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<meta name="ncbi_db" content="books" /><meta name="ncbi_pdid" content="book-part" /><meta name="ncbi_acc" content="NBK179827" /><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="/NBK179827/" /><meta name="ncbi_pagename" content="Optimization and characterization of an opioid kappa receptor (OPRK1) antagonist - 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>Optimization and characterization of an opioid kappa receptor (OPRK1) antagonist - 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="Optimization and characterization of an opioid kappa receptor (OPRK1) antagonist" /><meta name="citation_publisher" content="National Center for Biotechnology Information (US)" /><meta name="citation_date" content="2014/09/18" /><meta name="citation_author" content="Miguel Guerrero" /><meta name="citation_author" content="Mariagela Urbano" /><meta name="citation_author" content="Steven J Brown" /><meta name="citation_author" content="Charmagne Cayanan" /><meta name="citation_author" content="Jill Ferguson" /><meta name="citation_author" content="Michael Cameron" /><meta name="citation_author" content="Lakshmi A. Devi" /><meta name="citation_author" content="Ed Roberts" /><meta name="citation_author" content="Hugh Rosen" /><meta name="citation_pmid" content="24479196" /><meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK179827/" /><link rel="schema.DC" href="http://purl.org/DC/elements/1.0/" /><meta name="DC.Title" content="Optimization and characterization of an opioid kappa receptor (OPRK1) antagonist" /><meta name="DC.Type" content="Text" /><meta name="DC.Publisher" content="National Center for Biotechnology Information (US)" /><meta name="DC.Contributor" content="Miguel Guerrero" /><meta name="DC.Contributor" content="Mariagela Urbano" /><meta name="DC.Contributor" content="Steven J Brown" /><meta name="DC.Contributor" content="Charmagne Cayanan" /><meta name="DC.Contributor" content="Jill Ferguson" /><meta name="DC.Contributor" content="Michael Cameron" /><meta name="DC.Contributor" content="Lakshmi A. Devi" /><meta name="DC.Contributor" content="Ed Roberts" /><meta name="DC.Contributor" content="Hugh Rosen" /><meta name="DC.Date" content="2014/09/18" /><meta name="DC.Identifier" content="https://www.ncbi.nlm.nih.gov/books/NBK179827/" /><meta name="description" content="The opioid receptors are a subfamily of the family A G protein-coupled opioid receptor superfamily and consist of mu (OPRM1), delta (OPRD1), and kappa (OPRK1), all of which activate inhibitory G proteins. The dynorphins act as endogenous agonists of OPRK to activate a variety of signaling transduction pathways including those involving mitogen activated protein kinases (MAPK). Activation of OPRK leads to a number of physiological effects implicating a role for these receptors in addiction, dysphoria and reward. Hence, OPRK antagonists are being explored for their effects in the treatment of cocaine addiction, depression, and feeding behavior and have been proposed as a treatment for psychosis and schizophrenia. While a number of OPRK1 antagonists have been identified, all of the prototypic antagonists are very long-acting, exhibit unusual pharmacology, exhibit delayed onset of action, and are associated with serious safety concerns. Very few drug-like OPRK antagonists have been developed. New OPRK antagonists possessing novel scaffolds and improved selectivity are needed as pharmacological tools to better understand the OPRK- dynorphin system and as potential pharmacotherapies. The Scripps Research Institute Molecular Screening Center (SRIMSC), part of the Molecular Libraries Probe Production Centers Network (MLPCN), reports ML350 as a highly potent OPRK1 antagonist with an IC50 of 9-16 nM, with high selectivity (selectivities vs. OPRD1 and OPRM1 of 219-382–fold and 20-35–fold, respectively). ML350 was identified by high-throughput screening using a cell-based Tango™-format assay. A set of pharmacokinetic analyses show that ML350 has high passive membrane permeability, good brain penetration, no significant activity at three of four human cytochrome P450 subtypes, high binding for rodent plasma protein and modest binding for human plasma protein, and an encouraging in vivo pharmacokinetic profile in rats. ML350 was submitted to CEREP for broad panel screening against a panel of receptors, transporters, and ion channels; the data suggest that ML350 is generally inactive against a broad array of off targets and does not likely exert unwanted effects. Importantly, ML350 was shown to have a reversible analgesic effect when challenged with an OPRK agonist in a tail flick assay in mice. ML350 serves as a novel OPRK antagonist that can be developed as a therapeutic for the treatment of a variety of disorders involving the OPRK1-dynorphin system." /><meta name="og:title" content="Optimization and characterization of an opioid kappa receptor (OPRK1) antagonist" /><meta name="og:type" content="book" /><meta name="og:description" content="The opioid receptors are a subfamily of the family A G protein-coupled opioid receptor superfamily and consist of mu (OPRM1), delta (OPRD1), and kappa (OPRK1), all of which activate inhibitory G proteins. The dynorphins act as endogenous agonists of OPRK to activate a variety of signaling transduction pathways including those involving mitogen activated protein kinases (MAPK). Activation of OPRK leads to a number of physiological effects implicating a role for these receptors in addiction, dysphoria and reward. Hence, OPRK antagonists are being explored for their effects in the treatment of cocaine addiction, depression, and feeding behavior and have been proposed as a treatment for psychosis and schizophrenia. While a number of OPRK1 antagonists have been identified, all of the prototypic antagonists are very long-acting, exhibit unusual pharmacology, exhibit delayed onset of action, and are associated with serious safety concerns. Very few drug-like OPRK antagonists have been developed. New OPRK antagonists possessing novel scaffolds and improved selectivity are needed as pharmacological tools to better understand the OPRK- dynorphin system and as potential pharmacotherapies. The Scripps Research Institute Molecular Screening Center (SRIMSC), part of the Molecular Libraries Probe Production Centers Network (MLPCN), reports ML350 as a highly potent OPRK1 antagonist with an IC50 of 9-16 nM, with high selectivity (selectivities vs. OPRD1 and OPRM1 of 219-382–fold and 20-35–fold, respectively). ML350 was identified by high-throughput screening using a cell-based Tango™-format assay. A set of pharmacokinetic analyses show that ML350 has high passive membrane permeability, good brain penetration, no significant activity at three of four human cytochrome P450 subtypes, high binding for rodent plasma protein and modest binding for human plasma protein, and an encouraging in vivo pharmacokinetic profile in rats. ML350 was submitted to CEREP for broad panel screening against a panel of receptors, transporters, and ion channels; the data suggest that ML350 is generally inactive against a broad array of off targets and does not likely exert unwanted effects. Importantly, ML350 was shown to have a reversible analgesic effect when challenged with an OPRK agonist in a tail flick assay in mice. ML350 serves as a novel OPRK antagonist that can be developed as a therapeutic for the treatment of a variety of disorders involving the OPRK1-dynorphin system." /><meta name="og:url" content="https://www.ncbi.nlm.nih.gov/books/NBK179827/" /><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/ml350/" /><link rel="canonical" href="https://www.ncbi.nlm.nih.gov/books/NBK179827/" /><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="#__NBK179827_dtls__">Show details</a><div style="display:none" class="ui-widget" id="__NBK179827_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/ml351/" title="Previous page in this title">< Prev</a><a class="active page_link next" href="/books/n/mlprobe/ml349/" 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="_NBK179827_"><span class="title" itemprop="name">Optimization and characterization of an opioid kappa receptor (OPRK1) antagonist</span></h1><p class="contrib-group"><span itemprop="author">Miguel Guerrero</span>, <span itemprop="author">Mariagela Urbano</span>, <span itemprop="author">Steven J Brown</span>, <span itemprop="author">Charmagne Cayanan</span>, <span itemprop="author">Jill Ferguson</span>, <span itemprop="author">Michael Cameron</span>, <span itemprop="author">Lakshmi A. Devi</span>, <span itemprop="author">Ed Roberts</span>, and <span itemprop="author">Hugh Rosen</span>.</p><a data-jig="ncbitoggler" href="#__NBK179827_ai__" style="border:0;text-decoration:none">Author Information and Affiliations</a><div style="display:none" class="ui-widget" id="__NBK179827_ai__"><p class="contrib-group"><h4>Authors</h4><span itemprop="author">Miguel Guerrero</span>,<sup>*</sup> <span itemprop="author">Mariagela Urbano</span>,<sup>*</sup> <span itemprop="author">Steven J Brown</span>,<sup>*</sup> <span itemprop="author">Charmagne Cayanan</span>,<sup>*</sup> <span itemprop="author">Jill Ferguson</span>,<sup>*</sup> <span itemprop="author">Michael Cameron</span>,<sup>†</sup> <span itemprop="author">Lakshmi A. Devi</span>,<sup>‡</sup> <span itemprop="author">Ed Roberts</span>,<sup>*</sup> and <span itemprop="author">Hugh Rosen</span><sup>*</sup>.<sup><img src="/corehtml/pmc/pmcgifs/corrauth.gif" alt="corresponding author" /></sup></p><h4>Affiliations</h4><div class="affiliation"><sup>*</sup>
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The Scripps Research Institute, La Jolla CA</div><div class="affiliation"><sup>†</sup>
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The Scripps Research Institute, Jupiter, FL</div><div class="affiliation"><sup>‡</sup>
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Mount Sinai School of Medicine, New York, NY</div><div class="affiliation"><sup><img src="/corehtml/pmc/pmcgifs/corrauth.gif" alt="corresponding author" /></sup>Corresponding author:
