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<meta name="ncbi_db" content="books" /><meta name="ncbi_pdid" content="book-part" /><meta name="ncbi_acc" content="NBK158948" /><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="/NBK158948/" /><meta name="ncbi_pagename" content="Probing the CXCR6/CXCL16 Axis: Targeting Prevention of Prostate Cancer Metastasis - 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>Probing the CXCR6/CXCL16 Axis: Targeting Prevention of Prostate Cancer Metastasis - 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="Probing the CXCR6/CXCL16 Axis: Targeting Prevention of Prostate Cancer Metastasis" /><meta name="citation_publisher" content="National Center for Biotechnology Information (US)" /><meta name="citation_date" content="2014/05/13" /><meta name="citation_author" content="Paul M. Hershberger" /><meta name="citation_author" content="Satyamaheshwar Peddibhotla" /><meta name="citation_author" content="Eliot Sugarman" /><meta name="citation_author" content="Patrick Maloney" /><meta name="citation_author" content="Danielle Key" /><meta name="citation_author" content="Eigo Suyama" /><meta name="citation_author" content="Kevin Nguyen" /><meta name="citation_author" content="Stefan Vasile" /><meta name="citation_author" content="Martha Kraft" /><meta name="citation_author" content="Derek Stonich" /><meta name="citation_author" content="Arianna Mangravita-Novo" /><meta name="citation_author" content="Michael Vicchiarelli" /><meta name="citation_author" content="Palak Gosalia" /><meta name="citation_author" content="Monica Milewski" /><meta name="citation_author" content="Linda Li" /><meta name="citation_author" content="Michael Hedrick" /><meta name="citation_author" content="Qing Sun" /><meta name="citation_author" content="Eduard Sergienko" /><meta name="citation_author" content="Anton Cheltsov" /><meta name="citation_author" content="Sumeet Salanawil" /><meta name="citation_author" content="Jena Diwan" /><meta name="citation_author" content="Layton H. Smith" /><meta name="citation_author" content="Russell S. Taichman" /><meta name="citation_author" content="Thomas D.Y. Chung" /><meta name="citation_author" content="Anthony B. Pinkerton" /><meta name="citation_author" content="Siobhan Malany" /><meta name="citation_author" content="Gregory P. Roth" /><meta name="citation_pmid" content="24049849" /><meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK158948/" /><link rel="schema.DC" href="http://purl.org/DC/elements/1.0/" /><meta name="DC.Title" content="Probing the CXCR6/CXCL16 Axis: Targeting Prevention of Prostate Cancer Metastasis" /><meta name="DC.Type" content="Text" /><meta name="DC.Publisher" content="National Center for Biotechnology Information (US)" /><meta name="DC.Contributor" content="Paul M. Hershberger" /><meta name="DC.Contributor" content="Satyamaheshwar Peddibhotla" /><meta name="DC.Contributor" content="Eliot Sugarman" /><meta name="DC.Contributor" content="Patrick Maloney" /><meta name="DC.Contributor" content="Danielle Key" /><meta name="DC.Contributor" content="Eigo Suyama" /><meta name="DC.Contributor" content="Kevin Nguyen" /><meta name="DC.Contributor" content="Stefan Vasile" /><meta name="DC.Contributor" content="Martha Kraft" /><meta name="DC.Contributor" content="Derek Stonich" /><meta name="DC.Contributor" content="Arianna Mangravita-Novo" /><meta name="DC.Contributor" content="Michael Vicchiarelli" /><meta name="DC.Contributor" content="Palak Gosalia" /><meta name="DC.Contributor" content="Monica Milewski" /><meta name="DC.Contributor" content="Linda Li" /><meta name="DC.Contributor" content="Michael Hedrick" /><meta name="DC.Contributor" content="Qing Sun" /><meta name="DC.Contributor" content="Eduard Sergienko" /><meta name="DC.Contributor" content="Anton Cheltsov" /><meta name="DC.Contributor" content="Sumeet Salanawil" /><meta name="DC.Contributor" content="Jena Diwan" /><meta name="DC.Contributor" content="Layton H. Smith" /><meta name="DC.Contributor" content="Russell S. Taichman" /><meta name="DC.Contributor" content="Thomas D.Y. Chung" /><meta name="DC.Contributor" content="Anthony B. Pinkerton" /><meta name="DC.Contributor" content="Siobhan Malany" /><meta name="DC.Contributor" content="Gregory P. Roth" /><meta name="DC.Date" content="2014/05/13" /><meta name="DC.Identifier" content="https://www.ncbi.nlm.nih.gov/books/NBK158948/" /><meta name="description" content="Prostate cancer (PCa) has been cited as the second leading cause of cancer-related death in American men and its morbidity/mortality has increased globally in recent years. The high mortality rate is closely associated with the spread of malignant cells to various tissues especially to bone. Nearly 10% of patients whose conditions are diagnosed as PCa clinically present with bone metastasis and almost all patients who die of prostate cancer have skeletal involvement. Identifying new mechanisms that control bone metastasis is of great consequence to facilitate the design of therapeutics aimed at decreasing metastatic risk and/or resulting secondary complications. We completed an interrogation of the Molecular Libraries Small Molecule Repository (MLSMR) for antagonists of the CXCR6 receptor in cell-based functional assay. Here, we report for the first time a novel potent (140 nM IC50) small molecule CXCR6 antagonist that is selective (>79 μM IC50) against CXCR5, CXCR4, CCR6 and APJ receptors. It is non-promiscuous against 23 other G protein-coupled receptors (GPCRs), showing only moderate activity against 5-HT2B and DAT at 10 μM in competitive binding assays. This probe will assist in addressing a key hypothesis that the CXCR6/CXCL16 axis significantly contributes to PCa cell metastasis, proliferation and subsequent bone invasion. A small molecule antagonist would block cancer cell trafficking; hence mediate a metastatic event and disease progression. Access to pharmacologically available small molecule antagonists will ultimately enable our targeted studies in disease relevant models and allow for a more seamless translational advancement toward clinical applications." /><meta name="og:title" content="Probing the CXCR6/CXCL16 Axis: Targeting Prevention of Prostate Cancer Metastasis" /><meta name="og:type" content="book" /><meta name="og:description" content="Prostate cancer (PCa) has been cited as the second leading cause of cancer-related death in American men and its morbidity/mortality has increased globally in recent years. The high mortality rate is closely associated with the spread of malignant cells to various tissues especially to bone. Nearly 10% of patients whose conditions are diagnosed as PCa clinically present with bone metastasis and almost all patients who die of prostate cancer have skeletal involvement. Identifying new mechanisms that control bone metastasis is of great consequence to facilitate the design of therapeutics aimed at decreasing metastatic risk and/or resulting secondary complications. We completed an interrogation of the Molecular Libraries Small Molecule Repository (MLSMR) for antagonists of the CXCR6 receptor in cell-based functional assay. Here, we report for the first time a novel potent (140 nM IC50) small molecule CXCR6 antagonist that is selective (>79 μM IC50) against CXCR5, CXCR4, CCR6 and APJ receptors. It is non-promiscuous against 23 other G protein-coupled receptors (GPCRs), showing only moderate activity against 5-HT2B and DAT at 10 μM in competitive binding assays. This probe will assist in addressing a key hypothesis that the CXCR6/CXCL16 axis significantly contributes to PCa cell metastasis, proliferation and subsequent bone invasion. A small molecule antagonist would block cancer cell trafficking; hence mediate a metastatic event and disease progression. Access to pharmacologically available small molecule antagonists will ultimately enable our targeted studies in disease relevant models and allow for a more seamless translational advancement toward clinical applications." /><meta name="og:url" content="https://www.ncbi.nlm.nih.gov/books/NBK158948/" /><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/ml339/" /><link rel="canonical" href="https://www.ncbi.nlm.nih.gov/books/NBK158948/" /><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="#__NBK158948_dtls__">Show details</a><div style="display:none" class="ui-widget" id="__NBK158948_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/ml340/" title="Previous page in this title">< Prev</a><a class="active page_link next" href="/books/n/mlprobe/ml338/" 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="_NBK158948_"><span class="title" itemprop="name">Probing the CXCR6/CXCL16 Axis: Targeting Prevention of Prostate Cancer Metastasis</span></h1><p class="contrib-group"><span itemprop="author">Paul M. Hershberger</span>, <span itemprop="author">Satyamaheshwar Peddibhotla</span>, <span itemprop="author">Eliot Sugarman</span>, <span itemprop="author">Patrick Maloney</span>, <span itemprop="author">Danielle Key</span>, <span itemprop="author">Eigo Suyama</span>, <span itemprop="author">Kevin Nguyen</span>, <span itemprop="author">Stefan Vasile</span>, <span itemprop="author">Martha Kraft</span>, <span itemprop="author">Derek Stonich</span>, <span itemprop="author">Arianna Mangravita-Novo</span>, <span itemprop="author">Michael Vicchiarelli</span>, <span itemprop="author">Palak Gosalia</span>, <span itemprop="author">Monica Milewski</span>, <span itemprop="author">Linda Li</span>, <span itemprop="author">Michael Hedrick</span>, <span itemprop="author">Qing Sun</span>, <span itemprop="author">Eduard Sergienko</span>, <span itemprop="author">Anton Cheltsov</span>, <span itemprop="author">Sumeet Salanawil</span>, <span itemprop="author">Jena Diwan</span>, <span itemprop="author">Layton H. Smith</span>, <span itemprop="author">Russell S. Taichman</span>, <span itemprop="author">Thomas D.Y. Chung</span>, <span itemprop="author">Anthony B. Pinkerton</span>, <span itemprop="author">Siobhan Malany</span>, and <span itemprop="author">Gregory P. Roth</span>.</p><a data-jig="ncbitoggler" href="#__NBK158948_ai__" style="border:0;text-decoration:none">Author Information and Affiliations</a><div style="display:none" class="ui-widget" id="__NBK158948_ai__"><p class="contrib-group"><h4>Authors</h4><span itemprop="author">Paul M. Hershberger</span>,<sup>1</sup> <span itemprop="author">Satyamaheshwar Peddibhotla</span>,<sup>1</sup> <span itemprop="author">Eliot Sugarman</span>,<sup>1</sup> <span itemprop="author">Patrick Maloney</span>,<sup>1</sup> <span itemprop="author">Danielle Key</span>, <span itemprop="author">Eigo Suyama</span>,<sup>1</sup> <span itemprop="author">Kevin Nguyen</span>,<sup>1</sup> <span itemprop="author">Stefan Vasile</span>,<sup>1</sup> <span itemprop="author">Martha Kraft</span>,<sup>1</sup> <span itemprop="author">Derek Stonich</span>,<sup>2</sup> <span itemprop="author">Arianna Mangravita-Novo</span>,<sup>1</sup> <span itemprop="author">Michael Vicchiarelli</span>,<sup>1</sup> <span itemprop="author">Palak Gosalia</span>,<sup>2</sup> <span itemprop="author">Monica Milewski</span>,<sup>2</sup> <span itemprop="author">Linda Li</span>,<sup>2</sup> <span itemprop="author">Michael Hedrick</span>,<sup>2</sup> <span itemprop="author">Qing Sun</span>,<sup>2</sup> <span itemprop="author">Eduard Sergienko</span>,<sup>2</sup> <span itemprop="author">Anton Cheltsov</span>, <span itemprop="author">Sumeet Salanawil</span>,<sup>2</sup> <span itemprop="author">Jena Diwan</span>,<sup>2</sup> <span itemprop="author">Layton H. Smith</span>,<sup>1</sup> <span itemprop="author">Russell S. Taichman</span>,<sup>3</sup> <span itemprop="author">Thomas D.Y. Chung</span>,<sup>2</sup> <span itemprop="author">Anthony B. Pinkerton</span>,<sup>2</sup> <span itemprop="author">Siobhan Malany</span>,<sup>1</sup> and <span itemprop="author">Gregory P. Roth</span><sup>1</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, Orlando, Florida 32827, USA.</div><div class="affiliation"><sup>2</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>3</sup>
