-050), Penicillin-Streptomycin-Neomycin antibiotic
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mix (15140-122), 100X Penicillin-Streptomycin-Neomycin mix (15640-055), Sodium
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Pyruvate (11360-070), PBS without calcium or magnesium (14190-136), HEPES
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(15630-080), Trypsin/EDTA (25300-054), Zeocin (R250-01), Hygromycin (10687-010),
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Geneticin (10131-027), L-Glutamine (25030-081).</p><p>Probenecid was obtained from Sigma (P8761). S1P was obtained from Avanti Polar Lipids
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(860492P). Fatty Acid Free BSA was obtained from Calbiochem (NC9734015). 1536-well
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plates and 384-well plates were obtained from Greiner (789072 and 788092,
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respectively). T175 tissue culture flasks were obtained from Corning (431080).
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Charcoal/dextran treated fetal bovine serum (SH30068.03) and Bovine Growth Serum
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(SH30541.03) were obtained from Hyclone. U-2OS cells were obtained from ATCC
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(HTB-96). Cell Titer-Glo was obtained from Promega (G7572). Reagents for the Ricerca
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HitProfilingScreen + CYP450 were provided by Ricerca Biosciences, LLC.</p><div id="ml249.s4"><h3>2.1. Assays</h3><div id="ml249.s5"><h4>LC-MS/MS</h4><p>All analytical methods are in MRM mode where the parent ion is selected in Q1
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of the mass spectrometer. The parent ion is fragmented and a characteristic
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fragment ion is monitored in Q3. MRM mass spectroscopy methods are
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particularly sensitive because additional time is spent monitoring the
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desired ions and not sweeping a large mass range. Methods will be rapidly
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set up using Automaton<sup>®</sup> (Applied Biosystems), where the
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compounds are listed with their name and mass in an Excel datasheet.
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Compounds are submitted in a 96-well plate to the HPLC autosampler and are
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slowly injected without a column present. A narrow range centered on the
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indicated mass is scanned to detect the parent ion. The software then
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evaluates a few pre-selected parameters to determine conditions that
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maximize the signal for the parent ion. The molecule is then fragmented in
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the collision cell of the mass spectrometer and fragments with m/z larger
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than 70 but smaller than the parent mass are determined. Three separate
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collision energies are evaluated to fragment the parent ion and the largest
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three ions are selected. Each of these three fragment ions is further
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optimized and the best fragment is chosen. The software then inserts the
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optimized masses and parameters into a template method and saves it with a
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unique name that indicates the individual compound being optimized. Spectra
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for the parent ion and the fragmentation pattern are saved and can be
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reviewed later.</p></div><div id="ml249.s6"><h4>Solubility</h4><p>The solubility of compounds was tested in phosphate buffered saline, pH 7.4.
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Compounds were inverted for 24 hours in test tubes containing 1–2 mg
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of compound with 1 mL of PBS. The samples were centrifuged and analyzed by
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HPLC (Agilent 1100 with diode-array detector). Peak area was compared to a
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standard of known concentration. In cases when the concentration was too low
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for UV analysis or when the compound did not possess a good chromophore,
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LC-MS/MS analysis was used.</p></div><div id="ml249.s7"><h4>Stability</h4><p>Demonstration of stability in PBS was conducted under conditions likely to be
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experienced in a laboratory setting. The compound was dissolved in 1 mL of
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PBS at a concentration of 10 μM, unless its maximum solubility was
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insufficient to achieve this concentration. Low solubility compounds were
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tested between ten and fifty percent of their solubility limit. The solution
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was immediately aliquoted into seven standard polypropylene microcentrifuge
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tubes which were stored at ambient temperature in a block microcentrifuge
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tube holder. Individual tubes were frozen at −80°C at 0, 1,
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2, 4, 8, 24, and 48 hours. The frozen samples were thawed at room
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|
temperature and an equal volume of acetonitrile was added prior to
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determination of concentration by LC-MS/MS.</p></div><div id="ml249.s8"><h4>Determination of glutathione reactivity</h4><p>One μL of a 10 mM compound stock solution was added to 1 mL of a
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|
freshly prepared solution of 100 μM reduced glutathione. Final
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|
compound concentration is 10 μM unless solubility limited. The
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solution was allowed to incubate at 37°C for two hours prior to
|
|
being directly analyzed for glutathione adduct formation. LC-MS/MS analysis
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|
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
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|
neutral loss scan of −129 AMU to detect GSH adducts. This triggered
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|
positive ion enhanced resolution and enhanced product ion scans.</p></div><div id="ml249.s9"><h4>Primary, Confirmation, and Dose Response uHTS assays to identify S1P3
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|
receptor agonists (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/373" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 373</a> and <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/439" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 439</a>)</h4><p><b>Assay Overview:</b> The purpose of these assays was to identify
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compounds that act as agonists of the S1P3 receptor. A cell line containing
|
|
the human S1P3 receptor as well as the beta-lactamase (BLA) reporter-gene
|
|
under control of the nuclear factor of activated T-cells (NFAT) promoter was
|
|
used to measure S1P3 agonism. If the S1P3 receptor was stimulated by
|
|
agonist, transcription of the NFAT-BLA gene occurred via a Gα16
|
|
protein coupled signaling cascade. The amount of BLA activity was
|
|
proportional to the concentration of agonist. BLA activity was measured with
|
|
a fluorescent BLA substrate. The entire campaign was run with S1P as the
|
|
positive control. In this assay, S1P had a 50% effective
|
|
concentration (EC50) of approximately 200 nM. All data reported was
|
|
normalized on a per-plate basis to wells that contained cells in the
|
|
presence of 1 micromolar S1P (i.e. 100% activation). For the primary
|
|
screen (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/373" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 373</a>), all compounds were tested once at a 4.5 micromolar
|
|
final concentration, and for the confirmation screen (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/373" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID
|
|
373</a>), all compounds were tested in triplicate at the same
|
|
concentration. For the dose response screen (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/439" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID
|
|
439</a>), compounds were tested in triplicate in a ten-point 1:3
|
|
dilution series starting at a nominal concentration of 45 μM.</p><p><b>Protocol Summary:</b> A Chinese Hamster Ovary (CHO) cell line stably
|
|
transfected with human S1P3 receptor, nuclear factor of activated
|
|
T-cell-beta lactamase (NFAT-BLA) reporter construct and the Gα16
|
|
pathway coupling protein was used. Cells were cultured in T-175 sq cm flasks
|
|
at 37°C and 95% relative humidity (RH). The growth medium
|
|
consisted of Dulbecco's Modified Eagle's Media containing
|
|
10% v/v heat inactivated bovine growth serum, 0.1 mM NEAA, 1 mM
|
|
Sodium Pyruvate, 25 mM HEPES, 2 mg/mL 5 mM L-Glutamine, 0.2 mg/mL Hygromycin
|
|
B and 1× penicillin-streptomycin. Prior to the start of the assay,
|
|
cells were suspended to a concentration of 1 × 10<sup>6</sup>/mL in
|
|
phenol red free Dulbecco's Modified Eagle's Media containing
|
|
0.5% charcoal/dextran treated fetal bovine serum, 0.1 mM NEAA, 1 mM
|
|
Sodium Pyruvate, 25 mM HEPES, and 5 mM L-Glutamine. The assay began by
|
|
dispensing 5 mL of cell suspension to each test well of a 1536 well plate.
