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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 to Identify Novel Agonists of the Sphingosine 1-phosphate Receptor 4 (S1P4) - Probe Reports from the NIH Molecular Libraries Program - NCBI Bookshelf</title>
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<meta name="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 to Identify Novel Agonists of the Sphingosine 1-phosphate Receptor 4 (S1P4)">
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<meta name="citation_publisher" content="National Center for Biotechnology Information (US)">
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<meta name="citation_date" content="2011/12/12">
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<meta name="citation_author" content="Miguel Guerrero">
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<meta name="citation_author" content="Mariangela Urbano">
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<meta name="citation_author" content="Subash Velaparthi">
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<meta name="citation_author" content="Jian Zhao">
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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="Melissa Crisp">
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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="Michael Oldstone">
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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="22834032">
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<meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK98915/">
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<meta name="DC.Title" content="Probe Development Efforts to Identify Novel Agonists of the Sphingosine 1-phosphate Receptor 4 (S1P4)">
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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="Mariangela Urbano">
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<meta name="DC.Contributor" content="Subash Velaparthi">
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<meta name="DC.Contributor" content="Jian Zhao">
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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="Melissa Crisp">
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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="Michael Oldstone">
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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="2011/12/12">
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<meta name="DC.Identifier" content="https://www.ncbi.nlm.nih.gov/books/NBK98915/">
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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. S1P4 receptor is coupled to Gαi and Gαo G proteins and activates extracellular-signal-regulated kinases (ERK), Mitogen-activated protein kinases (MAPK) and Phospholipase C (PLC) downstream pathways. Inhibition of lymphocyte recirculation by nonselective S1P receptor agonists produces clinical immunosuppression preventing transplant rejection, but is associated with transient bradycardia. Understanding the contribution of individual receptors has been limited by the unavailability of selective agonists or antagonists for the 5 receptor subtypes. The Scripps Research Institute Molecular Screening Center (SRIMSC), part of the Molecular Libraries Probe Production Centers Network (MLPCN), identified a selective S1P4 agonist probe, ML178, by high-throughput screening using a cell-based Tango-format assay. ML178 activates S1P4 receptor with an EC50 of 46.3 nM, and is inactive as an agonist against other members of the receptor family, with EC50s > 50 μM against S1P1 receptor, S1P2 receptor, S1P3 receptor, and S1P5 receptor. ML178 is inhibited by an S1P4 receptor-selective antagonist with an IC50 of 0.83 μM, and is nontoxic to U2OS cells, with a CC50 of > 20 μM. Compound ML178 was submitted to Ricerca Biosciences, LLC target profiling against a panel of receptors, transporters, or ion channels; the data suggest that compound ML178 is generally inactive against a broad array of off targets and does not likely exert unwanted effects. ML178 is the first submicromolar, completely selective S1P4 receptor agonist to be identified.">
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<meta name="og:title" content="Probe Development Efforts to Identify Novel Agonists of the Sphingosine 1-phosphate Receptor 4 (S1P4)">
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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. S1P4 receptor is coupled to Gαi and Gαo G proteins and activates extracellular-signal-regulated kinases (ERK), Mitogen-activated protein kinases (MAPK) and Phospholipase C (PLC) downstream pathways. Inhibition of lymphocyte recirculation by nonselective S1P receptor agonists produces clinical immunosuppression preventing transplant rejection, but is associated with transient bradycardia. Understanding the contribution of individual receptors has been limited by the unavailability of selective agonists or antagonists for the 5 receptor subtypes. The Scripps Research Institute Molecular Screening Center (SRIMSC), part of the Molecular Libraries Probe Production Centers Network (MLPCN), identified a selective S1P4 agonist probe, ML178, by high-throughput screening using a cell-based Tango-format assay. ML178 activates S1P4 receptor with an EC50 of 46.3 nM, and is inactive as an agonist against other members of the receptor family, with EC50s > 50 μM against S1P1 receptor, S1P2 receptor, S1P3 receptor, and S1P5 receptor. ML178 is inhibited by an S1P4 receptor-selective antagonist with an IC50 of 0.83 μM, and is nontoxic to U2OS cells, with a CC50 of > 20 μM. Compound ML178 was submitted to Ricerca Biosciences, LLC target profiling against a panel of receptors, transporters, or ion channels; the data suggest that compound ML178 is generally inactive against a broad array of off targets and does not likely exert unwanted effects. ML178 is the first submicromolar, completely selective S1P4 receptor agonist to be identified.">
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id="jr-fip-info-p"><a id="jr-fip-prev" class="wsprkl btn" title="Jump to previuos 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="_NBK98915_"><span class="title" itemprop="name">Probe Development Efforts to Identify Novel Agonists of the Sphingosine
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1-phosphate Receptor 4 (S1P4)</span></h1><p class="contribs">Guerrero M, Urbano M, Velaparthi S, et al.</p><p class="fm-aai"><a href="#_NBK98915_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. S1P4 receptor is coupled to
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Gαi and Gαo G proteins and activates extracellular-signal-regulated
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kinases (ERK), Mitogen-activated protein kinases (MAPK) and Phospholipase C (PLC)
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downstream pathways. Inhibition of lymphocyte recirculation by nonselective S1P
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receptor agonists produces clinical immunosuppression preventing transplant
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rejection, but is associated with transient bradycardia. Understanding the
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contribution of individual receptors has been limited by the unavailability of
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selective agonists or antagonists for the 5 receptor subtypes. The Scripps Research
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Institute Molecular Screening Center (SRIMSC), part of the Molecular Libraries Probe
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Production Centers Network (MLPCN), identified a selective S1P4 agonist probe,
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<a href="/pcsubstance/?term=ML178[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML178</a>, by high-throughput screening using a cell-based Tango-format
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assay. <a href="/pcsubstance/?term=ML178[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML178</a> activates S1P4 receptor with an
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EC<sub>50</sub> of 46.3 nM, and is inactive as an agonist against other members
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of the receptor family, with EC<sub>50</sub>s > 50 μM against S1P1
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receptor, S1P2 receptor, S1P3 receptor, and S1P5 receptor. <a href="/pcsubstance/?term=ML178[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML178</a> is inhibited by an S1P4 receptor-selective antagonist with an
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IC<sub>50</sub> of 0.83 μM, and is nontoxic to U2OS cells, with a
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CC<sub>50</sub> of > 20 μM. Compound <a href="/pcsubstance/?term=ML178[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML178</a> was submitted to Ricerca Biosciences, LLC target profiling
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against a panel of receptors, transporters, or ion channels; the data suggest that
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compound <a href="/pcsubstance/?term=ML178[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML178</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=ML178[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML178</a> is the first submicromolar, completely selective S1P4 receptor
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agonist to be identified.</p></div><div class="h2"></div><p><b>Assigned Assay Grant #:</b> U01 <a href="/nuccore/3401208" class="bk_tag" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=nuccore">AI074564</a></p><p><b>Screening Center Name & PI:</b> Scripps Research Institute Molecular Screening Center, Scripps Research Institute Molecular Screening Center, H. Rosen, W. Roush</p><p><b>Chemistry Center Name & PI:</b> Scripps Research Institute Molecular Screening Center, Scripps Research Institute Molecular Screening Center, H. Rosen, W. Roush</p><p><b>Assay Submitter & Institution:</b> Michael B. Oldstone MD, Professor, The Scripps Research Institute</p><p><b>PubChem Summary Bioassay Identifier (AID):</b>
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<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1801" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1801</a></p><div id="ml178.s1"><h2 id="_ml178_s1_">Probe Structure & Characteristics</h2><div id="ml178.fu1" class="figure bk_fig"><div class="graphic"><img src="/books/NBK98915/bin/ml178fu1.jpg" alt="ML178." /></div><h3><span class="title"><a href="/pcsubstance/?term=ML178[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML178</a></span></h3></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml178tu1"><a href="/books/NBK98915/table/ml178.tu1/?report=objectonly" target="object" title="Table" class="img_link icnblk_img figpopup" rid-figpopup="figml178tu1" rid-ob="figobml178tu1"><img class="small-thumb" src="/books/NBK98915/table/ml178.tu1/?report=thumb" src-large="/books/NBK98915/table/ml178.tu1/?report=previmg" alt="Image " /></a><div class="icnblk_cntnt"><h4 id="ml178.tu1"><a href="/books/NBK98915/table/ml178.tu1/?report=objectonly" target="object" rid-ob="figobml178tu1">Table</a></h4></div></div></div><div id="ml178.s2"><h2 id="_ml178_s2_">Recommendations for scientific use of the probe</h2><p>Sphingosine 1-phosphate (S1P), a bioactive phospholipid released by activated blood platelets, influences multiple physiological systems, including endothelial integrity, lung epithelial integrity [<a class="bibr" href="#ml178.r1" rid="ml178.r1">1</a>, <a class="bibr" href="#ml178.r2" rid="ml178.r2">2</a>] and lymphocyte recirculation [<a class="bibr" href="#ml178.r3" rid="ml178.r3 ml178.r4 ml178.r5 ml178.r6 ml178.r7">3–7</a>], through five related high affinity G-protein coupled receptors [<a class="bibr" href="#ml178.r4" rid="ml178.r4">4</a>]. Understanding the contributions of individual S1P receptors to these physiological processes has been limited by the unavailability of selective agonists or antagonists for the 5 receptor subtypes. Following influenza infection, an excessive immune response on the part of the host can be detrimental by causing damage to tissues. Recently, modulation of S1P receptors locally in the lungs was shown to alter dendritic cell activation and accumulation in the mediastinal lymph nodes, resulting in blunted T cell responses and control of immunopathological features of influenza virus infection [<a class="bibr" href="#ml178.r8" rid="ml178.r8">8</a>]. The elucidation of the exact S1P receptors to be modulated for inhibition of pulmonary immune response is limited by the availability of specific probes that are water-soluble and suitable for local delivery in the airways. Reports showing that S1P5 receptor expression is very low in dendritic cells but that S1P4 receptor is highly expressed [<a class="bibr" href="#ml178.r9" rid="ml178.r9">9</a>], suggest that chemical activation of the S1P4 receptor subtype in the airways could be efficient at controlling the immunopathological response to viral infection. The availability of a selective S1P4 receptor antagonist may potentially provide a therapeutic tool for ensuring a balanced host immune response to influenza infection, in addition to serving as a useful tool for understanding S1P4 receptor biological function. This probe will be a useful tool for assays aiming to modulate S1P4 receptor signaling without affecting S1P1 receptor, and will be invaluable in research efforts designed to determine the effect of S1P4 signaling.</p></div><div id="ml178.s3"><h2 id="_ml178_s3_">1. Introduction</h2><p>Sphingosine 1-phosphate (S1P) is a bioactive phospholipid released by activated blood platelets that serves to influence heart rate [<a class="bibr" href="#ml178.r4" rid="ml178.r4">4</a>,<a class="bibr" href="#ml178.r10" rid="ml178.r10">10</a>], coronary artery caliber, endothelial integrity, lung epithelial integrity [<a class="bibr" href="#ml178.r1" rid="ml178.r1">1</a>,<a class="bibr" href="#ml178.r2" rid="ml178.r2">2</a>] and lymphocyte recirculation [<a class="bibr" href="#ml178.r3" rid="ml178.r3 ml178.r4 ml178.r5 ml178.r6 ml178.r7">3–7</a>] through five related high affinity G-protein coupled receptors [<a class="bibr" href="#ml178.r4" rid="ml178.r4">4</a>]. S1P4 receptor, the subject of this report, is coupled to Gαi and Gαo G proteins and activates ERK MAPK and PLC downstream pathways [<a class="bibr" href="#ml178.r11" rid="ml178.r11">11</a>]. Inhibition of lymphocyte recirculation by nonselective S1P receptor agonists produces clinical immunosuppression preventing transplant rejection, but is associated with transient bradycardia. In addition, it has been suggested that S1P4 expression and function may play a role in stimulating the ERK 1/2 pathway in a breast cancer cell line [<a class="bibr" href="#ml178.r12" rid="ml178.r12">12</a>]. Understanding the contribution of individual receptors has been limited by the unavailability of selective agonists or antagonists for the 5 receptor subtypes.</p><p>We have recently published a proof-of-concept paper showing that modulation of S1P receptors locally in the lungs during influenza infection altered dendritic cell (DC) activation and accumulation in the mediastinal lymph nodes, resulting in blunted T-cell response and allowing control of immunopathological features of the infection [<a class="bibr" href="#ml178.r8" rid="ml178.r8">8</a>]. Upon influenza virus infection, pulmonary DC become activated and migrate towards mediastinal lymph nodes to initiate the T-cell response. Influenza virus-specific T cells are rapidly induced and proliferate in mediastinal lymph node, then migrate to the infected sites, including the lungs, where they cause damage. While airway delivery of the broad S1P receptor agonist AFD-R, with activity on S1P1, S1P3, S1P4 and S1P5 receptors, but not S1P2 receptor, efficiently inhibited DC activation after influenza infection, specific chemical activation of the S1P1 receptor did not show any inhibitory effect. Moreover, we recently observed that the inhibitory effect of AFD-R was not diminished in S1P3-null mice. This series of experiments therefore rules out the involvement of S1P1, S1P2 and S1P3 receptors in the inhibition of the DC response to influenza virus infection. Based on the fact that S1P5 expression is very low in DC while S1P4 receptor was shown to be highly expressed [<a class="bibr" href="#ml178.r9" rid="ml178.r9">9</a>], we have hypothesized that chemical activation of S1P4 receptor in the airways could be effective at controlling the immunopathological response to viral infections. S1P4 receptor knockout mice exhibit limited phenotypes, but demonstrate that the receptor is important in megakaryocyte differentiation and platelet formation [<a class="bibr" href="#ml178.r13" rid="ml178.r13">13</a>]. Pandemic influenza represents a significant public health threat, with much of the morbidity and mortality associated with outbreaks such as H5N1 or the 1918 flu reflecting excessive immune damage to the lungs. Modulating this pathology could represent a promising novel therapeutic approach for influenza.</p><p>Several reports of compounds with S1P4 receptor agonist activity have been published (see reference 14 for a review). A patent report from Azzaoui et al (Novartis) claims a selective compound (compound <b>1</b> in <a class="figpopup" href="/books/NBK98915/table/ml178.t1/?report=objectonly" target="object" rid-figpopup="figml178t1" rid-ob="figobml178t1">Table 1</a>) [<a class="bibr" href="#ml178.r15" rid="ml178.r15">15</a>]. Oddly, these compounds were later reported by Novartis as partial agonists of the N-methyl-D-aspartate (NMDA) receptor complex with no mention of S1P4 receptor activity [<a class="bibr" href="#ml178.r16" rid="ml178.r16">16</a>]. Clemens et al report the synthesis of enantiomeric agonist compounds (compounds <b>2a</b> and <b>2b</b> in <a class="figpopup" href="/books/NBK98915/table/ml178.t1/?report=objectonly" target="object" rid-figpopup="figml178t1" rid-ob="figobml178t1">Table 1</a>) that incorporate a benzimidazole ring system that also have low nanomolar partial agonist activity on S1P5 receptor and is thus not selective for S1P4 [<a class="bibr" href="#ml178.r17" rid="ml178.r17">17</a>]. The constrained azacyclic FTY720 analogue reported by Hanessian (compound <b>3</b> in <a class="figpopup" href="/books/NBK98915/table/ml178.t1/?report=objectonly" target="object" rid-figpopup="figml178t1" rid-ob="figobml178t1">Table 1</a>) is a mixed S1P4 and S1P5 receptor agonist with single digit potencies in a calcium flux assay and thus is also not selective for S1P4 [<a class="bibr" href="#ml178.r18" rid="ml178.r18">18</a>]. Notably, all three compounds in the table below have zwitterionic headgroup mimetics of S1P and are unlike to provide selectivity and specificity required for elucidating specific S1P4 receptor-mediated biological function. A Japanese patent has been filed for a benzothiophene derivative as an S1P4 regulator [<a class="bibr" href="#ml178.r19" rid="ml178.r19">19</a>], but no further information is available about this compound. In summary, previously reported S1P4 receptor agonist compounds are unavailable, poorly characterized and/or not selective.