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<meta name="ncbi_db" content="books" /><meta name="ncbi_pdid" content="book-part" /><meta name="ncbi_acc" content="NBK280046" /><meta name="ncbi_domain" content="mlprobe" /><meta name="ncbi_report" content="record" /><meta name="ncbi_type" content="fulltext" /><meta name="ncbi_objectid" content="" /><meta name="ncbi_pcid" content="/NBK280046/" /><meta name="ncbi_pagename" content="Functional Antagonists of EBI-2 - Probe Reports from the NIH Molecular Libraries Program - NCBI Bookshelf" /><meta name="ncbi_bookparttype" content="chapter" /><meta name="ncbi_app" content="bookshelf" />
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<title>Functional Antagonists of EBI-2 - Probe Reports from the NIH Molecular Libraries Program - NCBI Bookshelf</title>
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<meta name="robots" content="INDEX,FOLLOW,NOARCHIVE" /><meta name="citation_inbook_title" content="Probe Reports from the NIH Molecular Libraries Program [Internet]" /><meta name="citation_title" content="Functional Antagonists of EBI-2" /><meta name="citation_publisher" content="National Center for Biotechnology Information (US)" /><meta name="citation_date" content="2015/01/16" /><meta name="citation_author" content="Robert Ardecky" /><meta name="citation_author" content="Eduard Sergienko" /><meta name="citation_author" content="Jiwen Zou" /><meta name="citation_author" content="Santhi Ganji" /><meta name="citation_author" content="Brock Brown" /><meta name="citation_author" content="Qing Sun" /><meta name="citation_author" content="Chen-Ting Ma" /><meta name="citation_author" content="Becky Hood" /><meta name="citation_author" content="Kevin Nguyen" /><meta name="citation_author" content="Stefan Vasile" /><meta name="citation_author" content="Eigo Suyama" /><meta name="citation_author" content="Arianna Mangravita-Novo" /><meta name="citation_author" content="Sumeet Salaniwal" /><meta name="citation_author" content="Paul Kung" /><meta name="citation_author" content="Layton H. Smith" /><meta name="citation_author" content="Thomas D.Y. Chung" /><meta name="citation_author" content="Michael R. Jackson" /><meta name="citation_author" content="Anthony B. Pinkerton" /><meta name="citation_author" content="Robert Rickert" /><meta name="citation_pmid" content="25834900" /><meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK280046/" /><link rel="schema.DC" href="http://purl.org/DC/elements/1.0/" /><meta name="DC.Title" content="Functional Antagonists of EBI-2" /><meta name="DC.Type" content="Text" /><meta name="DC.Publisher" content="National Center for Biotechnology Information (US)" /><meta name="DC.Contributor" content="Robert Ardecky" /><meta name="DC.Contributor" content="Eduard Sergienko" /><meta name="DC.Contributor" content="Jiwen Zou" /><meta name="DC.Contributor" content="Santhi Ganji" /><meta name="DC.Contributor" content="Brock Brown" /><meta name="DC.Contributor" content="Qing Sun" /><meta name="DC.Contributor" content="Chen-Ting Ma" /><meta name="DC.Contributor" content="Becky Hood" /><meta name="DC.Contributor" content="Kevin Nguyen" /><meta name="DC.Contributor" content="Stefan Vasile" /><meta name="DC.Contributor" content="Eigo Suyama" /><meta name="DC.Contributor" content="Arianna Mangravita-Novo" /><meta name="DC.Contributor" content="Sumeet Salaniwal" /><meta name="DC.Contributor" content="Paul Kung" /><meta name="DC.Contributor" content="Layton H. Smith" /><meta name="DC.Contributor" content="Thomas D.Y. Chung" /><meta name="DC.Contributor" content="Michael R. Jackson" /><meta name="DC.Contributor" content="Anthony B. Pinkerton" /><meta name="DC.Contributor" content="Robert Rickert" /><meta name="DC.Date" content="2015/01/16" /><meta name="DC.Identifier" content="https://www.ncbi.nlm.nih.gov/books/NBK280046/" /><meta name="description" content="A robust antibody response is essential for efficient identification and eradication of pathogenic microbes and toxins, whereas dysregulation of the antibody response can lead to autoimmune disease. Specific antigen encounter by B lymphocytes induces clonal expansion that encompasses several distinct stages of differentiation. During this differentiation process, a critical cell fate decision is made wherein some B cells will undergo terminal differentiation into antibody-producing cells, while a separate cohort will assume a distinct pathway of differentiation to become long-lived memory B cells. An exciting new development in the field is the revelation of a novel chemotactic axis involving the recognition of oxysterol compounds by the orphan G-protein-coupled receptor (GPCR), Epstein-Barr virus-induced gene 2 (EBI2). EBI2 is expressed on B cells and is highly induced upon activation. Recent gene targeting experiments revealed that EBI2-/- B cells exhibited defective migration, resulting in strongly impaired T cell-dependent antibody responses. Most recently, two research teams made the unlikely discovery that oxysterol compounds, previously known to bind nuclear receptors, are the physiologic ligands for EBI2. In order to investigate the importance of EBI2 in immune processes and potential as a drug target, selective and potent antagonists with good in vivo pharmacokinetics need to be developed. This probe report describes a potent functional antagonist of EBI-2, ML401 (CID 73169083, SID 173333998), which is potent (IC50 ∼ 1 nM), displays activity in a chemotaxis assay (IC50 ∼ 6 nM), and has a clean profile in a Eurofins/Ricerca panel as well as excellent rodent pharmacokinetics. As such ML401 should be a valuable tool to explore the in vivo effects of EBI-2 inhibition." /><meta name="og:title" content="Functional Antagonists of EBI-2" /><meta name="og:type" content="book" /><meta name="og:description" content="A robust antibody response is essential for efficient identification and eradication of pathogenic microbes and toxins, whereas dysregulation of the antibody response can lead to autoimmune disease. Specific antigen encounter by B lymphocytes induces clonal expansion that encompasses several distinct stages of differentiation. During this differentiation process, a critical cell fate decision is made wherein some B cells will undergo terminal differentiation into antibody-producing cells, while a separate cohort will assume a distinct pathway of differentiation to become long-lived memory B cells. An exciting new development in the field is the revelation of a novel chemotactic axis involving the recognition of oxysterol compounds by the orphan G-protein-coupled receptor (GPCR), Epstein-Barr virus-induced gene 2 (EBI2). EBI2 is expressed on B cells and is highly induced upon activation. Recent gene targeting experiments revealed that EBI2-/- B cells exhibited defective migration, resulting in strongly impaired T cell-dependent antibody responses. Most recently, two research teams made the unlikely discovery that oxysterol compounds, previously known to bind nuclear receptors, are the physiologic ligands for EBI2. In order to investigate the importance of EBI2 in immune processes and potential as a drug target, selective and potent antagonists with good in vivo pharmacokinetics need to be developed. This probe report describes a potent functional antagonist of EBI-2, ML401 (CID 73169083, SID 173333998), which is potent (IC50 ∼ 1 nM), displays activity in a chemotaxis assay (IC50 ∼ 6 nM), and has a clean profile in a Eurofins/Ricerca panel as well as excellent rodent pharmacokinetics. As such ML401 should be a valuable tool to explore the in vivo effects of EBI-2 inhibition." /><meta name="og:url" content="https://www.ncbi.nlm.nih.gov/books/NBK280046/" /><meta name="og:site_name" content="NCBI Bookshelf" /><meta name="og:image" content="https://www.ncbi.nlm.nih.gov/corehtml/pmc/pmcgifs/bookshelf/thumbs/th-mlprobe-lrg.png" /><meta name="twitter:card" content="summary" /><meta name="twitter:site" content="@ncbibooks" /><meta name="bk-non-canon-loc" content="/books/n/mlprobe/ml401/" /><link rel="canonical" href="https://www.ncbi.nlm.nih.gov/books/NBK280046/" /><link rel="stylesheet" href="/corehtml/pmc/css/figpopup.css" type="text/css" media="screen" /><link rel="stylesheet" href="/corehtml/pmc/css/bookshelf/2.26/css/books.min.css" type="text/css" /><link rel="stylesheet" href="/corehtml/pmc/css/bookshelf/2.26/css/books_print.min.css" type="text/css" media="print" /><style type="text/css">p a.figpopup{display:inline !important} .bk_tt {font-family: monospace} .first-line-outdent .bk_ref {display: inline} .body-content h2, .body-content .h2 {border-bottom: 1px solid #97B0C8} .body-content h2.inline {border-bottom: none} a.page-toc-label , .jig-ncbismoothscroll a {text-decoration:none;border:0 !important} .temp-labeled-list .graphic {display:inline-block !important} .temp-labeled-list img{width:100%}</style><script type="text/javascript" src="/corehtml/pmc/js/jquery.hoverIntent.min.js"> </script><script type="text/javascript" src="/corehtml/pmc/js/common.min.js?_=3.18"> </script><script type="text/javascript" src="/corehtml/pmc/js/large-obj-scrollbars.min.js"> </script><script type="text/javascript">window.name="mainwindow";</script><script type="text/javascript" src="/corehtml/pmc/js/bookshelf/2.26/book-toc.min.js"> </script><script type="text/javascript" src="/corehtml/pmc/js/bookshelf/2.26/books.min.js"> </script><meta name="book-collection" content="NONE" />
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<div class="pre-content"><div><div class="bk_prnt"><p class="small">NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health.</p><p>Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-. </p></div><div class="iconblock clearfix whole_rhythm no_top_margin bk_noprnt"><a class="img_link icnblk_img" title="Table of Contents Page" href="/books/n/mlprobe/"><img class="source-thumb" src="/corehtml/pmc/pmcgifs/bookshelf/thumbs/th-mlprobe-lrg.png" alt="Cover of Probe Reports from the NIH Molecular Libraries Program" height="100px" width="80px" /></a><div class="icnblk_cntnt eight_col"><h2>Probe Reports from the NIH Molecular Libraries Program [Internet].</h2><a data-jig="ncbitoggler" href="#__NBK280046_dtls__">Show details</a><div style="display:none" class="ui-widget" id="__NBK280046_dtls__"><div>Bethesda (MD): National Center for Biotechnology Information (US); 2010-.</div></div><div class="half_rhythm"><ul class="inline_list"><li style="margin-right:1em"><a class="bk_cntns" href="/books/n/mlprobe/">Contents</a></li></ul></div><div class="bk_noprnt"><form method="get" action="/books/n/mlprobe/" id="bk_srch"><div class="bk_search"><label for="bk_term" class="offscreen_noflow">Search term</label><input type="text" title="Search this book" id="bk_term" name="term" value="" data-jig="ncbiclearbutton" /> <input type="submit" class="jig-ncbibutton" value="Search this book" submit="false" style="padding: 0.1em 0.4em;" /></div></form></div></div><div class="icnblk_cntnt two_col"><div class="pagination bk_noprnt"><a class="active page_link prev" href="/books/n/mlprobe/ml400/" title="Previous page in this title">< Prev</a><a class="active page_link next" href="/books/n/mlprobe/ml404/" title="Next page in this title">Next ></a></div></div></div></div></div>
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<div class="main-content lit-style" itemscope="itemscope" itemtype="http://schema.org/CreativeWork"><div class="meta-content fm-sec"><h1 id="_NBK280046_"><span class="title" itemprop="name">Functional Antagonists of EBI-2</span></h1><p class="contrib-group"><span itemprop="author">Robert Ardecky</span>, <span itemprop="author">Eduard Sergienko</span>, <span itemprop="author">Jiwen Zou</span>, <span itemprop="author">Santhi Ganji</span>, <span itemprop="author">Brock Brown</span>, <span itemprop="author">Qing Sun</span>, <span itemprop="author">Chen-Ting Ma</span>, <span itemprop="author">Becky Hood</span>, <span itemprop="author">Kevin Nguyen</span>, <span itemprop="author">Stefan Vasile</span>, <span itemprop="author">Eigo Suyama</span>, <span itemprop="author">Arianna Mangravita-Novo</span>, <span itemprop="author">Sumeet Salaniwal</span>, <span itemprop="author">Paul Kung</span>, <span itemprop="author">Layton H. Smith</span>, <span itemprop="author">Thomas D.Y. Chung</span>, <span itemprop="author">Michael R. Jackson</span>, <span itemprop="author">Anthony B. Pinkerton</span>, and <span itemprop="author">Robert Rickert</span>.</p><a data-jig="ncbitoggler" href="#__NBK280046_ai__" style="border:0;text-decoration:none">Author Information and Affiliations</a><div style="display:none" class="ui-widget" id="__NBK280046_ai__"><p class="contrib-group"><h4>Authors</h4><span itemprop="author">Robert Ardecky</span>,<sup>1</sup> <span itemprop="author">Eduard Sergienko</span>,<sup>1</sup> <span itemprop="author">Jiwen Zou</span>,<sup>1</sup> <span itemprop="author">Santhi Ganji</span>,<sup>1</sup> <span itemprop="author">Brock Brown</span>,<sup>1</sup> <span itemprop="author">Qing Sun</span>,<sup>1</sup> <span itemprop="author">Chen-Ting Ma</span>,<sup>1</sup> <span itemprop="author">Becky Hood</span>,<sup>2</sup> <span itemprop="author">Kevin Nguyen</span>,<sup>2</sup> <span itemprop="author">Stefan Vasile</span>,<sup>2</sup> <span itemprop="author">Eigo Suyama</span>,<sup>2</sup> <span itemprop="author">Arianna Mangravita-Novo</span>,<sup>2</sup> <span itemprop="author">Sumeet Salaniwal</span>,<sup>1</sup> <span itemprop="author">Paul Kung</span>,<sup>1</sup> <span itemprop="author">Layton H. Smith</span>,<sup>2</sup> <span itemprop="author">Thomas D.Y. Chung</span>,<sup>1</sup> <span itemprop="author">Michael R. Jackson</span>,<sup>1</sup> <span itemprop="author">Anthony B. Pinkerton</span>,<sup>1</sup> and <span itemprop="author">Robert Rickert</span><sup>3</sup>.<sup><img src="/corehtml/pmc/pmcgifs/corrauth.gif" alt="corresponding author" /></sup></p><h4>Affiliations</h4><div class="affiliation"><sup>1</sup>
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Sanford-Burnham Center for Chemical Genomics at Sanford-Burnham Medical Research Institute, La Jolla, California 92037, USA</div><div class="affiliation"><sup>2</sup>
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Sanford-Burnham Center for Chemical Genomics at Sanford-Burnham Medical Research Institute, Orlando, Florida 32827, USA</div><div class="affiliation"><sup>3</sup>
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Infectious and Inflammatory Disease Center, Sanford-Burnham Medical Research Institute, La Jolla, California 92037, USA</div><div class="affiliation"><sup><img src="/corehtml/pmc/pmcgifs/corrauth.gif" alt="corresponding author" /></sup>Corresponding author: Anthony B. Pinkerton, Ph.D.