|
|
The cells were then allowed to incubate in the plates overnight at
|
|
37°C in 5% CO<sub>2</sub>. The next day, 25 nL of test
|
|
compound or DMSO control was added. The S1P positive control was also added
|
|
to the appropriate control wells to a final concentration of 1 μM.
|
|
Plates were then incubated at 37°C in 5% CO<sub>2</sub> for
|
|
4 hours. After the incubation, 1 μL/well of the GeneBLAzer's
|
|
fluorescent substrate mixture, prepared according to the
|
|
manufacturer's protocol and containing 200 mM probenicid was added.
|
|
After 2 hours of incubation at room temperature, plates were read on the
|
|
EnVision plate reader (PerkinElmer Lifesciences, Turku, Finland) at an
|
|
excitation wavelength 405 nm and emission wavelengths of 535 nm & 460
|
|
nm. <b>Assay Cutoff:</b> In the primary and confirmation screens,
|
|
compounds that exhibited 4.22% agonism or greater for S1P3 receptor
|
|
were considered active. In the dose response screen, compounds with EC50
|
|
> 10 μM were considered active.</p></div><div id="ml249.s10"><h4>Dose response assay to identify S1P3 receptor agonists with purchased
|
|
analogues (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1192" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1192</a>)</h4><p><b>Assay Overview:</b> The purpose of this assay was to determine dose
|
|
response curves for purchased structural analogues of an S1P3 agonist
|
|
(<a href="https://pubchem.ncbi.nlm.nih.gov/substance/7967985" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 7967985</a>) identified as active in previous experiments.
|
|
The assay was described above (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/373" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 373</a>). Compounds were
|
|
tested in quadruplicate in 384-well plates in a 10-point 1:3 dilution series
|
|
starting at a nominal test concentration of 10 micromolar.</p><p><b>Protocol Summary:</b> Cells were cultured as described above
|
|
(<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/373" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID
|
|
373</a>). Prior to the start of the assay, cells were suspended
|
|
at a concentration of 1 × 10<sup>6</sup>/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, followed by overnight
|
|
incubation at 37°C in 5% CO<sub>2</sub>. The next day, 50 nL
|
|
of test compound in DMSO, DMSO alone, or S1P (1 μM final nominal
|
|
concentration) was added to the appropriate wells. After 4 hours of
|
|
incubation, 2 μL/well of the GeneBLAzer fluorescent substrate
|
|
mixture, prepared according to the manufacturer's protocol and
|
|
containing 6 mM Probenicid, was added to all wells. The plates were then
|
|
incubated for 2 hours at room temperature. Plates were read on the EnVision
|
|
plate reader (PerkinElmer Lifesciences, Turku, Finland) at an excitation
|
|
wavelength of 405 nm and emission wavelengths of 590 nm and 460 nm.
|
|
<b>Assay Cutoff:</b> Compounds with EC50 ≤ 10 μM
|
|
were considered active.</p></div><div id="ml249.s11"><h4>Dose response assay to identify S1P3 receptor agonists with synthesized
|
|
compounds (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/540349" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 540349</a>)</h4><p><b>Assay Overview:</b> The purpose of this assay was to determine S1P3
|
|
receptor agonist dose response with synthesized compounds. This assay was
|
|
run as described above (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/373" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 373</a>). Compounds were tested in triplicate
|
|
using a 10-point 1:3 dilution series.</p><p><b>Protocol Summary:</b> Cells were cultured as described above
|
|
(<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/373" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID
|
|
373</a>). Prior to the start of the assay cells were suspended at
|
|
a concentration of 1.25 × 10<sup>6</sup>/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 mL of cell suspension to each well of a 384 well plate (8 ×
|
|
10<sup>3</sup> 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 micromolar final nominal concentration) in DMSO
|
|
was added to sample wells, and DMSO alone (0.5% final concentration)
|
|
was added to High Control wells. Next, S1P prepared in 2% BSA (0.7
|
|
micromolar final nominal concentration, corresponding to the EC80 of S1P)
|
|
was added to the appropriate 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
|
|
Probenicid, was added to all wells. The plates were then incubated for 2
|
|
hours at room temperature. Plates were read on the EnVision plate reader
|
|
(PerkinElmer Lifesciences, Turku, Finland) at an excitation wavelength of
|
|
405 nm and emission wavelengths of 535 nm and 460 nm. <b>Assay
|
|
Cutoff:</b> Compounds with EC50 ≤ 10 μM were
|
|
considered active.</p></div><div id="ml249.s12"><h4>Counterscreen dose response assay to identify S1P1 receptor agonists
|
|
(<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/540368" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 540368</a>)</h4><p><b>Assay Overview:</b> The purpose of this assay was to determine
|
|
whether powder samples of compounds identified as active for S1P3 agonist
|
|
were nonselective agonists as assayed by activation of S1P1. This assay uses
|
|
Tango S1P1-bla U2OS cells which express the human Endothelial
|
|
Differentiation Gene 1 (EDG1; S1P1) 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 agonists will activate S1P1 and
|
|
increase well FRET. Compounds were tested in triplicate using a 10-point,
|
|
1:3 dilution series.</p><p><b>Protocol Summary:</b> U2OS cells were cultured in T-175 sq cm flasks
|
|
at 37°C and 95% 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 micrograms/mL Zeocin, 50
|
|
micrograms/mL Hygromycin, and 100 micrograms/mL Geneticin. Prior to the
|
|
start of the assay, cells were suspended at a concentration of 2.75
|
|
× 10<sup>5</sup>/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 (1 × 10<sup>4</sup>
|
|
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, or S1P (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 Probenicid, was
|
|
added to all wells. After 2 hours of incubation at room temperature in the
|
|
dark, plates were read on the EnVision plate reader (PerkinElmer
|
|
Lifesciences, Turku, Finland) at an excitation wavelength of 405 nm and
|
|
emission wavelengths of 460 nm and 535 nm. <b>Assay Cutoff:</b>
|
|
Compounds with EC50 ≤ 10 μM were considered active.</p></div><div id="ml249.s13"><h4>Counterscreen dose response assay to identify S1P2 receptor agonists
|
|
(<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/540367" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 540367</a>)</h4><p><b>Assay Overview:</b> The purpose of this assay was to determine
|
|
whether powder samples of compounds identified as active for S1P3 agonist
|
|