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<span class="before-email-separator"></span><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="ude.sppircs@nesorh" class="oemail">ude.sppircs@nesorh</a></div></div><p class="small">Received: <span itemprop="datePublished">April 15, 2013</span>; Last Update: <span itemprop="dateModified">September 18, 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>The opioid receptors are a subfamily of the family A G protein-coupled opioid receptor superfamily and consist of mu (OPRM1), delta (OPRD1), and kappa (OPRK1), all of which activate inhibitory G proteins. The dynorphins act as endogenous agonists of OPRK to activate a variety of signaling transduction pathways including those involving mitogen activated protein kinases (MAPK). Activation of OPRK leads to a number of physiological effects implicating a role for these receptors in addiction, dysphoria and reward. Hence, OPRK antagonists are being explored for their effects in the treatment of cocaine addiction, depression, and feeding behavior and have been proposed as a treatment for psychosis and schizophrenia. While a number of OPRK1 antagonists have been identified, all of the prototypic antagonists are very long-acting, exhibit unusual pharmacology, exhibit delayed onset of action, and are associated with serious safety concerns. Very few drug-like OPRK antagonists have been developed. New OPRK antagonists possessing novel scaffolds and improved selectivity are needed as pharmacological tools to better understand the OPRK- dynorphin system and as potential pharmacotherapies. The Scripps Research Institute Molecular Screening Center (SRIMSC), part of the Molecular Libraries Probe Production Centers Network (MLPCN), reports <a href="/pcsubstance/?term=ML350[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML350</a> as a highly potent OPRK1 antagonist with an IC50 of 9-16 nM, with high selectivity (selectivities vs. OPRD1 and OPRM1 of 219-382–fold and 20-35–fold, respectively). <a href="/pcsubstance/?term=ML350[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML350</a> was identified by high-throughput screening using a cell-based Tango™-format assay. A set of pharmacokinetic analyses show that <a href="/pcsubstance/?term=ML350[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML350</a> has high passive membrane permeability, good brain penetration, no significant activity at three of four human cytochrome P450 subtypes, high binding for rodent plasma protein and modest binding for human plasma protein, and an encouraging <i>in vivo</i> pharmacokinetic profile in rats. <a href="/pcsubstance/?term=ML350[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML350</a> was submitted to CEREP for broad panel screening against a panel of receptors, transporters, and ion channels; the data suggest that <a href="/pcsubstance/?term=ML350[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML350</a> is generally inactive against a broad array of off targets and does not likely exert unwanted effects. Importantly, <a href="/pcsubstance/?term=ML350[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML350</a> was shown to have a reversible analgesic effect when challenged with an OPRK agonist in a tail flick assay in mice. <a href="/pcsubstance/?term=ML350[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML350</a> serves as a novel OPRK antagonist that can be developed as a therapeutic for the treatment of a variety of disorders involving the OPRK1-dynorphin system.</p></div><div class="h2"></div><p><b>Assigned Assay Grant #:</b> R03 NS053751</p><p><b>Screening Center Name & PI:</b> The Scripps Research Institute Molecular Screening Center (SRIMSC), Hugh Rosen</p><p><b>Chemistry Center Name & PI:</b> SRIMSC, Hugh Rosen</p><p><b>Assay Submitter & Institution:</b> Lakshmi Devi, Mount Sinai School of Medicine</p><p><b>PubChem Summary Bioassay Identifier (AID):</b>
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<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/652045" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">652045</a></p><div id="ml350.s1"><h2 id="_ml350_s1_">Probe Structure & Characteristics</h2><div id="ml350.f1" class="figure"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Image%20ml350f1&p=BOOKS&id=179827_ml350f1.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/NBK179827/bin/ml350f1.jpg" alt="Image ml350f1" class="tileshop" title="Click on image to zoom" /></a></div></div><div id="ml350.t1" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK179827/table/ml350.t1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml350.t1_lrgtbl__"><table><thead><tr><th id="hd_h_ml350.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CID/ML#</th><th id="hd_h_ml350.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Target Name</th><th id="hd_h_ml350.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">IC50 (nM) [SID, AID]</th><th id="hd_h_ml350.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Anti-target Name(s)</th><th id="hd_h_ml350.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">IC50 (μM) [SID, AID]</th><th id="hd_h_ml350.t1_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Fold Selective</th><th id="hd_h_ml350.t1_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Secondary Assay(s) Name: IC50/EC50 (nM) [SID, AID]</th></tr></thead><tbody><tr><td headers="hd_h_ml350.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CID 60156214/<a href="/pcsubstance/?term=ML350[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML350</a></td><td headers="hd_h_ml350.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b>OPRK1</b></td><td headers="hd_h_ml350.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">9.16 [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/144087319" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 144087319</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/652084" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 652084</a>]<br />16.4 nM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/144087319" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 144087319</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/652032" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 652032</a>]</td><td headers="hd_h_ml350.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b>OPRD1</b>; <b>OPRM1</b></td><td headers="hd_h_ml350.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b>OPRD1</b>: 3.5 uM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/144087319" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 144087319</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/652033" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 652033</a>]<br /><b>OPRM1</b>: 323 nM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/144087319" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 144087319</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/652034" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 652034</a>]</td><td headers="hd_h_ml350.t1_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b>OPRD1</b>: 219-382 fold<br /><b>OPRM1</b>: 20-35 fold</td><td headers="hd_h_ml350.t1_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b>Cytotoxicity:</b> [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/144087319" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 144087319</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/652086" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 652086</a>]<br /><b>Plasma protein binding:</b> [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/144087319" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 144087319</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/652078" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 652078</a>]<br /><b>Hepatic microsome stability:</b> [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/144087319" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 144087319</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/652086" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 652086</a>]<br /><b>Cytochrome P450 inhibition:</b> [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/144087319" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 144087319</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/652076" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 652076</a>]<br /><b>CEREP panel counterscreen:</b> [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/144087319" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 144087319</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/652083" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 652083</a>]<br /><b>CEREP hERG counterscreen:</b> [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/144087319" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 144087319</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/662075" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 662075</a>]<br /><b>Plasma and brain levels:</b> [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/144087319" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 144087319</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/662085" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 662085</a>]<br /><b>Tail Flick assay:</b> [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/144087319" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 144087319</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/652108" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 652108</a>]<br /><b>PAMPA permeability:</b> [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/144087319" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 144087319</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/652113" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 652113</a>]</td></tr></tbody></table></div></div></div><div id="ml350.s2"><h2 id="_ml350_s2_">Recommendations for scientific use of the probe</h2><p>Prototypic OPRK antagonists are very long-acting, exhibit delayed onset of action, and are associated with serious safety concerns. Very few drug-like OPRK antagonists have been developed. The phase I studies with PF-04455242 were terminated due to toxicity issues. Eli Lilly advanced LY2456302 to a phase I clinical trial study (oral treatment of alcohol dependence) to measure the occupancy of brain OPRK after single oral doses. The last update in <a href="http://ClinicalTrials.gov" ref="pagearea=body&targetsite=external&targetcat=link&targettype=uri">ClinicalTrials.gov</a> reported for this study is May 5, 2011; no information on any progress in further Phase I studies or progression to Phase II is available. Such a delay is unusual due to the costs involved. Hence, there is a need for additional drug-like highly potent and selective OPRK antagonists with a good safety profile and CNS exposure.