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<span class="before-email-separator"></span><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="gro.mahnrubdrofnas@notreknipa" class="oemail">gro.mahnrubdrofnas@notreknipa</a></div></div><p class="small">Received: <span itemprop="datePublished">April 15, 2014</span>; Last Update: <span itemprop="dateModified">January 16, 2015</span>.</p></div><div class="jig-ncbiinpagenav body-content whole_rhythm" data-jigconfig="allHeadingLevels: ['h2'],smoothScroll: false" itemprop="text"><div id="_abs_rndgid_" itemprop="description"><p>A robust antibody response is essential for efficient identification and eradication of pathogenic microbes and toxins, whereas dysregulation of the antibody response can lead to autoimmune disease. Specific antigen encounter by B lymphocytes induces clonal expansion that encompasses several distinct stages of differentiation. During this differentiation process, a critical cell fate decision is made wherein some B cells will undergo terminal differentiation into antibody-producing cells, while a separate cohort will assume a distinct pathway of differentiation to become long-lived memory B cells. An exciting new development in the field is the revelation of a novel chemotactic axis involving the recognition of oxysterol compounds by the orphan G-protein-coupled receptor (GPCR), Epstein-Barr virus-induced gene 2 (EBI2). EBI2 is expressed on B cells and is highly induced upon activation. Recent gene targeting experiments revealed that <i>EBI2</i><sup>-/-</sup> B cells exhibited defective migration, resulting in strongly impaired T cell-dependent antibody responses. Most recently, two research teams made the unlikely discovery that oxysterol compounds, previously known to bind nuclear receptors, are the physiologic ligands for EBI2. In order to investigate the importance of EBI2 in immune processes and potential as a drug target, selective and potent antagonists with good <i>in vivo</i> pharmacokinetics need to be developed. This probe report describes a potent functional antagonist of EBI-2, <a href="/pcsubstance/?term=ML401[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML401</a> (CID 73169083, <a href="https://pubchem.ncbi.nlm.nih.gov/substance/173333998" ref="pagearea=abstract&targetsite=entrez&targetcat=link&targettype=pubchem">SID 173333998</a>), which is potent (IC<sub>50</sub> ∼ 1 nM), displays activity in a chemotaxis assay (IC<sub>50</sub> ∼ 6 nM), and has a clean profile in a Eurofins/Ricerca panel as well as excellent rodent pharmacokinetics. As such <a href="/pcsubstance/?term=ML401[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML401</a> should be a valuable tool to explore the <i>in vivo</i> effects of EBI-2 inhibition.</p></div><div class="h2"></div><p><b>Assigned Assay Grant #:</b> 1 R03 MH097522-01</p><p><b>Screening Center Name & PI:</b> Sanford-Burnham Medical Research Institute & <i>Michael R. Jackson, Ph.D.</i></p><p><b>Chemistry Center Name & PI:</b> Sanford-Burnham Medical Research Institute & <i>Michael R. Jackson, Ph.D</i></p><p><b>Assay Submitter & Institution:</b> Robert Rickert, Ph.D., Sanford-Burnham Medical Research Institute, La Jolla, California 92037, USA.</p><p><b>PubChem Summary Bioassay Identifier (AID):</b>
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<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651641" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">651641</a></p><div id="ml401.s1"><h2 id="_ml401_s1_">Probe Structure & Characteristics</h2><p>This Center Probe Report describes <a href="/pcsubstance/?term=ML401[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML401</a>, a selective inhibitor of EBI-2. <a href="/pcsubstance/?term=ML401[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML401</a> has a central piperazine core. The chemical structure and data summary are shown in(<a class="figpopup" href="/books/NBK280046/table/ml401.t1/?report=objectonly" target="object" rid-figpopup="figml401t1" rid-ob="figobml401t1">Table 1</a>).</p><div id="ml401.f1" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Chemical%20Structure%20of%20ML401.&p=BOOKS&id=280046_ml401f1.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img src="/books/NBK280046/bin/ml401f1.jpg" alt="Chemical Structure of ML401." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="title">Chemical Structure of ML401</span></h3></div><div id="ml401.t1" class="table"><h3><span class="label">Table 1</span><span class="title">Potency and selectivity characteristics for probe ML401</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK280046/table/ml401.t1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml401.t1_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml401.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CID/ML#</th><th id="hd_h_ml401.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Target Name</th><th id="hd_h_ml401.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">IC<sub>50</sub> (nM)<br />[SID, AID]</th><th id="hd_h_ml401.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Anti-target Name(s)</th><th id="hd_h_ml401.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">IC<sub>50</sub> (μM)<br />[SID, AID]</th><th id="hd_h_ml401.t1_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Fold-Selective</th><th id="hd_h_ml401.t1_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Secondary Assay(s) Names<br />IC<sub>50</sub> (nM)<br />[SID, AID]</th></tr></thead><tbody><tr><td headers="hd_h_ml401.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CID 73169083<br /><a href="/pcsubstance/?term=ML401[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML401</a></td><td headers="hd_h_ml401.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">EBI-2</td><td headers="hd_h_ml401.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">1.03<br />[<a href="https://pubchem.ncbi.nlm.nih.gov/substance/173333998" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 173333998</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743408" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743408</a>]</td><td headers="hd_h_ml401.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><i>Eurofin panel</i></td><td headers="hd_h_ml401.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml401.t1_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml401.t1_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Chemotaxis<br />IC50 = 6.24<br />[<a href="https://pubchem.ncbi.nlm.nih.gov/substance/173333998" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 173333998</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/743410" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 743410</a>]</td></tr></tbody></table></div></div></div><div id="ml401.s2"><h2 id="_ml401_s2_">1. Recommendations for Scientific Use of the Probe</h2><p>A robust antibody response is essential for efficient identification and eradication of pathogenic microbes and toxins, whereas dysregulation of the antibody response can lead to autoimmune disease. Specific antigen encounter by B lymphocytes induces clonal expansion that encompasses several distinct stages of differentiation. The molecular cues that influence this cell fate decision into antibody-producing or long-lived memory B cells is of great importance for understanding vaccine efficacy and how/where B cell differentiation may go awry to cause autoimmunity. In this context, there is a growing appreciation of microenvironment-specific influences on B cell differentiation within the lymphoid tissue. Recently a novel chemotactic axis involving the recognition of oxysterol compounds by the orphan G-protein-coupled receptor (GPCR), Epstein-Barr virus-induced gene 2 (EBI2) (<a class="bk_pop" href="#ml401.r1">1</a>-<a class="bk_pop" href="#ml401.r4">4</a>) was discovered.