were nonselective agonists as assayed by activation of S1P2. 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 S1P1
|
|
agonists will activate S1P1 and increase well FRET. Compounds were tested in
|
|
quadruplicate at 50 micromolar.</p><p><b>Protocol Summary:</b> Cells were cultured as described above
|
|
(<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/373" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID
|
|
373</a>). Prior to assay, cells were suspended to a concentration
|
|
of 1.25 × 10<sup>6</sup>/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 × 10<sup>3</sup> cells/well) followed by incubation at
|
|
37°C in 5% CO<sub>2</sub> for 16 hours. To the appropriate
|
|
wells were then added 50 nL of test compound in DMSO (final nominal
|
|
concentration of 50 micromolar, final DMSO concentration of 0.5%) or
|
|
DMSO only (for high control wells) followed directly afterwards by 1
|
|
μL of S1P in 2% BSA (final concentration of 370 nanomolar,
|
|
i.e. a concentration that resulted in 80% activity). The high
|
|
control (EC80 challenge) and low control (100% antagonism) were
|
|
added to the appropriate control wells and plates were incubated again at
|
|
37°C in 5% CO<sub>2</sub> for 2 hours. The fluorogenic
|
|
LiveBLAzer substrate mixture with 10 mM Probenicid was prepared according to
|
|
the manufacturer's protocol and 2.2 μL 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> Where treatment with 50 μM compound did not
|
|
result in greater than 50% activation, the EC50 was determined
|
|
manually as greater than 50 μM. Compounds with an EC50 ≤ 50
|
|
μM were considered active.</p></div><div id="ml249.s14"><h4>Counterscreen dose response assay to identify S1P4 receptor agonists
|
|
(<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/540366" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 540366</a>)</h4><p><b>Assay Overview:</b> The purpose of this assay was to determine
|
|
whether powder samples of compounds identified as active for S1P3 agonist
|
|
were nonselective agonists as assayed by activation of S1P4. This assay uses
|
|
Tango S1P4-BLA U2OS cells which contain the human Endothelial
|
|
Differentiation Gene 6 (EDG6; S1P4) linked to a GAL4-VP16 transcription
|
|
factor via a TEV protease site. The cells also express a beta-arrestin/TEV
|
|
protease fusion protein and a beta-lactamase (BLA) reporter gene under the
|
|
control of a UAS response element. Stimulation of the 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 agonists will activate S1P1 and
|
|
increase well FRET. Compounds were tested in triplicate at 50
|
|
micromolar.</p><p><b>Protocol Summary:</b> U2OS cell were cultured as described above
|
|
(<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/540368" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 540368</a>). Prior to the start of the assay, cells were
|
|
suspended at a concentration of 2.5 × 10<sup>5</sup>/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, or S1P (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 Probenicid, was added to
|
|
all wells. After 2 hours of incubation at room temperature in the dark,
|
|
plates were read on the EnVision plate reader (PerkinElmer Lifesciences,
|
|
Turku, Finland) at an excitation wavelength of 405 nm and emission
|
|
wavelengths of 460 nm and 535 nm. <b>Assay Cutoff:</b> Where treatment
|
|
with 50 μM compound did not result in greater than 50%
|
|
activation, the EC50 was determined manually as greater than 50 μM.
|
|
Compounds with an EC50 ≤ 50 μM were considered active.</p></div><div id="ml249.s15"><h4>Counterscreen dose response assay to identify S1P5 receptor agonists
|
|
(<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/540369" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 540369</a>)</h4><p><b>Assay Overview:</b> The purpose of this assay was to determine
|
|
whether powder samples of compounds identified as active for S1P3 agonist
|
|
were nonselective agonists as assayed by activation of S1P4. 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 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
|
|
sextuplet using a 10-point, 1:3 dilution series starting at a nominal
|
|
concentration of 25 micromolar.</p><p><b>Protocol Summary:</b> U2OS cell were cultured as described above
|
|
(<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/540368" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 540368</a>). Prior to the start of the assay, cells were
|
|
suspended at a concentration of 2.75 × 10<sup>5</sup>/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 (1 × 10<sup>4</sup> 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, or S1P (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 Probenicid, was added to all wells. After 2 hours of
|
|
incubation at room temperature in the dark, plates were read on the EnVision
|
|
plate reader (PerkinElmer Lifesciences, Turku, Finland) at an excitation
|
|
wavelength of 405 nm and emission wavelengths of 460 nm and 535 nm.
|
|
<b>Assay Cutoff:</b> Compounds with EC50 ≤ 10 μM
|
|
were considered active.</p></div><div id="ml249.s16"><h4>Analysis of cytotoxicity (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/540344" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 540344</a>)</h4><p><b>Assay Overview:</b> The purpose of this assay was to determine
|
|
cytotoxicity of the S1P3 agonist compound CID 17253208, <a href="/pcsubstance/?term=ML249[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML249</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
|
|
micromolar.</p><p><b>Protocol Summary:</b> This assay was started by dispensing U2OS
|
|
cells in McCoy’s 5A medium plus 10% FBS, penicillin 100 U/ml
|
|
and streptomycin 100 ug/ml (20 μL, 4 × 10<sup>3</sup>
|
|
cells/well) into the wells of a 384-well plate. Eight 1:3 serial dilutions
|
|
of compound (100 μM in growth media) were made. 5μL 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, 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 micromolar were considered active (cytotoxic).</p></div><div id="ml249.s17"><h4>Competition binding assay with physiological ligand S1P (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/588327" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID
|
|
588327</a>)</h4><p><b>Assay Overview:</b> The purpose of this assay was to determine
|
|
whether <a href="/pcsubstance/?term=ML249[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML249</a> competes competitively or
|
|
noncompetitively for binding of [<sup>33</sup>P]S1P to cells
|
|
that contain the S1P3 receptor. In this assay, S1P3 Jump-In CHO cells
|
|
containing human S1P3 are incubated with a fixed concentration of
|
|
[<sup>33</sup>P]S1P and increasing concentrations of
|
|
S1P3 agonist. If the S1P3 agonist compound competes with the
|
|
[<sup>33</sup>P]S1P for binding to the S1P3 receptor,
|
|