</p><p>Selective OPRK antagonists are being explored for their effects in the treatment of a wide variety of areas including cocaine addiction [<a class="bk_pop" href="#ml350.r1">1</a>], depression [<a class="bk_pop" href="#ml350.r2">2</a>], and feeding behavior [<a class="bk_pop" href="#ml350.r3">3</a>] and have been proposed as a treatment for psychosis and schizophrenia [<a class="bk_pop" href="#ml350.r4">4</a>]. <a href="/pcsubstance/?term=ML350[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML350</a> will be used in a stress-induced migraine model assay that measures cutaneous (tactile) allodynia in rats as a measurable translational endpoint for headache-related pain [<a class="bk_pop" href="#ml350.r5">5</a>], an alcohol withdrawal assay that measures working memory performance and anxiety-like behavior in rats after withdrawal from alcohol [<a class="bk_pop" href="#ml350.r6">6</a>], and a cocaine reward assay that measures the effect on drug-seeking behavior in a cocaine addiction model.</p><p>The probe will be of use to researchers interested in the role of the OPRK in neuropathic pain, drug addiction, or affective psychiatric disorders.</p><p>New OPRK antagonists possessing novel scaffolds and improved selectivity are needed as pharmacological tools to better understand the OPRK-dynorphin system. Signaling through OPRK results in the activation of multiple signal transduction pathways including activation of PI3-kinase, PKCζ, and EERK-1 and ERK-2 [<a class="bk_pop" href="#ml350.r7">7</a>]. In addition, signaling through OPRK results in activation of the p38 MAPK [<a class="bk_pop" href="#ml350.r8">8</a>-<a class="bk_pop" href="#ml350.r15">15</a>] and the JNK1 [<a class="bk_pop" href="#ml350.r16">16</a>-<a class="bk_pop" href="#ml350.r17">17</a>] signaling pathways, and the JAK2/STAT3 and IRF2 signaling cascade [<a class="bk_pop" href="#ml350.r18">18</a>]. Different OPRK1 ligands are reported to activate distinct signal transduction pathways; this activation of multiple signal transduction pathways are thought to be due to ‘ligand-directed signal trafficking’ by OPRK1 [<a class="bk_pop" href="#ml350.r7">7</a>-<a class="bk_pop" href="#ml350.r17">17</a>, <a class="bk_pop" href="#ml350.r19">19</a>-<a class="bk_pop" href="#ml350.r21">21</a>]. It is thought that ligands that are selective for one pathway over the other will help elucidate the role of each pathway in specific responses. This will help in the development of reagents and/or therapeutics that can target pathway responsible for the wanted effects versus unwanted side effects.</p></div><div id="ml350.s3"><h2 id="_ml350_s3_">1. Introduction</h2><p>Opioids are the most widely used class of analgesics [<a class="bk_pop" href="#ml350.r22">22</a>]. The opioid receptors are a subfamily of the G protein-coupled opioid receptor (GPCR) superfamily and the three major types, mu (OPRM1), delta (OPRD1), and kappa (OPRK1) opioid receptors have been pharmacologically characterized and cloned [<a class="bk_pop" href="#ml350.r23">23</a>-<a class="bk_pop" href="#ml350.r25">25</a>]. All activate inhibitory G proteins. Signaling through OPRK results in the activation of a number of signal transduction pathways [<a class="bk_pop" href="#ml350.r7">7</a>-<a class="bk_pop" href="#ml350.r21">21</a>] and this, in turn, leads to a number of physiological effects implicating a role for these receptors in addiction, dysphoria and reward [<a class="bk_pop" href="#ml350.r1">1</a>-<a class="bk_pop" href="#ml350.r2">2</a>, <a class="bk_pop" href="#ml350.r4">4</a>]. Hence, kappa opioid antagonists are being explored for their effects in the treatment of cocaine addiction, depression, and feeding behavior and have been proposed as a treatment for psychosis and schizophrenia. While a number of OPRK1 antagonists have been identified, all of the prototypic antagonists very long-acting, exhibit unusual pharmacology, exhibit delayed onset of action, and are associated with serious safety concerns. Very few drug-like OPRK antagonists have been developed. New OPRK antagonists possessing novel scaffolds and improved selectivity are needed as pharmacological tools to better understand the OPRK system and as potential pharmacotherapies.</p><p><i><u>Neuropathic pain</u>:</i> Studies have identified a role for dynorphin and OPRK in neuropathic pain [<a class="bk_pop" href="#ml350.r26">26</a>]. The dynorphins act as endogenous agonists of OPRK1 [<a class="bk_pop" href="#ml350.r27">27</a>]. The OPRK1-dynorphin system mediates astrocyte proliferation through the activation of p38 MAPK that is required for the effects of neuropathic pain on analgesic responses [<a class="bk_pop" href="#ml350.r15">15</a>, <a class="bk_pop" href="#ml350.r28">28</a>]. Increased dynorphin expression in neuropathic pain leads to a sustained activation of OPRK [<a class="bk_pop" href="#ml350.r26">26</a>, <a class="bk_pop" href="#ml350.r29">29</a>-<a class="bk_pop" href="#ml350.r30">30</a>].</p><p>There is evidence that the endogenous dynorphin-derived opioids may produce either a sustained reduction or an increase in sensitivity to painful stimuli [<a class="bk_pop" href="#ml350.r15">15</a>, <a class="bk_pop" href="#ml350.r31">31</a>]. Thus, dynorphin can elicit multiple effects to modulate analgesic responses. Antinociceptive effects of intrathecal and systemic administration of selective OPRK1 agonists have been documented [<a class="bk_pop" href="#ml350.r31">31</a>-<a class="bk_pop" href="#ml350.r32">32</a>]. It has also been reported that the OPRK1 antagonist norbinaltorphimine (nor-BNI) significantly lowers pain thresholds and increases pain sensation after sciatic nerve ligation [<a class="bk_pop" href="#ml350.r33">33</a>]. Thus, the endogenous dynorphin-derived opioids may have both antinociceptive and pronociceptive actions. How sustained activation of opioid receptors by endogenous dynorphins contributes to the neuropathic pain state is not clear. One of the side effects of OPRK1-mediated analgesia is depression [<a class="bk_pop" href="#ml350.r30">30</a>, <a class="bk_pop" href="#ml350.r34">34</a>]; the development of novel analgesics that bypass this side effect would be therapeutically beneficial.</p><p><i><u>Drug addiction</u>:</i> About 90 million people worldwide suffer from drug addiction [<a class="bk_pop" href="#ml350.r35">35</a>]. A role for the OPRK1-dynorphin system in modulating drug addiction has been proposed; however its function appears to be diverse, and may modulate drug-seeking behavior depending on factors such as drug history, pattern of intake, and stress (for review see [<a class="bk_pop" href="#ml350.r36">36</a>-<a class="bk_pop" href="#ml350.r37">37</a>]). Multiple exposures to cocaine result in complex molecular changes in the brain, and, ultimately, in addiction [<a class="bk_pop" href="#ml350.r38">38</a>]. Although a single exposure to cocaine in rats does not affect brain dynorphin levels, repeated exposures increase dynorphin concentrations in the striatum and substantia nigra [<a class="bk_pop" href="#ml350.r39">39</a>].</p><p>The role of OPRK1 in cocaine addiction has been actively studied [<a class="bk_pop" href="#ml350.r40">40</a>-<a class="bk_pop" href="#ml350.r50">50</a>]. Results clearly demonstrate an activation of the OPRK1 system following chronic cocaine exposure. As OPRK1 stimulation in the brain produces aversive effects in animals and humans [<a class="bk_pop" href="#ml350.r51">51</a>-<a class="bk_pop" href="#ml350.r53">53</a>], it is likely that cocaine-induced up-regulation of OPRK1 in regions associated with reward might be part of a protective compensatory neuroadaptive mechanism to counteract the rewarding effect of cocaine and might contribute to the emergence of persistent dysphoria, an emotional state marked by anxiety, depression, and restlessness that is often reported in humans after the withdrawal of the drug [<a class="bk_pop" href="#ml350.r54">54</a>]. Studies with OPRK1 knockout mice [<a class="bk_pop" href="#ml350.r55">55</a>-<a class="bk_pop" href="#ml350.r62">62</a>] suggest that the receptor antagonists might be useful in preventing stress-induced relapse in cocaine-dependent individuals and therefore help to prevent drug use relapse.</p><p>It has been reported that chronic nicotine exposure affects OPRKs modulation of neurotransmission, leading to enhanced negative affect and increased anxiogenic effects [<a class="bk_pop" href="#ml350.r63">63</a>]. Furthermore, spontaneous nicotine withdrawal-induced anxiety-like behavior and somatic signs of withdrawal are blocked by pretreatment with the OPRK antagonists nor-BNI or JDTic [<a class="bk_pop" href="#ml350.r63">63</a>].</p><p>Several studies have implicated the role of OPRK1 signaling in stress-induced reoccurrence of ethanol self-administration [<a class="bk_pop" href="#ml350.r64">64</a>-<a class="bk_pop" href="#ml350.r65">65</a>]. In addition, OPRK1-dynorphin systems have been shown to be altered by opiate treatment in reward-related neural circuits in animal models [<a class="bk_pop" href="#ml350.r64">64</a>, <a class="bk_pop" href="#ml350.r66">66</a>-<a class="bk_pop" href="#ml350.r67">67</a>]. Taken together, these studies suggest that antagonism of OPRK1 may mitigate negative reinforcing behavioral states associated with drug withdrawal.</p><p><i><u>Affective psychiatric disorders</u>:</i> Affective psychiatric disorders comprise a worldwide health challenge; about 120 and 25 million people suffer from depression and schizophrenia, respectively [<a class="bk_pop" href="#ml350.r35">35</a>]. These afflictions are all characterized by changes in emotion, motivation, cognition, and stress reactivity. Imaging studies have consistently shown altered activity in the amygdala, hippocampus, basal ganglia, and prefrontal cortex of psychiatric patients [<a class="bk_pop" href="#ml350.r68">68</a>]; areas involved in stress responsiveness, emotional reactivity, goal-directed behavior, motivation, and executive function. OPRK1 and the dynorphin peptides are enriched in these brain regions, where they play a role in modulating neurotransmission. This has been an increasingly active area of research in recent years, and resulting data suggest that dysregulation of this system may contribute to the development and maintenance of various affective psychiatric disorders [<a class="bk_pop" href="#ml350.r69">69</a>]; for review, see [<a class="bk_pop" href="#ml350.r36">36</a>, <a class="bk_pop" href="#ml350.r70">70</a>]. However, solid evidence from clinical studies is lacking.