</p><p>EBI2 is expressed on B cells and is highly induced upon activation (<a class="bk_pop" href="#ml401.r5">5</a>). Recent gene targeting experiments revealed that <i>EBI2</i><sup>-/-</sup> B cells exhibited defective migration, resulting in strongly impaired T cell-dependent antibody responses (<a class="bk_pop" href="#ml401.r1">1</a>, <a class="bk_pop" href="#ml401.r2">2</a>). Most recently, two research teams made the unlikely discovery that oxysterol compounds, previously known to bind nuclear receptors, are the physiologic ligands for EBI2 (<a class="bk_pop" href="#ml401.r3">3</a>, <a class="bk_pop" href="#ml401.r4">4</a>). Although other closely related oxysterols showed some potency and binding capability to EBI2, the most potent endogenous EBI2 receptor ligand and activator was 7α,25-dihydroxycholesterol (7α,25-OHC) (<a class="bk_pop" href="#ml401.r4">4</a>). EBI2<sup>-/-</sup> B cells did not bind 7α,25-OHC and mice deficient for cholesterol 25-hydroxylase that is necessary to generate 7α,25-OHC display a phenotype similar to that of EBI2<sup>-/-</sup> mice (<a class="bk_pop" href="#ml401.r3">3</a>). The recent publication in July 2011 of the deorphanizing of this receptor is a major step forward towards understanding the role of EBI2 in immunobiology. In order to investigate the importance of EBI2 in immune processes and potential as a drug target, selective potent chemical probes that antagonize this receptor need to be identified and made freely available to the research community.</p><p>The proposed work enlists the MLPCN to screen chemical libraries to identify the first small molecule antagonists of EBI2, the probes sought will be identified and optimized based on their ability to antagonize this receptor in functional cellular systems, with the ultimate objective of generating compounds that will function <i>in vivo</i>. Armed with such tools a full examination of the consequences of antagonizing this receptor in well characterized ex vivo murine and human models of the immune system will be possible. Since EBI2 is required for the generation of antibody-producing cells, antagonists would be anticipated to not only blunt antibody production but may also have more broad reaching effects on inflammation. Prospectively, these probes could be used to investigate the role of this receptor in mouse models of autoantibody-dependent inflammatory disease such as rheumatoid arthritis and lupus, or more broadly applied to examine effects on systemic inflammation. Moreover, production of 7α,25-OHC occurs in many cell types and tissues, consistent a broader role in the regulation of inflammation. Of particular interest, studies in rat and human have revealed polymorphisms in the <i>Ebi2</i> promoter that are associated with multiple inflammatory disorders and type I diabetes (<a class="bk_pop" href="#ml401.r6">6</a>).</p><p><b><u>Prior Art:</u></b> A SciFinder search on April 27, 2012 showed one reference disclosing the structure and biological activity of several EBI2/GPR183 inverse agonists from a group at GSK (Benned-Jensen, et. al., Journal of Biological Chemistry, 2011, 29292-29302). The lead structure is shown in <a class="figpopup" href="/books/NBK280046/figure/ml401.f2/?report=objectonly" target="object" rid-figpopup="figml401f2" rid-ob="figobml401f2">Figure 1</a>. Although GSK682753A is quite potent as an inverse agonist on its constitutive endogenous activation, it has very poor microsomal and plasma stability (<i>vide infra</i>) and thus is unsuitable for <i>in vivo</i> proof of concept studies. Several structurally similar compounds are also disclosed in this paper. There are no other reports of EBI2/GPR183 antagonists.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml401f2" co-legend-rid="figlgndml401f2"><a href="/books/NBK280046/figure/ml401.f2/?report=objectonly" target="object" title="Figure 1" class="img_link icnblk_img figpopup" rid-figpopup="figml401f2" rid-ob="figobml401f2"><img class="small-thumb" src="/books/NBK280046/bin/ml401f2.gif" src-large="/books/NBK280046/bin/ml401f2.jpg" alt="Figure 1. EBI2 antagonist GSK682753." /></a><div class="icnblk_cntnt" id="figlgndml401f2"><h4 id="ml401.f2"><a href="/books/NBK280046/figure/ml401.f2/?report=objectonly" target="object" rid-ob="figobml401f2">Figure 1</a></h4><p class="float-caption no_bottom_margin">EBI2 antagonist GSK682753. </p></div></div><p>It is noteworthy that three pharmaceutical companies (Euroscreen, Novartis and Johnson & Johnson) have reported on the identification of 7α,25-OHC as the physiologic ligand of EBI2. Certainly, it would be expected that efforts are underway at these companies to develop EBI2 antagonists. However, compounds that they identify are unlikely to be published and available to academic investigators until intellectual property is secured and this typically creates a lag of 3-4 years, where potentially useful pharmacological tools would be unavailable to the research community, that could be used to elucidate the pharmacology and functional biology of this very newly deorphanzied GPCR. Although a compound functioning as an EBI2 inverse agonist on its constitutive endogenous activation has been reported by GSK (<a class="bk_pop" href="#ml401.r7">7</a>), no EBI2 antagonists that antagonize the binding and/or activation of EBI2 by 7α,25-OHC have been published. Indeed we have requested this compound GSK682753A from GSK, and are still awaiting a response. The antagonists that we identify will become available to the research community in 18 months and thereby will accelerate knowledge and the participation of new researchers on this important new receptor, and which would seed further discoveries of additional chemical probes and validation of their utility. As GPCRs are one of the most “druggable” target classes, we would fully expect that some of the probes arising from the MLPCN will become starting points for the development of potential therapies for immunological and inflammatory diseases.</p></div><div id="ml401.s3"><h2 id="_ml401_s3_">2. Materials and Methods</h2><div id="ml401.s4"><h3>2.1. Assays</h3><p><a class="figpopup" href="/books/NBK280046/table/ml401.t2/?report=objectonly" target="object" rid-figpopup="figml401t2" rid-ob="figobml401t2">Table 2</a> summarizes details for the assays that enabled this probe discovery project. A detailed description of the Primary and all assays can be found in the PubChem AIDs listed.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml401t2"><a href="/books/NBK280046/table/ml401.t2/?report=objectonly" target="object" title="Table 2" class="img_link icnblk_img figpopup" rid-figpopup="figml401t2" rid-ob="figobml401t2"><img class="small-thumb" src="/books/NBK280046/table/ml401.t2/?report=thumb" src-large="/books/NBK280046/table/ml401.t2/?report=previmg" alt="Table 2. Summary of Assays and AIDs." /></a><div class="icnblk_cntnt"><h4 id="ml401.t2"><a href="/books/NBK280046/table/ml401.t2/?report=objectonly" target="object" rid-ob="figobml401t2">Table 2</a></h4><p class="float-caption no_bottom_margin">Summary of Assays and AIDs. </p></div></div></div><div id="ml401.s5"><h3>2.2. Probe Chemical Characterization</h3><p><b>Chemical name of probe compound.</b> The IUPAC name of the probe is (<i>E</i>)-3-(4-bromophenyl)-1-(4-(4-chlorobenzyl)-piperazin-1-yl)prop-2-en-1-one. The actual batch prepared, tested and submitted to the MLSMR is archived as <a href="https://pubchem.ncbi.nlm.nih.gov/substance/173333998" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID173333998</a> corresponding to CID73169083. The probe <a href="/pcsubstance/?term=ML401[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML401</a> does not have any chiral centers.</p><div id="ml401.f3" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%202.%20Structure%20of%20ML401.&p=BOOKS&id=280046_ml401f3.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img src="/books/NBK280046/bin/ml401f3.jpg" alt="Figure 2. Structure of ML401." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 2</span><span class="title">Structure of ML401</span></h3></div><p><b>Solubility and Stability of</b>
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<a href="/pcsubstance/?term=ML401[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML401</a>