the radioactivity bound to the cells will decrease as the concentration of
|
|
cold S1P3 increases. If the S1P3 agonist compound does not compete with the
|
|
[<sup>33</sup>P]S1P for binding to the S1P3 receptor,
|
|
the radioactivity bound to the cells will remain unchanged as the
|
|
concentration of S1P3 agonist increases. S1P3 agonist compound was tested in
|
|
triplicate using a 10-point, 1:3 dilution series starting at a nominal
|
|
concentration of 10 micromolar (<b>Experiment 1</b>). An experiment to
|
|
demonstrate that cold S1P competes competitively for binding of
|
|
[<sup>33</sup>P]S1P to cells that contain the S1P3
|
|
receptor serves as a control (<b>Experiment 2</b>). In this assay,
|
|
S1P3 Jump-In Chinese Hamster Ovary (CHO) cells containing human S1P3 are
|
|
incubated with a fixed concentration of [<sup>33</sup>P]S1P
|
|
and increasing concentrations of cold S1P. If the cold S1P competes with the
|
|
[<sup>33</sup>P]S1P for binding to the S1P3 receptor,
|
|
the radioactivity bound to the cells will decrease as the concentration of
|
|
cold S1P3 increases. Cold S1P was tested in triplicate using a 10-point, 1:3
|
|
dilution series starting at a nominal concentration of 10 micromolar.</p><p><b>Protocol Summary:</b> S1P3 Jump-In CHO 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) (with GlutaMAX)
|
|
containing 10% v/v heat inactivated fetal bovine serum (dialyzed),
|
|
0.1 mM NEAA, 1 mM sodium pyruvate, 25 mM HEPES, and 1 ×
|
|
penicillin-streptomycin-neomycin. On the day before the assay, cells were
|
|
suspended at a concentration of 0.2 × 10<sup>6</sup>/mL in the
|
|
growth media and plated at 0.1 × 10<sup>6</sup>/well in a 24-well
|
|
plate. On the day of the assay, the growth medium was replaced with
|
|
serum-starvation medium consisted of Dulbecco's Modified
|
|
Eagle's Media (DMEM) (with GlutaMAX) containing 0.1 mM NEAA, 1 mM
|
|
sodium pyruvate, and no antibiotics. The cells were incubated at
|
|
37°C for 4 hours. After 4 hours of serum starvation, the medium was
|
|
removed and the cells were rinsed with 200 μL of ice-cold binding
|
|
buffer. The binding buffer consisted of 20 mM Tris-HCl (pH 7.5), 100 mM
|
|
NaCl, 15 mM NaF, 0.5 mM EDTA, 1 mM Na<sub>3</sub>VO<sub>4</sub>,
|
|
0.5% fatty acid-free BSA, and 1× protease inhibitor
|
|
cocktail. <b>Experiment 1:</b> For the S1P3 agonist competition assay,
|
|
30 μL of increasing concentrations of <a href="/pcsubstance/?term=ML249[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML249</a> (final <a href="/pcsubstance/?term=ML249[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML249</a> concentrations
|
|
of 0.001 nM to 10 μM) and 270 μL ice-cold binding buffer
|
|
containing [<sup>33</sup>P]S1P (final concentration of 0.1
|
|
nM) were added to the cells in each well. <b>Experiment 2:</b> For the
|
|
control assay, 30 μL of increasing concentrations of cold S1P (final
|
|
S1P concentrations of 0.001 nM to 10 μM) and 270 μL ice-cold
|
|
binding buffer containing [<sup>33</sup>P]S1P (final
|
|
concentration of 0.1 nM) were added to the cells in each well. For both
|
|
experiments, the cells were incubated at 4 °C for 30 minutes. The
|
|
cells were then washed three times with 500 μL ice-cold binding
|
|
buffer. The cells were lysed with 300 μL 0.5% SDS and
|
|
transferred to scintillation vials. 5 mL of scintillation cocktail was
|
|
dispensed into each vial and the vial vortexed. The <sup>33</sup>P
|
|
radioactivity (cpm) was counted for 5 minutes/vial in a Beckman LS 6000SC
|
|
counter.</p></div><div id="ml249.s18"><h4>Counterscreen panel assay for S1P3 agonists: Ricerca HitProfilingScreen
|
|
+ CYP450 (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/540351" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 540351</a>)</h4><p><b>Assay Overview:</b> The purpose of this panel of binding assays
|
|
performed by Ricerca Biosciences, LLC, was to identify a subset of potential
|
|
receptors, transporters, ion channels, etc. for which the S1P3 agonist
|
|
compound CID 17253208, <a href="/pcsubstance/?term=ML249[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML249</a>, displays
|
|
affinity.</p><p><b>Protocol Summary:</b> Assays for CYP450, 1A2; CYP450, 2C19; CYP450,
|
|
2C9; CYP450, 2D6; and CYP450, 3A4 were enzyme assays using human recombinant
|
|
insect Sf9 cells with 5 μM 3-cyano-7-ethoxycoumarin as substrate
|
|
(except for CYP450, 3A4, which used 50 μM
|
|
7-benzyloxy-4-(trifluoromethyl)-coumarin as substrate). Detection was based
|
|
on spectrofluorimetric quantitation of the enzymatic product produced.
|
|
Assays for the other targets were radioligand binding assays. <b>Assay
|
|
Cutoff:</b> A response of at least 50% inhibition or
|
|
stimulation was considered “active”. Negative inhibition
|
|
represents a stimulation of binding.</p></div></div><div id="ml249.s19"><h3>2.2. Probe Chemical Characterization</h3><div id="ml249.fu2" class="figure bk_fig"><div class="graphic"><img src="/books/NBK133421/bin/ml249fu2.jpg" alt="CID 17253208 SID 124360653 ML249." /></div><h3><span class="title">CID 17253208<br /><a href="https://pubchem.ncbi.nlm.nih.gov/substance/124360653" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID
|
|
124360653</a><br /><a href="/pcsubstance/?term=ML249[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML249</a></span></h3></div><p>The probe structure was verified by <sup>1</sup>H-NMR and LCMS. Compound purity
|
|
was assessed to be greater than 96% by <sup>1</sup>H-NMR and LC-MS
|
|
(<a class="figpopup" href="/books/NBK133421/figure/ml249.f1/?report=objectonly" target="object" rid-figpopup="figml249f1" rid-ob="figobml249f1">Figure 1</a>).</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml249f1" co-legend-rid="figlgndml249f1"><a href="/books/NBK133421/figure/ml249.f1/?report=objectonly" target="object" title="Figure 1" class="img_link icnblk_img figpopup" rid-figpopup="figml249f1" rid-ob="figobml249f1"><img class="small-thumb" src="/books/NBK133421/bin/ml249f1.gif" src-large="/books/NBK133421/bin/ml249f1.jpg" alt="Figure 1. LC-MS results for probe ML249." /></a><div class="icnblk_cntnt" id="figlgndml249f1"><h4 id="ml249.f1"><a href="/books/NBK133421/figure/ml249.f1/?report=objectonly" target="object" rid-ob="figobml249f1">Figure 1</a></h4><p class="float-caption no_bottom_margin">LC-MS results for probe ML249. </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 1.24 μM as determined by dilution of 10 mM stock
|
|
solution in DMSO into PBS. The probe has a half-life of > 48 hours in PBS
|
|