</p><p>There is increasing evidence for a potential involvement of OPRK1-dynorphin in schizophrenia; OPRK1 agonists appear to induce symptoms in humans and animals that are present in schizophrenia [<a class="bk_pop" href="#ml350.r70">70</a>-<a class="bk_pop" href="#ml350.r72">72</a>]. The potent OPRK1 agonist Salvinorin A produces hallucinations in humans, supporting the idea of a OPRK1-dynorphin involvement in disorders characterized by disturbed perception [<a class="bk_pop" href="#ml350.r72">72</a>]. Potential evidence for a role of dynorphin in psychotic disorders also comes from animal experiments. In rats, the selective OPRK1 agonist U- 50488H induced a dose-dependent reduction of pre-pulse inhibition [<a class="bk_pop" href="#ml350.r73">73</a>], which is seen as a readout of sensorimotor gating and is impaired in schizophrenics. Pre-pulse inhibition was restored by the selective OPRK1 antagonist nor-BNI [<a class="bk_pop" href="#ml350.r73">73</a>].</p><p>The role of OPRK1 in stress has been an active area of study, linking OPRK signaling and behavior [<a class="bk_pop" href="#ml350.r74">74</a>-<a class="bk_pop" href="#ml350.r75">75</a>]. Stress-induced opioid peptide release resulting in stress-induced analgesia through action at opioid receptors has been reported for all of the major opioid systems. It has been reported that OPRK1 activation after stress can also modulate numerous behaviors, including reward and depression [<a class="bk_pop" href="#ml350.r20">20</a>, <a class="bk_pop" href="#ml350.r76">76</a>-<a class="bk_pop" href="#ml350.r77">77</a>]. OPRK1 antagonist administration produces anxiolytic effects in several rat models of stress (elevated plus-maze, open-field, and fear potentiated startle paradigms) [<a class="bk_pop" href="#ml350.r78">78</a>], suggesting a role for endogenous dynorphin release in the expression of anxiety-like behavior in these stress models. OPRK1 antagonists produce effects similar to that of traditional anti-depressants [<a class="bk_pop" href="#ml350.r2">2</a>, <a class="bk_pop" href="#ml350.r51">51</a>, <a class="bk_pop" href="#ml350.r79">79</a>]. In an animal model widely used to model social defeat stress, wild-type mice treated with the selective OPRK1 antagonist Nor-BNI exhibit decreased social defeat postures [<a class="bk_pop" href="#ml350.r80">80</a>].</p><p>In the forced swim test, a rodent model of depression and a procedure that identifies in rats treatments with antidepressant efficacy in humans [<a class="bk_pop" href="#ml350.r81">81</a>], effects produced by OPRK1 agonists and antagonists have been interpreted as “prodepressive” and “anti-depressant”, respectively [<a class="bk_pop" href="#ml350.r2">2</a>, <a class="bk_pop" href="#ml350.r82">82</a>]. The antidepressant-like effects of OPRK1 antagonists have also been observed in other studies [<a class="bk_pop" href="#ml350.r51">51</a>, <a class="bk_pop" href="#ml350.r83">83</a>-<a class="bk_pop" href="#ml350.r84">84</a>]. Interestingly, standard antidepressant drugs often cause anxiety [<a class="bk_pop" href="#ml350.r85">85</a>-<a class="bk_pop" href="#ml350.r87">87</a>]. The antidepressant and anxiety-relieving effects of OPRK1 antagonists is notable, and suggests that this class drug might be particularly efficacious for the treatment of concomitant depressive and anxiety disorders [<a class="bk_pop" href="#ml350.r88">88</a>].</p><p>The physiological and pathophysiological mechanisms of OPRK1-dynorphin systems and their roles in neuropathic pain, drug addiction, and affective psychiatric disease in humans are active areas of study. The availability of new research tools such as potent and selective OPRK1 antagonists will facilitate understanding of the mechanisms involved in these processes and potentially have therapeutic value as novel therapies with an improved side effect profile to currently available drugs.</p><p>Several OPRK1 antagonists have been described in the literature. Norbinaltorphimine (nor-BNI) [<a class="bk_pop" href="#ml350.r89">89</a>-<a class="bk_pop" href="#ml350.r90">90</a>], 5′-guanidinonaltrindole (GNTI) [<a class="bk_pop" href="#ml350.r89">89</a>, <a class="bk_pop" href="#ml350.r91">91</a>], and JDTic [<a class="bk_pop" href="#ml350.r89">89</a>, <a class="bk_pop" href="#ml350.r92">92</a>] all exhibit a delay in the onset of action of approximately 24 hours [<a class="bk_pop" href="#ml350.r89">89</a>, <a class="bk_pop" href="#ml350.r93">93</a>-<a class="bk_pop" href="#ml350.r95">95</a>], and have very long lasting <i>in vivo</i> effects of up to 56 days [<a class="bk_pop" href="#ml350.r89">89</a>, <a class="bk_pop" href="#ml350.r95">95</a>-<a class="bk_pop" href="#ml350.r96">96</a>]. Due to their pharmacodynamics/pharmacokinetics and poor unbound brain/plasma ratio, morphine-like derivatives, nor-BNI and GNTI, are only used as pharmacological tools. The non-opioid compound JDTic exhibited a poor brain exposure but had an extraordinary persistence in brain (mean brain concentration declined by only 56% over 24 hours, and the drug was still detectable at 1 week in mice) despite its P-glycoprotein-mediated efflux, and its moderate lipophilicity and low affinity for cell homogenates. The presence of two basic nitrogens and entrapment in cellular compartments such as lysosomes have been proposed as a possible mechanism of this persistence [<a class="bk_pop" href="#ml350.r89">89</a>, <a class="bk_pop" href="#ml350.r97">97</a>]. A Phase 1 clinical trial of JDTic for cocaine dependence was terminated in 2012 due to adverse events. Several short-acting compounds from distinct chemotypes have been developed with greater CNS exposure compared to the prototypic ligands (nor-BNI, GNTI, JDTic). AstraZeneca disclosed a series of 8-azabicyclo[3.2.1]octan-3-yloxy-benzamides. AZ-MTAB was efficacious in animal models of mood disorders, but was associated with a significant hERG liability (IC50 of 260 nM) [<a class="bk_pop" href="#ml350.r97">97</a>-<a class="bk_pop" href="#ml350.r98">98</a>]. Another OPRK antagonist PF-04455242 [<a class="bk_pop" href="#ml350.r99">99</a>] was developed by Pfizer. However, on January 6, 2010 the phase 1 study for bipolar disorder and depression was terminated due to toxicology findings in animals exposed to PF-04455242 for three months. Eli Lilly identified a subnanomolar OPRK antagonist with selectivity of 21 and 135 over OPRM and OPRD respectively [<a class="bk_pop" href="#ml350.r100">100</a>]. In 2010 LY2456302 was advanced to Phase I clinical trial for the oral treatment of alcohol dependence. The clinical study was to assess the brain OPRK1 occupancy after single oral doses of LY2456302 as measured by positron emission tomography with radioligand LY2879788 (<sup>11</sup>C PKAB) in healthy subjects. The last update of the study was May 5, 2011, and no new studies for the compound are listed in <a href="http://ClinicalTrials.gov" ref="pagearea=body&targetsite=external&targetcat=link&targettype=uri">ClinicalTrials.gov</a>.</p><p>Even though a few drug-like OPRK antagonists with more favorable pharmacokinetics than nor-BNI, GNTI, JDTic have been developed, new OPRK antagonists possessing novel scaffolds and improved selectivity are needed both as pharmacological tools to better understand the OPRK1-dynorphin system and as potential pharmacotherapies.</p></div><div id="ml350.s4"><h2 id="_ml350_s4_">2. Materials and Methods</h2><div id="ml350.s5"><h3>2.1. Assays</h3><div id="ml350.s6"><h4>Probe Characterization Assays</h4><div id="ml350.s7"><h5>Solubility</h5><p>The solubility of compounds was tested in phosphate buffered saline, pH 7.4. Test tubes containing 1-2 mg compound in 1 mL PBS were inverted for 24. The samples were centrifuged and analyzed by HPLC (Agilent 1100 with diode-array detector). Peak area was compared to a standard of known concentration.</p></div><div id="ml350.s8"><h5>Stability</h5><p>Demonstration of stability in PBS was conducted under conditions likely to be experienced in a laboratory setting. The compound was dissolved in 1 mL of PBS at a concentration of 10 μM, unless its maximum solubility was insufficient to achieve this concentration. Low solubility compounds were tested between ten and fifty percent of their solubility limit. The solution was immediately aliquoted into seven standard polypropylene microcentrifuge tubes which were stored at ambient temperature in a block microcentrifuge tube holder. Individual tubes were frozen at -80 °C at 0, 1, 2, 4, 8, 24, and 48 hours. The frozen samples were thawed at room temperature and an equal volume of acetonitrile was added prior to determination of concentration by LC-MS/MS.</p></div><div id="ml350.s9"><h5>LC-MS/MS for stability assay</h5><p>All analytical methods are in MRM mode where the parent ion is selected in Q1 of the mass spectrometer. The parent ion is fragmented and a characteristic fragment ion is monitored in Q3. MRM mass spectroscopy methods are particularly sensitive because additional time is spent monitoring the desired ions and not sweeping a large mass range. Methods are rapidly set up using Automaton<sup>®</sup> (Applied Biosystems), where the compounds are listed with their name and mass in an Excel datasheet. Compounds are submitted in a 96-well plate to the HPLC autosampler and are slowly injected without a column present. A narrow range centered on the indicated mass is scanned to detect the parent ion. The software then evaluates a few pre-selected parameters to determine conditions that maximize the signal for the parent ion. The molecule is then fragmented in the collision cell of the mass spectrometer and fragments with m/z larger than 70 but smaller than the parent mass are determined. Three separate collision energies are evaluated to fragment the parent ion and the largest three ions are selected. Each of these three fragment ions is further optimized and the best fragment is chosen. The software then inserts the optimized masses and parameters into a template method and saves it with a unique name that indicates the individual compound being optimized. Spectra for the parent ion and the fragmentation pattern are saved and can be reviewed later.</p></div><div id="ml350.s10"><h5>Determination of glutathione reactivity</h5><p>One μL of a 10 mM compound stock solution was added to 1 mL of a freshly prepared solution of 50 μM reduced glutathione. Final compound concentration is 10 μM unless limited by solubility. The solution was allowed to incubate at 37°C for 6 hours prior to being directly analyzed for glutathione adduct formation. LC-MS/MS analysis of GSH adducts was performed on an API 4000 Q-TrapTM mass spectrometer equipped with a Turboionspray source (Applied Biosystems, Foster City, CA). Two methodologies were utilized: a negative precursor ion (PI) scan of m/z 272, corresponding to GSH fragmenting at the thioether bond, and a neutral loss scan of -129 AMU to detect GSH adducts. This triggered positive ion enhanced resolution and enhanced product ion scans [<a class="bk_pop" href="#ml350.r101">101</a>]).