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University of Michigan, Dept. of Periodontics & Oral Medicine, Ann Arbor, MI 49109, USA</div><div class="affiliation"><sup><img src="/corehtml/pmc/pmcgifs/corrauth.gif" alt="corresponding author" /></sup>Corresponding author: Siobhan Malany, 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@ynalams" class="oemail">gro.mahnrubdrofnas@ynalams</a></div></div><p class="small">Received: <span itemprop="datePublished">December 15, 2012</span>; Last Update: <span itemprop="dateModified">May 13, 2014</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>Prostate cancer (PCa) has been cited as the second leading cause of cancer-related death in American men and its morbidity/mortality has increased globally in recent years. The high mortality rate is closely associated with the spread of malignant cells to various tissues especially to bone. Nearly 10% of patients whose conditions are diagnosed as PCa clinically present with bone metastasis and almost all patients who die of prostate cancer have skeletal involvement. Identifying new mechanisms that control bone metastasis is of great consequence to facilitate the design of therapeutics aimed at decreasing metastatic risk and/or resulting secondary complications. We completed an interrogation of the Molecular Libraries Small Molecule Repository (MLSMR) for antagonists of the CXCR6 receptor in cell-based functional assay. Here, we report for the first time a novel potent (140 nM IC<sub>50</sub>) small molecule CXCR6 antagonist that is selective (>79 μM IC<sub>50</sub>) against CXCR5, CXCR4, CCR6 and APJ receptors. It is non-promiscuous against 23 other G protein-coupled receptors (GPCRs), showing only moderate activity against 5-HT2B and DAT at 10 μM in competitive binding assays. This probe will assist in addressing a key hypothesis that the CXCR6/CXCL16 axis significantly contributes to PCa cell metastasis, proliferation and subsequent bone invasion. A small molecule antagonist would block cancer cell trafficking; hence mediate a metastatic event and disease progression. Access to pharmacologically available small molecule antagonists will ultimately enable our targeted studies in disease relevant models and allow for a more seamless translational advancement toward clinical applications.</p></div><div class="h2"></div><p><b>Assigned Assay Grant #:</b> 1R03MH095589</p><p><b>Screening Center Name & PI:</b> Sanford-Burnham Medical Research Institute & John C. Reed, M.D., Ph.D.</p><p><b>Chemistry Center Name & PI:</b> Sanford-Burnham Medical Research Institute & John C. Reed, M.D., Ph.D.</p><p><b>Assay Submitter & Institution:</b> Gregory P. Roth, Ph.D., Sanford-Burnham Medical Research Institute Russell S. Taichman, Ph.D., University of Michigan (<i>Co-investigator</i>)</p><p><b>PubChem Summary Bioassay Identifier (AID):</b>
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<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602249" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">602249</a></p><div id="ml339.s1"><h2 id="_ml339_s1_">Probe Structure & Characteristics</h2><p>This Center Probe Report describes <a href="/pcsubstance/?term=ML339[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML339</a><b>,</b> a selective inhibitor of CXCR6. <a href="/pcsubstance/?term=ML339[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML339</a> has a [3.3.1] azabicyclononane chemical scaffold, with an amide substituent in the <i>exo</i>-orientation. The chemical structure and data summary (<a class="figpopup" href="/books/NBK158948/table/ml339.t1/?report=objectonly" target="object" rid-figpopup="figml339t1" rid-ob="figobml339t1">Table 1</a>).</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml339t1"><a href="/books/NBK158948/table/ml339.t1/?report=objectonly" target="object" title="Table 1" class="img_link icnblk_img figpopup" rid-figpopup="figml339t1" rid-ob="figobml339t1"><img class="small-thumb" src="/books/NBK158948/table/ml339.t1/?report=thumb" src-large="/books/NBK158948/table/ml339.t1/?report=previmg" alt="Table 1. Potency and selectivity characteristics for probe ML339 for antagonist activity." /></a><div class="icnblk_cntnt"><h4 id="ml339.t1"><a href="/books/NBK158948/table/ml339.t1/?report=objectonly" target="object" rid-ob="figobml339t1">Table 1</a></h4><p class="float-caption no_bottom_margin">Potency and selectivity characteristics for probe <i>ML339</i> for antagonist activity. </p></div></div><div id="ml339.fu1" class="figure bk_fig"><div class="graphic"><img src="/books/NBK158948/bin/ml339fu1.jpg" alt="Chemical Structure of ML339." /></div><h3><span class="title">Chemical Structure of <b>ML339</b></span></h3></div></div><div id="ml339.s2"><h2 id="_ml339_s2_">Recommendations for scientific use of the probe</h2><p>A selective antagonist of the CXCR6 receptor holds potential to advance the field of prostate cancer research. We anticipate that the probe will be used by our team and future investigators as a first small molecule pharmacological tool for investigating the precise role of the CXCR6/CXCL16 axis in PCa related metastatic and subsequent proliferative events <i>in vitro</i>.</p></div><div id="ml339.s3"><h2 id="_ml339_s3_">1. Introduction</h2><div id="ml339.s4"><h3>Specific Aims (<i>verbatim from original R03 application and status</i>)</h3><p><b>Aim 1</b> (Conduct a MLPCN Library Screen): We will complete a cell-based hCXCR6 HTS campaign to seek functional receptor antagonists.</p><ul><li class="half_rhythm"><div>We have completed all primary and secondary assay development and conducted a LOPAC1280 pilot screen, with good Z′ and S/B values. We have defined a suitable critical path plan for Probe development. We will assist the Sanford-Burnham MLPCN Center in reconfiguring the cell-based critical path functional assay parameters (primary and secondary) from 384 to 1536 well format. All cell lines are available in the assay provider’s lab and will be transferred to the MLPCN center under the standard NIH material transfer agreement. <i>This Aim has been completed.</i></div></li><li class="half_rhythm"><div>We will screen the MLSMR collection of 330,600 compounds according to the critical path workflow scheme and submit the assay protocol (AIDs) and screening data to PubChem in a timely manner. <i>The HTS campaign was complete and data loaded into PubChem – This Aim has been completed</i>.</div></li><li class="half_rhythm"><div>We will register our natural product extracts in PubChem and screen our library of 10,000 natural product extracts and peak fractions to expand novel chemical space outside of typical compounds available within the MLSMR. This will assure identification of suitable probe molecules after assay guided fractionation, dereplication, and isolation/structural identification of active components. <i>Please note, we were not able to register extracts in PubChem given CID and SID limitations. Efforts in exploring the natural product lead extract are ongoing and are outside of the MLPCN effort</i>.</div></li></ul><p><b>Aim 2</b> (Develop Probe Characterization Tertiary Assays): We will develop and validate hCXCR6 probe characterization assays.</p><ul><li class="half_rhythm"><div>We will develop new, and optimize existing, tertiary probe characterization assays including a membrane filtration radioligand binding assay (96 well), a Gαi coupled mechanism hCXCR6-based cAMP assay (96 well format), and <i>in vitro</i> chemotaxis and invasion assays using human and murine prostate cancer cells (LNCaP, LNCaP C4-2B, PC3, VCaP, RM1; 24 well format). <i>Assays have been developed; however, the testing of compounds in these assays are post-probe characterization as listed in the planned future studies section on p 17.</i></div></li><li class="half_rhythm"><div>Demonstrate assay consistency and robustness using an available anti-CXCL16 antibody as a positive antagonist control. <i>This has been completed</i>.</div></li></ul><p><b>Aim 3</b> (Characterize Probes): We will fully characterize resulting MLPCN hCXCR6 Probe(s) in the tertiary assays.</p><ul><li class="half_rhythm"><div>Probe candidate and key SAR-related molecules identified in Aim 1 will be evaluated and characterized utilizing the mechanistic assays developed and optimized in Aim 2. <i>All post-probe assay development has been completed as defined in the probe CPDP and related documents on file at the NIH. Given the fact that such assays are ‘post-probe’ characterization activities, this aspect of the program should be considered as ‘in progress’</i>.</div></li></ul><p>The approach described by these Specific Aims allowed for the successful screening and subsequent identification of novel small molecule hCXCR6 Probes. It is our expectation that validated and effective hCXCR6 chemokine receptor modulators will emerge, resulting in the downstream development of potential therapeutics for prostate cancer. Both orthosteric and allosteric receptor antagonists can be identified within this platform.</p></div><div id="ml339.s5"><h3>Background and Significance</h3><p>We have recently generated experimental data providing validation that inhibition of the CXCR6/CXCL16 axis may have therapeutic benefit in modulating prostate cancer (PCa) cell proliferation and metastasis with attending bone invasiveness. In addition, a robust screening platform suitable for HTS and secondary characterization has been developed.</p><p>PCa is the second leading cause of cancer death in American men and its morbidity has increased globally in recent years. The high mortality rate is closely associated with the spread of malignant cells to various tissues including bone. Nearly 10% of patients whose conditions are diagnosed as PCa initially present with bone metastasis and almost all patients who die of prostate cancers have skeletal involvement.<a class="bk_pop" href="#ml339.r1">1</a>–<a class="bk_pop" href="#ml339.r12">12</a> Identifying new mechanisms that control bone metastasis is of great consequence to facilitate the design of therapeutics aimed at decreasing metastatic risk and/or its complications. To address this unmet medical need, our team is actively engaged in exploring the chemical biology, medicinal chemistry, and therapeutic significance of modulating tumor cell trafficking and metastasis via chemokine receptor inhibition. The primary objective of this proposal is to use high throughput screening methods to identify small molecule antagonist probes that selectively inhibit CXCR6. Our team intends to address and explore a key hypothesis: The CXCR6/CXCL16 axis significantly contributes to PCa cell metastasis and subsequent bone invasion.<a class="bk_pop" href="#ml339.r13">13</a>–<a class="bk_pop" href="#ml339.r15">15</a> A small molecule antagonist would block cancer cell trafficking; hence mediate a metastatic event and disease progression to bone. Thus, access to pharmacologically available small molecule antagonists will ultimately enable our studies in disease relevant models and allow for a more seamless translational advance to clinical applications.</p><p>Although an understanding regarding the role of chemokines that regulate cell movement in the spleen and lymphatic system has emerged, considerably less is understood regarding the role of these protein ligands and their corresponding cellular receptors particularly in cancer metastasis, proliferation and secondary tissue invasiveness. To date, evidence is available that supports the role of the hCXCR4/CXCL12 axis in cancer progression and there are currently clinical studies targeting antagonism of the hCXCR4 receptor with a selective small molecule compound for a variety of metastatic events including PCa.<a class="bk_pop" href="#ml339.r16">16</a>–<a class="bk_pop" href="#ml339.r18">18</a> The CXCR4/CXCL12 axis only partially blocks metastatic behavior <i>in vitro</i>, and little is known about the relationship between PCa specific metastasis and other chemokine systems. Within the bone microenvironment hCXCR4 and CXCL12 appear to have conflicting roles. Genetic disruption of hCXCR4 enhances osteoclast activity and therefore stimulates tumor cell growth in bone yet data exists illustrating either growth stimulation or no effect of CXCL12 on osteoclasts thus indicating the role of this axis is not fully defined and understood. Recently, CXCL16 was identified as a ligand for hCXCR6 (STRL33), which is expressed by peripheral blood leukocytes.<a class="bk_pop" href="#ml339.r19">19</a> CXCL16 expression has been demonstrated in a variety of tissues and cells including activated endothelial cells. Additionally, CXCL16 has been shown to function as a potent and direct activator of NF-κB and induces κB-dependent pro-inflammatory gene transcription through interaction with heterotrimeric G-proteins triggering downstream PI3K, PDK-1, Akt, and IκB kinase (IKK) signal transduction events. Through a cytokine antibody array, Lu and coworkers<a class="bk_pop" href="#ml339.r15">15</a> reported that CXCL16 protein production was increased in aggressive PCa cells compared to the less aggressive PCa cells or benign prostate cells. We also found that both IL-1β and TNFα significantly induced CXCL16 production by LNCaP and PC3 cells, thereby indicating inflammatory cytokines may play a role in CXCL16 induction. These observations support the notion that hCXCL16/CXCR6 interactions may be important for PCa invasion and metastasis. As preliminary results, we have provided strong evidence that the hCXCR6/CXCL16 axis also plays a key role in PCa metastasis, tumor cell proliferation and invasiveness.</p><p><b>To date, no small molecule or antagonists are available to test our hypothesis.</b> Given the knowledge gap, we propose that the development of pharmacologically available small molecule antagonists to CXCR6 will lay the foundation for a novel approach to mediate PCa and its deadly metastases. We have shown that the expression of CXCR6 1) is correlated with disease progression, 2) regulates migration and bone invasiveness, 3) is involved in proliferation of human PCa.