at room temperature (100% compound remaining at 48 hours) (<a class="figpopup" href="/books/NBK133421/figure/ml249.f2/?report=objectonly" target="object" rid-figpopup="figml249f2" rid-ob="figobml249f2">Figure 2</a>).</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml249f2" co-legend-rid="figlgndml249f2"><a href="/books/NBK133421/figure/ml249.f2/?report=objectonly" target="object" title="Figure 2" class="img_link icnblk_img figpopup" rid-figpopup="figml249f2" rid-ob="figobml249f2"><img class="small-thumb" src="/books/NBK133421/bin/ml249f2.gif" src-large="/books/NBK133421/bin/ml249f2.jpg" alt="Figure 2. Stability of Probe ML249 in PBS." /></a><div class="icnblk_cntnt" id="figlgndml249f2"><h4 id="ml249.f2"><a href="/books/NBK133421/figure/ml249.f2/?report=objectonly" target="object" rid-ob="figobml249f2">Figure 2</a></h4><p class="float-caption no_bottom_margin">Stability of Probe ML249 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/NBK133421/table/ml249.t1/?report=objectonly" target="object" rid-figpopup="figml249t1" rid-ob="figobml249t1">Table 1</a>)
|
|
have been submitted to the SMR collection.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml249t1"><a href="/books/NBK133421/table/ml249.t1/?report=objectonly" target="object" title="Table 1" class="img_link icnblk_img figpopup" rid-figpopup="figml249t1" rid-ob="figobml249t1"><img class="small-thumb" src="/books/NBK133421/table/ml249.t1/?report=thumb" src-large="/books/NBK133421/table/ml249.t1/?report=previmg" alt="Table 1. Compounds submitted to the MLSMR." /></a><div class="icnblk_cntnt"><h4 id="ml249.t1"><a href="/books/NBK133421/table/ml249.t1/?report=objectonly" target="object" rid-ob="figobml249t1">Table 1</a></h4><p class="float-caption no_bottom_margin">Compounds submitted to the MLSMR. </p></div></div></div><div id="ml249.s20"><h3>2.3. Probe Preparation</h3><div id="ml249.f3" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%203.%20Synthesis%20scheme%20for%20ML249.&p=BOOKS&id=133421_ml249f3.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/NBK133421/bin/ml249f3.jpg" alt="Figure 3. Synthesis scheme for ML249." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 3</span><span class="title">Synthesis scheme for <a href="/pcsubstance/?term=ML249[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML249</a></span></h3></div><p>A mixture of carboxylic acid <b>1</b> and SOCl<sub>2</sub> in benzene was
|
|
refluxed for 3 hours. The solvent was successively removed under reduced
|
|
pressure. The residue was dissolved in CH<sub>2</sub>Cl<sub>2</sub> and the
|
|
mixture cooled to 0°C followed by sequential addition of DIPEA and
|
|
dicyclohexylamine. The reaction mixture was stirred at room temperature for 3
|
|
hours. The solvent was removed under reduced pressure, the crude diluted in
|
|
EtOAc and washed (2X) with brine. The organic phase was dried over sodium
|
|
sulfate and concentrated under reduced pressure. The crude was purified by
|
|
column chromatography (hexanes/EtOAc, 8:2) to furnish <a href="/pcsubstance/?term=ML249[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML249</a> in 90% yield.</p><p><sup>1</sup>H NMR (500 MHz, CDCl3): δ 6.03 (s, 1H), 3.85-3.76 (m, 1H),
|
|
3.14-3.02 (m, 1H), 2.59-2.44 (m, 2H), 2.09-2.00 (m, 1H), 1.86-1.71 (m, 7H),
|
|
1.64-1.42 (m, 7H), 1.26-1.15 (m, 4H), 1.10-0.95 (m, 4H); <sup>13</sup>C NMR (125
|
|
MHz, CDCl3): δ 174.71, 161.01, 160.03, 99.04, 59.26, 56.66, 31.38,
|
|
29.71, 26.55, 25.58, 25.29, 25.18, 8.59, 8.08. MS (EI) <i>m/z</i>: 317
|
|
(M<sup>+</sup>).</p></div></div><div id="ml249.s21"><h2 id="_ml249_s21_">3. Results</h2><div id="ml249.s22"><h3>3.1. Dose Response Curves for Probe</h3><p><a class="figpopup" href="/books/NBK133421/figure/ml249.f4/?report=objectonly" target="object" rid-figpopup="figml249f4" rid-ob="figobml249f4">Figure 4</a> shows the dose-response curve
|
|
for binding of <a href="/pcsubstance/?term=ML249[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML249</a> to CHO cells containing the human
|
|
S1P3 gene. <a href="/pcsubstance/?term=ML249[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML249</a> acts as a full agonist; the level of
|
|
activity achieved by increasing the dose of <a href="/pcsubstance/?term=ML249[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML249</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="figml249f4" co-legend-rid="figlgndml249f4"><a href="/books/NBK133421/figure/ml249.f4/?report=objectonly" target="object" title="Figure 4" class="img_link icnblk_img figpopup" rid-figpopup="figml249f4" rid-ob="figobml249f4"><img class="small-thumb" src="/books/NBK133421/bin/ml249f4.gif" src-large="/books/NBK133421/bin/ml249f4.jpg" alt="Figure 4. Dose-response curve for ML249." /></a><div class="icnblk_cntnt" id="figlgndml249f4"><h4 id="ml249.f4"><a href="/books/NBK133421/figure/ml249.f4/?report=objectonly" target="object" rid-ob="figobml249f4">Figure 4</a></h4><p class="float-caption no_bottom_margin">Dose-response curve for ML249. </p></div></div></div><div id="ml249.s23"><h3>3.2. Cellular Activity</h3><p><a href="/pcsubstance/?term=ML249[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML249</a> has been evaluated in a series of cell-based assays; the
|
|
primary screen [AIDs <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/373" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">373</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/439" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">439</a>, and <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/540349" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">540349</a>] and the S1P2 counterscreen [<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/540367" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID540367</a>] are CHO-based assays, and the S1P1
|
|
[<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/540368" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 540368</a>], S1P4 [<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/540366" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID
|
|
540366</a>], S1P5 [<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/540369" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 540369</a>], and
|
|
cytotoxicity [<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/540344" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 540344</a>] counterscreens are U2OS-based
|
|
assays.</p></div><div id="ml249.s24"><h3>3.3. Profiling Assays</h3><p>To date, the lead hit (CID 5309153) has been tested in 586 other bioassays
|
|
deposited in PubChem, and has shown activity in only 13 of those assays, three
|
|
of which are for the S1P3 receptor agonist project. The other ten assays give a
|
|
hit rate of 1.7%, indicating that this series is not generally active
|
|
across a broad range of cell-based and non-cell based assays.</p><p><a href="/pcsubstance/?term=ML249[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML249</a> was submitted to Ricerca Biosciences, LLC for
|
|
HitProfilingScreen + CYP450 (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/540351" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 540351</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=ML249[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML249</a> displays affinity.