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml178t1"><a href="/books/NBK98915/table/ml178.t1/?report=objectonly" target="object" title="Table 1" class="img_link icnblk_img figpopup" rid-figpopup="figml178t1" rid-ob="figobml178t1"><img class="small-thumb" src="/books/NBK98915/table/ml178.t1/?report=thumb" src-large="/books/NBK98915/table/ml178.t1/?report=previmg" alt="Table 1. Published S1P4 Receptor Agonists." /></a><div class="icnblk_cntnt"><h4 id="ml178.t1"><a href="/books/NBK98915/table/ml178.t1/?report=objectonly" target="object" rid-ob="figobml178t1">Table 1</a></h4><p class="float-caption no_bottom_margin">Published S1P4 Receptor Agonists. </p></div></div><p>In this report, we describe novel compounds with potent and selective S1P4 receptor agonist activity. This compound family does not have the charged head group moiety that characterizes compound developed in lead optimization approaches based upon S1P.</p></div><div id="ml178.s4"><h2 id="_ml178_s4_">2. Materials and Methods</h2><p>The following reagents were obtained from Invitrogen: Tango™ EDG6-bla U2OS cells (K1622), Tango™ EDG-1-BLA U2OS cells (part K1520), Tango™ EDG8-bla U2OS cells (K1518), GeneBLAzer FRET B/G Loading Kit (CCF4-AM) (part K1025), LiveBLAzer (K1096), Freestyle Expression Medium (12338-018), McCoy’s 5A Medium (modified) (1X) (16600-082), Dulbecco’s Modified Eagle’s Media with phenol red (11965-092), Dulbecco’s Modified Eagle’s Media without phenol red (21063-029), Fetal Bovine Serum, dialyzed (26400-036), NEAA (1114-050), Penicillin-Streptomycin-Neomycin antibiotic mix (15140-122), 100X Penicillin-Streptomycin-Neomycin mix (15640-055), Sodium Pyruvate (11360-070), PBS without calcium or magnesium (14190-136), HEPES (15630-080), Trypsin/EDTA (25300-054), Zeocin (R250-01), Hygromycin (10687-010), Geneticin (10131-027), L-Glutamine (25030-081).</p><p>Probenecid was obtained from Sigma (P8761). S1P was obtained from Avanti Polar Lipids (860492P). Fatty Acid Free BSA was obtained from Calbiochem (NC9734015). 1536-well plates and 384-well plates were obtained from Greiner (789072 and 788092, respectively). T175 tissue culture flasks were obtained from Corning (431080). Charcoal/dextran treated fetal bovine serum (SH30068.03) and Bovine Growth Serum (SH30541.03) were obtained from Hyclone. U-2OS cells were obtained from ATCC (HTB-96). Cell Titer-Glo was obtained from Promega (G7572). Reagents for the Ricerca HitProfilingScreen + CYP450 were provided by Ricerca Biosciences, LLC.</p><div id="ml178.s5"><h3>2.1. Assays</h3><div id="ml178.s6"><h4>LC-MS/MS</h4><p>All analytical methods were in MRM mode where the parent ion was selected in Q1 of the mass spectrometer. The parent ion was fragmented and a characteristic fragment ion monitored in Q3. MRM mass spectroscopy methods are particularly sensitive because additional time is spent monitoring the desired ions and not sweeping a large mass range. Methods were rapidly set up using Automaton<sup>®</sup> (Applied Biosystems), where the compounds were listed with their name and mass in an Excel datasheet. Compounds were submitted in a 96-well plate to the HPLC autosampler and slowly injected without a column present. A narrow range centered on the indicated mass was scanned to detect the parent ion. The software then evaluated a few pre-selected parameters to determine conditions that maximized the signal for the parent ion. The molecule was then fragmented in the collision cell of the mass spectrometer and fragments with m/z larger than 70 but smaller than the parent mass were determined. Three separate collision energies were evaluated to fragment the parent ion and the largest three ions were selected. Each of these three fragment ions was further optimized and the best fragment was chosen. The software then inserted the optimized masses and parameters into a template method and saved it with a unique name that indicated the individual compound being optimized. Spectra for the parent ion and the fragmentation pattern were saved and reviewable later.</p></div><div id="ml178.s7"><h4>Solubility</h4><p>The solubility of compounds was tested in phosphate buffered saline, pH 7.4. Compounds were inverted for 24 hours in test tubes containing 1–2 mg of compound with 1 mL of PBS. The samples were centrifuged and analyzed by HPLC (Agilent 1100 with diode-array detector). Peak area was compared to a standard of known concentration. In cases when the concentration was too low for UV analysis or when the compound did not possess a good chromophore, LC-MS/MS analysis was used.</p></div><div id="ml178.s8"><h4>Stability</h4><p>Demonstration of stability in PBS was conducted under conditions likely to be experienced in a laboratory setting. The compound was dissolved in 1 mL of PBS at a concentration of 10 μM, unless its maximum solubility was insufficient to achieve this concentration. Low solubility compounds were tested between ten and fifty percent of their solubility limit. The solution was immediately aliquoted into seven standard polypropylene microcentrifuge tubes which were stored at ambient temperature in a block microcentrifuge tube holder. Individual tubes were frozen at −80°C at 0, 1, 2, 4, 8, 24, and 48 hours. The frozen samples were thawed in a room temperature and an equal volume of acetonitrile was added prior to determination of concentration by LC-MS/MS.</p></div><div id="ml178.s9"><h4>Determination of glutathione reactivity</h4><p>One μL of a 10 mM compound stock solution was added to 1 mL of a freshly prepared solution of 100 μM reduced glutathione. Final compound concentration was 10 μM unless solubility limited. The solution was allowed to incubate at 37°C for two hours prior to being directly analyzed for glutathione adduct formation. LC-MS/MS analysis of GSH adducts was performed on an API 4000 Q-TrapTM mass spectrometer equipped with a Turboionspray source (Applied Biosystems, Foster City, CA). Two methodologies were utilized—a negative precursor ion (PI) scan of m/z 272, corresponding to GSH fragmenting at the thioether bond, and a neutral loss scan of −129 AMU to detect GSH adducts. This triggered positive ion enhanced resolution and enhanced product ion scans [<a class="bibr" href="#ml178.r20" rid="ml178.r20">20</a>,<a class="bibr" href="#ml178.r21" rid="ml178.r21">21</a>].</p></div><div id="ml178.s10"><h4>Primary uHTS assay to identify S1P4 receptor agonists (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1509" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1509</a>)</h4><p><b>Assay Overview:</b> The purpose of this assay was to identify compounds that act as agonists of the S1P4 receptor. This assay uses Tango™ S1P4-BLA U2OS cells which contain the human Endothelial Differentiation Gene 6 (EDG6; S1P4) linked to a GAL4-VP16 transcription factor via a TEV protease site. The cells also express a beta-arrestin/TEV protease fusion protein and a beta-lactamase (BLA) reporter gene under the control of a UAS response element. Stimulation of the S1P4 receptor by agonist causes migration of the fusion protein to the GPCR, and through proteolysis liberates GAL4-VP16 from the receptor. The liberated VP16-GAL4 migrates to the nucleus, where it induces transcription of the BLA gene. BLA expression is monitored by measuring fluorescence resonance energy transfer (FRET) of a cleavable, fluorogenic, cell-permeable BLA substrate. As designed, test compounds that act as S1P4 receptor agonists will activate S1P4 receptor and increase well FRET. Compounds were tested in singlicate at a final nominal concentration of 5 micromolar.</p><p><b>Protocol Summary:</b> U2OS cells were cultured in T-175 sq cm flasks at 37 degrees C and 95% relative humidity (RH). The growth media consisted of McCoy’s 5A Medium supplemented with 10% v/v dialyzed fetal bovine serum, 0.1 mM NEAA, 25 mM HEPES (pH 7.3), 1 mM sodium pyruvate, 100 U/mL penicillin-streptomycin-neomycin, 200 μg/mL zeocin, 50 μg/mL hygromycin, 100 μg/mL geneticin. Prior to the start of the assay, cells were suspended at a concentration of 250,000/mL in Assay Medium (Freestyle Expression Medium without supplements). The assay was started by dispensing 4 μL of cell suspension to each well, followed by overnight incubation at 37 degrees C in 5% CO<sub>2</sub> and 95% RH. The next day, 25 nL of test compound (5 μM final nominal concentration) in DMSO was added to sample wells, and DMSO alone (0.5 % final concentration) was added to low control wells. S1P prepared in 2% fatty-acid free BSA was added to the high control wells to a final concentration of 5 μM. Plates were then incubated at 37 degrees C in 5% CO<sub>2</sub> for 4 hrs. After the incubation, 1 μL of the LiveBLAzer FRET substrate mixture, prepared according to the manufacturer’s protocol and containing 10 mM Probenicid, was added to all wells. After 2 hours of incubation at room temperature in the dark, plates were read on the EnVision plate reader (PerkinElmer Lifesciences, Turku, Finland) at an excitation wavelength of 405 nm and emission wavelengths of 460 nm and 535 nm. <b>Assay Cutoff:</b> compounds that exhibited greater than 10.98% agonism for S1P4 receptor were considered active.</p></div><div id="ml178.s11"><h4>Confirmation uHTS assay to identify S1P4 receptor agonists (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1523" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1523</a>)</h4><p><b>Assay Overview:</b> The purpose of this assay was to confirm activity of compounds identified as active in the uHTS primary screen (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1509" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1509</a>). This assay was run as described above (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1509" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1509</a>). Compounds were tested in triplicate at a nominal concentration of 5 micromolar.</p><p><b>Protocol Summary:</b> The assay was performed as described above (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1509" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1509</a>). <b>Assay Cutoff:</b> compounds that exhibited greater than 10.98% agonism for S1P4 receptor were considered active.</p></div><div id="ml178.s12"><h4>Dose response uHTS assay to identify S1P4 receptor agonists (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1686" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1686</a>)</h4><p><b>Assay Overview:</b> The purpose of this assay was to determine dose response for compounds identified as active in the uHTS primary screen (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1509" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1509</a>) and that confirmed activity in the uHTS confirmation screen (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1523" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1523</a>). This assay was run as described above (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1509" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1509</a>). Compounds were tested in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 50 micromolar.</p><p><b>Protocol Summary:</b> The assay was performed as described above (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1509" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1509</a>). <b>Assay Cutoff:</b> compounds with an EC<sub>50</sub> equal to or less than 10 micromolar were considered active.</p></div><div id="ml178.s13"><h4>Counterscreen uHTS assay to identify S1P1 receptor agonists (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1563" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1563</a>)</h4><p><b>Assay Overview:</b> The purpose of this assay was to determine whether compounds identified as active in the primary uHTS assay (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1509" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1509</a>), and that confirmed activity in the uHTS confirmation screen (PubChem <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1523" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1523</a>), were nonselective agonists due to activation of the S1P1 receptor. This assay uses Tango™ S1P1-bla U2OS cells which express S1P1 (EDG1) linked to a GAL4-VP16 transcription factor via a TEV protease site. The cells also express a beta-arrestin/TEV protease fusion protein and a beta-lactamase (BLA) reporter gene under the control of a UAS response element. Stimulation of the S1P1 receptor by agonist causes migration of the fusion protein to the GPCR, and through proteolysis liberates GAL4-VP16 from the receptor. The liberated VP16-GAL4 migrates to the nucleus, where it induces transcription of the BLA gene. BLA expression is monitored by measuring fluorescence resonance energy transfer (FRET) of a cleavable, fluorogenic, cell-permeable BLA substrate. As designed, test compounds that act as S1P1 receptor agonists will activate S1P1 receptor and increase well FRET. Compounds were tested in triplicate at a final nominal concentration of 5 micromolar.</p><p><b>Protocol Summary:</b> Cells were cultured as described above (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1509" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1509</a>). Prior to the start of the assay, cells were suspended at a concentration of 625,000/mL in Assay Medium (Freestyle Expression Medium without supplements). The assay was started by dispensing 4 μL of cell suspension to each well, followed by overnight incubation at 37 degrees C in 5% CO<sub>2</sub> and 95% RH. The next day, 25 nL of test compound in DMSO (0.5 % final DMSO concentration), DMSO alone (low control), or S1P (high control; 5 μM final concentration) prepared in 2% fatty-acid free BSA, was added to the appropriate wells. Plates were then incubated at 37 degrees 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> compounds that exhibited greater than 20.14% agonism for S1P1 receptor were considered active.</p></div><div id="ml178.s14"><h4>Counterscreen dose response uHTS assay to identify S1P1 receptor agonists (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1701" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1701</a>)</h4><p><b>Assay Overview:</b> The purpose of this assay was to determine S1P1 agonist dose response for compounds identified as active in the uHTS S1P1 receptor counterscreen assay (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1563" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1563</a>). The assay was performed as described above (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1563" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1563</a>), except that compounds were tested in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 50 micromolar.</p><p><b>Protocol Summary:</b> The assay was performed as described above (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1563" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1563</a>). <b>Assay Cutoff:</b> compounds with an EC<sub>50</sub> equal to or less than 10 micromolar were considered active.</p></div><div id="ml178.s15"><h4>Dose response assay to identify S1P4 receptor agonists with purchased compounds (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504460" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504460</a>)</h4><p><b>Assay Overview:</b> The purpose of this assay was to determine S1P4 receptor agonist dose response with powder samples of purchased compounds. This assay was run as described above (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1509" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1509</a>). Compounds were tested in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 50 micromolar.</p><p><b>Protocol Summary:</b> Cells were cultured as described above (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1509" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1509</a>). Prior to the start of the assay, cells were suspended at a concentration of 1,000,000/mL in Assay Medium (Freestyle Expression Medium without supplements). The assay was started by dispensing 10 μL of cell suspension to each well, followed by overnight incubation at 37 degrees C in 5% CO<sub>2</sub> and 95% RH. The next day, 50 nL of test compound (50 μM final nominal concentration) in DMSO was added to sample wells, and DMSO alone (0.5 % final concentration) was added to low control wells. S1P prepared in 2% fatty-acid free BSA was also added to the high control wells to a final concentration of 6 μM. Plates were then incubated at 37 degrees C in 5% CO<sub>2</sub> for 4 hrs. 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 an EC<sub>50</sub> equal to or less than 10 micromolar were considered active.</p></div><div id="ml178.s16"><h4>Dose response assay to identify S1P4 receptor agonists with synthesized compounds (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463107" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 463107</a>)</h4><p><b>Assay Overview:</b> The purpose of this assay was to determine S1P4 receptor agonist dose response with synthesized compounds. This assay was run as described above (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1509" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1509</a>). Compounds were tested in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 50 micromolar.</p><p><b>Protocol Summary:</b> Cells were cultured as described above (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1509" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1509</a>). Prior to the start of the assay, cells were suspended at a concentration of 250,000/mL in Assay Medium (Freestyle Expression Medium without supplements). The assay was started by dispensing 4 μL of cell suspension to each well, followed by overnight incubation at 37 degrees C in 5% CO<sub>2</sub> and 95% RH. The next day, 25 nL of test compound in DMSO (0.5 % final DMSO concentration), DMSO alone (low control), or S1P (high control; 10 nM final nominal EC<sub>80</sub> concentration) prepared in 2% BSA was added to the appropriate wells. Plates were then incubated at 37 degrees C in 5% CO<sub>2</sub> for 4 hours. After the incubation, 1 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 an EC<sub>50</sub> equal to or less than 10 micromolar were considered active.