<a class="bk_pop" href="#ml339.r14">14</a>,<a class="bk_pop" href="#ml339.r15">15</a>,<a class="bk_pop" href="#ml339.r20">20</a> These data are relevant as they set the foundation for target validation and for moving forward with our proposed aims.</p></div><div id="ml339.s6"><h3>Prior Art</h3><p>A recent comprehensive search of the published scientific and patent literature on November 28, 2012 indicated that no small molecule CXCR6 antagonists have been reported. SciFinder was used to conduct the search. To follow up on the identification of MLS-0020556, a SciFinder substructure query not limited to CXCR6 relevance (<a class="figpopup" href="/books/NBK158948/table/ml339.t2/?report=objectonly" target="object" rid-figpopup="figml339t2" rid-ob="figobml339t2">Table 2</a>) was conducted on December 4, 2012. This search identified 76 compounds and 12 associated references. No relevance to CXCR6 activity was noted. Although these references are not considered prior art for this program, they are discussed here for completeness. Briefly, one patent<a class="bk_pop" href="#ml339.r21">21</a> issued to colleagues at SBMRI have identified MLS-0020645 as a 4.67 μM inhibitor of HePTP relevant to leukemia. This was a patent claiming methods of treatment and did not disclose further SAR or examples related to the azabicyclo[3.3.1] scaffold. This compound was present in our MLSMR screening collection edition and was found to be not active. Four patents, written in Japanese, disclosed structures that the same cohort appears to have summarized in a manuscript.<a class="bk_pop" href="#ml339.r22">22</a> Compounds disclosed were of interest due to gastrointestinal prokinetic and dopamine D2 antagonist activity. The compounds (D2 compounds, see <a class="figpopup" href="/books/NBK158948/table/ml339.t1/?report=objectonly" target="object" rid-figpopup="figml339t1" rid-ob="figobml339t1">Table 1</a>) contain the same azabicyclo[3.3.1] scaffold with bridgehead substitution involving an acid or ester linked via an alkyl chain. One additional reference was an analytical method development manuscript and the other four references disclosed different scaffolds and/or did not demonstrate utility of the azabicyclo[3.3.1] scaffold.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml339t2"><a href="/books/NBK158948/table/ml339.t2/?report=objectonly" target="object" title="Table 2" class="img_link icnblk_img figpopup" rid-figpopup="figml339t2" rid-ob="figobml339t2"><img class="small-thumb" src="/books/NBK158948/table/ml339.t2/?report=thumb" src-large="/books/NBK158948/table/ml339.t2/?report=previmg" alt="Table 2. Summary of Prior Art Search." /></a><div class="icnblk_cntnt"><h4 id="ml339.t2"><a href="/books/NBK158948/table/ml339.t2/?report=objectonly" target="object" rid-ob="figobml339t2">Table 2</a></h4><p class="float-caption no_bottom_margin">Summary of Prior Art Search. </p></div></div></div></div><div id="ml339.s7"><h2 id="_ml339_s7_">2. Materials and Methods</h2><p>Three key cell lines DiscoveRx) and their cognate ligands (R&D Systems) are utilized in this project:</p><div id="ml339.tu1" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK158948/table/ml339.tu1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml339.tu1_lrgtbl__"><table><tbody><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">PathHunter CHO-K1 hCXCR6 b-arrestin cell line (Cat# 93-0205C2)</td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">hCXCCL16 (Cat# 976-CX)</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">PathHunter CHO-K1 hCXCR5 b-arrestin cell line (Cat# 93-0204C2)</td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">hCXCL13 peptide (Cat# 801-CX)</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">PathHunter C2C12 hCXCR4 b-arrestin cell line (Cat# 93-0203C7)</td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">hCXCL12 peptide (Cat# 6448-SD-025</td></tr></tbody></table></div></div><div id="ml339.s8"><h3>2.1. Assays</h3><p><a class="figpopup" href="/books/NBK158948/table/ml339.t3/?report=objectonly" target="object" rid-figpopup="figml339t3" rid-ob="figobml339t3">Table 3</a> summarizes details for the assays that enabled this probe discovery project. A detailed description of the Primary assay can be found in the Appendix at end of the probe report and in the PubChem AIDs listed in <a class="figpopup" href="/books/NBK158948/table/ml339.t3/?report=objectonly" target="object" rid-figpopup="figml339t3" rid-ob="figobml339t3">Table 3</a>.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml339t3"><a href="/books/NBK158948/table/ml339.t3/?report=objectonly" target="object" title="Table 3" class="img_link icnblk_img figpopup" rid-figpopup="figml339t3" rid-ob="figobml339t3"><img class="small-thumb" src="/books/NBK158948/table/ml339.t3/?report=thumb" src-large="/books/NBK158948/table/ml339.t3/?report=previmg" alt="Table 3. Summary of Assays and AIDs." /></a><div class="icnblk_cntnt"><h4 id="ml339.t3"><a href="/books/NBK158948/table/ml339.t3/?report=objectonly" target="object" rid-ob="figobml339t3">Table 3</a></h4><p class="float-caption no_bottom_margin">Summary of Assays and AIDs. </p></div></div></div><div id="ml339.s9"><h3>2.2. Probe Chemical Characterization</h3><div id="ml339.s10"><h4>a. Chemical name of probe compound</h4><p>The IUPAC name of the probe is <i>N</i>-((1R,3s,5S)-9-(2-((2-chlorophenyl)amino)-2-oxoethyl)-9-azabicyclo[3.3.1]nonan-3-yl)-3,4,5-trimethoxybenzamide. The actual batch prepared, tested and submitted to the MLSMR is archived as <a href="https://pubchem.ncbi.nlm.nih.gov/substance/150864418" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID150864418</a> corresponding to CID60202254.</p></div><div id="ml339.s11"><h4>b. Probe chemical structure including stereochemistry if known</h4><p>The probe <a href="/pcsubstance/?term=ML339[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML339</a> is the <i>exo</i>-positional isomer. Although it possesses two stereogenic centers, it is an achiral molecule due to a plane of symmetry.</p><div id="ml339.f1" class="figure bk_fig"><div class="graphic"><img src="/books/NBK158948/bin/ml339f1.jpg" alt="Figure 1. Structure of ML339." /></div><h3><span class="label">Figure 1</span><span class="title">Structure of ML339</span></h3></div></div><div id="ml339.s12"><h4>c. Synthesis and Structural Verification Information of probe SID 150864418 corresponding to CID 60202254 (See <a class="figpopup" href="/books/NBK158948/figure/ml339.f6/?report=objectonly" target="object" rid-figpopup="figml339f6" rid-ob="figobml339f6">Scheme 1</a>)</h4><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml339f6" co-legend-rid="figlgndml339f6"><a href="/books/NBK158948/figure/ml339.f6/?report=objectonly" target="object" title="Scheme 1" class="img_link icnblk_img figpopup" rid-figpopup="figml339f6" rid-ob="figobml339f6"><img class="small-thumb" src="/books/NBK158948/bin/ml339f6.gif" src-large="/books/NBK158948/bin/ml339f6.jpg" alt="Scheme 1. Synthesis of ML339, conditions." /></a><div class="icnblk_cntnt" id="figlgndml339f6"><h4 id="ml339.f6"><a href="/books/NBK158948/figure/ml339.f6/?report=objectonly" target="object" rid-ob="figobml339f6">Scheme 1</a></h4><p class="float-caption no_bottom_margin">Synthesis of ML339, conditions. i) 3,4,5-trimethoxybenzoyl chloride, Et<sub>3</sub>N, CH<sub>2</sub>Cl<sub>2</sub>, 2 h, r.t.; ii) trifluoroacetic acid, CH<sub>2</sub>Cl<sub>2</sub>, 1 h, r.t.; iii) chloroacetyl chloride, DMAP (cat.), Et<sub>3</sub>N, CH<sub>2</sub>Cl<sub>2</sub>, 2 h, r.t., 99%; iv) (<i>B</i>), Et<sub>3</sub>N, CH<sub>2</sub>Cl<sub>2</sub>, 18 h, r.t., 24 % overall. <a href="/books/NBK158948/figure/ml339.f6/?report=objectonly" target="object" rid-ob="figobml339f6">(more...)</a></p></div></div><p>The <i>exo</i>-positional isomer of an analog (<b>7</b>) was confirmed by x-ray crystallography.<a class="bk_pop" href="#ml339.r23">23</a> This analog was synthesized from the same <i>exo</i>-building block as the probe, and there was no stereochemical ambiguity associated with the synthesis.</p><div id="ml339.f2" class="figure bk_fig"><div class="graphic"><img src="/books/NBK158948/bin/ml339f2.jpg" alt="Figure 2. X-ray Structure of 7." /></div><h3><span class="label">Figure 2</span><span class="title">X-ray Structure of <b>7</b></span></h3></div></div><div id="ml339.s13"><h4>d. Synthesis of ML339 and analogs</h4><p>For detailed procedures see <a href="#ml339.s17">Section 2.3</a>. Images of spectral data (<sup>1</sup>H-NMR, <sup>13</sup>C-NMR, and LC/MS) used to support the structural assignment of <a href="/pcsubstance/?term=ML339[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML339</a> can be found in <a href="#ml339.s52">Section 6.2</a> (Appendices & Supplementary Information).</p></div><div id="ml339.s14"><h4>d. If available from a vendor, please provide details</h4><p>This probe is not commercially available. A 25 mg sample of <a href="/pcsubstance/?term=ML339[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML339</a> synthesized at SBCCG has been deposited in the MLSMR (Evotec) (see Probe Submission <a class="figpopup" href="/books/NBK158948/table/ml339.t5/?report=objectonly" target="object" rid-figpopup="figml339t5" rid-ob="figobml339t5">Table 5</a>).</p></div><div id="ml339.s15"><h4>e. Calculated and known probe properties are shown in <a class="figpopup" href="/books/NBK158948/table/ml339.t4/?report=objectonly" target="object" rid-figpopup="figml339t4" rid-ob="figobml339t4">Table 4</a></h4><div id="ml339.t4" class="table"><h3><span class="label">Table 4</span><span class="title">CID 60202254 [ML339] MLS-0472139</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK158948/table/ml339.t4/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml339.t4_lrgtbl__"><table class="no_top_margin"><tbody><tr><th id="hd_b_ml339.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Molecular Weight</th><td headers="hd_b_ml339.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">502.00 [g/mol]</td></tr><tr><th id="hd_b_ml339.t4_1_1_2_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Molecular Formula</th><td headers="hd_b_ml339.t4_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">C<sub>26</sub>H<sub>32</sub>ClN<sub>3</sub>O<sub>5</sub></td></tr><tr><th id="hd_b_ml339.t4_1_1_3_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">AlogP</th><td headers="hd_b_ml339.t4_1_1_3_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">3.375</td></tr><tr><th id="hd_b_ml339.t4_1_1_4_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">H-Bond Donor</th><td headers="hd_b_ml339.t4_1_1_4_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">2</td></tr><tr><th id="hd_b_ml339.t4_1_1_5_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">H-Bond Acceptor</th><td headers="hd_b_ml339.t4_1_1_5_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">6</td></tr><tr><th id="hd_b_ml339.t4_1_1_6_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Rotatable Bond Count</th><td headers="hd_b_ml339.t4_1_1_6_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">8</td></tr><tr><th id="hd_b_ml339.t4_1_1_7_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Exact Mass</th><td headers="hd_b_ml339.t4_1_1_7_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">501.2030</td></tr><tr><th id="hd_b_ml339.t4_1_1_8_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">MonoIsotopic Mass</th><td headers="hd_b_ml339.t4_1_1_8_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">501.2030</td></tr><tr><th id="hd_b_ml339.t4_1_1_9_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Topological Polar Surface Area</th><td headers="hd_b_ml339.t4_1_1_9_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">89.1</td></tr><tr><th id="hd_b_ml339.t4_1_1_10_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Heavy Atom Count</th><td headers="hd_b_ml339.t4_1_1_10_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">35</td></tr><tr><th id="hd_b_ml339.t4_1_1_11_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Formal Charge</th><td headers="hd_b_ml339.t4_1_1_11_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0</td></tr><tr><th id="hd_b_ml339.t4_1_1_12_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Complexity</th><td headers="hd_b_ml339.t4_1_1_12_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">669</td></tr><tr><th id="hd_b_ml339.t4_1_1_13_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Isotope Atom Count</th><td headers="hd_b_ml339.t4_1_1_13_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0</td></tr><tr><th id="hd_b_ml339.t4_1_1_14_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Defined Atom StereoCenter Count</th><td headers="hd_b_ml339.t4_1_1_14_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0</td></tr><tr><th id="hd_b_ml339.t4_1_1_15_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Undefined Atom StereoCenter Count</th><td