</p></div></div><div id="ml350.s11"><h4>Primary Assays</h4><div id="ml350.s12"><h5>Primary HTS OPRK1 antagonists assay of Maybridge Collection (AID 652031, AID 652082, AID 65077)</h5><p><b>Assay Overview:</b> The purpose of this assay is to identify compounds from the Maybridge Library that act as antagonists of OPRK1. This assay uses Tango OPRK1-BLA U2OS cells which contain OPRK1 linked to a GAL4-VP16 transcription factor via a TEV protease site. The cells also express a beta-arrestin/TEV protease fusion protein and a beta-lactamase (BLA) reporter gene under the control of a UAS response element. Stimulation of the OPRK1 receptor by agonist U-50488 causes migration of the fusion protein to the GPCR, and through proteolysis liberates GAL4-VP16 from the receptor. The liberated VP16-GAL4 migrates to the nucleus, where it induces transcription of the BLA gene. BLA expression is monitored by measuring fluorescence resonance energy transfer (FRET) of a cleavable, fluorogenic, cell-permeable BLA substrate. As designed, test compounds that act as OPRK1 antagonists will inhibit OPRK1 activation and migration of the fusion protein, thus preventing proteolysis of GAL4-VP16 and BLA transcription, leading to no increase in well FRET. Compounds were tested in singlicate (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/652031" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 652031</a>) or triplicate (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/652082" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 652082</a>) at a final nominal concentration of 9 μM, or in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 50 micromolar (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/652077" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 652077</a>).</p><p><b>Protocol Summary:</b> U2OS cells were cultured in T-175 sq cm flasks at 37°C and 95% relative humidity (RH). The growth media consisted of McCoy's 5A Medium supplemented with 10% v/v dialyzed fetal bovine serum, 0.1 mM NEAA, 25 mM HEPES (pH 7.3), 1 mM sodium pyruvate, 100 U/mL penicillin-streptomycin, 200 μg/mL Zeocin, 50 μg/mL Hygromycin, and 100 μg/mL Geneticin. Prior to the start of the assay, cells were suspended at a concentration of 250,000/mL in Assay Medium (McCoy's 5A Medium supplemented with 10% v/v charcoal dextran stripped fetal bovine serum, 0.1 mM NEAA, 25 mM HEPES (pH 7.3), 1 mM sodium pyruvate, 100 U/mL penicillin-streptomyci). The assay was started by dispensing 10 μL of cell suspension to each well, followed by overnight incubation at 37°C in 5% CO<sub>2</sub> and 95% RH. The next day, 50 nL of test compound (9 μM final nominal concentration) in DMSO was added to sample wells, and DMSO alone (0.5 % final concentration) was added to control wells. Next, U-50488 in Assay Medium (8 nM final nominal EC80 concentration) was added to the appropriate wells. Plates were then incubated at 37°C in 5% CO<sub>2</sub> for 4 hours. After the incubation, 2.2 μL/well of the LiveBLAzer FRET substrate mixture, prepared according to the manufacturer's protocol and containing 10 mM Probenicid, was added to all wells. After 2 hours of incubation at room temperature in the dark, plates were read on the EnVision plate reader (PerkinElmer Lifesciences, Turku, Finland) at an excitation wavelength of 405 nm and emission wavelengths of 460 nm and 535 nm. <b>Assay Cutoff:</b> Compounds that inhibited OPRK >50% (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/652031" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AIDs 652031</a> and <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/652082" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">652082</a>), or with an IC50 of ≤10 μM (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/652077" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 652077</a>) were considered active.</p></div><div id="ml350.s13"><h5>S1P1 Counterscreen HTS assay of Maybridge Collection (AID 652087, AID 652079)</h5><p><b>Assay Overview:</b> The purpose of this assay is to determine whether compounds from the Maybridge Library that act as antagonists of OPRK1 are nonselective due to inhibition of S1P1. The Tango EDG-1-bla U2OS cells express S1P1 (EDG1) linked to a GAL4-VP16 transcription factor via a TEV protease site. The cells also express a beta-arrestin/TEV protease fusion protein and a beta-lactamase (BLA) reporter gene under the control of a UAS response element. Stimulation of the S1P1 receptor by agonist S1P causes migration of the fusion protein to the GPCR, and through proteolysis liberates GAL4-VP16 from the receptor. The liberated VP16-GAL4 migrates to the nucleus, where it induces transcription of the BLA gene. BLA expression is monitored by measuring fluorescence resonance energy transfer (FRET) of a cleavable, fluorogenic, cell-permeable BLA substrate. As designed, test compounds that act as S1P1 antagonists will inhibit S1P1 activation and migration of the fusion protein, thus preventing proteolysis of GAL4-VP16 and BLA transcription, leading to no increase in well FRET. Compounds were tested in triplicate at a final nominal concentration of 9 μM (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/652087" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 652087</a>) or in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 50 μM (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/652079" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 652079</a>).</p><p><b>Protocol Summary:</b> U2OS cells were cultured in T-175 sq cm flasks at 37°C and 95% RH. The growth media consisted of McCoy's 5A Medium supplemented with 10% v/v dialyzed fetal bovine serum, 0.1 mM NEAA, 25 mM HEPES (pH 7.3), 1 mM sodium pyruvate, 100 U/mL penicillin-streptomycin-neomycin, 200 μg/mL Zeocin, 50 μg/mL Hygromycin, and 100 μg/mL Geneticin. Prior to the start of the assay, cells were suspended at a concentration of 1,000,000/mL in Assay Medium (Freestyle Expression Medium without supplements). The assay was started by dispensing 10 μL of cell suspension to each well in 384-well plates, followed by overnight incubation at 37°C in 5% CO<sub>2</sub> and 95% RH. The next day, 50 nL of test compound in DMSO was added to sample wells, and DMSO alone (0.5 % final concentration) was added to control wells. Next, S1P prepared in 2% BSA (0.22 μM final nominal EC80 concentration) was added to the appropriate wells. Plates were then incubated at 37°C in 5% CO<sub>2</sub> for 4 hours. After the incubation, 2.2 μL/well of the LiveBLAzer FRET substrate mixture, prepared according to the manufacturer's protocol and containing 10 mM Probenicid, was added to all wells. After 2 hours of incubation at room temperature in the dark, plates were read on the EnVision plate reader (PerkinElmer Lifesciences, Turku, Finland) at an excitation wavelength of 405 nm and emission wavelengths of 460 nm and 535 nm. <b>Assay Cutoff:</b> Compounds that inhibited S1P1 >30% (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/652087" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID652087</a>) or with an IC50 of ≤10 μM (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/652079" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 652079</a>) were considered active.</p></div><div id="ml350.s14"><h5>OPRD1 Counterscreen HTS assay of Maybridge Collection (AID 652080)</h5><p><b>Assay Overview:</b> The purpose of this counterscreen assay is to test the selectivity of OPRK1 antagonist compounds against the OPRD1 receptor. This assay uses Tango OPRD1-bla U2OS cells which express OPRD1 linked to a GAL4-VP16 transcription factor via a TEV protease site. The cells also express a Beta-arrestin/TEV protease fusion protein and a Beta-lactamase (BLA) reporter gene under the control of a UAS response element. Stimulation of the OPRD1 receptor by agonist SNC80 causes migration of the Beta-arrestin fusion protein to the GPCR, and through proteolysis liberates GAL4-VP16 from the receptor. The liberated VP16-GAL4 migrates to the nucleus, where it induces transcription of the BLA gene. BLA expression is monitored by measuring fluorescence resonance energy transfer (FRET) of a cleavable, fluorogenic, cell-permeable BLA substrate. As designed, test compounds that act as OPRD1 antagonists will inhibit agonist activation and migration of the fusion protein, thus preventing proteolysis of GAL4-VP16 and BLA transcription, leading to no increase in well FRET. Compounds were tested in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 50 μM.</p><p><b>Protocol Summary:</b> The Tango OPRD1-U20S dividing cell line was routinely cultured in 150 mm dishes at 37°C, 5% CO<sub>2</sub> and 95% RH. The growth medium consisted of McCoys 5A Media supplemented with 10% v/v dialyzed fetal bovine serum, 25 mM HEPES, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, and 1X antibiotic mix (penicillin streptomycin). On day 1 of the assay, 16,000 cells in 10 μL of assay media (DMEM-Glutamax with sodium pyruvate, 10% fetal bovine serum stripped with charcoal-dextran, 25 mM HEPES, 0.1 mM non-essential amino acids, and antibiotic mix (penicillin streptomycin) were seeded into each well of a 384-well plate. 50 nl of test compound in DMSO were added to the appropriate wells and plates were incubated for 30 minutes at 37°C, 5% CO<sub>2</sub> and 95% RH. Next, 1.1 μL of 3.7 uM SNC80 (OPRD1 agonist EC80 Challenge; final concentration 370 nM) or DMSO in assay medium was added to appropriate wells and incubated 16-24 hours at 37°C, 5% CO<sub>2</sub> and 95% RH. On day 2, 2.5 μL of LiveBLazer trade mark FRET B/G (CCF4-AM) loading mix (prepared according to manufacturer's instructions; 6 μL solution A, 60 μL Solution B, 904 μL Solution C, and 30 μL Solution D) were added to each well, and plates incubated at room temperature in the dark for 2 hours. Well fluorescence was measured on Perkin Elmer's Envision using an Excitation filter 405 nm, Emission filters at 460 nm and 590 nm, bottom read. <b>Assay Cutoff:</b> Compounds that inhibited OPRD1 >30% were considered active.