</p></div><div id="ml178.s17"><h4>Counterscreen dose response assay to identify S1P1 receptor agonists (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463118" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 463118</a>)</h4><p><b>Assay Overview:</b> The purpose of this assay was to determine whether synthesized compounds identified as active in the previous assay (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463107" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 463107</a>) were nonselective agonists as assayed by activation of the S1P1 receptor. The assay was performed as described above (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1563" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1563</a>), except that compounds were tested in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 50 micromolar.</p><p><b>Protocol Summary:</b> Cells were cultured as described above (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1509" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1509</a>). Prior to the start of the assay, cells were suspended at a concentration of 275,000/mL in Assay Medium (Freestyle Expression Medium without supplements). The assay was started by dispensing 10 μL of cell suspension to each well of a 384-well plate (10,000 cells/well), followed by overnight incubation at 37 degrees C in 5% CO<sub>2</sub> and 95% RH. The next day, 50 nL of test compound in DMSO (0.5% final DMSO concentration), DMSO alone (low control), or S1P (high control; 40 nM final nominal EC<sub>80</sub> concentration) prepared in 2% BSA was added to the appropriate wells. Plates were then incubated at 37 degrees C in 5% CO<sub>2</sub> for 4 hours. After the incubation, 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 an EC<sub>50</sub> equal to or less than 10 micromolar were considered active.</p></div><div id="ml178.s18"><h4>Counterscreen dose response assay to identify S1P2 receptor agonists (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463122" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 463122</a>)</h4><p><b>Assay Overview:</b> The purpose of this assay was to determine whether synthesized compounds identified as active in a previous assay (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463107" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 463107</a>) were nonselective agonists as assayed by activation of the S1P2 receptor. A Chinese Hamster Ovary (CHO) cell line stably transfected with the human S1P2 receptor and a cAMP Response Element-beta lactamase (CRE-BLA) reporter construct was used to measure S1P2 agonism. Under normal conditions, S1P2 has low basal activity and therefore cells express low BLA levels. Stimulation of the S1P2 receptor by agonist increases BLA gene transcription. This increase is monitored by measuring fluorescence resonance energy transfer (FRET) of a cleavable fluorogenic cell-permeable BLA substrate. As designed, test compounds that act as S1P2 receptor agonists will activate S1P2 receptor and increase well FRET. Compounds were tested in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 50 micromolar.</p><p><b>Protocol Summary:</b> Cells were cultured in T-175 sq cm flasks at 37 degrees C and 95% relative humidity (RH). The growth media consisted of Dulbecco’s Modified Eagle’s Media (DMEM) supplemented with 10% v/v heat inactivated dialyzed fetal bovine serum, 0.1 mM NEAA, 1 mM sodium pyruvate, 25 mM HEPES, 5 mM L-glutamine, 2 mg/mL Geneticin and 1X antibiotic mix (mix of penicillin, streptomycin and neomycin). Prior to assay, cells were suspended to a concentration of 1,250,000/mL in assay media, which consisted of phenol red-free Dulbecco’s Modified Eagle’s Media supplemented with 2% charcoal/dextran-treated fetal bovine serum, 0.1 mM NEAA, 1 mM sodium pyruvate, 25 mM HEPES, 5 mM L-glutamine and 1X antibiotic mix (mix of penicillin, streptomycin and neomycin). The assay was initiated by dispensing 10 μL of cell suspension to each test well of a 384-well plate (6,000 cells/well) followed by incubation at 37 degrees C in 5% CO<sub>2</sub> for 16 hrs. To the appropriate wells were then added 50 nL of test compound in DMSO (final nominal concentration of 50 μM, final DMSO concentration of 0.5%) or DMSO only (low control). Next, 1 μL of S1P in 2% BSA (final concentration of 370 nM, i.e. a concentration that resulted in 80% activity) was added to the high control wells and plates were incubated again at 37 degrees C in 5% CO<sub>2</sub> for 2 hrs. The fluorogenic LiveBLAzer substrate mixture with 10 mM Probenicid was prepared according to the manufacturer’s protocol and 2.2 microliter of this mixture was then added to each well. After a further 2 hours of incubation at room temperature, plates were read on the EnVision plate reader (PerkinElmer Lifesciences, Turku, Finland) at an excitation wavelength of 405 nm and fluorescence emission wavelengths of 535 nm & 460 nm. <b>Assay Cutoff:</b> compounds with an EC<sub>50</sub> equal to or less than 10 micromolar were considered active.</p></div><div id="ml178.s19"><h4>Counterscreen dose response assay to identify S1P3 receptor agonists (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463123" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 463123</a>)</h4><p><b>Assay Overview:</b> The purpose of this assay was to determine whether synthesized compounds identified as active in a previous assay (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463107" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 463107</a>) were nonselective agonists as assayed by activation of the S1P3 receptor. In this assay, a CHO cell line containing human S1P3 receptor and the beta-lactamase (BLA) reporter gene under control of the nuclear factor of activated T-cells (NFAT) promoter was used to measure S1P3 receptor agonism by test compound. Stimulation of S1P3 receptor by S1P induces transcription of NFAT-BLA via a G-alpha16 protein coupled signaling cascade, and an increase in BLA activity. BLA activity is measured using a fluorescent BLA substrate. As designed, a compound that acts as a S1P3 receptor agonist will increase NFAT-BLA transcription and increase well fluorescence. Compounds were tested in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 50 micromolar.</p><p><b>Protocol Summary:</b> Cells were cultured in T-175 sq cm flasks at 37 degrees C and 95% RH. The growth media consisted of DMEM containing 10% v/v heat inactivated bovine growth serum, 0.1 mM NEAA, 1 mM sodium pyruvate, 25 mM HEPES, 5 mM L-glutamine, 2 mg/mL geneticin, 0.2 mg/mL hygromycin B, and 1x penicillin-streptomycin-neomycin. Prior to the start of the assay, cells were suspended at a concentration of 1,250,000/mL in phenol red-free DMEM supplemented as above, except with 0.5% charcoal/dextran-treated fetal bovine serum and no antibiotics. The assay was started by dispensing 10 μL of cell suspension to each well of a 384-well plate (8,000 cells/well), followed by overnight incubation at 37 degrees C in 5% CO<sub>2</sub> and 95% RH. The next day, 50 nL of test compound (50 μM final nominal concentration) in DMSO was added to sample wells, and DMSO alone (0.5 % final concentration) was added to low control wells. Next, S1P prepared in 2% BSA (0.7 μM final nominal concentration, corresponding to the EC<sub>80</sub> of S1P) was added to the high control wells. After 4 hours of incubation, 2.2 μL/well of the GeneBLAzer fluorescent substrate mixture, prepared according to the manufacturer’s protocol and containing 10 mM Probenicid, was added to all wells. The plates were then incubated for 2 hours at room temperature. Plates were read on the EnVision plate reader (PerkinElmer Lifesciences, Turku, Finland) at an excitation wavelength of 405 nm and emission wavelengths of 535 nm and 460 nm. <b>Assay Cutoff:</b> compounds with an EC<sub>50</sub> equal to or less than 10 micromolar were considered active.</p></div><div id="ml178.s20"><h4>Counterscreen dose response assay to identify S1P5 receptor agonists (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463129" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 463129</a>)</h4><p><b>Assay Overview:</b> The purpose of this assay was to determine whether synthesized compounds identified as active in a previous assay (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463107" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 463107</a>) were nonselective agonists as assayed by activation of the S1P5 receptor. This assay uses Tango™ S1P5-BLA U2OS cells which contain the human Endothelial Differentiation Gene 8 (EDG8; S1P5) linked to a GAL4-VP16 transcription factor via a TEV protease site. The cells also express a beta-arrestin/TEV protease fusion protein and a beta-lactamase (BLA) reporter gene under the control of a UAS response element. Stimulation of the S1P5 receptor by agonist causes migration of the fusion protein to the GPCR, and through proteolysis liberates GAL4-VP16 from the receptor. The liberated VP16-GAL4 migrates to the nucleus, where it induces transcription of the BLA gene. BLA expression is monitored by measuring fluorescence resonance energy transfer (FRET) of a cleavable, fluorogenic, cell-permeable BLA substrate. As designed, test compounds that act as S1P5 receptor agonists will stimulate migration of the fusion protein, thus increasing proteolysis of GAL4-VP16 and BLA transcription, leading to an increase in well FRET. Compounds were tested in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 50 micromolar.</p><p><b>Protocol Summary:</b> Cells were cultured as described above (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1509" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1509</a>). Prior to the start of the assay, cells were suspended at a concentration of 275,000/mL in Assay Medium (Freestyle Expression Medium without supplements). The assay was started by dispensing 10 μL of cell suspension to each well of a 384-well plate (10,000 cells/well), followed by overnight incubation at 37 degrees C in 5% CO<sub>2</sub> and 95% RH. The next day, 50 nL of test compound in DMSO (0.5% final DMSO concentration), DMSO alone (low control), or S1P (high control; 1.5 nM final nominal EC<sub>80</sub> concentration) prepared in 2% BSA was added to the appropriate wells. Plates were then incubated at 37 degrees C in 5% CO<sub>2</sub> for 4 hours. After the incubation, 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 an EC<sub>50</sub> equal to or less than 10 micromolar were considered active.</p></div><div id="ml178.s21"><h4>Analysis of cytotoxicity (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463119" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 463119</a>)</h4><p><b>Assay Overview:</b> The purpose of this assay was to determine cytotoxicity of a synthesized compound identified as active in a previous assay (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463107" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 463107</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 μg/mL (20 μL; 4,000 cells/well) into the wells of a 384-well plate. Eight 1:3 serial dilutions of compound (100 μM in growth media) were made. 5 μL of diluted compound or media were added to wells, giving final compound concentrations of 0–20 μM. The plate was incubated at 37 degrees 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 CC<sub>50</sub> equal to or less than 10 micromolar were considered active (cytotoxic).</p></div><div id="ml178.s22"><h4>Inhibition by S1P4-selective antagonist (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463225" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 463225</a>)</h4><p><b>Assay Overview:</b> The purpose of this assay is to determine whether a test compound with S1P4 agonist activity is inhibited by an S1P4-selective antagonist. This assay uses Tango™ S1P4-BLA U2OS cells which contain the human Endothelial Differentiation Gene 6 (EDG6; S1P4) linked to a GAL4-VP16 transcription factor via a TEV protease site. The cells also express a beta-arrestin/TEV protease fusion protein and a beta-lactamase (BLA) reporter gene under the control of a UAS response element. Stimulation of the S1P4 receptor by agonist causes migration of the fusion protein to the GPCR, and through proteolysis liberates GAL4-VP16 from the receptor. The liberated VP16-GAL4 migrates to the nucleus, where it induces transcription of the BLA gene. BLA expression is monitored by measuring fluorescence resonance energy transfer (FRET) of a cleavable, fluorogenic, cell-permeable BLA substrate. The presence of an S1P4-selective antagonist compound in the assay inhibits S1P4 activation and migration of the fusion protein, thus preventing proteolysis of GAL4-VP16 and BLA transcription, leading to no increase in well FRET. In the presence of a test agonist compound, the S1P4 receptor will be stimulated, leading to an increase in well FRET. Compound was tested in triplicate using a 6-point, 1:3 dilution series starting at a nominal 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/1509" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1509</a>). Prior to the start of the assay, cells were suspended at a concentration of 1,000,000/mL in Assay Medium (Freestyle Expression Medium without supplements). The assay was started by dispensing 10 μL of cell suspension to each well, followed by overnight incubation at 37 degrees C in 5% CO<sub>2</sub> and 95% RH. The next day, 50 nL of test compound in DMSO was added to sample wells, and DMSO alone (0.5% final concentration) was added to control wells. Next, 370 nM S1P4-selective antagonist SR2-304 was added to the appropriate wells. Plates were then incubated at 37 degrees C in 5% CO<sub>2</sub> for 4 hours. After the incubation, 2.2 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 an IC<sub>50</sub> equal to or less than 10 micromolar were considered active.</p></div><div id="ml178.s23"><h4>Counterscreen panel assay for S1P4 agonists: Ricerca HitProfilingScreen + CYP450 (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504400" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504400</a>)</h4><p><b>Assay Overview:</b> The purpose of this panel of binding assays performed by Ricerca Biosciences, LLC, is to identify a subset of potential receptors, transporters, ion channels, etc. for which the S1P4 agonist compound CID 44620892 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="ml178.s24"><h3>2.2. Probe Chemical Characterization</h3><div id="ml178.fu2" class="figure bk_fig"><div class="graphic"><img src="/books/NBK98915/bin/ml178fu5.jpg" alt="CID 44620892 SID 87544119 ML178." /></div><h3><span class="title">CID 44620892<br /><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544119" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 87544119</a><br /><a href="/pcsubstance/?term=ML178[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML178</a></span></h3></div><p>The probe structure was verified by <sup>1</sup>H-NMR and MS. Compound purity was assessed to be greater than 96% by <sup>1</sup>H-NMR (<a class="figpopup" href="/books/NBK98915/figure/ml178.f1/?report=objectonly" target="object" rid-figpopup="figml178f1" rid-ob="figobml178f1">Figure 1</a>) and LC-MS (<a class="figpopup" href="/books/NBK98915/figure/ml178.f2/?report=objectonly" target="object" rid-figpopup="figml178f2" rid-ob="figobml178f2">Figure 2</a>).</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml178f1" co-legend-rid="figlgndml178f1"><a href="/books/NBK98915/figure/ml178.f1/?report=objectonly" target="object" title="Figure 1" class="img_link icnblk_img figpopup" rid-figpopup="figml178f1" rid-ob="figobml178f1"><img class="small-thumb" src="/books/NBK98915/bin/ml178f1.gif" src-large="/books/NBK98915/bin/ml178f1.jpg" alt="Figure 1. 1H NMR spectrum of probe ML178." /></a><div class="icnblk_cntnt" id="figlgndml178f1"><h4 id="ml178.f1"><a href="/books/NBK98915/figure/ml178.f1/?report=objectonly" target="object" rid-ob="figobml178f1">Figure 1</a></h4><p class="float-caption no_bottom_margin"><sup>1</sup>H NMR spectrum of probe ML178. </p></div></div><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml178f2" co-legend-rid="figlgndml178f2"><a href="/books/NBK98915/figure/ml178.f2/?report=objectonly" target="object" title="Figure 2" class="img_link icnblk_img figpopup" rid-figpopup="figml178f2" rid-ob="figobml178f2"><img class="small-thumb" src="/books/NBK98915/bin/ml178f2.gif" src-large="/books/NBK98915/bin/ml178f2.jpg" alt="Figure 2. LC-MS results for probe ML178." /></a><div class="icnblk_cntnt" id="figlgndml178f2"><h4 id="ml178.f2"><a href="/books/NBK98915/figure/ml178.f2/?report=objectonly" target="object" rid-ob="figobml178f2">Figure 2</a></h4><p class="float-caption no_bottom_margin">LC-MS results for probe ML178. </p></div></div><p>MS (EI) <i>m/z</i> 439, 441, 443 (M+). <sup>1</sup>H NMR (400 MHz, CDCl<sub>3</sub>, TMS as internal standard): <i>δ</i> 7.37 (d, <i>J</i> = 8.4 Hz, 1H), 7.35 (d, <i>J</i> = 2.4 Hz, 1H), 7.25 (d, <i>J</i> = 1.8 Hz, 1H), 7.27 (d, <i>J</i> = 8.4 Hz, 1H), 7.18 (dd, <i>J<sub>1</sub></i>=2.4 Hz, <i>J<sub>2</sub></i>=8.7 Hz, 1H), 6.95 (d, <i>J</i> = 8.7 Hz, 1H), 4.39–4.46 (m, 4H).</p><p>Solubility in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM sodium phosphate dibasic, 2 mM potassium phosphate monobasic, pH 7.4) at room temperature (23 degrees C) was determined to be at least 10 μM as determined by dilution of 10 mM stock solution in DMSO into PBS. The probe has a half-life of 21 hours in PBS at room temperature (18% compound remaining at 48 hours) (<a class="figpopup" href="/books/NBK98915/figure/ml178.f3/?report=objectonly" target="object" rid-figpopup="figml178f3" rid-ob="figobml178f3">Figure 3</a>).</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml178f3" co-legend-rid="figlgndml178f3"><a href="/books/NBK98915/figure/ml178.f3/?report=objectonly" target="object" title="Figure 3" class="img_link icnblk_img figpopup" rid-figpopup="figml178f3" rid-ob="figobml178f3"><img class="small-thumb" src="/books/NBK98915/bin/ml178f3.gif" src-large="/books/NBK98915/bin/ml178f3.jpg" alt="Figure 3. Stability of Probe ML178 in PBS." /></a><div class="icnblk_cntnt" id="figlgndml178f3"><h4 id="ml178.f3"><a href="/books/NBK98915/figure/ml178.f3/?report=objectonly" target="object" rid-ob="figobml178f3">Figure 3</a></h4><p class="float-caption no_bottom_margin">Stability of Probe ML178 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 have been submitted to the SMR collection. Compound numbers refer to the