headers="hd_b_ml339.t4_1_1_15_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">2</td></tr><tr><th id="hd_b_ml339.t4_1_1_16_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Defined Bond StereoCenter Count</th><td headers="hd_b_ml339.t4_1_1_16_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0</td></tr><tr><th id="hd_b_ml339.t4_1_1_17_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Undefined Bond StereoCenter Count</th><td headers="hd_b_ml339.t4_1_1_17_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0</td></tr><tr><th id="hd_b_ml339.t4_1_1_18_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Covalently-Bonded Unit Count</th><td headers="hd_b_ml339.t4_1_1_18_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">1</td></tr></tbody></table></div></div></div><div id="ml339.s16"><h4>g. <a class="figpopup" href="/books/NBK158948/table/ml339.t5/?report=objectonly" target="object" rid-figpopup="figml339t5" rid-ob="figobml339t5">Table 5</a> summarizes the deposition of the Probe and 5 analogs</h4><div id="ml339.t5" class="table"><h3><span class="label">Table 5</span><span class="title">Probe and Analog Submissions to MLSMR (Evotec) for CXCR6 Probe</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK158948/table/ml339.t5/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml339.t5_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml339.t5_1_1_1_1" colspan="8" rowspan="1" style="text-align:center;vertical-align:middle;">Probe <a href="/pcsubstance/?term=ML339[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML339</a> – CID 60202254</th></tr><tr><th headers="hd_h_ml339.t5_1_1_1_1" id="hd_h_ml339.t5_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Probe/Analog</th><th headers="hd_h_ml339.t5_1_1_1_1" id="hd_h_ml339.t5_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS_ID (SBCCG)</th><th headers="hd_h_ml339.t5_1_1_1_1" id="hd_h_ml339.t5_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS_ID (MLSMR)</th><th headers="hd_h_ml339.t5_1_1_1_1" id="hd_h_ml339.t5_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CID</th><th headers="hd_h_ml339.t5_1_1_1_1" id="hd_h_ml339.t5_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SID</th><th headers="hd_h_ml339.t5_1_1_1_1" id="hd_h_ml339.t5_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Source (vendor or SBCCG syn)</th><th headers="hd_h_ml339.t5_1_1_1_1" id="hd_h_ml339.t5_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Amt (mg)</th><th headers="hd_h_ml339.t5_1_1_1_1" id="hd_h_ml339.t5_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Date ordered/Submitted</th></tr></thead><tbody><tr><td headers="hd_h_ml339.t5_1_1_1_1 hd_h_ml339.t5_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Probe<br /><a href="/pcsubstance/?term=ML339[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML339</a></td><td headers="hd_h_ml339.t5_1_1_1_1 hd_h_ml339.t5_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0472139</td><td headers="hd_h_ml339.t5_1_1_1_1 hd_h_ml339.t5_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS004576089</td><td headers="hd_h_ml339.t5_1_1_1_1 hd_h_ml339.t5_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">60202254</td><td headers="hd_h_ml339.t5_1_1_1_1 hd_h_ml339.t5_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/150864418" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">150864418</a></td><td headers="hd_h_ml339.t5_1_1_1_1 hd_h_ml339.t5_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SBCCG syn</td><td headers="hd_h_ml339.t5_1_1_1_1 hd_h_ml339.t5_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">25.1</td><td headers="hd_h_ml339.t5_1_1_1_1 hd_h_ml339.t5_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">10/31/2012</td></tr><tr><td headers="hd_h_ml339.t5_1_1_1_1 hd_h_ml339.t5_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Analog 1</td><td headers="hd_h_ml339.t5_1_1_1_1 hd_h_ml339.t5_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0020556</td><td headers="hd_h_ml339.t5_1_1_1_1 hd_h_ml339.t5_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS004576090</td><td headers="hd_h_ml339.t5_1_1_1_1 hd_h_ml339.t5_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">653186</td><td headers="hd_h_ml339.t5_1_1_1_1 hd_h_ml339.t5_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/144220951" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">144220951</a></td><td headers="hd_h_ml339.t5_1_1_1_1 hd_h_ml339.t5_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SBCCG syn</td><td headers="hd_h_ml339.t5_1_1_1_1 hd_h_ml339.t5_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">20.2</td><td headers="hd_h_ml339.t5_1_1_1_1 hd_h_ml339.t5_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">10/31/2012</td></tr><tr><td headers="hd_h_ml339.t5_1_1_1_1 hd_h_ml339.t5_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Analog 2</td><td headers="hd_h_ml339.t5_1_1_1_1 hd_h_ml339.t5_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0472135</td><td headers="hd_h_ml339.t5_1_1_1_1 hd_h_ml339.t5_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS004576091</td><td headers="hd_h_ml339.t5_1_1_1_1 hd_h_ml339.t5_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">60202215</td><td headers="hd_h_ml339.t5_1_1_1_1 hd_h_ml339.t5_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/144220963" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">144220963</a></td><td headers="hd_h_ml339.t5_1_1_1_1 hd_h_ml339.t5_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SBCCG syn</td><td headers="hd_h_ml339.t5_1_1_1_1 hd_h_ml339.t5_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">20.2</td><td headers="hd_h_ml339.t5_1_1_1_1 hd_h_ml339.t5_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">10/31/2012</td></tr><tr><td headers="hd_h_ml339.t5_1_1_1_1 hd_h_ml339.t5_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Analog 3</td><td headers="hd_h_ml339.t5_1_1_1_1 hd_h_ml339.t5_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0472137</td><td headers="hd_h_ml339.t5_1_1_1_1 hd_h_ml339.t5_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS004576092</td><td headers="hd_h_ml339.t5_1_1_1_1 hd_h_ml339.t5_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">60202210</td><td headers="hd_h_ml339.t5_1_1_1_1 hd_h_ml339.t5_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/144220965" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">144220965</a></td><td headers="hd_h_ml339.t5_1_1_1_1 hd_h_ml339.t5_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SBCCG syn</td><td headers="hd_h_ml339.t5_1_1_1_1 hd_h_ml339.t5_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">20.1</td><td headers="hd_h_ml339.t5_1_1_1_1 hd_h_ml339.t5_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">10/31/2012</td></tr><tr><td headers="hd_h_ml339.t5_1_1_1_1 hd_h_ml339.t5_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Analog 4</td><td headers="hd_h_ml339.t5_1_1_1_1 hd_h_ml339.t5_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0472119</td><td headers="hd_h_ml339.t5_1_1_1_1 hd_h_ml339.t5_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS004576093</td><td headers="hd_h_ml339.t5_1_1_1_1 hd_h_ml339.t5_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">60202260</td><td headers="hd_h_ml339.t5_1_1_1_1 hd_h_ml339.t5_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/144220947" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">144220947</a></td><td headers="hd_h_ml339.t5_1_1_1_1 hd_h_ml339.t5_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SBCCG syn</td><td headers="hd_h_ml339.t5_1_1_1_1 hd_h_ml339.t5_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">19.1</td><td headers="hd_h_ml339.t5_1_1_1_1 hd_h_ml339.t5_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">10/31/2012</td></tr><tr><td headers="hd_h_ml339.t5_1_1_1_1 hd_h_ml339.t5_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Analog 5</td><td headers="hd_h_ml339.t5_1_1_1_1 hd_h_ml339.t5_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0472007</td><td headers="hd_h_ml339.t5_1_1_1_1 hd_h_ml339.t5_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS004576094</td><td headers="hd_h_ml339.t5_1_1_1_1 hd_h_ml339.t5_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">60156312</td><td headers="hd_h_ml339.t5_1_1_1_1 hd_h_ml339.t5_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/144088977" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">144088977</a></td><td headers="hd_h_ml339.t5_1_1_1_1 hd_h_ml339.t5_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SBCCG syn</td><td headers="hd_h_ml339.t5_1_1_1_1 hd_h_ml339.t5_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">21.7</td><td headers="hd_h_ml339.t5_1_1_1_1 hd_h_ml339.t5_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">10/31/2012</td></tr></tbody></table></div></div></div></div><div id="ml339.s17"><h3>2.3. Probe Preparation</h3><div id="ml339.s18"><h4>Experimental</h4><p><i>Conditions for the synthesis of MLS-0472139: exo</i>-3-Amino-<i>N</i>-Boc-9-azabicyclo[3.3.1]nonane (CAS #1363380-67-9, 150 mg, 0.62 mmol) was dissolved in 5 mL of dichloromethane before charging with triethylamine (0.26 mL, 1.87 mmol) and 3,4,5-trimethoxybenzoyl chloride (173 mg, 0.75 mmol). After 2 h, the volatiles were evaporated <i>in vacuo</i> and the residue treated with 5 mL of water and extracted with 3× 3 mL ethyl acetate. Concentration of the organics returned crude <i>exo</i>-(1R,3S,5S)-<i>tert</i>-butyl 3-(3,4,5-trimethoxybenzamido)-9-azabicyclo[3.3.1]nonane-9-carboxylate (<b>A</b>), which was used without purification. <sup>1</sup>H NMR (500 MHz, CDCl<sub>3</sub>) δ 6.97 (s, 2H), 5.70 (d, <i>J</i> = 8.1 Hz, 1H), 5.00 (dq, <i>J</i> = 12.4, 6.1 Hz, 1H), 4.40 (br. s, 2H), 3.94 (s, 6H), 3.90 (s, 3H), 3.13 (qd, <i>J</i> = 7.3, 4.8 Hz, 1H), 2.16 (dd, <i>J</i> = 13.1, 5.9 Hz, 2H), 2.11 – 1.98 (m, 1H), 1.88 (tt, <i>J</i> = 13.3, 5.9 Hz, 2H), 1.80 – 1.63 (m, 4H), 1.50 (s, 9H). MS (ESI+ve): Calculated for C<sub>23</sub>H<sub>35</sub>N<sub>2</sub>O<sub>6</sub>, [M+H] = 435.24, observed [M+H] = 435.29.</p><p><b>A</b>, (0.62 mmol from the previous step) was dissolved in 4 mL of dichloromethane and treated with 4 mL of trifluoroacetic acid. After stirring for 1 h, the mixture was concentrated to afford exo-<i>N</i>-((1R,3S,5S)-9-azabicyclo[3.3.1]nonan-3-yl)-3,4,5-trimethoxybenzamide (<b>B</b>), as a colorless oil. The material was understood to be the trifluoroacetate salt, partly contaminated with residual trifluoracetic acid, and was carried forward without purification. <sup>1</sup>H NMR (500 MHz, CDCl<sub>3</sub>) δ 7.08 (s, 2H), 5.10 (dd, <i>J</i> = 13.3, 6.4 Hz, 1H), 3.98 – 3.90 (m., 2H), 3.93 (s, 9H), 3.24 (qd, <i>J</i> = 7.3, 4.8 Hz, 1H), 2.50 – 2.27 (m, 4H), 2.27 – 2.03 (m, 4H), 2.03 – 1.85 (m, 1H). MS (ESI+ve): Calculated for C<sub>18</sub>H<sub>27</sub>N<sub>2</sub>O<sub>4</sub>, [M+H] = 335.41, observed [M+H] = 335.22.</p><p>A solution of 2-chloroacetyl chloride (0.33 mL, 4.15 mmol) in 4 mL dichloromethane was treated with a solution of 2-chloroaniline (0.43 mL, 4.1 mmol), <i>N,N</i>-dimethylpyridin-4-amine (5 mg, cat.) and triethylamine (0.58 mL, 4.16 mmol) in 4 mL dichloromethane dropwise under a nitrogen atmosphere. The mixture was stirred for 2 h and the solvent was removed <i>in vacuo</i>. The residue was taken up in 50 mL ethyl acetate and was washed with aqueous sodium bisulfate. The organic phase was dried over sodium sulfate and concentrated <i>in vacuo</i> to provide 2-chloro-<i>N</i>-(2-chlorophenyl)acetamide <b>C</b> (832 mg, 99%) as a tan solid which was used without further purification. <sup>1</sup>H NMR (500 MHz, CDCl<sub>3</sub>) δ 8.96 (s, 1H), 8.39 (dd, <i>J</i> = 8.3, 1.2 Hz, 1H), 7.44 (dd, <i>J</i> = 8.0, 1.4 Hz, 1H), 7.36 – 7.31 (m, 1H), 7.13 (td, <i>J</i> = 7.9, 1.5 Hz, 1H), 4.27 (s, 2H).</p><p>Crude <b>B</b> from the previous step (max 0.62 mmol) was dissolved in 1.5 mL of dichloromethane before charging with triethylamine (0.88 mL, 6.29 mmol) and solid <b>C</b> from the previous step (192 mg, 0.94 mmol). After stirring for 18 h, the mixture was concentrated and the residue was purified via silica gel flash chromatography, eluting with 0–60% ethyl acetate / hexane, to afford 74 mg (24 % overall yield for the 3 step sequence) of <i>N</i>-((1R,3S,5S)-9-(2-((2-chlorophenyl)amino)-2-oxoethyl)-9-azabicyclo[3.3.1]nonan-3-yl)-3,4,5-trimethoxybenzamide, <b>18 (</b><a href="/pcsubstance/?term=ML339[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML339</a><b>)</b>. Melting Point: decomposition > 160 °C; <sup>1</sup>H NMR (500 MHz, DMSO-<i>d</i><sub>6</sub>) δ 10.26 (s, 1H), 8.41 (dd, <i>J</i> = 8.2, 1.6 Hz, 1H), 8.04 (d, <i>J</i> = 7.9 Hz, 1H), 7.53 (dd, <i>J</i> = 8.0, 1.4 Hz, 1H), 7.36 (td, <i>J</i> = 8.4, 7.9, 1.5 Hz, 1H), 7.17 – 7.09 (m, 3H), 4.80 – 4.66 (m, 1H), 3.84 (s, 6H), 3.70 (s, 3H), 3.48 (s, 2H), 3.00 (s, 2H), 2.11 – 1.99 (m, 2H), 1.99 – 1.88 (m, 2H), 1.88 – 1.80 (m, 1H), 1.78 – 1.66 (m, 5H); <sup>13</sup>C NMR (125 MHz, DMSO-<i>d</i><sub>6</sub>) δ 169.51, 164.90, 152.50, 139.92, 134.50, 130.00, 129.26, 127.95, 124.60, 121.89, 120.24, 104.88, 60.06, 57.28, 56.06, 52.39, 42.65, 31.82, 29.18, 19.64; HRMS (ESI+ve): Calculated for C<sub>26</sub>H<sub>32</sub>ClN<sub>3</sub>O<sub>5</sub>, [M+H] = 502.2103, observed [M+H] = 502.2099.