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<b>in PBS at room temperature.</b> The stability of <a href="/pcsubstance/?term=ML401[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML401</a> was investigated (<a class="figpopup" href="/books/NBK280046/figure/ml401.f4/?report=objectonly" target="object" rid-figpopup="figml401f4" rid-ob="figobml401f4">Figure 3</a>) in aqueous buffers at room temperature by monitoring the amount of starting <a href="/pcsubstance/?term=ML401[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML401</a> apparently remaining after incubation at room temperature in either PBS (pH 7.4) or 1:1 PBS:acetonitrile (v/v). <a href="/pcsubstance/?term=ML401[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML401</a> was stable in PBS:acetonitrile with 100% remaining after 48 hrs. The apparently lower stability (48.74%) in neat PBS is a reflection of low solubility. Thus, the stability value is artificially low due to compound precipitation <i>vs.</i> degradation. As noted in the <i>Summary of in vitro ADME/T properties</i> (<a href="#ml401.s12">Section 3.3</a>, <a class="figpopup" href="/books/NBK280046/table/ml401.t3/?report=objectonly" target="object" rid-figpopup="figml401t3" rid-ob="figobml401t3">Table 5</a>) <a href="/pcsubstance/?term=ML401[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML401</a> has low solubility (0.76/0.53/0.49 μg/mL) at pH 5.0, 6.2 and 7.4 in pION buffer and comparable solubility in PBS at pH 7.4. While the scaffold structure represented by <a href="/pcsubstance/?term=ML401[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML401</a> contains a Michael acceptor, it shows no reactivity when incubated with 1 mM glutathione for 20 hrs at 37°C in PBS buffer (data not shown, but there is essentially no loss of <a href="/pcsubstance/?term=ML401[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML401</a> or presence of a GSH adduct; the control ethacrynic acid was completely reacted as expected). We also note that the probe and close analogs display excellent plasma and microsomal stability as well as rodent pharmacokinetics and thus it appears that the compounds are not reactive in physiological conditions. <a class="figpopup" href="/books/NBK280046/table/ml401.t4/?report=objectonly" target="object" rid-figpopup="figml401t4" rid-ob="figobml401t4">Table 3</a> summarizes the deposition of the Probe and 5 analogs.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml401f4" co-legend-rid="figlgndml401f4"><a href="/books/NBK280046/figure/ml401.f4/?report=objectonly" target="object" title="Figure 3" class="img_link icnblk_img figpopup" rid-figpopup="figml401f4" rid-ob="figobml401f4"><img class="small-thumb" src="/books/NBK280046/bin/ml401f4.gif" src-large="/books/NBK280046/bin/ml401f4.jpg" alt="Figure 3. Stability of ML401 in 1×." /></a><div class="icnblk_cntnt" id="figlgndml401f4"><h4 id="ml401.f4"><a href="/books/NBK280046/figure/ml401.f4/?report=objectonly" target="object" rid-ob="figobml401f4">Figure 3</a></h4><p class="float-caption no_bottom_margin">Stability of ML401 in 1×. </p></div></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml401t4"><a href="/books/NBK280046/table/ml401.t4/?report=objectonly" target="object" title="Table 3" class="img_link icnblk_img figpopup" rid-figpopup="figml401t4" rid-ob="figobml401t4"><img class="small-thumb" src="/books/NBK280046/table/ml401.t4/?report=thumb" src-large="/books/NBK280046/table/ml401.t4/?report=previmg" alt="Table 3. Probe and Analog Submissions to MLSMR (Evotec) for EBI2inhibitors." /></a><div class="icnblk_cntnt"><h4 id="ml401.t4"><a href="/books/NBK280046/table/ml401.t4/?report=objectonly" target="object" rid-ob="figobml401t4">Table 3</a></h4><p class="float-caption no_bottom_margin">Probe and Analog Submissions to MLSMR (Evotec) for EBI2inhibitors. </p></div></div></div><div id="ml401.s6"><h3>2.3. Probe Preparation</h3><p>This probe is not commercially available. A 27 mg sample of <a href="/pcsubstance/?term=ML401[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML401</a> synthesized at SBCCG according to <a class="figpopup" href="/books/NBK280046/figure/ml401.f5/?report=objectonly" target="object" rid-figpopup="figml401f5" rid-ob="figobml401f5">Scheme 1</a>, has been deposited in the MLSMR (Evotec) (see Probe Submission <a class="figpopup" href="/books/NBK280046/table/ml401.t4/?report=objectonly" target="object" rid-figpopup="figml401t4" rid-ob="figobml401t4">Table 3</a>).</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml401f5" co-legend-rid="figlgndml401f5"><a href="/books/NBK280046/figure/ml401.f5/?report=objectonly" target="object" title="Scheme1" class="img_link icnblk_img figpopup" rid-figpopup="figml401f5" rid-ob="figobml401f5"><img class="small-thumb" src="/books/NBK280046/bin/ml401f5.gif" src-large="/books/NBK280046/bin/ml401f5.jpg" alt="Scheme1. Synthesis of ML401, conditions: a. CDI, THF 2 to 3 hrs at RT (79%); b. DCM, TFA 0 °C; c. 4-Cl benzyl bromide, K2CO3, Acetone, 50°C 2 hours (58%)." /></a><div class="icnblk_cntnt" id="figlgndml401f5"><h4 id="ml401.f5"><a href="/books/NBK280046/figure/ml401.f5/?report=objectonly" target="object" rid-ob="figobml401f5">Scheme1</a></h4><p class="float-caption no_bottom_margin">Synthesis of ML401, conditions: <i>a.</i> CDI, THF 2 to 3 hrs at RT (79%); <i>b.</i> DCM, TFA 0 °C; <i>c.</i> 4-Cl benzyl bromide, K<sub>2</sub>CO<sub>3</sub>, Acetone, 50°C 2 hours (58%). </p></div></div><div id="ml401.s7"><h4>Preparation of (E)-tert-butyl 4-(3-(4-bromophenyl)acryloyl)piperazine-1-carboxylate (3)</h4><div id="ml401.f6" class="figure"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Image%20ml401f6&p=BOOKS&id=280046_ml401f6.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img src="/books/NBK280046/bin/ml401f6.jpg" alt="Image ml401f6" class="tileshop" title="Click on image to zoom" /></a></div></div><p>4-Bromo Cinnamic acid <b>1</b> (200 mg, 0.880 mmol) was dissolved in 10 ml of THF and after the addition of CDI (213.9 mg, 1.3 mmol), the mixture was stirred at room temperature for 30 minutes. Boc-Piperazine <b>2</b> (196.6 mg 1.055 mmol) was added to the resulting mixture and the reaction mixture was stirred for 2 hours at room temperature. When the reaction was determined to be complete by HPLC, the reaction mixture was concentrated under reduced pressure and the resulting solid was dissolved in EtOAc. The solution was washed with saturated aqueous NaHCO<sub>3</sub>, washed with saturated aqueous NaCl and dried over Na<sub>2</sub>SO<sub>4</sub>. The resulting organic layer was concentrated under reduced pressure afforded the title compound <b>3</b> (275 mg, 79%) as a white solid which was used directly into the next step without purification. <sup>1</sup>H NMR (400 MHz, DMSO<i>-d</i><sub>6</sub>) δ 1.42 (s, 9H), 3.75 (m, 4H), 4.45 (m, 2H), 7.87 (d, 2H), 8.15 (d, 2H), MS (EI) m/z 397 (M+1).</p></div><div id="ml401.s8"><h4>Preparation of (E)-3-(4-bromophenyl)-1-(4-2, 2, 2-trifluroacetyl) piprazin-1-yl) prop-2-en-1one (4)</h4><div id="ml401.f7" class="figure"><div class="graphic"><img src="/books/NBK280046/bin/ml401f7.jpg" alt="Image ml401f7" /></div></div><p>(E)-tert-butyl4-(3-(4-bromophenyl)acryloyl)piperazine-1-carboxylate <b>3</b> (200 mg, 0.505 mmol) was dissolved in dichloromethane (3 ml) and cooled to 0 °C using an ice-water bath. Trifluoro acetic acid (1.2 mL) was slowly added to the resulting mixture and the reaction was stirred for one hour at room temperature. When the reaction was determined to be complete by HPLC, the reaction mixture was concentrated under reduced pressure. The resulting mixture was washed with toluene twice to remove excess of TFA and the organic layer was concentrated under reduced pressure to yielded the TFA salt of product <b>4</b> (379 mg), MS (EI) m/z 297 (M+1).