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<b><a class="figpopup" href="/books/NBK98915/table/ml178.t4/?report=objectonly" target="object" rid-figpopup="figml178t4" rid-ob="figobml178t4">SAR Table</a></b>.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml178tu2"><a href="/books/NBK98915/table/ml178.tu2/?report=objectonly" target="object" title="Table" class="img_link icnblk_img figpopup" rid-figpopup="figml178tu2" rid-ob="figobml178tu2"><img class="small-thumb" src="/books/NBK98915/table/ml178.tu2/?report=thumb" src-large="/books/NBK98915/table/ml178.tu2/?report=previmg" alt="Image " /></a><div class="icnblk_cntnt"><h4 id="ml178.tu2"><a href="/books/NBK98915/table/ml178.tu2/?report=objectonly" target="object" rid-ob="figobml178tu2">Table</a></h4></div></div></div><div id="ml178.s25"><h3>2.3. Probe Preparation</h3><div id="ml178.f4" class="figure bk_fig"><div class="graphic"><img src="/books/NBK98915/bin/ml178f4.jpg" alt="Figure 4. Synthesis scheme for ML178." /></div><h3><span class="label">Figure 4</span><span class="title">Synthesis scheme for <a href="/pcsubstance/?term=ML178[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML178</a></span></h3><div class="caption"><p>Reagents and conditions: i.- <b>1</b> (1 equiv), 1,2-dibromoethane (3 equiv), K<sub>2</sub>CO<sub>3</sub> (3 equiv), DMSO, 56 °C, overnight, 75%. ii.- <b>2</b> (1.0 equiv), <b>3</b> (1.5 equiv), K<sub>2</sub>CO<sub>3</sub> (1.5 equiv), DMSO, 70 °C, overnight, 86%.</p></div></div><p>To a solution of 1,2-dibromoethane (0.92 mmol, 79 μL) in DMSO (3 mL) was added at room temperature in one portion K<sub>2</sub>CO<sub>3</sub> (0.92 mmol, 43 mg) followed by portion-wise addition of <b>1</b> (0.306 mmol, 50 mg), the mixture was stirred overnight at 70 degrees C. The mixture was diluted with ethyl acetate (15 mL) and washed with 5% NaHCO<sub>3</sub> solution (10 mL × 3), water (10 mL) and brine (10 mL). The organic phase was dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography (20% EtOAc/Hexane) to afford <b>2</b> (62 mg, 75%). MS (EI) <i>m/z</i> 268, 270 (M<sup>+</sup>).</p><p>To a solution of <b>2</b> (0.23 mmol, 62 mg) in DMSO (2 mL) were added K<sub>2</sub>CO<sub>3</sub> (0.35 mmol, 48 mg) and <b>3</b> (0.35 mmol, 88 mg), the reaction was stirred overnight at 70 degrees C. The mixture was diluted with ethyl acetate (15 mL) and washed with 5% NaHCO<sub>3</sub> solution (10 mL × 3), water (10 mL) and brine (10 mL). The organic phase was dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography (30% EtOAc/Hexane) to afford <b>4</b> (87 mg, 86%). MS (EI) <i>m/z</i> 439, 441, 443 (M<sup>+</sup>). <sup>1</sup>H NMR (300 MHz, CDCl<sub>3</sub>): <i>δ</i> 7.37 (d, <i>J</i> = 8.4 Hz, 1H), 7.35 (d, <i>J</i> = 2.4 Hz, 1H), 7.25 (d, <i>J</i> = 1.8 Hz, 1H), 7.27 (d, <i>J</i> = 8.4 Hz, 1H), 7.18 (dd, <i>J<sub>1</sub>=</i>2.4 Hz, <i>J<sub>2</sub></i>=8.7 Hz, 1H), 6.95 (d, <i>J</i> = 8.7 Hz, 1H), 4.39–4.46 (m, 4H).</p><p>Stability test: 1.2 mgs of compound <b>4</b> were dissolved in DMSO (2 mL) and stirred at room temperature over a period of 24 hours. LC-MS analysis of <b>4</b> showed no appreciable decomposition in this period of time.</p></div></div><div id="ml178.s26"><h2 id="_ml178_s26_">3. Results</h2><p>S1P4 is natively coupled to G<sub>i</sub> or G<sub>12/13</sub> [<a class="bibr" href="#ml178.r22" rid="ml178.r22">22</a>]. The Tango assays are based on beta-arrestin recruitment, a response that occurs subsequent to G-protein activation and G protein-coupled receptor kinase-directed phosphorylation of intracellular residues on the GPCR. The Tango beta-lactamase reporter technology is well described in the literature and has been used in high-throughput screening campaigns for a range of target classes, including GPCRs [<a class="bibr" href="#ml178.r23" rid="ml178.r23 ml178.r24 ml178.r25">23–25</a>]. Numerous assays based on beta-arrestin recruitment have been used for GPCR screening (e.g. PubChem AIDs <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2764" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2764</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/493098" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">493098</a>, and <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504459" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">504459</a>).</p><p>Compound <b>1</b> (see <b><a class="figpopup" href="/books/NBK98915/table/ml178.t4/?report=objectonly" target="object" rid-figpopup="figml178t4" rid-ob="figobml178t4">SAR Table</a></b>)
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a: 1) activates S1P4 receptor (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463107" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 463107</a>) with an EC<sub>50</sub> of 46.3 nM, 2) is inhibited by an S1P4 receptor-selective antagonist with an IC<sub>50</sub> of 0.83 μM (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463225" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 463225</a>), 3) is inactive as an agonist against other members of the receptor family, with EC<sub>50</sub>s > 50 μM against S1P1 receptor (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463118" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 463118</a>), S1P2 receptor (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463122" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 463122</a>), S1P3 receptor (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/3463123" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID3463123</a>), and S1P5 receptor (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463129" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID463129</a>), and 4) is nontoxic to U2OS cells (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463119" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 463119</a>), with a CC<sub>50</sub> of > 20 μM.</p><div id="ml178.s27"><h3>3.1. Summary of Screening Results</h3><p>In the uHTS primary assay (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1509" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1509</a>), 218K compounds were screened at 5 μM. A total of 770 compounds (0.35%) were active, passing the set threshold of 10.98% S1P4 receptor agonism. For the uHTS confirmation assay, (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1523" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1523</a>), 737 active compounds were retested in triplicate, and 53 compounds (7.19%) were confirmed as active. In a uHTS counterscreen assay against the S1P1 receptor (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1563" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1563</a>), 28 of these compounds were found to be inactive against S1P1 receptor. Out of the 53 compounds confirmed as active in the confirmation assay (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1523" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1523</a>), 46 were available from the NIH small molecule repository. These 46 compounds were tested in uHTS dose response assays for S1P4 receptor agonism (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1686" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1686</a>) and for S1P1 receptor agonism (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1563" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1563</a>), and 11 were found to be selective for S1P4 receptor—having an EC<sub>50</sub> of less than 10 μM for S1P4 receptor and an EC<sub>50</sub> of greater than 10 μM for S1P1 receptor. S1P4 receptor agonists considered for probe optimization are shown in <a class="figpopup" href="/books/NBK98915/table/ml178.t2/?report=objectonly" target="object" rid-figpopup="figml178t2" rid-ob="figobml178t2">Table 2</a>.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml178t2"><a href="/books/NBK98915/table/ml178.t2/?report=objectonly" target="object" title="Table 2" class="img_link icnblk_img figpopup" rid-figpopup="figml178t2" rid-ob="figobml178t2"><img class="small-thumb" src="/books/NBK98915/table/ml178.t2/?report=thumb" src-large="/books/NBK98915/table/ml178.t2/?report=previmg" alt="Table 2. Selected S1P4 Receptor Agonists from the Primary Screening Campaign." /></a><div class="icnblk_cntnt"><h4 id="ml178.t2"><a href="/books/NBK98915/table/ml178.t2/?report=objectonly" target="object" rid-ob="figobml178t2">Table 2</a></h4><p class="float-caption no_bottom_margin">Selected S1P4 Receptor Agonists from the Primary Screening Campaign. </p></div></div><div id="ml178.f5" class="figure bk_fig"><div class="graphic"><img src="/books/NBK98915/bin/ml178f5.jpg" alt="Figure 5. S1P4 agonist HTS overview." /></div><h3><span class="label">Figure 5</span><span class="title">S1P4 agonist HTS overview</span></h3></div><p>Despite the liability of an aromatic nitro moiety, <a href="https://pubchem.ncbi.nlm.nih.gov/substance/22409359" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 22409359</a> is active in only eight out of 315 PubChem assays, three of which are for the S1P4 receptor agonist. Interestingly, <a href="https://pubchem.ncbi.nlm.nih.gov/substance/17406856" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 17406856</a> is similar to compounds claimed in an Actelion patent (US 2008/0146629) describing thazolidin-4-one derivatives as immunosuppressant S1P1 receptor agonists. This lead molecule provides a validated scaffold for development of S1P4 receptor specific agonists. This report focuses on the lead compound CID 4780551.</p><p>The Scripps Molecular Libraries Chemistry Core purchased 37 similar compounds to evaluate S1P4 receptor agonist activity. The goal was to identify the essential functional groups of the lead molecule. Results are reported in <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504460" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504460</a>; while most of the compounds purchased were not active, some useful information was obtained from this effort. Compound <b>8</b> in <b>SAR</b>
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<a class="figpopup" href="/books/NBK98915/table/ml178.t4/?report=objectonly" target="object" rid-figpopup="figml178t4" rid-ob="figobml178t4">Table 4</a> comes from the replacement of the glycol linker with 2-hydroxyacetamide and resulted in a 10-fold loss of activity. Further probe optimization was accomplished with a set of synthesized compounds (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463107" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 463107</a>). To investigate the impact of the linker length, analogues <b>6</b> and <b>22</b> were synthesized and found inactive. Length as well as electronic properties in the linker is essential for the potency. When 2,4-dichloro substituents were removed from ring A, the potency was lost completely (<b>19</b>). Moreover, replacement of the clorines with methyl or methoxy groups (<b>compounds 9, 10</b>) resulted in more than 20-fold loss of potency. The symmetrical tri-chlorosubstituted <b>3</b> was 3-fold less potent, presumably for steric reasons. Monosubstituted phenyl rings <b>17</b>, <b>7</b>, <b>13</b>, <b>21</b> were found inactive. Taken together, these data suggest that 2,4-disubstitution is an essential structural requirement for the potency. Replacement of the 2-nitro group in ring C with bromine or chlorine (compounds <b>1</b>, <b>4, 7</b>) improved potency ~3 fold. Removal of the 6-methyl group (compound <b>11)</b> resulted in more than 100-fold loss of potency. Interestingly, replacement of the 6-methyl group with fluoromethyl or bromine had no appreciable impact on the potency (compounds <b>1</b>, <b>7</b>). These data indicated that substituents on the 6-position might be involved in a lipophilic interaction.</p><p>A set of compounds with excellent potency in the S1P4 receptor agonist assay was selected to test agonism in counterscreen assays for the S1P1, S1P2, S1P3, and S1P5 receptors (AIDs <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463118" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">463118</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463122" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">463122</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463123" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">463123</a>, and <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463129" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">463129</a>, respectively). Results are summarized in <a class="figpopup" href="/books/NBK98915/table/ml178.t3/?report=objectonly" target="object" rid-figpopup="figml178t3" rid-ob="figobml178t3">Table 3</a>. Notably, all the selected compounds displayed high selectivity for the S1P4 receptor <i>versus</i> the other subtypes. Compound <b>1</b> was available in amounts sufficient for profiling and on that basis was selected as an exemplar of the potent set of SAR agonists and designated as the probe (<a href="/pcsubstance/?term=ML178[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML178</a>) in this report. In an assay run to assess cytotoxicity (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463119" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 463119</a>), compound <b>1</b> had a CC<sub>50</sub> of > 20 μM.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml178t3"><a href="/books/NBK98915/table/ml178.t3/?report=objectonly" target="object" title="Table 3" class="img_link icnblk_img figpopup" rid-figpopup="figml178t3" rid-ob="figobml178t3"><img class="small-thumb" src="/books/NBK98915/table/ml178.t3/?report=thumb" src-large="/books/NBK98915/table/ml178.t3/?report=previmg" alt="Table 3. Specificity of selected S1P4 receptor agonist compounds against S1P1, S1P2, S1P3, and S1P5 receptors." /></a><div class="icnblk_cntnt"><h4 id="ml178.t3"><a href="/books/NBK98915/table/ml178.t3/?report=objectonly" target="object" rid-ob="figobml178t3">Table 3</a></h4><p class="float-caption no_bottom_margin">Specificity of selected S1P4 receptor agonist compounds against S1P1, S1P2, S1P3, and S1P5
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receptors. Compound numbers in column 1 refer to the entry numbers in SAR Table 4 </p></div></div></div><div id="ml178.s28"><h3>3.2. Dose Response Curves for Probe</h3><div id="ml178.f6" class="figure bk_fig"><div class="graphic"><img src="/books/NBK98915/bin/ml178f6.jpg" alt="Figure 6. Dose response curve for probe ML178." /></div><h3><span class="label">Figure 6</span><span class="title">Dose response curve for probe <a href="/pcsubstance/?term=ML178[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML178</a></span></h3></div></div><div id="ml178.s29"><h3>3.3. Scaffold/Moiety Chemical Liabilities</h3><p>The probe has minimal chemical liabilities. Successful substitution of an undesired nitro group in the original hit by a less reactive halogen substituent and enhanced potency improved the probe’s potential utility in functional cell-based assays. Increased potency, solubility and stability are desired to facilitate <i>in vivo</i> studies.</p></div><div id="ml178.s30"><h3>3.4. SAR Table</h3><p>The original screening hit CID 4780551 can be conceptualized as three components consisting of Ring A, linker B, and Ring C. <a class="figpopup" href="/books/NBK98915/table/ml178.t4/?report=objectonly" target="object" rid-figpopup="figml178t4" rid-ob="figobml178t4">Table 4</a> shows structures of compounds used for probe optimization.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml178t4"><a href="/books/NBK98915/table/ml178.t4/?report=objectonly" target="object" title="Table 4" class="img_link icnblk_img figpopup" rid-figpopup="figml178t4" rid-ob="figobml178t4"><img class="small-thumb" src="/books/NBK98915/table/ml178.t4/?report=thumb" src-large="/books/NBK98915/table/ml178.t4/?report=previmg" alt="Table 4. SAR Table for S1P4 Receptor Agonist Probe Optimization. Fragment point of attachment of is indicated with an *." /></a><div class="icnblk_cntnt"><h4 id="ml178.t4"><a href="/books/NBK98915/table/ml178.t4/?report=objectonly" target="object" rid-ob="figobml178t4">Table 4</a></h4><p class="float-caption no_bottom_margin">SAR Table for S1P4 Receptor Agonist Probe Optimization. Fragment point of attachment of is indicated with an <sup>*</sup>. </p></div></div></div><div id="ml178.s31"><h3>3.5. Cellular Activity</h3><p><a href="/pcsubstance/?term=ML178[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML178</a> was evaluated for cell toxicity (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463119" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 463119</a>), and its CC<sub>50</sub> was determined to be greater than 20 μM. This result is expected since cytotoxic compounds would disrupt signaling. The ligand binding domain of S1P4 receptor is extracellular and an agonist is not required to cross the plasma membrane.</p></div><div id="ml178.s32"><h3>3.6. Profiling Assays</h3><p>To date, the lead hit (CID 4780551) has been tested in 316 other bioassays deposited in PubChem, and has shown activity in only eight of those assays, three of which are for the S1P4 receptor agonist project. The other five assays give a hit rate of 1.6%, indicating that this series is not generally active across a broad range of cell-based and non-cell based assays.</p><p>Compound <a href="/pcsubstance/?term=ML178[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML178</a> was submitted to Ricerca Biosciences, LLC for HitProfilingScreen + CYP450 (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504400" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504400</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=ML178[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML178</a> displays affinity. Out of 35 targets tested, four (CYP450 1A2, CYP450 2C19, norepinephrine transporter NET, and dihydropyridine calcium channel L-type) resulted in ≥ 50% inhibition of activity. These data suggest that compound <a href="/pcsubstance/?term=ML178[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML178</a> is generally inactive against a broad array of off targets and does not likely exert unwanted effects.</p></div></div><div id="ml178.s33"><h2 id="_ml178_s33_">4. Discussion</h2><p><a href="/pcsubstance/?term=ML178[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML178</a> activates S1P4 receptor in a cell-based Tango-format assay (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463107" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 463107</a>) with an EC<sub>50</sub> of 46.3 nM; is inhibited by an S1P4 receptor-selective antagonist with an IC<sub>50</sub> of 0.83 μM (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463225" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 463225</a>); is inactive as an agonist against other members of the receptor family, with EC<sub>50</sub>s > 50 μM against S1P1 receptor (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463118" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 463118</a>), S1P2 receptor (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463122" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 463122</a>), S1P3 receptor (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/3463123" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID3463123</a>), and S1P5 receptor (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463129" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID463129</a>), and is nontoxic to U2OS cells (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463119" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 463119</a>), with a CC<sub>50</sub> of > 20 μM. The Tango assay is not able to distinguish partial agonists from full agonists [<a class="bibr" href="#ml178.r26" rid="ml178.r26">26</a>]. <a href="/pcsubstance/?term=ML178[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML178</a> is stable in DMSO for chemical handling, and has good biological availability with a half-life of 21 hours in PBS.