</p></div></div></div><div id="ml339.s19"><h2 id="_ml339_s19_">3. Results</h2><div id="ml339.s20"><h3>3.1. Summary of Screening Results</h3><div id="ml339.s21"><h4>Results of Screening and Hit Validation</h4><p>The screening funnel results are summarized in <a class="figpopup" href="/books/NBK158948/figure/ml339.f3/?report=objectonly" target="object" rid-figpopup="figml339f3" rid-ob="figobml339f3">Figure 3</a>. We completed a full-deck cell-based screen of the available MLSMR library of ~360,000 compounds at 20 μM final library compound concentration at 1% (v/v) final DMSO concentration for inhibitors of the human chemokine (C-X-C motif) receptor 6 (CXCR6) in CHO-K1 cells using a β-Arrestin-2 readout. Cells were pre-treated with compound before stimulation with of the endogenous CXCL16 ligand or addition of DMSO only for 90 minutes. Inhibition of β-Arrestin recruitment was measured using the β-galactosidase PathHunter Enzyme Complementation Assay Platform (DiscoveRx). The assay was validated using anti-hCXCR16 as an antagonist positive control tested in the presence of an EC<sub>80</sub> concentration of hCXCL16 (IC<sub>50</sub> = 2.0 nM; % response = 100%). <a class="figpopup" href="/books/NBK158948/figure/ml339.f3/?report=objectonly" target="object" rid-figpopup="figml339f3" rid-ob="figobml339f3">Figure 3</a> summarizes the hit triage and prosecution. An initial 1305 hits were obtained at hit threshold of >40% inhibitory activity. Cheminformatic filtering was applied to eliminate known PAINS<a class="bk_pop" href="#ml339.r24">24</a> (<u>P</u>an <u>A</u>ssay <u>I</u>nterference Compound<u>s</u>) and promiscuous compounds. Additionally, our experienced project chemists visually inspected the chemical structures of the filtered hits and eliminated any compounds they judged were <i>a priori</i> undesirable, intractable or were previously problematic in other screens and projects. The final number of hits thus selected for reorder was 929. We requested all of these as “cherry-picks” of fresh DMSO stock solutions from the NIH MLSMR (Molecular Libraries Small Molecule Repository) managed by Evotec (South San Francisco, CA) and received 854 of them for a 91.9% order fulfillment.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml339f3" co-legend-rid="figlgndml339f3"><a href="/books/NBK158948/figure/ml339.f3/?report=objectonly" target="object" title="Figure 3" class="img_link icnblk_img figpopup" rid-figpopup="figml339f3" rid-ob="figobml339f3"><img class="small-thumb" src="/books/NBK158948/bin/ml339f3.gif" src-large="/books/NBK158948/bin/ml339f3.jpg" alt="Figure 3. Summary of Screening Triage." /></a><div class="icnblk_cntnt" id="figlgndml339f3"><h4 id="ml339.f3"><a href="/books/NBK158948/figure/ml339.f3/?report=objectonly" target="object" rid-ob="figobml339f3">Figure 3</a></h4><p class="float-caption no_bottom_margin">Summary of Screening Triage. </p></div></div><p>Of the 854 received cherry-picks, 563 confirmed in duplicate at the original 20 μM test concentration and 366 compounds showed IC<sub>50</sub> < 20 μM inhibition of CXCR6 receptor β-Arrestin recruitment. 176 compounds showed activity in a β-galactosidase enzyme assay using the same PathHunter reagents and were eliminated as interfering with the protein complementation system. The remaining 190 compounds were tested against two additional GPCRs using the same β-Arrestin platform from DiscoveRx, the human CCR6 chemokine receptor and the human non-related Apelin (APJ) receptor to determine overall GPCR selectivity. A single compound showed initial 6 μM IC<sub>50</sub> activity in the primary CXCR6 assay and was devoid of activity at inhibiting either the CCR6 or APJ receptors. This compound represented a chemically tractable scaffold and was advanced through multiple rounds of medicinal chemistry and structure-activity relationship (SAR) studies. These studies resulted in one first in class small molecule that was potent in antagonizing β-arrestin recruitment of the CXCR6 receptor and was inactive in at the CCR6, CXCR5, CXCR4 and APJ receptors. The synthetic strategies for this scaffold are described further.</p></div></div><div id="ml339.s22"><h3>3.2. Dose Response Curves for Probe</h3><p>The multiple dose response titrations in <a class="figpopup" href="/books/NBK158948/figure/ml339.f4/?report=objectonly" target="object" rid-figpopup="figml339f4" rid-ob="figobml339f4">Figure 4</a> of the probe <a href="/pcsubstance/?term=ML339[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML339</a> against an EC<sub>80</sub> concentration of the natural ligand for hCXCR6, hCXCR4, hCXCR5 and hCCR6 receptors highlight the high degree of selectivity the compound has for the human CXCR6 receptor.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml339f4" co-legend-rid="figlgndml339f4"><a href="/books/NBK158948/figure/ml339.f4/?report=objectonly" target="object" title="Figure 4" class="img_link icnblk_img figpopup" rid-figpopup="figml339f4" rid-ob="figobml339f4"><img class="small-thumb" src="/books/NBK158948/bin/ml339f4.gif" src-large="/books/NBK158948/bin/ml339f4.jpg" alt="Figure 4. Specificity of ML339 Inhibition for the hCXCR6 receptor vs. human hCXCR4, CXCR5 and CCR6 receptors." /></a><div class="icnblk_cntnt" id="figlgndml339f4"><h4 id="ml339.f4"><a href="/books/NBK158948/figure/ml339.f4/?report=objectonly" target="object" rid-ob="figobml339f4">Figure 4</a></h4><p class="float-caption no_bottom_margin">Specificity of ML339 Inhibition for the hCXCR6 receptor <i>vs</i>. human hCXCR4, CXCR5 and CCR6 receptors. </p></div></div></div><div id="ml339.s23"><h3>3.3. Scaffold/Moiety Chemical Liabilities</h3><p><a href="/pcsubstance/?term=ML339[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML339</a> contains three methoxy groups that may compromise metabolic stability. This is consistent with the low metabolic stability in human and mouse liver microsomes see during <i>in vitro</i> ADME/T profiling (see <a class="figpopup" href="/books/NBK158948/table/ml339.t10/?report=objectonly" target="object" rid-figpopup="figml339t10" rid-ob="figobml339t10">Table 10</a>).</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml339t10"><a href="/books/NBK158948/table/ml339.t10/?report=objectonly" target="object" title="Table 10" class="img_link icnblk_img figpopup" rid-figpopup="figml339t10" rid-ob="figobml339t10"><img class="small-thumb" src="/books/NBK158948/table/ml339.t10/?report=thumb" src-large="/books/NBK158948/table/ml339.t10/?report=previmg" alt="Table 10. Summary of in vitro ADME Properties of probe ML339." /></a><div class="icnblk_cntnt"><h4 id="ml339.t10"><a href="/books/NBK158948/table/ml339.t10/?report=objectonly" target="object" rid-ob="figobml339t10">Table 10</a></h4><p class="float-caption no_bottom_margin">Summary of <i>in vitro</i> ADME Properties of probe <i>ML339</i>. </p></div></div></div><div id="ml339.s24"><h3>3.4. SAR Analysis</h3><p>The screening program identified MLS-0020556 as a singleton hit. Given the ambiguous database structure, the <i>exo</i>/<i>endo</i> positional amide isomer question required resolution. This defined several <b>Key Questions</b> which our SAR program has addressed successfully. These included:</p><ol><li class="half_rhythm"><div>Are there commercially available analogs with similar activity?</div></li><li class="half_rhythm"><div>Is the [3.3.1]azabicyclononane required for activity?</div></li><li class="half_rhythm"><div>In the hit MLS-0020556, is the reverse amide substituent in the <i>exo</i> or <i>endo</i> position?</div></li><li class="half_rhythm"><div>If the [3.3.1] system is required or preferred, is the exo or endo position required or preferred?</div></li><li class="half_rhythm"><div>Is the 3,4,5-trimethoxy aryl motif required?</div></li><li class="half_rhythm"><div>Does substitution of the benzamide group appended to the bridgehead nitrogen influence activity?</div></li></ol><p><a class="figpopup" href="/books/NBK158948/table/ml339.t6/?report=objectonly" target="object" rid-figpopup="figml339t6" rid-ob="figobml339t6">Tables 6</a>, <a class="figpopup" href="/books/NBK158948/table/ml339.t7/?report=objectonly" target="object" rid-figpopup="figml339t7" rid-ob="figobml339t7">7</a>, and <a class="figpopup" href="/books/NBK158948/table/ml339.t8/?report=objectonly" target="object" rid-figpopup="figml339t8" rid-ob="figobml339t8">8</a> summarize SAR, which addresses these questions.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml339t6"><a href="/books/NBK158948/table/ml339.t6/?report=objectonly" target="object" title="Table 6" class="img_link icnblk_img figpopup" rid-figpopup="figml339t6" rid-ob="figobml339t6"><img class="small-thumb" src="/books/NBK158948/table/ml339.t6/?report=thumb" src-large="/books/NBK158948/table/ml339.t6/?report=previmg" alt="Table 6. SAR of Purchased Analogs." /></a><div class="icnblk_cntnt"><h4 id="ml339.t6"><a href="/books/NBK158948/table/ml339.t6/?report=objectonly" target="object" rid-ob="figobml339t6">Table 6</a></h4><p class="float-caption no_bottom_margin">SAR of Purchased Analogs. </p></div></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml339t7"><a href="/books/NBK158948/table/ml339.t7/?report=objectonly" target="object" title="Table 7" class="img_link icnblk_img figpopup" rid-figpopup="figml339t7" rid-ob="figobml339t7"><img class="small-thumb" src="/books/NBK158948/table/ml339.t7/?report=thumb" src-large="/books/NBK158948/table/ml339.t7/?report=previmg" alt="Table 7. SAR of Alternative Scaffolds." /></a><div class="icnblk_cntnt"><h4 id="ml339.t7"><a href="/books/NBK158948/table/ml339.t7/?report=objectonly" target="object" rid-ob="figobml339t7">Table 7</a></h4><p class="float-caption no_bottom_margin">SAR of Alternative Scaffolds. </p></div></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml339t8"><a href="/books/NBK158948/table/ml339.t8/?report=objectonly" target="object" title="Table 8" class="img_link icnblk_img figpopup" rid-figpopup="figml339t8" rid-ob="figobml339t8"><img class="small-thumb" src="/books/NBK158948/table/ml339.t8/?report=thumb" src-large="/books/NBK158948/table/ml339.t8/?report=previmg" alt="Table 8. SAR of exo-[3.3.1] Scaffold Compounds." /></a><div class="icnblk_cntnt"><h4 id="ml339.t8"><a href="/books/NBK158948/table/ml339.t8/?report=objectonly" target="object" rid-ob="figobml339t8">Table 8</a></h4><p class="float-caption no_bottom_margin">SAR of <i>exo</i>-[3.3.1] Scaffold Compounds. </p></div></div><p><a class="figpopup" href="/books/NBK158948/table/ml339.t6/?report=objectonly" target="object" rid-figpopup="figml339t6" rid-ob="figobml339t6">Table 6</a> addresses <b>Key Question #1</b>. Briefly, this table showed that MLS-0020556 (Entry <b>1</b>) was, in fact, a singleton hit. Five commercially available structural analogs (Entries <b>2–6</b>) were identified and purchased along with a new batch of Entry <b>1</b> powder. The newly purchased analogs included the unsubstituted case (Entry <b>2</b>) as well as cases with either fewer methoxy groups (Entries <b>4</b> and <b>6</b>) or with substitution on the phenyl ring linked to the bridgehead (Entries <b>3–6</b>). Test results showed no activity for Entries <b>2–6</b>, but reconfirmed the activity of the parent hit (Entry <b>1</b>). None of the compounds in <a class="figpopup" href="/books/NBK158948/table/ml339.t6/?report=objectonly" target="object" rid-figpopup="figml339t6" rid-ob="figobml339t6">Table 6</a> were active in the CCR6 or CXCR5 counterscreens. These data prompted us to explore the importance of the [3.3.1] system and address the <i>exo vs. endo</i> question.</p><p><b>Key Questions #2, #3 and #4</b> are addressed in <a class="figpopup" href="/books/NBK158948/table/ml339.t7/?report=objectonly" target="object" rid-figpopup="figml339t7" rid-ob="figobml339t7">Table 7</a>. Overall, this table shows that the exo-[3.3.1] scaffold is required. Entries <b>7</b> and <b>8</b> were initially synthesized from a building block which was a 1:1 <i>exo</i> / <i>endo</i> mixture, and the two positional isomers were isolated by flash chromatography. As only the <i>exo</i> building block was commercially available, this separation was necessary to obtain both positional isomers from the same synthetic sequence. Subsequently, another batch of MLS-0020556 (Entry <b>13</b>, <a class="figpopup" href="/books/NBK158948/table/ml339.t8/?report=objectonly" target="object" rid-figpopup="figml339t8" rid-ob="figobml339t8">Table 8</a>) was synthesized from the pure <i>exo</i> building block. As this sample was identical to the Entry <b>7</b> compound, Entry <b>7</b>, as well as the parent singleton hit (<a class="figpopup" href="/books/NBK158948/table/ml339.t6/?report=objectonly" target="object" rid-figpopup="figml339t6" rid-ob="figobml339t6">Table 6</a>, Entry <b>1</b>) was known to be <i>exo</i> and the Entry <b>8</b> compound, by default, was known to be <i>endo</i>. Entry <b>7</b> was further characterized by x-ray crystallography to unambiguously establish the <i>exo</i> positional isomer. Entries <b>9</b> and <b>10</b> were synthesized from the commercially available pure <i>exo</i> and pure <i>endo</i> building blocks. Comparison of Entries <b>7</b>, <b>9</b>, and <b>11</b> (n = 3, 2, 0, respectively, with no other changes) revealed about 4 times more potency for the [3.3.1] scaffold than for the corresponding [3.2.1] variant. The monocyclic analog (Entry <b>11</b>) was inactive. Comparison of Entries <b>7</b> and <b>8</b> showed that, for the [3.3.1] scaffold, the <i>exo</i> positional isomer was required for activity. The same trend was noted for the less active [3.2.1] scaffold (Entries <b>9</b> and <b>10</b>). None of the compounds in <a class="figpopup" href="/books/NBK158948/table/ml339.t7/?report=objectonly" target="object" rid-figpopup="figml339t7" rid-ob="figobml339t7">Table 7</a> were active in the APJ counterscreen. These data prompted us to explore the SAR at positions R<sub>1</sub> and R<sub>2</sub>.