</p></div><div id="ml350.s15"><h5>OPRM1 Counterscreen HTS assay of Maybridge Collection (AID 652081)</h5><p><b>Assay Overview:</b> The purpose of this counterscreen assay is to test the selectivity of OPRK1 antagonist compounds against the OPRM1 receptor. The assay monitors GPCR-Beta-arrestin proximity using low affinity fragment complementation of beta-galactosidase (beta-gal). The reconstituted holoenzyme catalyzes the hydrolysis of a substrate which yields a chemiluminescent signal. This assay employs U2OS cells which express OPRM1 fused to a beta-gal peptide fragment (enzyme donor), and beta-arrestin fused to the complementary beta-gal fragment (enzyme acceptor). Cells are incubated with test compounds and an agonist DAMGO (EC80 challenge), followed by measurement of well luminescence. As designed, compounds that inhibit OPRM1 will decrease the level of beta-arrestin recruitment elicited by DAMGO, resulting in a decrease in the level of reconstitution of the beta-gal holoenzyme, and decreased well luminescence. Compounds were tested in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 50 uM.</p><p><b>Protocol Summary:</b> The PathHunter<sup>®</sup> DiscoverX OPRM1-U20S cell line was routinely cultured in 150 mm dishes at 37°C, 5% CO<sub>2</sub> and 95% RH. The growth medium consisted of DMEM/F12 1:1 Media supplemented with 10% v/v heat inactivated fetal bovine serum, 25 mM HEPES, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 1x antibiotic mix (penicillin streptomycin). On Day 1 of the assay, 5000 cells in 20 μL of assay buffer (Discover X's Cell Plating Reagent 5) were seeded into each well of a 384-well plate, and incubated 16-24 hours at 37°C, 5% CO<sub>2</sub> and 95% RH. On Day 2, 100 nL of test compound in DMSO were added to the appropriate wells and plates were incubated for 30 minutes at 37°C, 5% CO<sub>2</sub> and 95% RH. Next, 2.2 μL of DAMGO OPRM1 agonist (EC80 Challenge; 1.8 μL of 3.7 μM DAMGO and 0.4 μL assay buffer; final assay concentration 303 nM) or DMSO in assay media were added. After incubation for 3 hours at 37°C, 5% CO<sub>2</sub> and 95% RH, 10 μL of Path Hunter Detection Mix prepared according to manufacturer's protocol; 1 part Galacton Star:5 parts Emerald II:19 parts PH Cell Assay Buffer) was added to each well, and plates were incubated at room temperature in the dark for 1 hour. Well luminescence was measured on Perkin Elmer's Envision. <b>Assay Cutoff:</b> Compounds that inhibited OPRM1 >30% were considered active.</p></div></div><div id="ml350.s16"><h4>Secondary Assays</h4><div id="ml350.s17"><h5>OPRK1 antagonist assay of SAR compounds (AID 652032, AID 652084)</h5><p><b>Assay Overview:</b> The purpose of this assay is to confirm the potency of test synthesized compounds. The assay is as described above (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/652031" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 652031</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/652082" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 652082</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/65077" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 65077</a>). Compounds were tested triplicate (except for <a href="https://pubchem.ncbi.nlm.nih.gov/substance/144087324" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 144087324</a>, which was tested in quadruplicate) using a 10-point, 1:3 dilution series starting at a nominal concentration of 50 μM (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/652032" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 652032</a>) or in either quadulplicate or octuplet using a 12-point, 1:3 dilution series starting at a nominal concentration of 10 μM (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/652084" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 652084</a>).</p><p><b>Protocol Summary:</b> The Tango OPRK1-U20S dividing cell line was cultured as described above (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/652031" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AIDs 652031</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/652082" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">652082</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/65077" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">65077</a>). On day 1 of the assay, 16,000 cells in 10 μL of assay media (DMEM-Glutamax with sodium pyruvate, 10% fetal bovine serum stripped with charcoal-dextran, 25 mM HEPES, 0.1 mM non-essential amino acids, and antibiotic mix (penicillin streptomycin) were seeded into each well of a 384-well plate. On Day 2, 50 nL of test compound in DMSO were added to the appropriate wells and plates were incubated for 30 minutes at 37°C, 5% CO<sub>2</sub> and 95% RH. Next, 0.6 uL of 111 nM <a href="/nuccore/1277101" class="bk_tag" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=nuccore">U50488</a> (OPRK1 agonist EC80 Challenge; final concentration 6 nM) or DMSO in assay medium was added to appropriate wells and incubated 4 hours at 37°C, 5% CO<sub>2</sub> and 95% RH. 2.5 μL of LiveBLazer (trade mark) FRET B/G (CCF4-AM) loading mix (prepared according to manufacturer's instructions; 6 μL solution A, 60 μL Solution B, 904 μL Solution C, and 30 μL Solution D) were added to each well, and plates incubated at room temperature in the dark for 2 hours. Well fluorescence was measured on Perkin Elmer's Envision using an Excitation filter 409 nm, Emission filters at 460 nm and 590 nm, bottom read. <b>Assay Cutoff:</b> Compounds with an IC50 of ≤10 μM were considered active.</p></div><div id="ml350.s18"><h5>OPRD1 Counterscreen assay of SAR compounds (AID 652033)</h5><p><b>Assay Overview:</b> The assay is as described above (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/652080" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 652080</a>). Compounds were tested in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 50 μM.</p><p><b>Protocol Summary:</b> The Tango OPRD1-U20S dividing cell line was cultured and the assay was performed as described above (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/652080" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 652080</a>). <b>Assay Cutoff:</b> Compounds with an IC50 of ≤10 μM were considered active.</p></div><div id="ml350.s19"><h5>OPRM1 Counterscreen assay of SAR compounds (AID 652034)</h5><p><b>Assay Overview:</b> The assay is as described above (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/652081" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 652081</a>). Compounds were tested in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 50 μM.</p><p><b>Protocol Summary:</b> The PathHunter<sup>®</sup> DiscoverX OPRM1-U20S cell line was cultured and the assay was performed as described above (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/652034" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 652034</a>). <b>Assay Cutoff:</b> Compounds with an IC50 of ≤10 μM were considered active.</p></div><div id="ml350.s20"><h5>Cytotoxicity assay (AID 652086)</h5><p><b>Assay Overview:</b> The purpose of this assay is to determine cytotoxicity of a powder compound that inhibits OPRK1. In this assay, U2OS cells are incubated with test compound, followed by determination of cell viability. The assay utilizes the CellTiter-Glo luminescent reagent to measure intracellular ATP in viable cells. Luciferase present in the reagent catalyzes the oxidation of beetle luciferin to oxyluciferin and light in the presence of cellular ATP. Well luminescence is directly proportional to ATP levels and cell viability. As designed, compounds that reduce cell viability will reduce ATP levels, luciferin oxidation and light production, resulting in decreased well luminescence. Compounds were tested in quadruplicate in a 12-point 1:3 dilution series starting at a nominal test concentration of 10 μM.</p><p><b>Protocol Summary:</b> This assay was started by dispensing OPRK1-bla U20S cells in McCoy's 5A medium plus 10% FBS, penicillin 100 U/mL and streptomycin 100 μg/mL (20 μL, 4000 cells/well) into the wells of a 384-well plate. Twelve 1:3 serial dilutions of compound (100 μM in growth media) were made. 5 μL of diluted compound or media were added to wells. The plate was incubated at 37°C in a humidified incubator for 24 hours, then equilibrated to room temperature for 30 minutes. 25 μL CellTitre-Glo reagent was added to each well, followed by incubation of the plate in the dark for 10 minutes. Well luminescence was measured on the Envision plate reader. <b>Assay Cutoff:</b> Compounds with a CC50 value ≤10 μM were considered active (cytotoxic).</p></div><div id="ml350.s21"><h5>Pharmacokinetic assay: plasma protein binding (AID 652078)</h5><p><b>Assay Overview:</b> The purpose of this assay is to assess the percentages of a lead OPRK1 antagonist test compound that bind to human, mouse, and rat plasma proteins.</p><p><b>Protocol Summary:</b> Plasma protein binding to human, mouse, and rat plasma was evaluated using equilibrium dialysis (Pierce RED system). Compound (1.0 μM) was added to the plasma compartment and after eight hours the concentration of drug was measured in the plasma and buffer compartments by LC-MS/MS. The concentration in the buffer compartment is considered to be the free fraction of compound. <b>Assay Cutoff:</b> For each plasma species, compounds that exhibited > 50% binding to plasma protein were considered active.</p></div><div id="ml350.s22"><h5>Pharmacokinetic assay: hepatic microsome stability (AID 652088)</h5><p><b>Assay Overview:</b> The purpose of this assay is to assess the stability of a lead OPRK1 antagonist test compound in the presence of pooled human, rat, mouse, monkey, and dog microsomes.</p><p><b>Protocol Summary:</b> Test compound was incubated (separately) with 0.2 mg/mL pooled human, mouse, rat, monkey, and dog hepatic microsomes and cofactors to determine the rate of metabolism. Samples were collected at multiple time points and the concentration of test compound was determined using HPLC coupled to a triple quadrupole mass spectrometer (LC/MS-MS), allowing the calculation of half-life for each compound. <b>Assay Cutoff:</b> For each microsome species, compounds with a half-life of > 60 minutes were considered active.</p></div><div id="ml350.s23"><h5>Pharmacokinetic assay: cytochrome P450 inhibition (AID 652076)</h5><p><b>Assay Overview:</b> The purpose of this assay is to obtain information regarding potential drug-drug interactions for a lead OPRK1 antagonist test compound. Inhibition of the four major human isoforms of cytochrome P450 are evaluated by following the metabolism of specific marker substrates CYP1A2, CYP2C9, CYP2D6, and CYP3A4 in the presence test compound.