</p><div id="ml178.s34"><h3>4.1. Comparison to existing art and how the new probe is an improvement</h3><p>There are no S1P4 selective antagonist compounds currently available. Previously reported S1P4 receptor agonist compounds are unavailable, poorly characterized, and not selective. Dose response assays against all five S1P receptors demonstrate that compound <b>1</b> is the first submicromolar, completely selective S1P4 receptor agonist to be identified. A direct comparison of EC<sub>50</sub> values between <a href="/pcsubstance/?term=ML178[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML178</a> and prior art compounds in a [γ-<sup>35</sup>S]GTP assay would require the development of a cell line overexpressing S1P4 and the development of that assay, which was beyond the scope of this report.</p></div><div id="ml178.s35"><h3>4.2. Mechanism of Action</h3><p>The S1P5 receptor counterscreen assay is in the same Tango format as the S1P4 receptor assay used for the primary screening based upon arrestin recruitment to activated receptor. From this we infer that the probe compound directly interacts and activates the S1P4 receptor. Work is ongoing in the assay provider’s lab with this probe to elucidate the signaling pathway activated in native, S1P4 receptor-expressing cells.</p></div><div id="ml178.s36"><h3>4.3. Planned Future Studies</h3><p>Medicinal chemistry to enhance solubility and potency are planned. In the extended probe development period, our aim will be to develop these further using traditional MedChem techniques and scaffold hopping methods to produce at least a tenfold increase in potency while maintaining selectivity. Specially, with the current SAR, we will vary the subsituents at the allowed positions on rings A and C. In particular, elements that may be undesirable <i>in vivo</i> such as brominated pyridines will be replaced with suitable bio-isosteres. The pharmacokinetic (PK) properties of the lead compounds will also be optimized using a series of <i>in vitro</i> and <i>in vivo</i> (mouse) PK studies to identify a compound that is suitable for use in animal studies. The most promising compounds will be tested for selectivity against the panel of off-target proteins including GPCRs and ion channels.</p><p>Compounds with improved solubility and potency will be useful for mechanism of action studies planned by Dr. Oldstone, including the role of S1P4 receptor in dendritic cell migration, production of pro-inflammatory cytokines, and the effect of S1P4-specific agonist compounds on the response to influenza in mice.</p></div></div><div id="ml178.s37"><h2 id="_ml178_s37_">5. References</h2><dl class="temp-labeled-list"><dl class="bkr_refwrap"><dt>1.</dt><dd><div class="bk_ref" id="ml178.r1">Marsolais D, Hahm B, Walsh K, Edelmann K, MacGavern D, Hatta Y, Kawaoka Y, Rosen H, Oldstones MBA. A critical role for the sphingosine analog AAL-R in dampening the cytokine response during influenza virus infection. <span><span class="ref-journal">Proc. Natl. Acad. Sci. </span>2009;<span class="ref-vol">106</span>(5):1560–1565.</span> [<a href="/pmc/articles/PMC2635800/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2635800</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/19164548" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19164548</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>2.</dt><dd><div class="bk_ref" id="ml178.r2">Marsolais D, Rosen H. Chemical modulators of S1P receptors as barrier-oriented therapeutic molecules. <span><span class="ref-journal">Nat. Rev. Drug Disc. </span>2009;<span class="ref-vol">8</span>(4):297–307.</span> [<a href="/pmc/articles/PMC4455967/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC4455967</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/19300460" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19300460</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>3.</dt><dd><div class="bk_ref" id="ml178.r3">Sanna MG, Liao J, Jo E, Alfonso C, Ahn MY, Peterson MS, Webb B, Lefebvre S, Chun J, Gray N, Rosen H. Sphingosine 1-phosphate (S1P) receptor subtypes S1P1 and S1P3, respectively, regulate lymphocyte recirculation and heart rate. <span><span class="ref-journal">J Biol Chem. </span>2004;<span class="ref-vol">279</span>(14):13839–13848.</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>4.</dt><dd><div class="bk_ref" id="ml178.r4">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>(2):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>5.</dt><dd><div class="bk_ref" id="ml178.r5">Wei SH, Rosen H, Matheu MP, Sanna MG, Wang SK, Jo E, Wong CH, Parker I, Cahalan MD. Sphingosine 1-phosphate type 1 receptor agonism inhibits transendothelial migration of medullary T cells to lymphatic sinuses. <span><span class="ref-journal">Nat Immunol. </span>2005;<span class="ref-vol">6</span>(12):1228–1235.</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>6.</dt><dd><div class="bk_ref" id="ml178.r6">Jo E, Sanna MG, Gonzalez-Cabrera PJ, Thangada S, Tigyi G, Osborne D, Hla T, Parrill AL, Rosen H. S1P1-selective in vivo-active agonists from high-throughput screening: off-the-shelf chemical probes of receptor interactions, signaling, and fate. <span><span class="ref-journal">Chem Biol. </span>2005;<span class="ref-vol">12</span>(6):703–715.</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="ml178.r7">Alfonso C, McHeyzer-Williams MG, Rosen H. CD69 down-modulation and inhibition of thymic egress by short- and long-term selective chemical agonism of sphingosine 1-phosphate receptors. <span><span class="ref-journal">Eur J Immunol. </span>2006;<span class="ref-vol">36</span>(1):149–159.</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>8.</dt><dd><div class="bk_ref" id="ml178.r8">Marsolais D, Hahm B, Edelmann KH, Walsh KB, Guerrero M, Hatta Y, Kawaoka Y, Roberts E, Oldstone MB, Rosen H. Local not systemic modulation of dendritic cell S1P receptors in lung blunts virus-specific immune responses to influenza. <span><span class="ref-journal">Mol Pharmacol. </span>2008;<span class="ref-vol">74</span>(3):896–903.</span> [<a href="/pmc/articles/PMC2574812/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2574812</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/18577684" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18577684</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>9.</dt><dd><div class="bk_ref" id="ml178.r9">Maeda Y, Matsuyuki H, Shimano K, Kataoka H, Sugahara K, Chiba K. Migration of CD4 T cells and dendritic cells toward sphingosine 1-phosphate (S1P) is mediated by different receptor subtypes: S1P regulates the functions of murine mature dendritic cells via S1P receptor type 3. <span><span class="ref-journal">J Immunol. </span>2007;<span class="ref-vol">178</span>(6):3437–3446.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/17339438" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17339438</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>10.</dt><dd><div class="bk_ref" id="ml178.r10">Sanna MG, Liao J, Jo E, Alfonso C, Ahn MY, Peterson MS, Webb B, Lefebvre S, Chun J, Gray N, Rosen H. Sphingosine 1-phosphate (S1P) receptor subtypes S1P1 and S1P3, respectively, regulate lymphocyte recirculation and heart rate. <span><span class="ref-journal">J Biol Chem. </span>2004;<span class="ref-vol">279</span>(14):13839–13848.</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>11.</dt><dd><div class="bk_ref" id="ml178.r11">Toman RE, Spiegel S. Lysophospholipid receptors in the nervous system. <span><span class="ref-journal">Neurochem Res. </span>2002;<span class="ref-vol">27</span>(7–8):619–627.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/12374197" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 12374197</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>12.</dt><dd><div class="bk_ref" id="ml178.r12">Long JS, Fujiwara Y, Edwards J, Tannahill CL, Tigyi G, Pyne S, Pyne NJ. Sphingosine 1-phosphate receptor 4 uses HER2 (ERBB2) to regulate extracellular signal regulated kinase-1/2 in MDA-MB-453 breast cancer cells. <span><span class="ref-journal">J Biol Chem. </span>2010 Nov 12;<span class="ref-vol">285</span>(46):35957–35966.</span> [<a href="/pmc/articles/PMC2975218/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2975218</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/20837468" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 20837468</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>13.</dt><dd><div class="bk_ref" id="ml178.r13">Golfier S, Kondo S, Schulze T, Takeuchi T, Vassileva G, Achtman AH, Gräler MH, Abbondanzo SJ, Wiekowski M, Kremmer E, Endo Y, Lira SA, Bacon KB, Lipp M. Shaping of terminal megakaryocyte differentiation and proplatelet development by sphingosine-1-phosphate receptor S1P4. <span><span class="ref-journal">FASEB J. </span>2010 Aug 9;</span> [Epub ahead of print] [<a href="https://pubmed.ncbi.nlm.nih.gov/20686109" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 20686109</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>14.</dt><dd><div class="bk_ref" id="ml178.r14">Lynch KR, Macdonald TL. Sphingosine 1-phosphate chemical biology. <span><span class="ref-journal">Biochim Biophys Acta. </span>2008;<span class="ref-vol">1781</span>(9):508–512.</span> [<a href="/pmc/articles/PMC3412363/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3412363</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/18638568" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18638568</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>15.</dt><dd><div class="bk_ref" id="ml178.r15">Azzaoui K, Bouhelal R, Buehlmayer P, Guerini D, Koller M. Indol-alanine derivatives as selective S1P4-agonists. 7,754,896. <span><span class="ref-journal">U.S. Patent. </span></span> filed January 20, 2005, and issued July 13, 2010.</div></dd></dl><dl class="bkr_refwrap"><dt>16.</dt><dd><div class="bk_ref" id="ml178.r16">Urwyler S, Floersheim P, Roy BL, Koller M. Drug design, in vitro pharmacology, and structure-activity relationships of 3-acylamino-2-aminopropionic acid derivatives, a novel class of partial agonists at the glycine site on the N-methyl-D-aspartate (NMDA) receptor complex. <span><span class="ref-journal">J Med Chem. </span>2009;<span class="ref-vol">52</span>(16):5093–5107.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/19642674" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19642674</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>17.</dt><dd><div class="bk_ref" id="ml178.r17">Clemens JJ, Davis MD, Lynch KR, Macdonald TL. Synthesis of benzimidazole based analogues of sphingosine-1-phosphate: discovery of potent, subtype-selective S1P4 receptor agonists. <span><span class="ref-journal">Bioorg Med Chem Lett. </span>2004;<span class="ref-vol">14</span>(19):4903–4906.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/15341948" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 15341948</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>18.</dt><dd><div class="bk_ref" id="ml178.r18">Hanessian S, Charron G, Billich A, Guerini D. Constrained azacyclic analogues of the immunomodulatory agent FTY720 as molecular probes for sphingosine 1-phosphate receptors. <span><span class="ref-journal">Bioorg Med Chem Lett. </span>2007;<span class="ref-vol">17</span>(2):491–494.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/17070046" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17070046</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>19.</dt><dd><div class="bk_ref" id="ml178.r19">Yasushi K, Tomohiko E. (Kyorin Pharmaceutical Co, Ltd., Japan). Benzothiophene derivative and its addition salt and Sphingosine-1-phorphoric acid (S1P4) receptor regulator. 2008-2080334. <span><span class="ref-journal">Japanese Patent. </span></span> filed February 23, 2007, and issued November 9, 2008.</div></dd></dl><dl class="bkr_refwrap"><dt>20.</dt><dd><div class="bk_ref" id="ml178.r20">Li X, He Y, Ruiz CH, Koenig M, Cameron MD. Characterization of dasatinib and its structural analogs as CYP3A4 mechanism-based inactivators and the proposed bioactivation pathways. <span><span class="ref-journal">Drug Metab Dispos. </span>2009;<span class="ref-vol">37</span>(6):1242–1250.</span> [<a href="/pmc/articles/PMC3202349/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3202349</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/19282395" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19282395</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>21.</dt><dd><div class="bk_ref" id="ml178.r21">Li X, Kamenecka TM, Cameron MD. Bioactivation of the epidermal growth factor receptor inhibitor gefitinib: implications for pulmonary and hepatic toxicities. <span><span class="ref-journal">Chem Res Toxicol. </span>2009;<span class="ref-vol">22</span>(10):1736–1742.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/19803472" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19803472</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>22.</dt><dd><div class="bk_ref" id="ml178.r22">Rosen H, Gonzalez-Cabrera PJ, Sanna MG, Brown S. Sphingosine 1-phosphate receptor signaling. <span><span class="ref-journal">Annu Rev Biochem. </span>2009;<span class="ref-vol">78</span>:743–768.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/19231986" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19231986</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>23.</dt><dd><div class="bk_ref" id="ml178.r23">Zlokarnik G, Negulescu PA, Knapp TE, Mere L, Burres N, Feng L, Whitney M, Roemer K, Tsien RY. Quantitation of transcription and clonal selection of single living cells with beta-lactamase as reporter. <span><span class="ref-journal">Science. </span>1998;<span class="ref-vol">279</span>(5347):84–88.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/9417030" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 9417030</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>24.</dt><dd><div class="bk_ref" id="ml178.r24">Kunapuli P, Ransom R, Murphy K, Pettibone D, Kerby J, Grimwood S, Zuck P, Hodder P, Lacson R, Hoffman I, Inglese J, Strulovici B. Development of an intact cell reporter gene beta-lactamase assay for G protein-coupled receptors. <span><span class="ref-journal">Analytical Biochem. </span>2003;<span class="ref-vol">314</span>(1):16–29.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/12633598" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 12633598</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>25.</dt><dd><div class="bk_ref" id="ml178.r25">Xing H, Tran HC, Knapp TE, Negulescu PA, Pollok BA. A fluorescent reporter assay for the detection of ligands acting through G1 protein-coupled receptors. <span><span class="ref-journal">J. Receptor & Signal Transduction Research. </span>2000;<span class="ref-vol">20</span>(4):189–210.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/11192018" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 11192018</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>26.</dt><dd><div class="bk_ref" id="ml178.r26">van der Lee MM, Blomenröhr M, van der Doelen AA, Wat JW, Smits N, Hanson BJ, van Koppen CJ, Zaman GJ. Pharmacological characterization of receptor redistribution and beta-arrestin recruitment assays for the cannabinoid receptor 1. <span><span class="ref-journal">J Biomol Screen. </span>2009;<span class="ref-vol">14</span>(7):811–823.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/19520790" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19520790</span></a>]</div></dd></dl></dl></div><div id="bk_toc_contnr"></div></div></div><div class="fm-sec"><h2 id="_NBK98915_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">Mariangela Urbano</span>,<sup>*</sup> <span itemprop="author">Subash Velaparthi</span>,<sup>*</sup> <span itemprop="author">Jian Zhao</span>,<sup>*</sup> <span itemprop="author">Marie-Therese Schaeffer</span>,<sup>†</sup> <span itemprop="author">Steven J Brown</span>,<sup>†</sup> <span itemprop="author">Melissa Crisp</span>,<sup>‡</sup> <span itemprop="author">Jill Ferguson</span>,<sup>†</sup> <span itemprop="author">Edward Roberts</span>,<sup>*</sup> <span itemprop="author">Michael Oldstone</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">October 12, 2010</span>; Last Update: <span itemprop="dateModified">December 12, 2011</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, Urbano M, Velaparthi S, et al. Probe Development Efforts to Identify Novel Agonists of the Sphingosine 1-phosphate Receptor 4 (S1P4) 2010 Oct 12 [Updated 2011 Dec 12]. 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/ml180/?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/ml177/?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="figobml178fu1"><div id="ml178.fu1" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK98915/bin/ml178fu1.jpg" alt="ML178." /></div><h3><span class="title"><a href="/pcsubstance/?term=ML178[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML178</a></span></h3></div></article><article data-type="table-wrap" id="figobml178tu1"><div id="ml178.tu1" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK98915/table/ml178.tu1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml178.tu1_lrgtbl__"><table><thead><tr><th id="hd_h_ml178.tu1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">CID/ML#</th><th id="hd_h_ml178.tu1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Target Name</th><th id="hd_h_ml178.tu1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">EC<sub>50</sub> (nM) [SID, AID]</th><th id="hd_h_ml178.tu1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Anti-target Name</th><th id="hd_h_ml178.tu1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">EC<sub>50</sub> (μM) [SID, AID]</th><th id="hd_h_ml178.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Fold Selective</th><th id="hd_h_ml178.tu1_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Secondary Assay(s) Name: EC<sub>50</sub> (μM) [SID, AID]</th></tr></thead><tbody><tr><td headers="hd_h_ml178.tu1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">CID 44620892/<a href="/pcsubstance/?term=ML178[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML178</a></td><td headers="hd_h_ml178.tu1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">S1P4</td><td headers="hd_h_ml178.tu1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">46.3 [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544119" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 87544119</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463107" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 463107</a>]</td><td headers="hd_h_ml178.tu1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">S1P1</td><td headers="hd_h_ml178.tu1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">>50 [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544119" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 87544119</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463118" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 463118</a>]</td><td headers="hd_h_ml178.