</p><p><b>Key Questions #5 and #6</b> are addressed in <a class="figpopup" href="/books/NBK158948/table/ml339.t8/?report=objectonly" target="object" rid-figpopup="figml339t8" rid-ob="figobml339t8">Table 8</a>. Overall, this table shows good opportunity for improving potency via modification of R<sub>1</sub>, suggests requirement of the 3,4,5-trimethoxy substitution pattern at R<sub>2</sub>, and identifies the new probe <a href="/pcsubstance/?term=ML339[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML339</a>. All compounds in <a class="figpopup" href="/books/NBK158948/table/ml339.t8/?report=objectonly" target="object" rid-figpopup="figml339t8" rid-ob="figobml339t8">Table 8</a> were prepared from the commercially available pure <i>exo</i> building block. A synthesized batch of the unsubstituted R<sub>1</sub> = R<sub>2</sub> = H case (Entry <b>12</b>) reconfirmed the inactivity of the purchased batch (<a class="figpopup" href="/books/NBK158948/table/ml339.t6/?report=objectonly" target="object" rid-figpopup="figml339t6" rid-ob="figobml339t6">Table 6</a>, Entry <b>2</b>), and proved retroactively that the purchased Entry <b>2</b> material was, in fact, the <i>exo</i> positional isomer. The SAR at the R<sub>2</sub> position is clearly seen by comparing the set of five R<sub>2</sub> = 3,4,5-trimethoxy cases (Entries <b>13–17</b>) with the corresponding five 3,4-dimethoxy (Entries <b>20–24</b>), 3,5-dimethoxy (Entries <b>25–29</b>), 3-methoxy (Entries <b>30–34</b>), 4-methoxy (Entries <b>35–39</b>), and 3,4-methyleneoxy (Entries <b>40–44</b>) cases. For these analogous sets, activity was only observed for the trimethoxy cases. At the R<sub>2</sub> position, 4-Cl was about two-fold better than the 4-H case (Entries <b>15</b> and <b>13</b>), whereas 4-F, 4-Me, and 4-OMe were about two to five fold worse than no substitution (Entries <b>14</b>, <b>16</b>, <b>17</b>, and <b>13</b>). Substitution at the 2-position was markedly better than at the 4-position, with the 2-OMe and 2-Cl cases both being 10-fold better than the corresponding 4-OMe and 4-Cl cases (Entries <b>19</b>, <b>18</b>, <b>17</b>, and <b>15</b>). The 2-Cl case (Entry <b>18</b>) was clearly the best of all, and was therefore scaled-up to be nominated as <a href="/pcsubstance/?term=ML339[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML339</a>. The data for the submitted scaled-up 2<sup>nd</sup> batch (IC<sub>50</sub> = 0.14 μM; % response 100%) are also shown in <a class="figpopup" href="/books/NBK158948/table/ml339.t1/?report=objectonly" target="object" rid-figpopup="figml339t1" rid-ob="figobml339t1">Table 1</a>. None of the compounds in <a class="figpopup" href="/books/NBK158948/table/ml339.t8/?report=objectonly" target="object" rid-figpopup="figml339t8" rid-ob="figobml339t8">Table 8</a> were active in the CCR6, CXCR5 or APJ counterscreens when tested.</p></div><div id="ml339.s25"><h3>3.5. Cellular Activity</h3><p>The murine assay was validated using anti-mCXCR16 as an antagonist positive control tested in the presence of an EC<sub>80</sub> concentration of mCXCL16 (IC<sub>50</sub> = 1.5 nM; % response = 100%). Probe <a href="/pcsubstance/?term=ML339[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML339</a> was shown to be 100-fold less active at the murine CXCR6 receptor using the DiscoveRx β-arrestin cell-based assay format (<a class="figpopup" href="/books/NBK158948/table/ml339.t9/?report=objectonly" target="object" rid-figpopup="figml339t9" rid-ob="figobml339t9">Table 9</a>). This is an interesting observation because we previously demonstrated that the human and murine ligands have activity within 2-fold at each respective species-based receptor in this assay format (as agonist). Within the CPDP, we elected to state that a suitable chemical probe would have equipotent to 3-fold species variance based on comparing assay development EC<sub>50</sub> values between the human and murine CXCR6 receptors and ligands (agonist mode). The probe <a href="/pcsubstance/?term=ML339[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML339</a> will now be able to assist in determining whether activity at hCXCR6 is based on a species specific allosteric <i>vs.</i> orthosteric functional inactivation of the receptor. While the need for activity at mCXCR6 and stated 3-fold stringency is <u>not critical</u> for our human cell <i>in vitro</i> PCa chemotaxis and related post-probe studies, it would assure the final probe series will be useful to future Investigators interested in the role of CXCR6 in murine lymphatic cells for immunology-based studies. Although this observation limits the current probe utility outside of immunology, it fully is adequate for planned post probe PCa-based human cell optimization and for use in associated murine xenograft studies. Furthermore, the set of compounds shown in <a class="figpopup" href="/books/NBK158948/table/ml339.t9/?report=objectonly" target="object" rid-figpopup="figml339t9" rid-ob="figobml339t9">Table 9</a> gave no response when screened against CXCR5 (<a class="figpopup" href="/books/NBK158948/table/ml339.t8/?report=objectonly" target="object" rid-figpopup="figml339t8" rid-ob="figobml339t8">Table 8</a>) and CXCR4, which underscore their selectivity. Thus, a suitable ‘first in class’ pharmacological tool, useful for exploring the role of CXCR6 related PCa, has been discovered.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml339t9"><a href="/books/NBK158948/table/ml339.t9/?report=objectonly" target="object" title="Table 9" class="img_link icnblk_img figpopup" rid-figpopup="figml339t9" rid-ob="figobml339t9"><img class="small-thumb" src="/books/NBK158948/table/ml339.t9/?report=thumb" src-large="/books/NBK158948/table/ml339.t9/?report=previmg" alt="Table 9. SAR of Selected Compounds as Mouse CXCR6 Antagonists." /></a><div class="icnblk_cntnt"><h4 id="ml339.t9"><a href="/books/NBK158948/table/ml339.t9/?report=objectonly" target="object" rid-ob="figobml339t9">Table 9</a></h4><p class="float-caption no_bottom_margin">SAR of Selected Compounds as Mouse CXCR6 Antagonists. </p></div></div></div><div id="ml339.s26"><h3>3.6. Profiling Assays</h3><p>As a <i>pro forma</i> activity, the SBCCG is committed to profiling all final probe(s) compound(s) 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=ML339[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML339</a> was evaluated in a detailed <i>in vitro</i> pharmacology screen as shown in <a class="figpopup" href="/books/NBK158948/table/ml339.t10/?report=objectonly" target="object" rid-figpopup="figml339t10" rid-ob="figobml339t10">Table 10</a>:</p><p><a href="/pcsubstance/?term=ML339[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML339</a> can achieve concentrations >10× IC<sub>50</sub> in aqueous buffer between a pH range of 5.0–7.4.</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=ML339[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML339</a> exhibits good permeability with increased pH 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=ML339[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML339</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=ML339[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML339</a> showed good stability in human plasma, and moderate stability in 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=ML339[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML339</a> is almost completely metabolized in human and mouse liver microsomes within 1 hour.</p><p><a href="/pcsubstance/?term=ML339[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML339</a> shows no toxicity (>50 μM) towards immortalized Fa2-N4 human hepatocytes.</p><div id="ml339.s27"><h4>Profiling against other GPCRs</h4><p>The probe, <a href="/pcsubstance/?term=ML339[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML339</a> (CID 60202254), was submitted to the Psychoactive Drug Screening Program (PDSP) at the University of North Carolina (Bryan Roth, PI) and the data against a GPCR binding assay panel is shown in <a class="figpopup" href="/books/NBK158948/figure/ml339.f5/?report=objectonly" target="object" rid-figpopup="figml339f5" rid-ob="figobml339f5">Figure 5</a>. Preliminary results indicate <a href="/pcsubstance/?term=ML339[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML339</a> shows a low level of promiscuity across a range of GPCRs, with the only activity appearing against 5-HT2B and DAT. It is not known whether these activities in binding assays are translated into functional modification of the activities of these receptors.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml339f5" co-legend-rid="figlgndml339f5"><a href="/books/NBK158948/figure/ml339.f5/?report=objectonly" target="object" title="Figure 5" class="img_link icnblk_img figpopup" rid-figpopup="figml339f5" rid-ob="figobml339f5"><img class="small-thumb" src="/books/NBK158948/bin/ml339f5.gif" src-large="/books/NBK158948/bin/ml339f5.jpg" alt="Figure 5. Profile of ML339 against the Psychoactive Drug Screening Program (PDSP) % inhibition at 10 μM." /></a><div class="icnblk_cntnt" id="figlgndml339f5"><h4 id="ml339.f5"><a href="/books/NBK158948/figure/ml339.f5/?report=objectonly" target="object" rid-ob="figobml339f5">Figure 5</a></h4><p class="float-caption no_bottom_margin">Profile of ML339 against the Psychoactive Drug Screening Program (PDSP) % inhibition at 10 μM. </p></div></div></div></div></div><div id="ml339.s28"><h2 id="_ml339_s28_">4. Discussion</h2><div id="ml339.s29"><h3>4.1. Comparison to existing art and how the new probe is an improvement</h3><p><a href="/pcsubstance/?term=ML339[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML339</a> is the first reported selective small molecule CXCR6 antagonist. It represents a meaningful improvement in potency over the HTS hit compound, maintains good GPCR selectivity, and offers an additional starting point for lead optimization efforts and eventual <i>in vivo</i> studies.</p></div><div id="ml339.s30"><h3>4.3. Planned Future Studies</h3><p>Representative samples and probe <a href="/pcsubstance/?term=ML339[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML339</a> are under evaluation in a radioligand binding assay format along with chemotaxis in PC3 cells in the Taichman laboratory. Future, post-probe research may include additional SAR, functional assay, pharmacology, and <i>in vivo</i> experiments. Future rounds of SAR may include further exploration of the R<sub>2</sub> substituent, alternatives to the trimethoxy benzamide at R<sub>1</sub>, and modification of the glycine-like linking unit between the bridgehead and the R<sub>2</sub> amide group. Consistent with the goal of achieving proof of concept in an <i>in vivo</i> model, structural perturbations which may enhance metabolic stability and activity against mouse CXCR6 will be emphasized. Profiling assays may be expanded to include radioligand binding, calcium mobilization, cAMP, and chemotaxis. Pharmacological studies may be extended to include multipoint time-based profiling of chemical and metabolic stability, including metabolite identification.</p></div></div><div id="ml339.s31"><h2 id="_ml339_s31_">5. References</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="ml339.r1">Coleman RE. Clinical features of metastatic bone disease and risk of skeletal morbidity. <span><span class="ref-journal">Clin Cancer Res. </span>2006;<span class="ref-vol">12</span>:6243s–6240s.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/17062708" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17062708</span></a>]</div></dd><dt>2.</dt><dd><div class="bk_ref" id="ml339.r2">Wang J, Shiozawa Y, Wang J, Wang Y, Jung Y, Pienta KJ, Mehra R, Loberg R, Taichman RS. The role of CXCR7/RDC1 as a chemokine receptor for CXCL12/SDF-1 in prostate cancer. <span><span class="ref-journal">J Biol Chem. </span>2008;<span class="ref-vol">283</span>:4283–4294.