</p><p><b>Protocol Summary:</b> The metabolism of CYP1A2 (phenaceten demethylated to acetaminophen), CYP2C9 (tolbutamide hydroxylated to hydroxytolbutamide), CYP2D6 (bufuralol hydroxylated to 4′-hydroxybufuralol), and CYP3A4 (midazolam hydroxylated to 1′-hydroxymidazolam) in the presence or absence of 10 μM test compound were evaluated. The concentration of each marker substrate is approximately its Km. Specific inhibitors for each isoform were used to validate the system. Compound concentrations were determined by LC-MS/MS. <b>Assay Cutoff:</b> For each P450 isoform, compounds that inhibited ≤50% were considered active.</p></div><div id="ml350.s24"><h5>CEREP broad panel counterscreen of receptors, transporters and ion channels (AID 652083)</h5><p><b>Assay Overview:</b> The purpose of this panel of binding assays performed by CEREP is to identify a subset of potential receptors, transporters, ion channels, etc. for which a lead OPRK1 antagonist compound displays affinity. Assays were run in duplicate.</p><p><b>Protocol Summary:</b> The panel assays were radioligand binding assays. Specific ligand binding to the targets was defined as the difference between the total binding in the presence of 10 μM test compound and the nonspecific binding determined in the presence of 10 μM compound and an excess of labeled ligand. The results are expressed as a percent inhibition of control specific binding. <b>Assay Cutoff:</b> For each assay target, inhibition of ≥50% was considered active.</p></div></div><div id="ml350.s25"><h4>CEREP hERG counterscreen assay (AID 652075)</h4><p><b>Assay Overview:</b> The purpose of this binding assay performed by CEREP is to determine whether a lead OPRK1 antagonist compound has activity against the potassium voltage-gated channel, hERG. The assay was run in duplicate.</p><p><b>Protocol Summary:</b> The assay was a radioligand binding assay. Specific ligand binding to the target was defined as the difference between the total binding in the presence of 10 μM test compound and the nonspecific binding determined in the presence of 10 μM compound and an excess of unlabeled ligand. The results are expressed as a percent inhibition of control specific binding. <b>Assay Cutoff:</b> Inhibition of ≥50% was considered active.</p><div id="ml350.s26"><h5><i>In vivo</i> pharmacokinetic assay: plasma and brain levels (AID 652085)</h5><p><b>Assay Overview:</b> The purpose of this assay is to assess the level of a lead OPRK1 antagonist compound in mouse plasma and brain at 30 minutes and 120 minutes after dosing.</p><p><b>Protocol Summary:</b> Compounds were dosed IP at 10 mg/kg in a 1 mg/ml solution containing 1 part DMSO, 1 part Tween 80, and 8 parts water into C57Bl6 mice (n = 6). Blood and brain were taken at 30 minutes and 120 minutes. Blood was collected into EDTA-containing tubes and plasma was generated using standard centrifugation techniques. Brain was homogenized and proteins were precipitated with acetonitrile and compound concentrations were determined by LC-MS/MS. Data were fit by WinNonLin using a noncompartmental model and compound concentration in plasma and brain is calculated. <b>Assay Cutoff:</b> Compounds that exhibited a brain to plasma ratio of > 1 were considered active.</p></div><div id="ml350.s27"><h5><i>In vivo</i> pharmacokinetic assay: parallel artificial membrane permeability assay (PAMPA) (AID 652113)</h5><p><b>Assay Overview:</b> The purpose of this assay is to assess the permeability of a lead OPRK1 antagonist test compound using a commercial Parallel Artificial Membrane Permeability Assay (PAMPA) kit.</p><p><b>Protocol Summary:</b> An assessment of permeability was done using a commercial PAMPA kit. Compound was evaluated over a range of concentrations in 300 μL of PBS containing the compound in a well of the receiver plate, which is coupled to the bottom donor plate. The plates were allowed to incubate at room temperature. After 5 hours, aliquots were taken from the donor and receiver plates and the concentration of drug was determined. Propanolol and antiprine were used as positive controls; BHF177 and BLK998 were used as negative controls. Compound permeability was calculated.</p></div><div id="ml350.s28"><h5><i>In vivo</i> tail flick assay (AID 652108)</h5><p><b>Assay Overview:</b> The purpose of this assay is to assess the effect of a lead OPRK antagonist test compound in the Tail Flick assay in mice. The Tail Flick assay is a pain receptive assay in which a mouse is placed within a restraining tube with its tail protruding. The tail is placed on a level surface, radiant heat is applied to the tail and the latency of the mouse to remove its tail from the heat is recorded. This latency is used as a measure to indicate neurological pathology. In this assay, the mice are administered an OPRK1 agonist (U-69593) and test compound, and the ability of test compound to block the analgesic effect of the agonist compound is measured.</p><p><b>Protocol Summary:</b> This assay was performed by the Mouse Behavioral Assessment Core of The Scripps Research Institute. Ten mice each were pre-treated with test compound (administered i.p. at 10 mg/kg), OPRK1 antagonist NOR-BNI (administered s.c. 10 mg/kg), or vehicle. Mice were subsequently challenged with OPRK1 agonist U-69593 (administered i.p. at 2 mg/kg) at one hour, 24 hours, and 1 week post pre-treatment. After each agonist challenge, each moue was tested by application of a heat source three times and the latency time of the mouse to remove its tail from the heat was measured and reported in seconds.</p></div></div></div><div id="ml350.s29"><h3>2.2. Probe Chemical Characterization</h3><div id="ml350.f2" class="figure"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Image%20ml350f2&p=BOOKS&id=179827_ml350f2.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/NBK179827/bin/ml350f2.jpg" alt="Image ml350f2" class="tileshop" title="Click on image to zoom" /></a></div></div><p>The probe structure was verified by <sup>1</sup>H and <sup>13</sup>C NMR (see <a href="#ml350.s30">Section 2.3</a>) and high resolution LC-MS (<a class="figpopup" href="/books/NBK179827/figure/ml350.f3/?report=objectonly" target="object" rid-figpopup="figml350f3" rid-ob="figobml350f3">Figure 1</a>). Purity was assessed to be greater than 95% by LC-MS.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml350f3" co-legend-rid="figlgndml350f3"><a href="/books/NBK179827/figure/ml350.f3/?report=objectonly" target="object" title="Figure 1" class="img_link icnblk_img figpopup" rid-figpopup="figml350f3" rid-ob="figobml350f3"><img class="small-thumb" src="/books/NBK179827/bin/ml350f3.gif" src-large="/books/NBK179827/bin/ml350f3.jpg" alt="Figure 1. LC/MS analysis of ML350." /></a><div class="icnblk_cntnt" id="figlgndml350f3"><h4 id="ml350.f3"><a href="/books/NBK179827/figure/ml350.f3/?report=objectonly" target="object" rid-ob="figobml350f3">Figure 1</a></h4><p class="float-caption no_bottom_margin">LC/MS analysis of ML350. </p></div></div><p>Solubility (at room temperature) for <a href="/pcsubstance/?term=ML350[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML350</a> in PBS was determined to be 557 μM. Solubility in water and saline was determined to be 1.1 mM. <a href="/pcsubstance/?term=ML335[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML335</a> has a half-life of >48 hours in PBS at room temperature (79% compound remaining at 48 hours) (<a class="figpopup" href="/books/NBK179827/figure/ml350.f4/?report=objectonly" target="object" rid-figpopup="figml350f4" rid-ob="figobml350f4">Figure 2</a>).</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml350f4" co-legend-rid="figlgndml350f4"><a href="/books/NBK179827/figure/ml350.f4/?report=objectonly" target="object" title="Figure 2" class="img_link icnblk_img figpopup" rid-figpopup="figml350f4" rid-ob="figobml350f4"><img class="small-thumb" src="/books/NBK179827/bin/ml350f4.gif" src-large="/books/NBK179827/bin/ml350f4.jpg" alt="Figure 2. Stability of ML350 (CYM50202) in PBS." /></a><div class="icnblk_cntnt" id="figlgndml350f4"><h4 id="ml350.f4"><a href="/books/NBK179827/figure/ml350.f4/?report=objectonly" target="object" rid-ob="figobml350f4">Figure 2</a></h4><p class="float-caption no_bottom_margin">Stability of ML350 (CYM50202) in PBS. </p></div></div><p>No Michael acceptor adducts were observed when a sample of the probe was incubated with 50 μM glutathione and analyzed by LC-MS.</p><p>The following compounds have been submitted to the SMR collection (<a class="figpopup" href="/books/NBK179827/table/ml350.t2/?report=objectonly" target="object" rid-figpopup="figml350t2" rid-ob="figobml350t2">Table 1</a>).</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml350t2"><a href="/books/NBK179827/table/ml350.t2/?report=objectonly" target="object" title="Table 1" class="img_link icnblk_img figpopup" rid-figpopup="figml350t2" rid-ob="figobml350t2"><img class="small-thumb" src="/books/NBK179827/table/ml350.t2/?report=thumb" src-large="/books/NBK179827/table/ml350.t2/?report=previmg" alt="Table 1. Compounds submitted to the SMR collection (2-21-2013)." /></a><div class="icnblk_cntnt"><h4 id="ml350.t2"><a href="/books/NBK179827/table/ml350.t2/?report=objectonly" target="object" rid-ob="figobml350t2">Table 1</a></h4><p class="float-caption no_bottom_margin">Compounds submitted to the SMR collection (2-21-2013). </p></div></div></div><div id="ml350.s30"><h3>2.3. Probe Preparation</h3><div id="ml350.f5" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%203.%20Synthetic%20scheme%20for%20ML350%20(CYM50202).&p=BOOKS&id=179827_ml350f5.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/NBK179827/bin/ml350f5.jpg" alt="Figure 3. Synthetic scheme for ML350 (CYM50202)." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 3</span><span class="title">Synthetic scheme for ML350 (CYM50202)</span></h3></div><p>To a stirred solution of trans-rac-<i>N</i>-Boc-4amino-3-hydroxy piperidine <b>I</b> and cyclohexanone in DCE were added NaBH(OAc)<sub>3</sub> and AcOH. The reaction mixture was stirred overnight at room temperature. The mixture was diluted with ethyl acetate and washed with brine (2X). The organic phase was concentrated, and the product <b>II</b> purified by column chromatography using CH<sub>2</sub>Cl<sub>2</sub>/MeOH (9:1).