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">> 1000</td><td headers="hd_h_ml178.tu1_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><b>S1P1 Counterscreen:</b> >50 [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544119" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 87544119</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463118" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 463118</a>]<br /><b>S1P2 Counterscreen:</b> >50 [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544119" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 87544119</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463122" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 463122</a>]<br /><b>S1P3 Counterscreen:</b> >50 [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544119" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 87544119</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463123" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 463123</a>]<br /><b>S1P5 Counterscreen:</b> >50 [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544119" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 87544119</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463129" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 463129</a>]<br /><b>Cytotoxicity:</b> CC<sub>50</sub>>20 [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544119" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 87544119</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463119" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 463119</a>]<br /><b>Inhibition by antagonist:</b> IC<sub>50</sub>≤ 10 μM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544119" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 87544119</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463225" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 463225</a>]</td></tr></tbody></table></div></div></article><article data-type="table-wrap" id="figobml178t1"><div id="ml178.t1" class="table"><h3><span class="label">Table 1</span><span class="title">Published S1P4 Receptor Agonists</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK98915/table/ml178.t1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml178.t1_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml178.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Compound number</th><th id="hd_h_ml178.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Structure</th><th id="hd_h_ml178.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Published EC<sub>50</sub> for S1P4 Receptor (assay type)</th><th id="hd_h_ml178.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Selective?</th><th id="hd_h_ml178.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Reference</th></tr><tr><th headers="hd_h_ml178.t1_1_1_1_1 hd_h_ml178.t1_1_1_1_2 hd_h_ml178.t1_1_1_1_3 hd_h_ml178.t1_1_1_1_4 hd_h_ml178.t1_1_1_1_5" id="hd_h_ml178.t1_1_1_2_1" colspan="5" rowspan="1" style="text-align:left;vertical-align:middle;">
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<span class="hr"></span></th></tr></thead><tbody><tr><td headers="hd_h_ml178.t1_1_1_1_1 hd_h_ml178.t1_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b>1</b></td><td headers="hd_h_ml178.t1_1_1_1_2 hd_h_ml178.t1_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK98915/bin/ml178fu2.jpg" alt="Image ml178fu2.jpg" /></div></td><td headers="hd_h_ml178.t1_1_1_1_3 hd_h_ml178.t1_1_1_2_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">10 nM (calcium mobilization)<br />250 nM ([γ-<sup>35</sup>S]GTP)</td><td headers="hd_h_ml178.t1_1_1_1_4 hd_h_ml178.t1_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Yes</td><td headers="hd_h_ml178.t1_1_1_1_5 hd_h_ml178.t1_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a class="bibr" href="#ml178.r15" rid="ml178.r15">15</a></td></tr><tr><td headers="hd_h_ml178.t1_1_1_1_1 hd_h_ml178.t1_1_1_2_1 hd_h_ml178.t1_1_1_1_2 hd_h_ml178.t1_1_1_1_3 hd_h_ml178.t1_1_1_1_4 hd_h_ml178.t1_1_1_1_5" colspan="5" rowspan="1" style="text-align:left;vertical-align:middle;">
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<span class="hr"></span></td></tr><tr><td headers="hd_h_ml178.t1_1_1_1_1 hd_h_ml178.t1_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b>2a</b></td><td headers="hd_h_ml178.t1_1_1_1_2 hd_h_ml178.t1_1_1_2_1" rowspan="2" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK98915/bin/ml178fu3.jpg" alt="Image ml178fu3.jpg" /></div></td><td headers="hd_h_ml178.t1_1_1_1_3 hd_h_ml178.t1_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">36 nM</td><td headers="hd_h_ml178.t1_1_1_1_4 hd_h_ml178.t1_1_1_2_1" rowspan="2" colspan="1" style="text-align:center;vertical-align:middle;">No; low nM partial agonist on S1P5</td><td headers="hd_h_ml178.t1_1_1_1_5 hd_h_ml178.t1_1_1_2_1" rowspan="2" colspan="1" style="text-align:center;vertical-align:middle;"><a class="bibr" href="#ml178.r17" rid="ml178.r17">17</a></td></tr><tr><td headers="hd_h_ml178.t1_1_1_1_1 hd_h_ml178.t1_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b>2b</b><br /><b>enantiomers</b></td><td headers="hd_h_ml178.t1_1_1_1_3 hd_h_ml178.t1_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">48 nM<br />([γ-<sup>35</sup>S]GTP)</td></tr><tr><td headers="hd_h_ml178.t1_1_1_1_1 hd_h_ml178.t1_1_1_2_1 hd_h_ml178.t1_1_1_1_2 hd_h_ml178.t1_1_1_1_3 hd_h_ml178.t1_1_1_1_4 hd_h_ml178.t1_1_1_1_5" colspan="5" rowspan="1" style="text-align:left;vertical-align:middle;">
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<span class="hr"></span></td></tr><tr><td headers="hd_h_ml178.t1_1_1_1_1 hd_h_ml178.t1_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b>3</b></td><td headers="hd_h_ml178.t1_1_1_1_2 hd_h_ml178.t1_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK98915/bin/ml178fu4.jpg" alt="Image ml178fu4.jpg" /></div></td><td headers="hd_h_ml178.t1_1_1_1_3 hd_h_ml178.t1_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">17 nM (calcium mobilization)</td><td headers="hd_h_ml178.t1_1_1_1_4 hd_h_ml178.t1_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">No; 5 nM EC<sub>50</sub> on S1P5</td><td headers="hd_h_ml178.t1_1_1_1_5 hd_h_ml178.t1_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a class="bibr" href="#ml178.r18" rid="ml178.r18">18</a></td></tr></tbody></table></div></div></article><article data-type="fig" id="figobml178fu2"><div id="ml178.fu2" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK98915/bin/ml178fu5.jpg" alt="CID 44620892 SID 87544119 ML178." /></div><h3><span class="title">CID 44620892<br /><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544119" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 87544119</a><br /><a href="/pcsubstance/?term=ML178[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML178</a></span></h3></div></article><article data-type="fig" id="figobml178f1"><div id="ml178.f1" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK98915/bin/ml178f1.jpg" alt="Figure 1. 1H NMR spectrum of probe ML178." /></div><h3><span class="label">Figure 1</span><span class="title"><sup>1</sup>H NMR spectrum of probe <a href="/pcsubstance/?term=ML178[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML178</a></span></h3></div></article><article data-type="fig" id="figobml178f2"><div id="ml178.f2" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK98915/bin/ml178f2.jpg" alt="Figure 2. LC-MS results for probe ML178." /></div><h3><span class="label">Figure 2</span><span class="title">LC-MS results for probe <a href="/pcsubstance/?term=ML178[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML178</a></span></h3></div></article><article data-type="fig" id="figobml178f3"><div id="ml178.f3" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK98915/bin/ml178f3.jpg" alt="Figure 3. Stability of Probe ML178 in PBS." /></div><h3><span class="label">Figure 3</span><span class="title">Stability of Probe <a href="/pcsubstance/?term=ML178[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML178</a> in PBS</span></h3></div></article><article data-type="table-wrap" id="figobml178tu2"><div id="ml178.tu2" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK98915/table/ml178.tu2/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml178.tu2_lrgtbl__"><table><thead><tr><th id="hd_h_ml178.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Designation</th><th id="hd_h_ml178.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Compound Number</th><th id="hd_h_ml178.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CID</th><th id="hd_h_ml178.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SID</th><th id="hd_h_ml178.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SRID</th><th id="hd_h_ml178.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS</th></tr></thead><tbody><tr><td headers="hd_h_ml178.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b>Screening Hit</b></td><td headers="hd_h_ml178.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b>2</b></td><td headers="hd_h_ml178.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CID 4780551</td><td headers="hd_h_ml178.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544106" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 87544106</a></td><td headers="hd_h_ml178.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-01000708838-2</td><td headers="hd_h_ml178.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS003115015</td></tr><tr><td headers="hd_h_ml178.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b>Analog 1</b></td><td headers="hd_h_ml178.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b>5</b></td><td headers="hd_h_ml178.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CID 44620883</td><td headers="hd_h_ml178.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544111" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 87544111</a></td><td headers="hd_h_ml178.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-02000000296-1</td><td headers="hd_h_ml178.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS003115016</td></tr><tr><td headers="hd_h_ml178.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b>Analog 2</b></td><td headers="hd_h_ml178.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b>6</b></td><td headers="hd_h_ml178.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CID 44620891</td><td headers="hd_h_ml178.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544112" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 87544112</a></td><td headers="hd_h_ml178.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-02000000297-1</td><td headers="hd_h_ml178.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS003115017</td></tr><tr><td headers="hd_h_ml178.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b>Analog 3</b></td><td headers="hd_h_ml178.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b>3</b></td><td headers="hd_h_ml178.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CID 44620882</td><td headers="hd_h_ml178.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544118" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 87544118</a></td><td headers="hd_h_ml178.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-02000000303-1</td><td headers="hd_h_ml178.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS003115018</td></tr><tr><td headers="hd_h_ml178.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b>Probe</b></td><td headers="hd_h_ml178.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b>1</b></td><td headers="hd_h_ml178.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CID 44620892</td><td headers="hd_h_ml178.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544119" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 87544119</a></td><td headers="hd_h_ml178.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-02000000304-1</td><td headers="hd_h_ml178.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS003115019</td></tr><tr><td headers="hd_h_ml178.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b>Analog 5</b></td><td headers="hd_h_ml178.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b>4</b></td><td headers="hd_h_ml178.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CID 44620895</td><td headers="hd_h_ml178.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544121" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 87544121</a></td><td headers="hd_h_ml178.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-02000000306-1</td><td headers="hd_h_ml178.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS003115020</td></tr></tbody></table></div></div></article><article data-type="fig" id="figobml178f4"><div id="ml178.f4" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK98915/bin/ml178f4.jpg" alt="Figure 4. Synthesis scheme for ML178." /></div><h3><span class="label">Figure 4</span><span class="title">Synthesis scheme for <a href="/pcsubstance/?term=ML178[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML178</a></span></h3><div class="caption"><p>Reagents and conditions: i.- <b>1</b> (1 equiv), 1,2-dibromoethane (3 equiv), K<sub>2</sub>CO<sub>3</sub> (3 equiv), DMSO, 56 °C, overnight, 75%. ii.- <b>2</b> (1.0 equiv), <b>3</b> (1.5 equiv), K<sub>2</sub>CO<sub>3</sub> (1.5 equiv), DMSO, 70 °C, overnight, 86%.</p></div></div></article><article data-type="fig" id="figobml178f5"><div id="ml178.f5" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK98915/bin/ml178f5.jpg" alt="Figure 5. S1P4 agonist HTS overview." /></div><h3><span class="label">Figure 5</span><span class="title">S1P4 agonist HTS overview</span></h3></div></article><article data-type="table-wrap" id="figobml178t2"><div id="ml178.t2" class="table"><h3><span class="label">Table 2</span><span class="title">Selected S1P4 Receptor Agonists from the Primary Screening Campaign</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK98915/table/ml178.t2/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml178.t2_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml178.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SID</th><th id="hd_h_ml178.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Structure</th><th id="hd_h_ml178.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">S1P4 EC<sub>50</sub> (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1686" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1686</a>), S1P1 EC<sub>50</sub> (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1701" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1701</a>)</th></tr></thead><tbody><tr><td headers="hd_h_ml178.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/22409359" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 22409359</a></td><td headers="hd_h_ml178.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK98915/bin/ml178fu6.jpg" alt="Image ml178fu6.jpg" /></div></td><td headers="hd_h_ml178.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.529 μM, 49.8 μM</td></tr><tr><td headers="hd_h_ml178.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/17406856" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 17406856</a></td><td headers="hd_h_ml178.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK98915/bin/ml178fu7.jpg" alt="Image ml178fu7.jpg" /></div></td><td headers="hd_h_ml178.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.844 μM, 1.18 μM</td></tr></tbody></table></div></div></article><article data-type="table-wrap" id="figobml178t3"><div id="ml178.t3" class="table"><h3><span class="label">Table 3</span><span class="title">Specificity of selected S1P4 receptor agonist compounds against S1P1, S1P2, S1P3, and S1P5
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receptors</span></h3><div class="caption"><p>Compound numbers in column 1 refer to the entry numbers in <a class="figpopup" href="/books/NBK98915/table/ml178.t4/?report=objectonly" target="object" rid-figpopup="figml178t4" rid-ob="figobml178t4">SAR Table 4</a></p></div><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK98915/table/ml178.t3/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml178.t3_lrgtbl__"><table><thead><tr><th id="hd_h_ml178.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Compound Number</th><th id="hd_h_ml178.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">CID</th><th id="hd_h_ml178.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">SID</th><th id="hd_h_ml178.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Structure</th><th id="hd_h_ml178.t3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">S1P4 EC50 (μM) [<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463107" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 463107</a>]</th><th id="hd_h_ml178.t3_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">S1P1 EC50 (μM) [<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463118" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 463118</a>]</th><th id="hd_h_ml178.t3_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">S1P2 EC50 (μM) [<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463122" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 463122</a>]</th><th id="hd_h_ml178.t3_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">S1P3 EC50 (μM) [<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463123" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 463123</a>]</th><th id="hd_h_ml178.t3_1_1_1_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">S1P5 EC50 (μM) [<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463129" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 463129</a>]</th></tr></thead><tbody><tr><td headers="hd_h_ml178.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b>2</b></td><td headers="hd_h_ml178.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">4780551</td><td headers="hd_h_ml178.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544106" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87544106</a></td><td headers="hd_h_ml178.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK98915/bin/ml178fu35.jpg" alt="Image ml178fu35.jpg" /></div></td><td headers="hd_h_ml178.t3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.162</td><td headers="hd_h_ml178.t3_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">> 50</td><td headers="hd_h_ml178.t3_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">> 50</td><td headers="hd_h_ml178.t3_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">> 50</td><td headers="hd_h_ml178.t3_1_1_1_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">> 50</td></tr><tr><td headers="hd_h_ml178.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b>3</b></td><td headers="hd_h_ml178.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">44620882</td><td headers="hd_h_ml178.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544118" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87544118</a></td><td headers="hd_h_ml178.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK98915/bin/ml178fu36.jpg" alt="Image ml178fu36.jpg" /></div></td><td headers="hd_h_ml178.t3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.57</td><td headers="hd_h_ml178.t3_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">> 50</td><td headers="hd_h_ml178.t3_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">> 50</td><td headers="hd_h_ml178.t3_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">> 50</td><td headers="hd_h_ml178.t3_1_1_1_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">> 50</td></tr><tr><td headers="hd_h_ml178.