|
|
Out of 35 targets tested, six (CYP450 2C19, CYP450 3A4, norepinephrine
|
|
transporter NET, cannabinoid CB<sub>1</sub>, histamine H<sub>1</sub>, and sodium
|
|
channel site 2) resulted in borderline (≥ 50%) inhibition, and
|
|
one (nicotinic acetylcholine) resulted in borderline (≥ 50%)
|
|
activation when tested at 30 μM. The range of Ricerca activities was 51
|
|
to 74%. If these compounds have ‘normal’ dose response
|
|
curves the IC50s will clearly be in the double digit micromolar range. These
|
|
data suggest that <a href="/pcsubstance/?term=ML249[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML249</a> is generally inactive against a broad
|
|
array of off-targets and does not likely exert unwanted effects.</p></div></div><div id="ml249.s25"><h2 id="_ml249_s25_">4. Discussion</h2><div id="ml249.s26"><h3>4.1. Comparison to Existing Art and How the New Probe is an Improvement</h3><p>No submicromolar, completely selective S1P3 selective agonist compounds are
|
|
currently available. Previously reported S1P3 receptor agonist compounds are
|
|
micromolar agonists and/or possess unknown selectivity. Dose response assays
|
|
against all five S1P receptors demonstrate that <a href="/pcsubstance/?term=ML249[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML249</a> is the first submicromolar, completely selective S1P3
|
|
receptor agonist to be identified.</p></div></div><div id="ml249.s27"><h2 id="_ml249_s27_">5. References</h2><dl class="temp-labeled-list"><dl class="bkr_refwrap"><dt>1.</dt><dd><div class="bk_ref" id="ml249.r1">Goetzl EJ, Wang W, McGiffert C, Liao JJ, Huang MC. Sphingosine 1-phosphate as an
|
|
intracellular messenger and extracellular mediator in
|
|
immunity. <span><span class="ref-journal">Acta Paediatr. </span>2007 Suppl
|
|
96:49–52.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/17391442" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17391442</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>2.</dt><dd><div class="bk_ref" id="ml249.r2">Spiegel S, Milstien S. Sphingosine-1-phosphate: an
|
|
enigmatic signalling lipid. <span><span class="ref-journal">Nat Rev Mol Cell
|
|
Biol. </span>2003;<span class="ref-vol">4</span>:397–407.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/12728273" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 12728273</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>3.</dt><dd><div class="bk_ref" id="ml249.r3">Forrest M, Sun SY, Hajdu R, Bergstrom J, Card D, Doherty G, Hale J, Keohane C, Meyers C, Milligan J, Mills S, Nomura N, Rosen H, Rosenbach M, Shei GJ, Singer II, Tian M, West S, White V, Xie J, Proia RL, Mandala S. Immune cell regulation and
|
|
cardiovascular effects of sphingosine 1-phosphate receptor agonists in
|
|
rodents are mediated via distinct receptor
|
|
subtypes. <span><span class="ref-journal">J Pharmacol Exp
|
|
Ther. </span>2004;<span class="ref-vol">309</span>:758–768.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/14747617" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 14747617</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>4.</dt><dd><div class="bk_ref" id="ml249.r4">Sanna MG, Liao J, Jo E, Alfonso C, Ahn MY, Peterson MS, Webb B, Lefebvre S, Chun J, Gray N, Rosen H. Sphingosine 1-phosphate (S1P)
|
|
receptor subtypes S1P1 and S1P3, respectively, regulate lymphocyte
|
|
recirculation and heart rate. <span><span class="ref-journal">J Biol
|
|
Chem. </span>2004;<span class="ref-vol">279</span>:13839–13848.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/14732717" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 14732717</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>5.</dt><dd><div class="bk_ref" id="ml249.r5">Alfonso C, McHeyzer-Williams MG, Rosen H. CD69 down-modulation and
|
|
inhibition of thymic egress by short- and long-term selective chemical
|
|
agonism of sphingosine 1-phosphate receptors. <span><span class="ref-journal">Eur
|
|
J
|
|
Immunol. </span>2006;<span class="ref-vol">36</span>:149–159.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/16342326" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 16342326</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>6.</dt><dd><div class="bk_ref" id="ml249.r6">Jo E, Sanna MG, Gonzalez-Cabrera PJ, Thangada S, Tigyi G, Osborne DA, Hla T, Parrill AL, Rosen H. S1P1-selective in vivo-active
|
|
agonists from high-throughput screening: off-the-shelf chemical probes
|
|
of receptor interactions, signaling, and
|
|
fate. <span><span class="ref-journal">Chem
|
|
Biol. </span>2005;<span class="ref-vol">12</span>:703–715.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/15975516" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 15975516</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>7.</dt><dd><div class="bk_ref" id="ml249.r7">Wei SH, Rosen H, Matheu MP, Sanna MG, Wang SK, Jo E, Wong CH, Parker I, Cahalan MD. Sphingosine 1-phosphate type
|
|
1 receptor agonism inhibits transendothelial migration of medullary T
|
|
cells to lymphatic sinuses. <span><span class="ref-journal">Nat
|
|
Immunol. </span>2005;<span class="ref-vol">6</span>:1228–1235.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/16273098" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 16273098</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>8.</dt><dd><div class="bk_ref" id="ml249.r8">Hla T. Signaling and biological
|
|
actions of sphingosine 1-phosphate. <span><span class="ref-journal">Pharmacol
|
|
Res. </span>2003;<span class="ref-vol">47</span>:401–407.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/12676514" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 12676514</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>9.</dt><dd><div class="bk_ref" id="ml249.r9">Mandala S, Hajdu R, Bergstrom J, Quackenbush E, Xie J, Milligan J, Thornton R, Shei GJ, Card D, Keohane C, Rosenbach M, Hale J, Lynch CL, Rupprecht K, Parsons W, Rosen H. Alteration of lymphocyte
|
|
trafficking by sphingosine-1-phosphate receptor
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agonists. <span><span class="ref-journal">Science. </span>2002;<span class="ref-vol">296</span>:346–349.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/11923495" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 11923495</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>10.</dt><dd><div class="bk_ref" id="ml249.r10">Sanchez T, Hla T. Structural and functional
|
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characteristics of S1P receptors. <span><span class="ref-journal">J Cell
|
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Biochem. </span>2004;<span class="ref-vol">92</span>:913–922.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/15258915" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 15258915</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>11.</dt><dd><div class="bk_ref" id="ml249.r11">Lee MJ, Thangada S, Claffey KP, Ancellin N, Liu CH, Kluk M, Volpi M, Sha'afi RI, Hla T. Vascular endothelial cell
|
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adherens junction assembly and morphogenesis induced by
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sphingosine-1-phosphate. <span><span class="ref-journal">Cell. </span>1999;<span class="ref-vol">99</span>:301–312.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/10555146" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 10555146</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>12.</dt><dd><div class="bk_ref" id="ml249.r12">Ohmori T, Yatomi Y, Okamoto H, Miura Y, Rile G, Satoh K, Ozaki Y. G(i)-mediated Cas tyrosine
|
|
phosphorylation in vascular endothelial cells stimulated with
|
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sphingosine 1-phosphate: possible involvement in cell motility
|
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enhancement in cooperation with Rho-mediated
|
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pathways. <span><span class="ref-journal">J Biol
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Chem. </span>2001;<span class="ref-vol">276</span>:5274–5280.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/11056155" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 11056155</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>13.</dt><dd><div class="bk_ref" id="ml249.r13">Wang F, Van Brocklyn JR, Hobson JP, Movafagh S, Zukowska-Grojec Z, Milstien S, Spiegel S. Sphingosine 1-phosphate
|
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stimulates cell migration through a G(i)-coupled cell surface receptor.