</p></div><div id="ml401.s9"><h4>Preparation of (E)-3-(4-bromophenyl)-1-(4-(4-chlorobenzyl)piperazin-1-yl)prop-2-en-1one (5)</h4><div id="ml401.f8" class="figure"><div class="graphic"><img src="/books/NBK280046/bin/ml401f8.jpg" alt="Image ml401f8" /></div></div><p>(E)-3-(4-bromophenyl)-1-(4-2,2,2-trifluroacetyl)piprazin-1-yl)prop-2-en-1one 4 (100 mg, 0.244 mmol) was dissolved in acetone (5 mL), potassium carbonate (112 mg, 0.812 mmol) was added followed by 4-chlorobenzyl bromide (50 mg, 0.244 mmol). The resulting reaction mixture was stirred at 50 °C for about 2 hours. When the reaction was determined to be complete by HPLC, the reaction mixture was cooled to room temperature and concentrated under reduced pressure. The resulting precipitate was dissolved in EtOAc (20 ml). Water (15 mL) was added and the two layers were separated. The aqueous phase was extracted with EtOAc (3 × 20 mL), the collected organic phases were washed with brine (10 mL), dried over Na<sub>2</sub>SO<sub>4</sub> and the solvent was evaporated to give crude product which was purified using HPLC with acetonitrile and water (20:100 gradient) to yield the desired product <b>5</b> (60 mg, 58 %). <sup>1</sup>H NMR (400 MHz, DMSO<i>-d</i><sub>6</sub>) δ 2.35 (d, 4H), 3.49 (s, 2H), 3.55 (s, 2H), 3.68 (s, 2H), 7.35 (m, 6H), 7.58 (d, 2H), 7.66 (d, 2H), <sup>13</sup>C NMR (400 MHz, DMSO<i>-d</i><sub>6</sub>) δ(ppm) 41.8, 45.0, 52.1, 53.1, 60.8, 119.1, 122.7, 128.2, 130.0, 130.7, 131.60, 134.4, 136.9, 140.2, 164.2, MS (EI) m/z 420 (M+1).</p><div id="ml401.f9" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%204A.%201H%20NMR%20Spectrum%20of%20ML401%20(500%20MHz%2C%20CDCl3).&p=BOOKS&id=280046_ml401f9.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img src="/books/NBK280046/bin/ml401f9.jpg" alt="Figure 4A. 1H NMR Spectrum of ML401 (500 MHz, CDCl3)." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 4A</span><span class="title"><sup>1</sup>H NMR Spectrum of ML401 (500 MHz, CDCl<sub>3</sub>)</span></h3></div><div id="ml401.f10" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%204B.%2013C%20NMR%20Spectrum%20of%20ML401%20(125%20MHz%2C%20CDCl3).&p=BOOKS&id=280046_ml401f10.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img src="/books/NBK280046/bin/ml401f10.jpg" alt="Figure 4B. 13C NMR Spectrum of ML401 (125 MHz, CDCl3)." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 4B</span><span class="title"><sup>13</sup>C NMR Spectrum of ML401 (125 MHz, CDCl<sub>3</sub>)</span></h3></div><div id="ml401.f11" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%204C.%20LC%2FMS%20for%20ML401.&p=BOOKS&id=280046_ml401f11.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img src="/books/NBK280046/bin/ml401f11.jpg" alt="Figure 4C. LC/MS for ML401." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 4C</span><span class="title">LC/MS for ML401</span></h3></div><div id="ml401.f12" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%204D.%20MS%20for%20ML401.&p=BOOKS&id=280046_ml401f12.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img src="/books/NBK280046/bin/ml401f12.jpg" alt="Figure 4D. MS for ML401." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 4D</span><span class="title">MS for ML401</span></h3></div></div></div></div><div id="ml401.s10"><h2 id="_ml401_s10_">3. Results</h2><div id="ml401.s11"><h3>3.1 & 3.2. Response Curves for Probe & Cellular Activity</h3><p>The dose response curves of <a href="/pcsubstance/?term=ML401[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML401</a> for antagonism in the β-arrestin and chemotaxis assays are shown in <a class="figpopup" href="/books/NBK280046/figure/ml401.f13/?report=objectonly" target="object" rid-figpopup="figml401f13" rid-ob="figobml401f13">Figure 5</a>. There is only a 5-fold loss of potency in chemotaxis.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml401f13" co-legend-rid="figlgndml401f13"><a href="/books/NBK280046/figure/ml401.f13/?report=objectonly" target="object" title="Figure 5" class="img_link icnblk_img figpopup" rid-figpopup="figml401f13" rid-ob="figobml401f13"><img class="small-thumb" src="/books/NBK280046/bin/ml401f13.gif" src-large="/books/NBK280046/bin/ml401f13.jpg" alt="Figure 5. Potency of ML401 inhibition in EBI2 beta-arrestin and RS11846 cell chemotaxis assays." /></a><div class="icnblk_cntnt" id="figlgndml401f13"><h4 id="ml401.f13"><a href="/books/NBK280046/figure/ml401.f13/?report=objectonly" target="object" rid-ob="figobml401f13">Figure 5</a></h4><p class="float-caption no_bottom_margin">Potency of ML401 inhibition in EBI2 beta-arrestin and RS11846 cell chemotaxis assays. Error bars are standard deviation of quadruplicate runs tested on at least two separate days. </p></div></div></div><div id="ml401.s12"><h3>3.3. Profiling Assays</h3><p>As a <i>pro forma</i> activity, the SBCCG is committed to profiling all final probe compounds and in certain cases key informative analogs in the PanLabs full panel as negotiated by the MLPCN network. Additional commercial profiling services will be considered for funding by SBCCG as deemed appropriate and informative. <a href="/pcsubstance/?term=ML401[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML401</a> was evaluated in a detailed <i>in vitro</i> pharmacology screen as shown in <a class="figpopup" href="/books/NBK280046/table/ml401.t3/?report=objectonly" target="object" rid-figpopup="figml401t3" rid-ob="figobml401t3">Table 5</a>:</p><div id="ml401.t3" class="table"><h3><span class="label">Table 5</span><span class="title">Summary of <i>in vitro</i> ADME Properties of EBI2 inhibitor probe ML401 (MLS-0472527)</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK280046/table/ml401.t3/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml401.t3_lrgtbl__"><table class="no_top_margin"><tbody><tr><td colspan="3" rowspan="1" style="text-align:left;vertical-align:middle;"><b>Aqueous Solubility</b> in pION's buffer (μg/mL) pH 5.0/6.2/7.4</td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">0.76/0.53/0.49</td></tr><tr><td colspan="3" rowspan="1" style="text-align:left;vertical-align:middle;"><b>Aqueous Solubility</b> in 1× PBS, pH 7.4 (μg/mL)</td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">0.04</td></tr><tr><td colspan="3" rowspan="1" style="text-align:left;vertical-align:middle;"><b>Chemical Stability</b> in 1× PBS pH 7.4 / with 50% ACN (% remaining after 48 hrs)</td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">100</td></tr><tr><td colspan="3" rowspan="1" style="text-align:left;vertical-align:middle;"><b>PAMPA Permeability</b>, P<sub>e</sub> (×10<sup>-6</sup> cm/s) Donor pH: 5.0 / 6.2 / 7.4 Acceptor pH: 7.4</td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">1858/1180/1577</td></tr><tr><td colspan="2" rowspan="2" style="text-align:left;vertical-align:middle;"><b>Plasma Protein Binding</b> (% Bound)</td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Human 1 μM / 10 μM</td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">98.89/99.15</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Mouse 1 μM / 10 μM</td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">98.91/99.30</td></tr><tr><td colspan="3" rowspan="1" style="text-align:left;vertical-align:middle;"><b>Plasma Stability</b> (% Remaining at 3 hrs) Human/Mouse</td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">73.55/83.64</td></tr><tr><td colspan="3" rowspan="1" style="text-align:left;vertical-align:middle;"><b>Hepatic Microsome Stability</b> (% Remaining at 1hr) Human/Mouse</td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">51.87/36.56</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;"><b>Cytotoxicity</b></td><td colspan="2" rowspan="1" style="text-align:left;vertical-align:middle;">Fa2N-4 Immortalized Human Hepatocytes LC<sub>50</sub> (μM)</td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">>50</td></tr></tbody></table></div></div><p><a href="/pcsubstance/?term=ML401[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML401</a> achieved modest concentrations 18-20 × IC<sub>50</sub> in aqueous buffer between a pH range of 5.0-7.4. The solubility was comparable in PBS.</p><p>The PAMPA (<b>P</b>arallel <b>A</b>rtificial <b>M</b>embrane <b>P</b>ermeability <b>A</b>ssay) assay is used as an <i>in vitro</i> model of passive, transcellular permeability. An artificial membrane immobilized on a filter is placed between a donor and acceptor compartment. At the start of the test, drug is introduced in the donor compartment. Following the permeation period, the concentration of drug in the donor and acceptor compartments is measured using UV spectroscopy. Consistent with its solubility data, <a href="/pcsubstance/?term=ML401[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML401</a> exhibited excellent permeability across the pH range of the donor compartment.