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b>1</b></td><td headers="hd_h_ml178.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">44620892</td><td headers="hd_h_ml178.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544119" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87544119</a></td><td headers="hd_h_ml178.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK98915/bin/ml178fu37.jpg" alt="Image ml178fu37.jpg" /></div></td><td headers="hd_h_ml178.t3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.046</td><td headers="hd_h_ml178.t3_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">> 50</td><td headers="hd_h_ml178.t3_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">> 50</td><td headers="hd_h_ml178.t3_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">> 50</td><td headers="hd_h_ml178.t3_1_1_1_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">> 50</td></tr><tr><td headers="hd_h_ml178.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b>4</b></td><td headers="hd_h_ml178.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">44620895</td><td headers="hd_h_ml178.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544121" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87544121</a></td><td headers="hd_h_ml178.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK98915/bin/ml178fu38.jpg" alt="Image ml178fu38.jpg" /></div></td><td headers="hd_h_ml178.t3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.052</td><td headers="hd_h_ml178.t3_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">> 50</td><td headers="hd_h_ml178.t3_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">> 50</td><td headers="hd_h_ml178.t3_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">> 50</td><td headers="hd_h_ml178.t3_1_1_1_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">> 50</td></tr><tr><td headers="hd_h_ml178.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b>7</b></td><td headers="hd_h_ml178.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">44620894</td><td headers="hd_h_ml178.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544123" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87544123</a></td><td headers="hd_h_ml178.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK98915/bin/ml178fu39.jpg" alt="Image ml178fu39.jpg" /></div></td><td headers="hd_h_ml178.t3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.045</td><td headers="hd_h_ml178.t3_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">> 50</td><td headers="hd_h_ml178.t3_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">> 50</td><td headers="hd_h_ml178.t3_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">> 50</td><td headers="hd_h_ml178.t3_1_1_1_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">> 50</td></tr></tbody></table></div></div></article><article data-type="fig" id="figobml178f6"><div id="ml178.f6" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK98915/bin/ml178f6.jpg" alt="Figure 6. Dose response curve for probe ML178." /></div><h3><span class="label">Figure 6</span><span class="title">Dose response curve for probe <a href="/pcsubstance/?term=ML178[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML178</a></span></h3></div></article><article data-type="table-wrap" id="figobml178t4"><div id="ml178.t4" class="table"><h3><span class="label">Table 4</span><span class="title">SAR Table for S1P4 Receptor Agonist Probe Optimization. Fragment point of attachment of is indicated with an <sup>*</sup></span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK98915/table/ml178.t4/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml178.t4_lrgtbl__"><table class="no_margin"><thead><tr><th id="hd_h_ml178.t4_1_1_1_1" colspan="5" rowspan="1" style="text-align:center;vertical-align:middle;">SAR Analysis for S1P4</th><th id="hd_h_ml178.t4_1_1_1_2" colspan="3" rowspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK98915/bin/ml178fu8.jpg" alt="Image ml178fu8.jpg" /></div></th><th id="hd_h_ml178.t4_1_1_1_3" colspan="4" rowspan="1" style="text-align:center;vertical-align:middle;">Potency (μM) mean ± S.E.M.</th><th id="hd_h_ml178.t4_1_1_1_4" rowspan="3" colspan="1" headers="hd_h_ml178.t4_1_1_1_4" style="text-align:center;vertical-align:middle;">Target to Antitarget Fold Selectivity</th></tr><tr><th headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_1" id="hd_h_ml178.t4_1_1_2_1" rowspan="2" colspan="1" style="text-align:center;vertical-align:bottom;">Entry</th><th headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_2" id="hd_h_ml178.t4_1_1_2_2" rowspan="2" colspan="1" style="text-align:center;vertical-align:bottom;">CID</th><th headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_3" id="hd_h_ml178.t4_1_1_2_3" rowspan="2" colspan="1" style="text-align:center;vertical-align:bottom;">SID</th><th headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_4" id="hd_h_ml178.t4_1_1_2_4" rowspan="2" colspan="1" style="text-align:center;vertical-align:bottom;">Center Internal Number</th><th headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_5" id="hd_h_ml178.t4_1_1_2_5" rowspan="2" colspan="1" style="text-align:center;vertical-align:bottom;">P/S<sup>†</sup></th><th headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_6" id="hd_h_ml178.t4_1_1_2_6" rowspan="2" colspan="1" style="text-align:center;vertical-align:bottom;">Ring A</th><th headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_7" id="hd_h_ml178.t4_1_1_2_7" rowspan="2" colspan="1" style="text-align:center;vertical-align:bottom;">Linker B (oriented toward ring substituents)</th><th headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_8" id="hd_h_ml178.t4_1_1_2_8" rowspan="2" colspan="1" style="text-align:center;vertical-align:bottom;">Ring C</th><th headers="hd_h_ml178.t4_1_1_1_3" id="hd_h_ml178.t4_1_1_2_9" colspan="2" rowspan="1" style="text-align:center;vertical-align:bottom;">Target S1P4</th><th headers="hd_h_ml178.t4_1_1_1_3" id="hd_h_ml178.t4_1_1_2_10" colspan="2" rowspan="1" style="text-align:center;vertical-align:bottom;">Antitarget S1P1</th></tr><tr><th headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_9" id="hd_h_ml178.t4_1_1_3_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">n<sup>**</sup></th><th headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_9" id="hd_h_ml178.t4_1_1_3_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">EC50</th><th headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_10" id="hd_h_ml178.t4_1_1_3_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">n<sup>**</sup></th><th headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_10" id="hd_h_ml178.t4_1_1_3_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">EC50</th></tr></thead><tbody><tr><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">44620892</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544119" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87544119</a></td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-02000000304-1</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">S</td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK98915/bin/ml178fu9.jpg" alt="Image ml178fu9.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK98915/bin/ml178fu10.jpg" alt="Image ml178fu10.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK98915/bin/ml178fu11.jpg" alt="Image ml178fu11.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_9 hd_h_ml178.t4_1_1_3_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_9 hd_h_ml178.t4_1_1_3_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.046</td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_10 hd_h_ml178.t4_1_1_3_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_10 hd_h_ml178.t4_1_1_3_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">> 50</td><td headers="hd_h_ml178.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">> 1000</td></tr><tr><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_1" rowspan="2" colspan="1" style="text-align:center;vertical-align:middle;">2</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_2" rowspan="2" colspan="1" style="text-align:center;vertical-align:middle;">4780551</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/22409359" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">22409359</a></td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-01000708838-1</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_5" rowspan="2" colspan="1" style="text-align:center;vertical-align:middle;">S</td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_6" rowspan="2" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK98915/bin/ml178fu9.jpg" alt="Image ml178fu9.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_7" rowspan="2" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK98915/bin/ml178fu10.jpg" alt="Image ml178fu10.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_8" rowspan="2" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK98915/bin/ml178fu12.jpg" alt="Image ml178fu12.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_9 hd_h_ml178.t4_1_1_3_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_9 hd_h_ml178.t4_1_1_3_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.529</td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_10 hd_h_ml178.t4_1_1_3_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_10 hd_h_ml178.t4_1_1_3_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">49.8</td><td headers="hd_h_ml178.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">94</td></tr><tr><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544106" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87544106</a></td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-01000708838-2</td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_9 hd_h_ml178.t4_1_1_3_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_9 hd_h_ml178.t4_1_1_3_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.162</td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_10 hd_h_ml178.t4_1_1_3_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_10 hd_h_ml178.t4_1_1_3_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">> 50</td><td headers="hd_h_ml178.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">> 300</td></tr><tr><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">44620882</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544118" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87544118</a></td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-02000000303-2</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">S</td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK98915/bin/ml178fu13.jpg" alt="Image ml178fu13.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK98915/bin/ml178fu10.jpg" alt="Image ml178fu10.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK98915/bin/ml178fu12.jpg" alt="Image ml178fu12.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_9 hd_h_ml178.t4_1_1_3_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_9 hd_h_ml178.t4_1_1_3_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.57</td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_10 hd_h_ml178.t4_1_1_3_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_10 hd_h_ml178.t4_1_1_3_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">> 50</td><td headers="hd_h_ml178.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">> 88</td></tr><tr><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">4</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">44620895</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544121" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87544121</a></td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-02000000306</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">S</td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK98915/bin/ml178fu9.jpg" alt="Image ml178fu9.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK98915/bin/ml178fu10.jpg" alt="Image ml178fu10.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK98915/bin/ml178fu14.jpg" alt="Image ml178fu14.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_9 hd_h_ml178.t4_1_1_3_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_9 hd_h_ml178.t4_1_1_3_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.052</td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_10 hd_h_ml178.t4_1_1_3_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_10 hd_h_ml178.t4_1_1_3_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">> 50</td><td headers="hd_h_ml178.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">> 960</td></tr><tr><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">5</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">44620883</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544111" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87544111</a></td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-02000000296-1</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">S</td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK98915/bin/ml178fu9.jpg" alt="Image ml178fu9.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK98915/bin/ml178fu10.jpg" alt="Image ml178fu10.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK98915/bin/ml178fu15.jpg" alt="Image ml178fu15.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_9 hd_h_ml178.t4_1_1_3_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_9 hd_h_ml178.t4_1_1_3_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">29.8</td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_10 hd_h_ml178.t4_1_1_3_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_10 hd_h_ml178.t4_1_1_3_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></td><td headers="hd_h_ml178.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></td></tr><tr><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">6</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">44620891</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544112" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87544112</a></td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-02000000297-1</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">S</td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK98915/bin/ml178fu9.jpg" alt="Image ml178fu9.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK98915/bin/ml178fu16.jpg" alt="Image ml178fu16.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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|
<div class="graphic"><img src="/books/NBK98915/bin/ml178fu12.jpg" alt="Image ml178fu12.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_9 hd_h_ml178.t4_1_1_3_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_9 hd_h_ml178.t4_1_1_3_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">> 50</td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_10 hd_h_ml178.t4_1_1_3_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_10 hd_h_ml178.t4_1_1_3_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></td><td headers="hd_h_ml178.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></td></tr><tr><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">7</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">44620894</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544123" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87544123</a></td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-02000000308-1</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">S</td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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|
<div class="graphic"><img src="/books/NBK98915/bin/ml178fu9.jpg" alt="Image ml178fu9.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
|
|
<div class="graphic"><img src="/books/NBK98915/bin/ml178fu10.jpg" alt="Image ml178fu10.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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|
<div class="graphic"><img src="/books/NBK98915/bin/ml178fu17.jpg" alt="Image ml178fu17.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_9 hd_h_ml178.t4_1_1_3_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_9 hd_h_ml178.t4_1_1_3_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.045</td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_10 hd_h_ml178.t4_1_1_3_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_10 hd_h_ml178.t4_1_1_3_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></td><td headers="hd_h_ml178.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></td></tr><tr><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">8</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3418421</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/88095679" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">88095679</a></td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-02000000317-2</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">P</td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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|
<div class="graphic"><img src="/books/NBK98915/bin/ml178fu18.jpg" alt="Image ml178fu18.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
|
|
<div class="graphic"><img src="/books/NBK98915/bin/ml178fu19.jpg" alt="Image ml178fu19.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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|
<div class="graphic"><img src="/books/NBK98915/bin/ml178fu20.jpg" alt="Image ml178fu20.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_9 hd_h_ml178.t4_1_1_3_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_9 hd_h_ml178.t4_1_1_3_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3.64</td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_10 hd_h_ml178.t4_1_1_3_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_10 hd_h_ml178.t4_1_1_3_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></td><td headers="hd_h_ml178.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></td></tr><tr><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">9</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">44620884</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544114" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87544114</a></td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-02000000299-1</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">S</td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
|
|
<div class="graphic"><img src="/books/NBK98915/bin/ml178fu21.jpg" alt="Image ml178fu21.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
|
|
<div class="graphic"><img src="/books/NBK98915/bin/ml178fu10.jpg" alt="Image ml178fu10.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
|
|