|
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Potential involvement in angiogenesis. <span><span class="ref-journal">J Biol
|
|
Chem. </span>1999;<span class="ref-vol">274</span>:35343–35350.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/10585401" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 10585401</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>14.</dt><dd><div class="bk_ref" id="ml249.r14">Murakami A, Takasugi H, Ohnuma S, Koide Y, Sakurai A, Takeda S, Hasegawa T, Sasamori J, Konno T, Hayashi K, Watanabe Y, Mori K, Sato Y, Takahashi A, Mochizuki N, Takakura N. Sphingosine 1-phosphate (S1P)
|
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regulates vascular contraction via S1P3 receptor: investigation based on
|
|
a new S1P3 receptor antagonist. <span><span class="ref-journal">Mol
|
|
Pharmacol. </span>2010;<span class="ref-vol">77</span>:704–713.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/20097776" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 20097776</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>15.</dt><dd><div class="bk_ref" id="ml249.r15">Niessen F, Schaffner F, Furlan-Freguia C, Pawlinski R, Bhattacharjee G, Chun J, Derian CK, Andrade-Gordon P, Rosen H, Ruf W. Dendritic cell PAR1-S1P3
|
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signalling couples coagulation and
|
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inflammation. <span><span class="ref-journal">Nature. </span>2008;<span class="ref-vol">452</span>:654–658.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/18305483" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18305483</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>16.</dt><dd><div class="bk_ref" id="ml249.r16">Fujiwara Y, Osborne DA, Walker MD, Wang DA, Bautista DA, Liliom K, Van Brocklyn JR, Parrill AL, Tigyi G. Identification of the
|
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hydrophobic ligand binding pocket of the S1P1
|
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receptor. <span><span class="ref-journal">J Biol
|
|
Chem. </span>2007;<span class="ref-vol">282</span>:2374–2385.</span> [<a href="/pmc/articles/PMC3446783/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3446783</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/17114791" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17114791</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>17.</dt><dd><div class="bk_ref" id="ml249.r17">Burford NT, Watson J, Bertekap R, Alt A. Strategies for the
|
|
identification of allosteric modulators of G-protein-coupled
|
|
receptors. <span><span class="ref-journal">Biochem
|
|
Pharmacol. </span>2011;<span class="ref-vol">81</span>:691–702.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/21184747" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 21184747</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>18.</dt><dd><div class="bk_ref" id="ml249.r18">Christopoulos A. Allosteric binding sites on
|
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cell-surface receptors: novel targets for drug
|
|
discovery. <span><span class="ref-journal">Nat Rev Drug
|
|
Discov. </span>2002;<span class="ref-vol">1</span>:198–210.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/12120504" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 12120504</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>19.</dt><dd><div class="bk_ref" id="ml249.r19">May LT, Avlani VA, Sexton PM, Christopoulos A. Allosteric modulation of G
|
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protein-coupled receptors. <span><span class="ref-journal">Curr Pharm
|
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Des. </span>2004;<span class="ref-vol">10</span>:2003–2013.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/15279541" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 15279541</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>20.</dt><dd><div class="bk_ref" id="ml249.r20">Urwyler S. Allosteric modulation of
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family C G-protein-coupled receptors: from molecular insights to
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therapeutic perspectives. <span><span class="ref-journal">Pharmacol
|
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Rev. </span>2011;<span class="ref-vol">63</span>:59–126.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/21228259" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 21228259</span></a>]</div></dd></dl></dl></div><div id="bk_toc_contnr"></div></div></div><div class="fm-sec"><h2 id="_NBK133421_pubdet_">Publication Details</h2><h3>Author Information and Affiliations</h3><p class="contrib-group"><h4>Authors</h4><span itemprop="author">Miguel Guerrero</span>,<sup>*</sup> <span itemprop="author">Ramulu Poddutoori</span>,<sup>*</sup> <span itemprop="author">Fernando Pinacho-Crisostomo</span>,<sup>*</sup> <span itemprop="author">Marie-Therese Schaeffer</span>,<sup>†</sup> <span itemprop="author">Steven J Brown</span>,<sup>†</sup> <span itemprop="author">Timothy Spicer</span>,<sup>‡</sup> <span itemprop="author">Peter Chase</span>,<sup>‡</sup> <span itemprop="author">Jill Ferguson</span>,<sup>†</sup> <span itemprop="author">Edward Roberts</span>,<sup>*</sup> <span itemprop="author">Germana Sanna</span>,<sup>†</sup> <span itemprop="author">Peter Hodder</span>,<sup>‡</sup> and <span itemprop="author">Hugh Rosen</span><sup>*</sup>.<sup><img src="/corehtml/pmc/pmcgifs/corrauth.gif" alt="corresponding author" /></sup></p><h4>Affiliations</h4><div class="affiliation"><sup>*</sup>
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Department of Chemistry, The Scripps Research Institute, La
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Jolla CA 92037</div><div class="affiliation"><sup>†</sup>
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Department of Chemical Physiology, The Scripps Research
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Institute Molecular Screening Center, The Scripps Research Institute, La Jolla, CA
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92037</div><div class="affiliation"><sup>‡</sup>
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Department of Chemistry, The Scripps Research Institute,
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Jupiter, FL, 33458</div><div class="affiliation"><sup>§</sup>
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Department of Immunology and Microbial Science, The Scripps
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Research Institute, La Jolla CA 92037</div><div class="affiliation"><sup><img src="/corehtml/pmc/pmcgifs/corrauth.gif" alt="corresponding author" /></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><h3>Publication History</h3><p class="small">Received: <span itemprop="datePublished">August 31, 2011</span>; Last Update: <span itemprop="dateModified">February 25, 2013</span>.</p><h3>Copyright</h3><div><div class="half_rhythm"><a href="/books/about/copyright/">Copyright Notice</a></div></div><h3>Publisher</h3><p>National Center for Biotechnology Information (US), Bethesda (MD)</p><h3>NLM Citation</h3><p>Guerrero M, Poddutoori R, Pinacho-Crisostomo F, et al. Probe Development Efforts for an Allosteric Agonist of the Sphingosine 1-phosphate Receptor 3 (S1P3) 2011 Aug 31 [Updated 2013 Feb 25]. 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></p></div><div class="small-screen-prev"><a href="/books/n/mlprobe/ml250/?report=reader"><svg xmlns="http://www.w3.org/2000/svg" viewBox="0 0 100 100" preserveAspectRatio="none"><path d="M75,30 c-80,60 -80,0 0,60 c-30,-60 -30,0 0,-60"></path><text x="20" y="28" textLength="60" style="font-size:25px">Prev</text></svg></a></div><div class="small-screen-next"><a href="/books/n/mlprobe/ml248/?report=reader"><svg xmlns="http://www.w3.org/2000/svg" viewBox="0 0 100 100" preserveAspectRatio="none"><path d="M25,30c80,60 80,0 0,60 c30,-60 30,0 0,-60"></path><text x="20" y="28" textLength="60" style="font-size:25px">Next</text></svg></a></div></article><article data-type="fig" id="figobml249fu1"><div id="ml249.fu1" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK133421/bin/ml249fu1.jpg" alt="ML249." /></div><h3><span class="title"><a href="/pcsubstance/?term=ML249[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML249</a></span></h3></div></article><article data-type="table-wrap" id="figobml249tu1"><div id="ml249.tu1" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK133421/table/ml249.tu1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml249.tu1_lrgtbl__"><table><thead><tr><th id="hd_h_ml249.tu1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">CID/ML#</th><th id="hd_h_ml249.tu1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Target Name</th><th id="hd_h_ml249.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_ml249.tu1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Anti-target Name</th><th id="hd_h_ml249.tu1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">EC50 (μM) [SID,
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AID]</th><th id="hd_h_ml249.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Fold Selective</th><th id="hd_h_ml249.tu1_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Secondary Assay(s) Name: EC50 (μM)
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[SID, AID]</th></tr></thead><tbody><tr><td headers="hd_h_ml249.tu1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">17253208/<a href="/pcsubstance/?term=ML249[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML249</a></td><td headers="hd_h_ml249.tu1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">S1P3</td><td headers="hd_h_ml249.tu1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">72.3–132 nM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/124360653" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 124360653</a>; <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/540349" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID
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540349</a>]</td><td headers="hd_h_ml249.tu1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">S1P1</td><td headers="hd_h_ml249.tu1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">28.1 μM–38.5 μM
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[<a href="https://pubchem.ncbi.nlm.nih.gov/substance/124360653" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 124360653</a>;