</p><p>Plasma protein binding is a measure of a drug's efficiency to bind to the proteins within blood plasma. The less bound a drug is, the more efficiently it can traverse cell membranes or diffuse. Highly plasma protein bound drugs are confined to the vascular space, thereby having a relatively low volume of distribution. In contrast, drugs that remain largely unbound in plasma are generally available for distribution to other organs and tissues. <a href="/pcsubstance/?term=ML401[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML401</a> was highly plasma protein bound.</p><p>Plasma stability is a measure of the stability of small molecules and peptides in plasma and is an important parameter, which can strongly influence the <i>in vivo</i> efficacy of a test compound. Drug candidates are exposed to enzymatic processes (proteinases, esterases) in plasma, and they can undergo intramolecular rearrangement or bind irreversibly (covalently) to proteins. <a href="/pcsubstance/?term=ML401[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML401</a> showed very good stability in both human plasma and mouse plasma.</p><p>The microsomal stability assay is commonly used to rank compounds according to their metabolic stability. This assay addresses the pharmacologic question of how long the parent compound will remain circulating in plasma within the body. <a href="/pcsubstance/?term=ML401[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML401</a> showed moderate stability in both human and mouse liver microsomes after 1 hour.</p><p><a href="/pcsubstance/?term=ML401[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML401</a> showed no toxicity (>50 μM) towards immortalized Fa2-N4 human hepatocytes and non-cytotoxic in MTT (CellTiter96, Promega) in both LnCap and IMR-32 cells (>50 μM).</p><div id="ml401.s13"><h4>Counterscreening</h4><p>Lastly, we also subjected <a href="/pcsubstance/?term=ML401[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML401</a> to a lead profiling panel, looking at its cross reactivity across a range of enzymes and receptors (<a class="figpopup" href="/books/NBK280046/figure/ml401.f14/?report=objectonly" target="object" rid-figpopup="figml401f14" rid-ob="figobml401f14">Figure 6</a>). <a href="/pcsubstance/?term=ML401[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML401</a> was found to have minimal promiscuity, with no activity on 5 CYPs that were tested. Modest activity (in the high μM range) was found across several targets but this was not viewed as an issue with single digit nM activity on EBI-2.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml401f14" co-legend-rid="figlgndml401f14"><a href="/books/NBK280046/figure/ml401.f14/?report=objectonly" target="object" title="Figure 6" class="img_link icnblk_img figpopup" rid-figpopup="figml401f14" rid-ob="figobml401f14"><img class="small-thumb" src="/books/NBK280046/bin/ml401f14.gif" src-large="/books/NBK280046/bin/ml401f14.jpg" alt="Figure 6. Eurofins Panel for ML401." /></a><div class="icnblk_cntnt" id="figlgndml401f14"><h4 id="ml401.f14"><a href="/books/NBK280046/figure/ml401.f14/?report=objectonly" target="object" rid-ob="figobml401f14">Figure 6</a></h4><p class="float-caption no_bottom_margin">Eurofins Panel for ML401. </p></div></div></div></div></div><div id="ml401.s14"><h2 id="_ml401_s14_">4. Discussion</h2><div id="ml401.s15"><h3>4.1. Comparison to existing art and how the new probe is an improvement</h3><p>The only disclosed EBI-2 antagonists are three compounds described in a paper from GSK<sup>7</sup> However, in our laboratories all of the compounds from the paper displayed very poor microsomal and plasma stability (<5% remaining after 1 hr incubation in both human and mouse plasma and microsomes). Thus, a compound that allows for the interrogation of the <i>in vivo</i> effects of EBI-2 antagonism is an important advance.</p></div></div><div id="ml401.s16"><h2 id="_ml401_s16_">5. References</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="ml401.r1">Gatto D, Paus D, Basten A, Mackay CR, Brink R. Guidance of B cells by the orphan G protein-coupled receptor EBI2 shapes humoral immune responses. <span><span class="ref-journal">Immunity. </span>2009;<span class="ref-vol">31</span>(2):259–69.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/19615922" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19615922</span></a>]</div></dd><dt>2.</dt><dd><div class="bk_ref" id="ml401.r2">Pereira JP, Kelly LM, Xu Y, Cyster JG. EBI2 mediates B cell segregation between the outer and centre follicle. <span><span class="ref-journal">Nature. </span>2009;<span class="ref-vol">460</span>(7259):1122–6.</span> [<a href="/pmc/articles/PMC2809436/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2809436</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/19597478" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19597478</span></a>]</div></dd><dt>3.</dt><dd><div class="bk_ref" id="ml401.r3">Hannedouche S, Zhang J, Yi T, Shen W, Nguyen D, Pereira JP, et al. Oxysterols direct immune cell migration via EBI2. <span><span class="ref-journal">Nature. </span>2011;<span class="ref-vol">475</span>(7357):524–7.</span> [<a href="/pmc/articles/PMC4297623/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC4297623</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/21796212" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 21796212</span></a>]</div></dd><dt>4.</dt><dd><div class="bk_ref" id="ml401.r4">Liu C, Yang XV, Wu J, Kuei C, Mani NS, Zhang L, et al. Oxysterols direct B-cell migration through EBI2. <span><span class="ref-journal">Nature. </span>2011;<span class="ref-vol">475</span>(7357):519–23.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/21796211" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 21796211</span></a>]</div></dd><dt>5.</dt><dd><div class="bk_ref" id="ml401.r5">Chan TD, Gardam S, Gatto D, Turner VM, Silke J, Brink R. <em>In vivo</em> control of B-cell survival and antigen-specific B-cell responses. <span><span class="ref-journal">Immunological Reviews. </span>2010;<span class="ref-vol">237</span>(1):90–103.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/20727031" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 20727031</span></a>]</div></dd><dt>6.</dt><dd><div class="bk_ref" id="ml401.r6">Heinig M, Petretto E, Wallace C, Bottolo L, Rotival M, Lu H, et al. A trans-acting locus regulates an anti-viral expression network and type 1 diabetes risk. <span><span class="ref-journal">Nature. </span>2010;<span class="ref-vol">467</span>(7314):460–4.</span> [<a href="/pmc/articles/PMC3657719/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3657719</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/20827270" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 20827270</span></a>]</div></dd><dt>7.</dt><dd><div class="bk_ref" id="ml401.r7">Benned-Jensen T, Smethurst C, Holst PJ, Page KR, Sauls H, Sivertsen B, et al. Ligand Modulation of the Epstein-Barr Virus-induced Seven-transmembrane Receptor EBI2: IDENTIFICATION OF A POTENT AND EFFICACIOUS INVERSE AGONIST. <span><span class="ref-journal">The Journal of Biological Chemistry. </span>2011;<span class="ref-vol">286</span>(33):29292–302.</span> [<a href="/pmc/articles/PMC3190735/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3190735</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/21673108" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 21673108</span></a>]</div></dd><dt>8.</dt><dd><div class="bk_ref" id="ml401.r8">Lapinsh M, Gutcaits A, Prusis P, Post C, Lundstedt T, Wikberg JE. Classification of G-protein coupled receptors by alignment-independent extraction of principal chemical properties of primary amino acid sequences. <span><span class="ref-journal">Protein science : a publication of the Protein Society. </span>2002;<span class="ref-vol">11</span>(4):795–805.