<div class="graphic"><img src="/books/NBK98915/bin/ml178fu20.jpg" alt="Image ml178fu20.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_9 hd_h_ml178.t4_1_1_3_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_9 hd_h_ml178.t4_1_1_3_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3.9</td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_10 hd_h_ml178.t4_1_1_3_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_10 hd_h_ml178.t4_1_1_3_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></td><td headers="hd_h_ml178.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></td></tr><tr><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">10</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">44620886</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544115" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87544115</a></td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-02000000300-1</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">S</td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
|
|
<div class="graphic"><img src="/books/NBK98915/bin/ml178fu22.jpg" alt="Image ml178fu22.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
|
|
<div class="graphic"><img src="/books/NBK98915/bin/ml178fu10.jpg" alt="Image ml178fu10.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
|
|
<div class="graphic"><img src="/books/NBK98915/bin/ml178fu20.jpg" alt="Image ml178fu20.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_9 hd_h_ml178.t4_1_1_3_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_9 hd_h_ml178.t4_1_1_3_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">7.2</td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_10 hd_h_ml178.t4_1_1_3_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_10 hd_h_ml178.t4_1_1_3_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></td><td headers="hd_h_ml178.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></td></tr><tr><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">11</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">44620893</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544122" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87544122</a></td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-02000000307-1</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">S</td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
|
|
<div class="graphic"><img src="/books/NBK98915/bin/ml178fu9.jpg" alt="Image ml178fu9.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
|
|
<div class="graphic"><img src="/books/NBK98915/bin/ml178fu10.jpg" alt="Image ml178fu10.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
|
|
<div class="graphic"><img src="/books/NBK98915/bin/ml178fu23.jpg" alt="Image ml178fu23.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_9 hd_h_ml178.t4_1_1_3_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_9 hd_h_ml178.t4_1_1_3_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">12</td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_10 hd_h_ml178.t4_1_1_3_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_10 hd_h_ml178.t4_1_1_3_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></td><td headers="hd_h_ml178.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></td></tr><tr><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">12</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3879894</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/88095661" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">88095661</a></td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-01000055180-5</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">P</td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK98915/bin/ml178fu24.jpg" alt="Image ml178fu24.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK98915/bin/ml178fu25.jpg" alt="Image ml178fu25.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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|
<div class="graphic"><img src="/books/NBK98915/bin/ml178fu20.jpg" alt="Image ml178fu20.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_9 hd_h_ml178.t4_1_1_3_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_9 hd_h_ml178.t4_1_1_3_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">23.3</td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_10 hd_h_ml178.t4_1_1_3_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_10 hd_h_ml178.t4_1_1_3_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></td><td headers="hd_h_ml178.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></td></tr><tr><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">13</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">4260746</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/88095659" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">88095659</a></td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-01000055073-3</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">P</td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK98915/bin/ml178fu26.jpg" alt="Image ml178fu26.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK98915/bin/ml178fu25.jpg" alt="Image ml178fu25.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK98915/bin/ml178fu20.jpg" alt="Image ml178fu20.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_9 hd_h_ml178.t4_1_1_3_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_9 hd_h_ml178.t4_1_1_3_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">> 50</td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_10 hd_h_ml178.t4_1_1_3_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_10 hd_h_ml178.t4_1_1_3_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></td><td headers="hd_h_ml178.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></td></tr><tr><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">14</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2800221</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/88095671" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">88095671</a></td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-02000000311-1</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">P</td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK98915/bin/ml178fu27.jpg" alt="Image ml178fu27.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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|
<div class="graphic"><img src="/books/NBK98915/bin/ml178fu28.jpg" alt="Image ml178fu28.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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|
<div class="graphic"><img src="/books/NBK98915/bin/ml178fu20.jpg" alt="Image ml178fu20.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_9 hd_h_ml178.t4_1_1_3_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_9 hd_h_ml178.t4_1_1_3_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">> 50</td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_10 hd_h_ml178.t4_1_1_3_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_10 hd_h_ml178.t4_1_1_3_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></td><td headers="hd_h_ml178.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></td></tr><tr><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">15</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">44620890</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544107" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87544107</a></td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-02000000292-1</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">S</td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK98915/bin/ml178fu12.jpg" alt="Image ml178fu12.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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|
<div class="graphic"><img src="/books/NBK98915/bin/ml178fu25.jpg" alt="Image ml178fu25.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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|
<div class="graphic"><img src="/books/NBK98915/bin/ml178fu20.jpg" alt="Image ml178fu20.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_9 hd_h_ml178.t4_1_1_3_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_9 hd_h_ml178.t4_1_1_3_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">> 50</td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_10 hd_h_ml178.t4_1_1_3_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_10 hd_h_ml178.t4_1_1_3_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></td><td headers="hd_h_ml178.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></td></tr><tr><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">16</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">7567697</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544108" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87544108</a></td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-02000000293-1</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">S</td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK98915/bin/ml178fu12.jpg" alt="Image ml178fu12.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK98915/bin/ml178fu16.jpg" alt="Image ml178fu16.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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|
<div class="graphic"><img src="/books/NBK98915/bin/ml178fu29.jpg" alt="Image ml178fu29.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_9 hd_h_ml178.t4_1_1_3_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_9 hd_h_ml178.t4_1_1_3_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">> 50</td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_10 hd_h_ml178.t4_1_1_3_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_10 hd_h_ml178.t4_1_1_3_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></td><td headers="hd_h_ml178.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></td></tr><tr><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">17</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">44620880</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544109" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87544109</a></td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-02000000294-1</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">S</td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK98915/bin/ml178fu30.jpg" alt="Image ml178fu30.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK98915/bin/ml178fu10.jpg" alt="Image ml178fu10.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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|
<div class="graphic"><img src="/books/NBK98915/bin/ml178fu29.jpg" alt="Image ml178fu29.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_9 hd_h_ml178.t4_1_1_3_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_9 hd_h_ml178.t4_1_1_3_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">> 50</td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_10 hd_h_ml178.t4_1_1_3_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_10 hd_h_ml178.t4_1_1_3_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></td><td headers="hd_h_ml178.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></td></tr><tr><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">18</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3879894</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544105" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87544105</a></td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-01000055180-3</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">S</td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK98915/bin/ml178fu31.jpg" alt="Image ml178fu31.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK98915/bin/ml178fu10.jpg" alt="Image ml178fu10.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK98915/bin/ml178fu29.jpg" alt="Image ml178fu29.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_9 hd_h_ml178.t4_1_1_3_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_9 hd_h_ml178.t4_1_1_3_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">> 50</td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_10 hd_h_ml178.t4_1_1_3_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_10 hd_h_ml178.t4_1_1_3_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></td><td headers="hd_h_ml178.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></td></tr><tr><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">19</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">44620888</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544110" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87544110</a></td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-02000000295-1</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">S</td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK98915/bin/ml178fu32.jpg" alt="Image ml178fu32.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK98915/bin/ml178fu10.jpg" alt="Image ml178fu10.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK98915/bin/ml178fu29.jpg" alt="Image ml178fu29.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_9 hd_h_ml178.t4_1_1_3_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_9 hd_h_ml178.t4_1_1_3_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">> 50</td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_10 hd_h_ml178.t4_1_1_3_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_10 hd_h_ml178.t4_1_1_3_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></td><td headers="hd_h_ml178.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></td></tr><tr><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">20</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">44620885</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544113" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87544113</a></td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-02000000298-1</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">S</td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK98915/bin/ml178fu9.jpg" alt="Image ml178fu9.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK98915/bin/ml178fu10.jpg" alt="Image ml178fu10.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK98915/bin/ml178fu29.jpg" alt="Image ml178fu29.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_9 hd_h_ml178.t4_1_1_3_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_9 hd_h_ml178.t4_1_1_3_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">> 50</td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_10 hd_h_ml178.t4_1_1_3_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_10 hd_h_ml178.t4_1_1_3_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></td><td headers="hd_h_ml178.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></td></tr><tr><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">21</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">44620889</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544116" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87544116</a></td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-02000000301-1</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">S</td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK98915/bin/ml178fu33.jpg" alt="Image ml178fu33.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK98915/bin/ml178fu10.jpg" alt="Image ml178fu10.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK98915/bin/ml178fu29.jpg" alt="Image ml178fu29.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_9 hd_h_ml178.t4_1_1_3_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_9 hd_h_ml178.t4_1_1_3_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">> 50</td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_10 hd_h_ml178.t4_1_1_3_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_10 hd_h_ml178.t4_1_1_3_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></td><td headers="hd_h_ml178.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></td></tr><tr><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">22</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">44620887</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544117" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87544117</a></td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-02000000302-1</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">S</td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK98915/bin/ml178fu9.jpg" alt="Image ml178fu9.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
|
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<div class="graphic"><img src="/books/NBK98915/bin/ml178fu28.jpg" alt="Image ml178fu28.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK98915/bin/ml178fu29.jpg" alt="Image ml178fu29.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_9 hd_h_ml178.t4_1_1_3_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_9 hd_h_ml178.t4_1_1_3_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">> 50</td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_10 hd_h_ml178.t4_1_1_3_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_10 hd_h_ml178.t4_1_1_3_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></td><td headers="hd_h_ml178.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></td></tr><tr><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">23</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">44620881</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544124" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87544124</a></td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-02000000309-1</td><td headers="hd_h_ml178.t4_1_1_1_1 hd_h_ml178.t4_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">S</td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
|
|
<div class="graphic"><img src="/books/NBK98915/bin/ml178fu9.jpg" alt="Image ml178fu9.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
|
|
<div class="graphic"><img src="/books/NBK98915/bin/ml178fu10.jpg" alt="Image ml178fu10.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_2 hd_h_ml178.t4_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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|
<div class="graphic"><img src="/books/NBK98915/bin/ml178fu34.jpg" alt="Image ml178fu34.jpg" /></div></td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_9 hd_h_ml178.t4_1_1_3_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_9 hd_h_ml178.t4_1_1_3_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">> 50</td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_10 hd_h_ml178.t4_1_1_3_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></td><td headers="hd_h_ml178.t4_1_1_1_3 hd_h_ml178.t4_1_1_2_10 hd_h_ml178.t4_1_1_3_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></td><td headers="hd_h_ml178.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></td></tr></tbody></table></div><div class="tblwrap-foot"><div><dl class="temp-labeled-list small"><dl class="bkr_refwrap"><dt>†</dt><dd><div id="ml178.tfn1"><p class="no_margin">P = Purchased; S = Synthesized</p></div></dd></dl><dl class="bkr_refwrap"><dt>**</dt><dd><div id="ml178.tfn2"><p class="no_margin">n = Number of Replicates</p></div></dd></dl></dl></div></div></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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