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<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/540368" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 540368</a>]</td><td headers="hd_h_ml249.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">213–1370</td><td headers="hd_h_ml249.tu1_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;"><b>S1P1 Counterscreen:</b>
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28.1–38.5 μM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/124360653" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 124360653</a>; <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/540368" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID
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540368</a>]<br /><b>S1P2
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Counterscreen:</b> >50 μM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/124360653" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 124360653</a>; <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/540367" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID
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540367</a>]<br /><b>S1P4
|
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Counterscreen:</b> >50 μM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/124360653" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 124360653</a>; <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/540366" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID
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540366</a>]<br /><b>S1P5
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Counterscreen:</b> >50 μM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/124360653" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 124360653</a>; <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/540369" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID
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540369</a>]<br /><b>Competition Binding
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Assay:</b> NA [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/124360653" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 124360653</a>;
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<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/588327" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID
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588327</a>]<br /><b>Cytotoxicity:</b>
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CC50 > 10 μM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/124360653" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 124360653</a>; <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/540344" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID
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540344</a>]<br /><b>Ricerca
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Profiling:</b> NA [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/124360653" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 124360653</a>;
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<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/540351" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 540351</a>]</td></tr></tbody></table></div></div></article><article data-type="fig" id="figobml249fu2"><div id="ml249.fu2" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK133421/bin/ml249fu2.jpg" alt="CID 17253208 SID 124360653 ML249." /></div><h3><span class="title">CID 17253208<br /><a href="https://pubchem.ncbi.nlm.nih.gov/substance/124360653" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID
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124360653</a><br /><a href="/pcsubstance/?term=ML249[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML249</a></span></h3></div></article><article data-type="fig" id="figobml249f1"><div id="ml249.f1" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%201.%20LC-MS%20results%20for%20probe%20ML249.&p=BOOKS&id=133421_ml249f1.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 data-src="/books/NBK133421/bin/ml249f1.jpg" alt="Figure 1. LC-MS results for probe ML249." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 1</span><span class="title">LC-MS results for probe <a href="/pcsubstance/?term=ML249[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML249</a></span></h3></div></article><article data-type="fig" id="figobml249f2"><div id="ml249.f2" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%202.%20Stability%20of%20Probe%20ML249%20in%20PBS.&p=BOOKS&id=133421_ml249f2.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 data-src="/books/NBK133421/bin/ml249f2.jpg" alt="Figure 2. Stability of Probe ML249 in PBS." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 2</span><span class="title">Stability of Probe <a href="/pcsubstance/?term=ML249[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML249</a> in
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PBS</span></h3></div></article><article data-type="table-wrap" id="figobml249t1"><div id="ml249.t1" class="table"><h3><span class="label">Table 1</span><span class="title">Compounds submitted to the MLSMR</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK133421/table/ml249.t1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml249.t1_lrgtbl__"><table class="no_top_margin"><tbody><tr><th id="hd_b_ml249.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Designation</th><th id="hd_b_ml249.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CID</th><th id="hd_b_ml249.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SID</th><th id="hd_b_ml249.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SRID</th><th id="hd_b_ml249.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS</th></tr><tr><th id="hd_b_ml249.t1_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Probe</th><td headers="hd_b_ml249.t1_1_1_2_1 hd_b_ml249.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">17253208</td><td headers="hd_b_ml249.t1_1_1_2_1 hd_b_ml249.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/124360653" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">124360653</a></td><td headers="hd_b_ml249.t1_1_1_2_1 hd_b_ml249.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-03000000611-2</td><td headers="hd_b_ml249.t1_1_1_2_1 hd_b_ml249.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS003675929</td></tr><tr><th id="hd_b_ml249.t1_1_1_3_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Analog 1</th><td headers="hd_b_ml249.t1_1_1_3_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">25110500</td><td headers="hd_b_ml249.t1_1_1_3_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/124360663" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">124360663</a></td><td headers="hd_b_ml249.t1_1_1_3_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-03000000625-2</td><td headers="hd_b_ml249.t1_1_1_3_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS003675925</td></tr><tr><th id="hd_b_ml249.t1_1_1_4_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Analog 2</th><td headers="hd_b_ml249.t1_1_1_4_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">25110499</td><td headers="hd_b_ml249.t1_1_1_4_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/12436066" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">12436066</a></td><td headers="hd_b_ml249.t1_1_1_4_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-03000000624-2</td><td headers="hd_b_ml249.t1_1_1_4_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS003675926</td></tr><tr><th id="hd_b_ml249.t1_1_1_5_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Analog 3</th><td headers="hd_b_ml249.t1_1_1_5_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">17253281</td><td headers="hd_b_ml249.t1_1_1_5_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/124360671" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">124360671</a></td><td headers="hd_b_ml249.t1_1_1_5_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-03000000651-2</td><td headers="hd_b_ml249.t1_1_1_5_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS003675927</td></tr><tr><th id="hd_b_ml249.t1_1_1_6_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Analog 4</th><td headers="hd_b_ml249.t1_1_1_6_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">25110484</td><td headers="hd_b_ml249.t1_1_1_6_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/124360649" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">124360649</a></td><td headers="hd_b_ml249.t1_1_1_6_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-03000000605-2</td><td headers="hd_b_ml249.t1_1_1_6_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS003675928</td></tr><tr><th id="hd_b_ml249.t1_1_1_7_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Analog 5</th><td headers="hd_b_ml249.t1_1_1_7_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">25110498</td><td headers="hd_b_ml249.t1_1_1_7_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/124360661" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">124360661</a></td><td headers="hd_b_ml249.t1_1_1_7_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-03000000623-2</td><td headers="hd_b_ml249.t1_1_1_7_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS003675930</td></tr></tbody></table></div></div></article><article data-type="fig" id="figobml249f3"><div id="ml249.f3" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%203.%20Synthesis%20scheme%20for%20ML249.&p=BOOKS&id=133421_ml249f3.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 data-src="/books/NBK133421/bin/ml249f3.jpg" alt="Figure 3. Synthesis scheme for ML249." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 3</span><span class="title">Synthesis scheme for <a href="/pcsubstance/?term=ML249[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML249</a></span></h3></div></article><article data-type="fig" id="figobml249f4"><div id="ml249.f4" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK133421/bin/ml249f4.jpg" alt="Figure 4. Dose-response curve for ML249." /></div><h3><span class="label">Figure 4</span><span class="title">Dose-response curve for <a href="/pcsubstance/?term=ML249[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML249</a></span></h3></div></article></div><div id="jr-scripts"><script src="/corehtml/pmc/jatsreader/ptpmc_3.22/js/libs.min.js"> </script><script src="/corehtml/pmc/jatsreader/ptpmc_3.22/js/jr.min.js"> </script></div></div>
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