</span> [<a href="/pmc/articles/PMC2373523/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2373523</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/11910023" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 11910023</span></a>]</div></dd><dt>9.</dt><dd><div class="bk_ref" id="ml401.r9">Birkenbach M, Josefsen K, Yalamanchili R, Lenoir G, Kieff E. Epstein-Barr virus-induced genes: first lymphocyte-specific G protein-coupled peptide receptors. <span><span class="ref-journal">Journal of Virology. </span>1993;<span class="ref-vol">67</span>(4):2209–20.</span> [<a href="/pmc/articles/PMC240341/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC240341</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/8383238" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 8383238</span></a>]</div></dd><dt>10.</dt><dd><div class="bk_ref" id="ml401.r10">Joost P, Methner A. Phylogenetic analysis of 277 human G-protein-coupled receptors as a tool for the prediction of orphan receptor ligands. <span><span class="ref-journal">Genome biology. </span>2002;<span class="ref-vol">3</span>(11):RESEARCH0063.</span> [<a href="/pmc/articles/PMC133447/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC133447</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/12429062" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 12429062</span></a>]</div></dd><dt>11.</dt><dd><div class="bk_ref" id="ml401.r11">Yoshida R, Imai T, Hieshima K, Kusuda J, Baba M, Kitaura M, et al. Molecular cloning of a novel human CC chemokine EBI1-ligand chemokine that is a specific functional ligand for EBI1, CCR7. <span><span class="ref-journal">The Journal of Biological Chemistry. </span>1997;<span class="ref-vol">272</span>(21):13803–9.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/9153236" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 9153236</span></a>]</div></dd><dt>12.</dt><dd><div class="bk_ref" id="ml401.r12">Norregaard K, Benned-Jensen T, Rosenkilde MM. EBI2, GPR18 and GPR17--three structurally related, but biologically distinct 7TM receptors. <span><span class="ref-journal">Curr Top Med Chem. </span>2011;<span class="ref-vol">11</span>(6):618–28.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/21261596" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 21261596</span></a>]</div></dd></dl></div><div id="bk_toc_contnr"></div></div></div>
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<div xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance"></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Views</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="PDF_download" id="Shutter"></a></div><div class="portlet_content"><ul xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="simple-list"><li><a href="/books/NBK280046/?report=reader">PubReader</a></li><li><a href="/books/NBK280046/?report=printable">Print View</a></li><li><a data-jig="ncbidialog" href="#_ncbi_dlg_citbx_NBK280046" data-jigconfig="width:400,modal:true">Cite this Page</a><div id="_ncbi_dlg_citbx_NBK280046" style="display:none" title="Cite this Page"><div class="bk_tt">Ardecky R, Sergienko E, Zou J, et al. Functional Antagonists of EBI-2. 2014 Apr 15 [Updated 2015 Jan 16]. In: Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-. <span class="bk_cite_avail"></span></div></div></li></ul></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>In this Page</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="page-toc" id="Shutter"></a></div><div class="portlet_content"><ul xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="simple-list"><li><a href="#ml401.s1" ref="log$=inpage&link_id=inpage">Probe Structure & Characteristics</a></li><li><a href="#ml401.s2" ref="log$=inpage&link_id=inpage">Recommendations for Scientific Use of the Probe</a></li><li><a href="#ml401.s3" ref="log$=inpage&link_id=inpage">Materials and Methods</a></li><li><a href="#ml401.s10" ref="log$=inpage&link_id=inpage">Results</a></li><li><a href="#ml401.s14" ref="log$=inpage&link_id=inpage">Discussion</a></li><li><a href="#ml401.s16" ref="log$=inpage&link_id=inpage">References</a></li></ul></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Related information</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="discovery_db_links" id="Shutter"></a></div><div class="portlet_content"><ul><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pmc&DbFrom=books&Cmd=Link&LinkName=books_pmc_refs&IdsFromResult=3412086" ref="log$=recordlinks">PMC</a><div class="brieflinkpop offscreen_noflow">PubMed Central citations</div></li><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pcsubstance&DbFrom=books&Cmd=Link&LinkName=books_pcsubstance&IdsFromResult=3412086" ref="log$=recordlinks">PubChem Substance</a><div class="brieflinkpop offscreen_noflow">Related PubChem Substances</div></li><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pubmed&DbFrom=books&Cmd=Link&LinkName=books_pubmed_refs&IdsFromResult=3412086" ref="log$=recordlinks">PubMed</a><div class="brieflinkpop offscreen_noflow">Links to PubMed</div></li></ul></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Similar articles in PubMed</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="PBooksDiscovery_RA" id="Shutter"></a></div><div class="portlet_content"><ul><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/23481574" ref="ordinalpos=1&linkpos=1&log$=relatedreviews&logdbfrom=pubmed"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> B cell localization: regulation by EBI2 and its oxysterol ligand.</a><span class="source">[Trends Immunol. 2013]</span><div class="brieflinkpop offscreen_noflow"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> B cell localization: regulation by EBI2 and its oxysterol ligand.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Gatto D, Brink R. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Trends Immunol. 2013 Jul; 34(7):336-41. Epub 2013 Mar 5.</em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/24810762" ref="ordinalpos=1&linkpos=2&log$=relatedreviews&logdbfrom=pubmed"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> Oxysterol-EBI2 signaling in immune regulation and viral infection.</a><span class="source">[Eur J Immunol. 2014]</span><div class="brieflinkpop offscreen_noflow"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> Oxysterol-EBI2 signaling in immune regulation and viral infection.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Daugvilaite V, Arfelt KN, Benned-Jensen T, Sailer AW, Rosenkilde MM. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Eur J Immunol. 2014 Jul; 44(7):1904-12. Epub 2014 Jun 20.</em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/22930711" ref="ordinalpos=1&linkpos=3&log$=relatedarticles&logdbfrom=pubmed">Identification of structural motifs critical for epstein-barr virus-induced molecule 2 function and homology modeling of the ligand docking site.</a><span class="source">[Mol Pharmacol. 2012]</span><div class="brieflinkpop offscreen_noflow">Identification of structural motifs critical for epstein-barr virus-induced molecule 2 function and homology modeling of the ligand docking site.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Zhang L, Shih AY, Yang XV, Kuei C, Wu J, Deng X, Mani NS, Mirzadegan T, Sun S, Lovenberg TW, et al. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Mol Pharmacol. 2012 Dec; 82(6):1094-103. Epub 2012 Aug 28.</em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/24678947" ref="ordinalpos=1&linkpos=4&log$=relatedarticles&logdbfrom=pubmed">Identification and characterization of small molecule modulators of the Epstein-Barr virus-induced gene 2 (EBI2) receptor.</a><span class="source">[J Med Chem. 2014]</span><div class="brieflinkpop offscreen_noflow">Identification and characterization of small molecule modulators of the Epstein-Barr virus-induced gene 2 (EBI2) receptor.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Gessier F, Preuss I, Yin H, Rosenkilde MM, Laurent S, Endres R, Chen YA, Marsilje TH, Seuwen K, Nguyen DG, et al. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">J Med Chem. 2014 Apr 24; 57(8):3358-68. 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