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/18057003" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18057003</span></a>]</div></dd><dt>3.</dt><dd><div class="bk_ref" id="ml339.r3">Sun YX, Pedersen EA, Shiozawa Y, Havens AM, Jung Y, Wang J, Pienta KJ, Taichman RS. CD26/dipeptidyl peptidase IV regulates prostate cancer metastasis by degrading SDF-1/CXCL12. <span><span class="ref-journal">Clin Exp Metastasis. </span>2008;<span class="ref-vol">25</span>:765–776.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/18563594" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18563594</span></a>]</div></dd><dt>4.</dt><dd><div class="bk_ref" id="ml339.r4">Wang J, Wang J, Dai J, Jung Y, Wei CL, Wang Y, Havens AM, Hogg PJ, Keller ET, Pienta KJ, Nor JE, Wang CY, Taichman RS. A glycolytic mechanism regulating an angiogenic switch in prostate cancer. <span><span class="ref-journal">Cancer Res. </span>2007;<span class="ref-vol">67</span>:149–159.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/17210694" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17210694</span></a>]</div></dd><dt>5.</dt><dd><div class="bk_ref" id="ml339.r5">Sun YX, Fang M, Wang J, Cooper CR, Pienta KJ, Taichman RS. Expression and activation of alpha v beta 3 integrins by SDF-1/CXC12 increases the aggressiveness of prostate cancer cells. <span><span class="ref-journal">Prostate. </span>2007;<span class="ref-vol">67</span>:61–73.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/17034033" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17034033</span></a>]</div></dd><dt>6.</dt><dd><div class="bk_ref" id="ml339.r6">Keller ET, Dai J, Escara-Wilke J, Hall CL, Ignatoski K, Taichman RS, Keller J. New trends in the treatment of bone metastasis. <span><span class="ref-journal">J Cell Biochem. </span>2007;<span class="ref-vol">102</span>:1095–1102.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/17955492" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17955492</span></a>]</div></dd><dt>7.</dt><dd><div class="bk_ref" id="ml339.r7">Taichman RS, Loberg RD, Mehra R, Pienta KJ. The evolving biology and treatment of prostate cancer. <span><span class="ref-journal">J Clin Invest. </span>2007;<span class="ref-vol">117</span>:2351–2361.</span> [<a href="/pmc/articles/PMC1952634/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC1952634</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/17786228" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17786228</span></a>]</div></dd><dt>8.</dt><dd><div class="bk_ref" id="ml339.r8">Havens AM, Jung Y, Sun YX, Taichman RS. The role of sialomucin CD164 (MGC-24v or endolyn) in prostate cancer metastasis. <span><span class="ref-journal">BMC Cancer. </span>2006;<span class="ref-vol">6</span>:195.</span> [<a href="/pmc/articles/PMC1557671/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC1557671</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/16859559" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 16859559</span></a>]</div></dd><dt>9.</dt><dd><div class="bk_ref" id="ml339.r9">Wang J, Loberg R, Taichman RS. The pivotal role of CXCL12 (SDF-1)/CXCR4 axis in bone metastasis. <span><span class="ref-journal">Cancer Metastasis Rev. </span>2006;<span class="ref-vol">25</span>:573–587.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/17165132" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17165132</span></a>]</div></dd><dt>10.</dt><dd><div class="bk_ref" id="ml339.r10">Sun YX, Schneider A, Jung Y, Wang J, Dai J, Wang J, Cook K, Osman NI, Koh-Paige AJ, Shim H, Pienta KJ, Keller ET, McCauley LK, Taichman RS. Skeletal Localization and Neutralization of the SDF-1(CXCL12)/CXCR4 Axis Blocks Prostate Cancer Metastasis and Growth in Osseous Sites <em>In vivo</em>. <span><span class="ref-journal">Journal of Bone & Mineral Research. </span>2005:318–329.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/15647826" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 15647826</span></a>]</div></dd><dt>11.</dt><dd><div class="bk_ref" id="ml339.r11">Sun Y-X, Wang J, Shelburne CE, Lopatin DE, Chinnaiyan AM, Pienta KJ, Rubin MA, Taichman RS. The expression of CXCR4 and CXCL12 (SDF-1) in human prostate cancers (PCa) <em>in vivo</em>. <span><span class="ref-journal">J Cell Biochem. </span>2003;<span class="ref-vol">89</span>:462–473.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/12761880" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 12761880</span></a>]</div></dd><dt>12.</dt><dd><div class="bk_ref" id="ml339.r12">Taichman RS, Cooper C, Keller ET, Pienta KJ, Taichman N, McCauley LK. Use of the Stromal Cell derived Factor-1/CXCR4 Pathway in Prostate Cancer Metastasis to Bone. <span><span class="ref-journal">Cancer Res. </span>2002;<span class="ref-vol">62</span>:1832–1837.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/11912162" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 11912162</span></a>]</div></dd><dt>13.</dt><dd><div class="bk_ref" id="ml339.r13">Chandrasekar B, Bysani S, Mummidi S. CXCL16 signals via Gi, phosphatidylinositol 3-kinase, Akt, I kappa B kinase, and nuclear factor-kappa B and induces cell-cell adhesion and aortic smooth muscle cell proliferation. <span><span class="ref-journal">J Biol Chem. </span>2004;<span class="ref-vol">279</span>:3188–3196.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/14625285" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 14625285</span></a>]</div></dd><dt>14.</dt><dd><div class="bk_ref" id="ml339.r14">Wang J, Lu Y, Wang J, Koch AE, Zhang J, Taichman RS. CXCR6 induces prostate cancer progression by the AKT/mammalian target of rapamycin signaling pathway. <span><span class="ref-journal">Cancer Res. </span>2008;<span class="ref-vol">68</span>:10367–10376.</span> [<a href="/pmc/articles/PMC2884407/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2884407</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/19074906" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19074906</span></a>]</div></dd><dt>15.</dt><dd><div class="bk_ref" id="ml339.r15">Lu Y, Wang J, Xu Y, Koch AE, Cai Z, Chen X, Galson DL, Taichman RS, Zhang J. CXCL16 Functions as a Novel Chemotactic Factor for Prostate Cancer Cells <em>In vitro</em>. <span><span class="ref-journal">Mol Cancer Res. </span>2008;<span class="ref-vol">6</span>:546–554.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/18344492" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18344492</span></a>]</div></dd><dt>16.</dt><dd><div class="bk_ref" id="ml339.r16">Mori T, Koizumi M, Toyda E, Ito D, Kami K, Masui T, Fujimoto K, Tamamura H, Hiramatsu K, Fujii N, Imamura M. CXCR4 antagonist inhibits stromal cell-derived factor 1-induced migration and invasion of human pancreatic cancer. <span><span class="ref-journal">Mol Cancer Ther. </span>2004;<span class="ref-vol">3</span>:29–37.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/14749473" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 14749473</span></a>]</div></dd><dt>17.</dt><dd><div class="bk_ref" id="ml339.r17">Hatse S, Princen K, Bridger G, De Clercq E, Schols D. Chemokine receptor inhibition by AMD3100 is strictly confined to CXCR4. <span><span class="ref-journal">FEBS. </span>2002;<span class="ref-vol">527</span>:255–262.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/12220670" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 12220670</span></a>]</div></dd><dt>18.</dt><dd><div class="bk_ref" id="ml339.r18">Hirbe AC, Morgan EA, Weilbaecher KN. The CXCR4/SDF-1 chemokine axis: a potential therapeutic target for bone metastases? <span><span class="ref-journal">Curr Pharm Des. </span>2010;<span class="ref-vol">16</span>:11284–1290.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/20166978" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 20166978</span></a>]</div></dd><dt>19.</dt><dd><div class="bk_ref" id="ml339.r19">Matloubian M, David A, Engel S, Ryan JE, Cyster JG. A transmembrane CXC chemokine is a ligand for HIV-coreceptor Bonzo. <span><span class="ref-journal">Nature Immunol. </span>2000;<span class="ref-vol">1</span>:298–304.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/11017100" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 11017100</span></a>]</div></dd><dt>20.</dt><dd><div class="bk_ref" id="ml339.r20">Hu W, Zhen X, Xiong B, Wang B, Zhang W, Zhou W. CXCR6 is expressed in human prostate cancer <em>in vivo</em> and is involved in the <em>in vitro</em> invasion of PC3 and LNCap cells. <span><span class="ref-journal">Cancer Sci. </span>2008;<span class="ref-vol">99</span>:1362–1369.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/18452560" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18452560</span></a>]</div></dd><dt>21.</dt><dd><div class="bk_ref" id="ml339.r21">Mustelin Tomas, Tautz Lutz, Cosford Nicholas, David Peter, Sergienko Eduard. Methods using HePTP inhibitors for treating leukemia and myelodysplastic syndrome, and methods for identifying agents for treating these diseases. US 20090105240. <span>2009</span></div></dd><dt>22.</dt><dd><div class="bk_ref" id="ml339.r22">Sakaguchi Jun, Iwasaki Nobuhiko, Iwanaga Yuji, Saito Takaharu, Takahara Eiji, Kato Hideo, Hanaoka Miyoji. Synthesis and gastrointestinal prokinetic activity of novel benzamide derivatives with amphoteric side chains. <span><span class="ref-journal">Chem Pharm Bull. </span>2001;<span class="ref-vol">49</span>(4):424–36.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/11310669" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 11310669</span></a>]</div></dd><dt>23.</dt><dd><div class="bk_ref" id="ml339.r23"><span class="ref-journal">Dr Maren Pink, Indiana University Molecular Structure Center.</span> <a href="http://www.iumsc.indiana.edu/aboutIUMSC/index.html" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">http://www<wbr style="display:inline-block"></wbr>.iumsc.indiana<wbr style="display:inline-block"></wbr>.edu/aboutIUMSC/index.html</a>.</div></dd><dt>24.</dt><dd><div class="bk_ref" id="ml339.r24">Baell Jonathan B, Holloway Georgina A. New substructure filters for removal of pan assay interference compounds (PAINS) from screening libraries and for their exclusion in bioassays. <span><span class="ref-journal">J Med Chem. </span>2010;<span class="ref-vol">53</span>:2719–2740.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/20131845" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 20131845</span></a>]</div></dd></dl></div><div id="ml339.s32"><h2 id="_ml339_s32_">6. Supplementary Information</h2><div id="ml339.s33"><h3>6.1. Assay Details</h3><div id="ml339.s34"><h4>Materials</h4><p>PathHunter CHO-K1 hCXCR6 b-arrestin cell line (DiscoveRx, Cat# 93-0205C2)</p><p>PathHunter CHO-K1 hCXCR5 b-arrestin cell line (DiscoveRx, Cat# 93-0204C2)</p><p>PathHunter C2C12 hCXCR4 b-arrestin cell line (DiscoveRx, Cat# 93-0203C7)</p><p>F12 nutrient mix HAMs (Invitrogen, Cat# 11765)</p><p>DMEM (with glutamine) (Mediatech, Cat# 11765-054)</p><p>CP4 and CP 10 Plating Media (DiscoverX)</p><p>hCXCCL16 (R&D Systems, Cat# 976-CX)</p><p>hCXCL13 peptide (R&D Systems, Cat# 801-CX)</p><p>hCXCL12 peptide (R&D Systems, Cat# <a href="http://www.rndsystems.com/Products/6448-SD" ref="pagearea=body&targetsite=external&targetcat=link&targettype=uri">6448-SD-025</a>)</p><p>Fetal Bovine Serum, heat-inactivated (Hyclone, Cat# SH30396)</p><p>100X Penicillin/Streptomycin Solution (Invitrogen, Cat#15140-122)</p><p>Hygromycin B (Roche, Cat# 10843555001)</p><p>Geneticin (MPBiomedicals, Cat# 1672548)</p><p>Trypsin-EDTA 0.25% (Invitrogen, Cat# 25200-056)</p><p>Cell Dissociation Buffer (Invitrogen, Cat# 13151)</p><p>DPBS (Hyclone, Cat# 30028.02)</p><p>T225 TC Flask (Nunc, Cat# 159934)</p><p>Cell strainer, 40 μm (BD, Cat# 352340)</p><p>1536-well, white, solid-bottom, Kalypsys compatible, TC plate (Corning)</p><p>PathHunter Detection Reagents (DiscoveRx, Cat# 93-0001)</p><p>Galacton Star</p><p>Emerald 11</p><p>Cell Assay Buffer</p></div><div id="ml339.s35"><h4>Reagents</h4><div id="ml339.s36"><h5>Growth Media for CXCR6 and CXCR5</h5><p>F12 nutrient mix HAMs supplemented with 10% hi-FBS, 1X Penicillin/Streptomycin; selection reagents: 300 μg/ml Hygromycin B, 800 μg/ml Geneticin</p></div><div id="ml339.s37"><h5>Growth Media for CXCR4</h5><p>DMEM with 20% hi-FBS, 1X Penicillin/Streptomycin; selection reagents: 300ug/ml Hygromycin B, 800ug/ml Geneticin</p></div><div id="ml339.s38"><h5>Assay/plating Media for CXCR6 and CXCR5</h5><p>Same as Growth Media without the selection reagents</p></div><div id="ml339.s39"><h5>Plating Media for CXCR4</h5><p>DiscoveRx CP9 with 20% FCS</p></div><div id="ml339.s40"><h5>Assay Media for CXCR4</h5><p>DiscoveRx CP4 plating medium serum free</p></div><div id="ml339.s41"><h5>Negative Control</h5><p>Growth Media with agonist</p></div><div id="ml339.s42"><h5>Detection Reagent</h5><p>Use the following ratio to prepare the detection reagent:</p><p>Galacton Star: Emerald II: Assay Buffer = 1 : 5 : 19</p></div></div><div id="ml339.s43"><h4>uHTS Procedures for CXCR6 Dose-response in 1536-well</h4><div id="ml339.s44"><h5>Day1 Cell Seeding</h5><ol><li class="half_rhythm"><div>Plate 800 cells/well in 3 μL of assay media into columns 1–48 of a 1536-well assay plate, using combi dispenser.</div></li><li class="half_rhythm"><div>Centrifuge plates at 500 rpm for 1 minute on a Vspin centrifuge.</div></li><li class="half_rhythm"><div>Put Kalypsys lids on and incubate overnight at 37°C, 100% relative humidity, 5% CO<sub>2</sub> for 16–18 hours.</div></li></ol></div><div id="ml339.s45"><h5>Day 2 Compound Addition</h5><ol><li class="half_rhythm"><div>Centrifuge compound plates at 500 rpm for 1 minute on a Vspin centrifuge.</div></li><li class="half_rhythm"><div>Using LabCyte Echo, transfer 40–2.5 nl from a 10 mM and 0.312 mM Echo qualified plate containing DPI liquid reordered test into assay plate Col. 5 – 44 (final concentration of test compounds is 79 μM to 0.156 μM, 0.8% DMSO). Add 40 nL DMSO to positive and negative control wells in Columns 1 – 4 and 45–48</div></li><li class="half_rhythm"><div>Immediately following compound/DMSO transfer via the Echo, using the Biotek Dispenser, transfer 2 μL/well of Assay media to Col. 1–2 for the positive control.</div></li><li class="half_rhythm"><div>Using the Biotek Dispenser, add 2 μL/well of 25 nM CXCL16 (FAC = 10 nM) in assay media to Col. 3–48 for the negative control and test compounds.</div></li><li class="half_rhythm"><div>Centrifuge plates at 1000 rpm for 1 minute on a Vspin centrifuge.</div></li><li class="half_rhythm"><div>Incubate plates at 25°C in the dark for 90 minutes.</div></li><li class="half_rhythm"><div>Following 90 min incubation, deliver 3.0 μL of Detection Reagent solution to each assay plate (Columns 1 – 48) using a Biotek dispenser.</div></li><li class="half_rhythm"><div>Centrifuge plates at 2000 rpm for 3 minute on a Vspin centrifuge.</div></li><li class="half_rhythm"><div>Incubate plates for 60 minutes at 25°C in the dark.</div></li><li class="half_rhythm"><div>Read plates using the Envision plate reader using a luminescence protocol.</div></li></ol></div></div><div id="ml339.s46"><h4>Procedure for CXCR5 Dose-response in 384-well</h4><div id="ml339.s47"><h5>Day1 Cell Seeding</h5><ol><li class="half_rhythm"><div>Plate 2500 cells/well in 20 μL of assay media into columns 1–24 of a 384-well assay plate, using Biotek dispenser.</div></li><li class="half_rhythm"><div>Centrifuge plates at 500 rpm for 1 minute on a Vspin centrifuge.</div></li><li class="half_rhythm"><div>Incubate overnight at 37°C, 100% relative humidity, 5% CO2 for 16–18 hours.</div></li></ol></div><div id="ml339.s48"><h5>Day2 Compound Addition</h5><ol><li class="half_rhythm"><div>Centrifuge compound plates at 500 rpm for 1 minute on a Vspin centrifuge.</div></li><li class="half_rhythm"><div>Using LabCyte Echo 555, transfer 200 nL of DMSO to positive and negative control wells in columns 1 – 2 and 23–24, respectively. Using a dose response protocol, transfer compounds from 10mM and 0.312 mM Echo qualified plates into assay plate columns –3–22. (Final concentrations range 66 μM to 0.128 μM, 10 doses, with 0.66% DMSO.)</div></li><li class="half_rhythm"><div>Immediately following compound/DMSO transfer via the Echo, using the Biotek Dispenser, transfer 10 μL/well of Assay media to Col. 1–2 for the positive control wells and 10 μL/well of 225 nM CXCL13 (FAC = 75 nM) in assay media to Col. 3–24 for the negative control and test compound wells.</div></li><li class="half_rhythm"><div>Centrifuge plates at 500 rpm for 1 min on a Vspin centrifuge</div></li><li class="half_rhythm"><div>Incubate plates at 25°C in the dark for 90 minutes.</div></li><li class="half_rhythm"><div>Following 90 min incubation, deliver 15 μL of Detection Reagent solution to each assay plate (Columns 1 – 24) using a Biotek dispenser.</div></li><li class="half_rhythm"><div>Centrifuge plates at 2000 rpm for 2 min on a Vspin centrifuge.</div></li><li class="half_rhythm"><div>Incubate plates for 60 minutes at 25°C in the dark.</div></li><li class="half_rhythm"><div>Read plates using the Envision using a luminescence protocol.</div></li></ol></div></div><div id="ml339.s49"><h4>Procedure for CXCR4 Dose-response in 384-well</h4><div id="ml339.s50"><h5>Day1 Cell Seeding</h5><ol><li class="half_rhythm"><div>Plate 5000 cells/well in 12 μL of plating media into columns 1–24 of a 384-well assay plate, using Biotek dispenser.</div></li><li class="half_rhythm"><div>Centrifuge plates at 500 rpm for 1 minute on a Vspin centrifuge.</div></li><li class="half_rhythm"><div>Incubate overnight at 37°C, 100% relative humidity, 5% CO<sub>2</sub> for 16–18 hours.</div></li></ol></div><div id="ml339.s51"><h5>Day2 Compound Addition</h5><ol><li class="half_rhythm"><div class="half_rhythm">Wash plate with CP4 media and incubate 3h at 37°C.</div></li><li class="half_rhythm"><div class="half_rhythm">Using LabCyte Echo 555, transfer 160 nL of DMSO to positive and negative control wells in columns 1 – 2 and 23–24, respectively. Using a dose response protocol, transfer compounds from 10mM and 0.312 mM Echo qualified plates into assay plate columns 3–22. (Final concentrations range 79 μM to 0.154 μM, 10 doses, with 0.8% DMSO.)</div></li><li class="half_rhythm"><div class="half_rhythm">Immediately following compound/DMSO transfer via the Echo, using the Biotek Dispenser, transfer 8 μL/well of Assay media to Col. 1–2 for the positive control wells.</div><div class="half_rhythm">and 10 μL/well of 225 nM CXCL12 (FAC = 75 nM) in assay media to Col. 3–24 for the negative control and test compound wells.</div></li><li class="half_rhythm"><div class="half_rhythm">Centrifuge plates at 500 rpm for 1 min on a Vspin centrifuge.</div></li><li class="half_rhythm"><div class="half_rhythm">Incubate plates at 25°C in the dark for 90 minutes.</div></li><li class="half_rhythm"><div class="half_rhythm">Following 90 min incubation, deliver 12 μL of Detection Reagent solution to each assay plate (Columns 1 – 24) using a Biotek dispenser</div></li><li class="half_rhythm"><div class="half_rhythm">Centrifuge plates at 2000 rpm for 2 min on a Vspin centrifuge</div></li><li class="half_rhythm"><div class="half_rhythm">Incubate plates for 60 minutes at 25°C in the dark.</div></li><li class="half_rhythm"><div class="half_rhythm">Read plates using the Envision using a luminescence protocol.</div></li></ol></div></div></div><div id="ml339.s52"><h3>6.2. <sup>1</sup>H NMR and LC-MS spectra of ML339</h3><div id="ml339.fu2" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=1H%20NMR%20Spectrum%20of%20ML339%20(500%20MHz%2C%20DMSO-d6).&p=BOOKS&id=158948_ml339fu10.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/NBK158948/bin/ml339fu10.jpg" alt="1H NMR Spectrum of ML339 (500 MHz, DMSO-d6)." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="title"><sup>1</sup>H NMR Spectrum of ML339 (500 MHz, DMSO-<i>d</i><sub>6</sub>)</span></h3></div><div id="ml339.fu3" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=13C%20NMR%20Spectrum%20of%20ML339%20(125%20MHz%2C%20DMSO-d6).&p=BOOKS&id=158948_ml339fu11.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/NBK158948/bin/ml339fu11.jpg" alt="13C NMR Spectrum of ML339 (125 MHz, DMSO-d6)." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="title"><sup>13</sup>C NMR Spectrum of ML339 (125 MHz, DMSO-<i>d</i><sub>6</sub>)</span></h3><div class="caption"><p>LC/MS for ML339.</p></div></div><div id="ml339.fu4" class="figure"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Image%20ml339fu12&p=BOOKS&id=158948_ml339fu12.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/NBK158948/bin/ml339fu12.jpg" alt="Image ml339fu12" class="tileshop" title="Click on image to zoom" /></a></div></div><div id="ml339.fu5" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=MS%20spectrum%20for%20M339.&p=BOOKS&id=158948_ml339fu13.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/NBK158948/bin/ml339fu13.jpg" alt="MS spectrum for M339." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="title">MS spectrum for M339</span></h3></div></div></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/NBK158948/?report=reader">PubReader</a></li><li><a href="/books/NBK158948/?report=printable">Print View</a></li><li><a data-jig="ncbidialog" href="#_ncbi_dlg_citbx_NBK158948" data-jigconfig="width:400,modal:true">Cite this Page</a><div id="_ncbi_dlg_citbx_NBK158948" style="display:none" title="Cite this Page"><div class="bk_tt">Hershberger PM, Peddibhotla S, Sugarman E, et al. Probing the CXCR6/CXCL16 Axis: Targeting Prevention of Prostate Cancer Metastasis. 2012 Dec 15 [Updated 2014 May 13]. In: Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-. <span class="bk_cite_avail"></span></div></div></li><li><a href="/books/NBK158948/pdf/Bookshelf_NBK158948.pdf">PDF version of this page</a> (685K)</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="#ml339.s1" ref="log$=inpage&link_id=inpage">Probe Structure & Characteristics</a></li><li><a href="#ml339.s2" ref="log$=inpage&link_id=inpage">Recommendations for scientific use of the probe</a></li><li><a href="#ml339.s3" ref="log$=inpage&link_id=inpage">Introduction</a></li><li><a href="#ml339.s7" ref="log$=inpage&link_id=inpage">Materials and Methods</a></li><li><a href="#ml339.s19" ref="log$=inpage&link_id=inpage">Results</a></li><li><a href="#ml339.s28" ref="log$=inpage&link_id=inpage">Discussion</a></li><li><a href="#ml339.s31" ref="log$=inpage&link_id=inpage">References</a></li><li><a href="#ml339.s32" ref="log$=inpage&link_id=inpage">Supplementary Information</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=3054230" ref="log$=recordlinks">PMC</a><div class="brieflinkpop offscreen_noflow">PubMed Central citations</div></li><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pcassay&DbFrom=books&Cmd=Link&LinkName=books_pcassay_probe&IdsFromResult=3054230" ref="log$=recordlinks">PubChem BioAssay for Chemical Probe</a><div class="brieflinkpop offscreen_noflow">PubChem BioAssay records reporting screening data for the development of the chemical probe(s) described in this book chapter</div></li><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pcsubstance&DbFrom=books&Cmd=Link&LinkName=books_pcsubstance&IdsFromResult=3054230" ref="log$=recordlinks">PubChem Substance</a><div class="brieflinkpop offscreen_noflow">Related PubChem Substances</div></li><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pubmed&DbFrom=books&Cmd=Link&LinkName=books_pubmed_refs&IdsFromResult=3054230" ref="log$=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/26799186" ref="ordinalpos=1&linkpos=1&log$=relatedarticles&logdbfrom=pubmed">CXCR6-CXCL16 axis promotes prostate cancer by mediating cytoskeleton rearrangement via Ezrin activation and αvβ3 integrin clustering.</a><span class="source">[Oncotarget. 2016]</span><div class="brieflinkpop offscreen_noflow">CXCR6-CXCL16 axis promotes prostate cancer by mediating cytoskeleton rearrangement via Ezrin activation and αvβ3 integrin clustering.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Singh R, Kapur N, Mir H, Singh N, Lillard JW Jr, Singh S. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Oncotarget. 2016 Feb 9; 7(6):7343-53. </em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/20646641" ref="ordinalpos=1&linkpos=2&log$=relatedarticles&logdbfrom=pubmed">[Role of CXCL16/CXCR6 axis in the metastasis of human prostate cancer].</a><span class="source">[Zhonghua Yi Xue Za Zhi. 2010]</span><div class="brieflinkpop offscreen_noflow">[Role of CXCL16/CXCR6 axis in the metastasis of human prostate cancer].<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Zhou WH, Hu WD, Wu ZQ, Zheng XM, Wang BC. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Zhonghua Yi Xue Za Zhi. 2010 Apr 13; 90(14):947-51. </em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/20122997" ref="ordinalpos=1&linkpos=3&log$=relatedreviews&logdbfrom=pubmed"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> CXCR6/CXCL16 functions as a regulator in metastasis and progression of cancer.</a><span class="source">[Biochim Biophys Acta. 2010]</span><div class="brieflinkpop offscreen_noflow"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> CXCR6/CXCL16 functions as a regulator in metastasis and progression of cancer.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Deng L, Chen N, Li Y, Zheng H, Lei Q. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Biochim Biophys Acta. 2010 Aug; 1806(1):42-9. Epub 2010 Feb 1.</em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/21468586" ref="ordinalpos=1&linkpos=4&log$=relatedarticles&logdbfrom=pubmed">Clinical significance of CXCL16/CXCR6 expression in patients with prostate cancer.</a><span class="source">[Mol Med Rep. 2011]</span><div class="brieflinkpop offscreen_noflow">Clinical significance of CXCL16/CXCR6 expression in patients with prostate cancer.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Ha HK, Lee W, Park HJ, Lee SD, Lee JZ, Chung MK. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Mol Med Rep. 2011 May-Jun; 4(3):419-24. 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