</p><p>To a solution of <b>II</b> in CH<sub>2</sub>Cl<sub>2</sub> was added TFA and the reaction mixture was stirred for 30 minutes at room temperature. The mixture was concentrated under reduced pressure. The residue was dissolved in ethanol followed by the addition of DIPEA and pyridine <b>III</b>. The reaction mixture was heated at 145°C for 35 minutes under microwave irradiation. The crude was concentrated under reduced pressure and the product purified by HPLC furnishing the pure compound <a href="/pcsubstance/?term=ML350[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML350</a> (<a href="/protein/992538227/?report=GenPept" class="bk_tag" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=genpept">CYM50202</a>; CID 60156214).</p><p><sup>1</sup>H NMR and <sup>13</sup>C NMR of methyl 5-bromo-2-(4-(cyclohexylamino)-3-hydroxypiperidin-1-yl)nicotinate are as follows: <sup>1</sup>H NMR (600 MHz, CDCl<sub>3</sub>): <i>δ</i> 8.20 (s, 1H), 8.07 (dd, <i>J</i> = 9.5, 2.4 Hz, 1H), 3.95-3.90 (m, 2H), 3.83 (s, 3H), 3.79 (d, <i>J</i> = 13.3 Hz, 1H), 3.15 (bs, 2H), 2.89 (t, <i>J</i> = 12.4 Hz, 1H), 2.81 (t, <i>J</i> = 12.1 Hz, 1H), 2.07-2.02 (m, 3H), 1.90-1.82 (m, 3H), 1.67 (d, <i>J</i> = 12.4 Hz, 1H), 1.51 (q, <i>J</i> = 11.5 Hz, 1H), 11.4 (q, <i>J</i> = 11.4 Hz, 1H), 1.29-1.16 (m, 3H); <sup>13</sup>C NMR (125 MHz, CDCl<sub>3</sub>): <i>δ</i> 166.63, 157.96, 152.16, 143.80, 115.87, 110.44, 68.27, 59.70, 56.08, 55.39, 53.34, 48.62, 30.61, 29.16, 27.24, 25.67, 25.47, 25.31. MS (EI) <i>m/z</i>: 412, 414 (M+H). The purity was assessed to be greater than 95% by LC-MS.</p></div></div><div id="ml350.s31"><h2 id="_ml350_s31_">3. Results</h2><div id="ml350.s32"><h3>3.1. Summary of Screening Results</h3><p>Prior to implementing the OPRM1-OPRD1 agonist HTS screen of the MLPCN library, a series of pilot screens of the Maybridge Library was done at the SRIMSC Screening Center (<a class="figpopup" href="/books/NBK179827/figure/ml350.f6/?report=objectonly" target="object" rid-figpopup="figml350f6" rid-ob="figobml350f6">Figure 4</a>). Screens for each opioid receptor were run in agonist and antagonist mode. An OPRK1 antagonist screen with an activity cutoff of >50% inhibition identified 72 hits out of 16,000 compounds screened (1X%INH; <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/652031" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 652031</a>). A confirmation OPRK1 antagonist screen (3X%INH; <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/652082" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 652082</a>) and an S1P1 counterscreen (3X%INH; <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/652087" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 652087</a>) identified 11 compounds that confimed OPRK1 antagonism and were inactive at S1P1; these were repurchased as powders. These 11 compounds were screened in four dose response assays: OPRK1 antagonist (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/652077" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 652077</a>), OPRM1 antagonist counterscreen (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/652081" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 652081</a>), OPRD1 antagonist counterscreen (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/652080" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 652080</a>), and S1P1 antagonist counterscreen (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/652079" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 652079</a>). From these screens a hit compound that appeared to exhibit potency and selectivity as an OPRK antagonist was identified (CID 2796048) (<a class="figpopup" href="/books/NBK179827/figure/ml350.f7/?report=objectonly" target="object" rid-figpopup="figml350f7" rid-ob="figobml350f7">Figure 5</a>). Structural integrity and potency of CID 2796048 were verified by independent synthesis. Chemical archeology was performed on and around the structure. Medicinal chemistry optimization by SAR by purchase and synthesis was begun.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml350f6" co-legend-rid="figlgndml350f6"><a href="/books/NBK179827/figure/ml350.f6/?report=objectonly" target="object" title="Figure 4" class="img_link icnblk_img figpopup" rid-figpopup="figml350f6" rid-ob="figobml350f6"><img class="small-thumb" src="/books/NBK179827/bin/ml350f6.gif" src-large="/books/NBK179827/bin/ml350f6.jpg" alt="Figure 4. Flow chart describing Maybridge screening results." /></a><div class="icnblk_cntnt" id="figlgndml350f6"><h4 id="ml350.f6"><a href="/books/NBK179827/figure/ml350.f6/?report=objectonly" target="object" rid-ob="figobml350f6">Figure 4</a></h4><p class="float-caption no_bottom_margin">Flow chart describing Maybridge screening results. </p></div></div><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml350f7" co-legend-rid="figlgndml350f7"><a href="/books/NBK179827/figure/ml350.f7/?report=objectonly" target="object" title="Figure 5" class="img_link icnblk_img figpopup" rid-figpopup="figml350f7" rid-ob="figobml350f7"><img class="small-thumb" src="/books/NBK179827/bin/ml350f7.gif" src-large="/books/NBK179827/bin/ml350f7.jpg" alt="Figure 5. Compound identified from Maybridge library; CID 2796048." /></a><div class="icnblk_cntnt" id="figlgndml350f7"><h4 id="ml350.f7"><a href="/books/NBK179827/figure/ml350.f7/?report=objectonly" target="object" rid-ob="figobml350f7">Figure 5</a></h4><p class="float-caption no_bottom_margin">Compound identified from Maybridge library; CID 2796048. </p></div></div></div><div id="ml350.s33"><h3>3.2. Dose Response Curve for Probe</h3><div id="ml350.f8" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%206.%20Dose%20response%20curve%20for%20ML350.&p=BOOKS&id=179827_ml350f8.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/NBK179827/bin/ml350f8.jpg" alt="Figure 6. Dose response curve for ML350." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 6</span><span class="title">Dose response curve for ML350</span></h3></div></div><div id="ml350.s34"><h3>3.3. Scaffold/Moiety Chemical Liabilities</h3><p>A resolution of the trans-diastereomeric mixture will need to be performed in order to establish the individual antagonist activity and PK profile. The methyl ester metabolic “soft spot” may underlie rapid metabolism. Addressing these issues is fundamental in further development of this chemotype.</p></div><div id="ml350.s35"><h3>3.4. SAR Table</h3><p>The original screening hit CID 2796048 can be conceptualized as consisting of three regions A, B, and C. <a class="figpopup" href="/books/NBK179827/table/ml350.t3/?report=objectonly" target="object" rid-figpopup="figml350t3" rid-ob="figobml350t3">Table 2</a> shows structures of compounds used for probe optimization.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml350t3"><a href="/books/NBK179827/table/ml350.t3/?report=objectonly" target="object" title="Table 2" class="img_link icnblk_img figpopup" rid-figpopup="figml350t3" rid-ob="figobml350t3"><img class="small-thumb" src="/books/NBK179827/table/ml350.t3/?report=thumb" src-large="/books/NBK179827/table/ml350.t3/?report=previmg" alt="Table 2. SAR Table for optimization of antagonist probe ML350 for OPRK." /></a><div class="icnblk_cntnt"><h4 id="ml350.t3"><a href="/books/NBK179827/table/ml350.t3/?report=objectonly" target="object" rid-ob="figobml350t3">Table 2</a></h4><p class="float-caption no_bottom_margin">SAR Table for optimization of antagonist probe ML350 for OPRK. </p></div></div><p>The HTS hit compound (entry <b>1</b>) was purchased (<a href="https://pubchem.ncbi.nlm.nih.gov/substance/26534319" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 26534319</a>) with confirmed IC50s of 410 nM at OPRK1, 4.59 μM at OPRM1 and no demonstrated OPRD1 activity at concentrations up to 50 μM. Compound <b>1</b> does not contain reactive functional groups, is chemically amiable and is moderately and highly selective against the OPRM1 and OPRD1, thus making this compound suitable for medicinal chemistry optimization.</p><p><i><u>Pyridine region (A)</u>:</i> We started our SAR studies by modifying the two metabolic soft spots (ester groups) in region A using the piperidine-4-yl and cyclohexylamine as regions B and C. 4-cyclohexylamine piperidine was the constant moiety to simplify the synthetic work and allow rapid screening of region A. First, we sought to minimize the structure of the hit (Andrews' analysis) by reducing the number of substituents on the pyridine ring. Interestingly, the 5-acetamide (entry <b>2</b>) was found less that 2-fold less potent than compound <b>1</b>. Changing the acetamide group for halogens led to an interesting series of compounds. The increase in potency was inversely related to the electronegativity of the halogen atoms: the fluoride (entry <b>6</b>) was ∼16-fold less potent than compound <b>1</b>, while the iodine (entry <b>3</b>), bromide (entry <b>4</b>) and chloride (entry <b>5</b>) were ∼5-, ∼3-, ∼2.5-fold more potent, respectively. Conversely, the selectivity against the OPRM1 increased directly proportionally to their electronegativity: iodide, bromide and chloride were ∼6-, 7.5- and 12.5-fold selective against the OPRM. Interestingly the selectivity against the OPRD1 was higher for the bromide (143-fold) followed by the iodide (32-fold) and chloride (25-fold). Installing a phenyl ring (entry <b>7</b>) on the 5-position led to complete loss of potency. Replacing the methyl ester of compound <b>4</b> for an isopropyl ester furnished compound <b>8</b> with ∼3-fold loss in potency at the OPRK1. A decrease in selectivity against the OPRD (7.5-fold) and an increase in selectivity against the OPRM1 (50-fold) were observed, indicating that steric factors at the 3-position of ring A are important for modulating potency and selectivity. A 39-fold loss in potency was observed for the carboxylic acid (entry <b>9</b>) compared to compound <b>4</b>.</p><p><i><u>Cyclohexylamine region (C)</u>:</i> The cyclohexylamine analog of compound <b>1</b> (entry <b>10</b>) was ∼6-fold less potent at OPRK1. To continue our SAR studies we selected the methyl 5-bromopyridine-3-carboxylate moiety from compound <b>4</b> due to its potency (19.5-fold more potent than compound <b>10</b>) and selectivity profile. The 1,2-<i>trans</i>-aminoalcohol (entry <b>11</b>), 1,2-<i>cis</i>-aminoalcohol (entry <b>12</b>) and diastereomeric 1,3-aminoalcohol (entry <b>13</b>) were ∼2-, ∼4-, and ∼5-fold less potent than the un-substituted compound <b>4</b>, respectively. The selectivity against the OPRM1 of <i>trans</i>- and <i>cis</i>-aminoalchol were 12- and 33-fold, respectively, while the selectivity of the 1,3-aminoalcohol was 25-fold. Drastic loss of potency resulted from the noncyclic 1,2-aminoalcohol (entry <b>14</b>). The diasteromeric 2-methoxycycloxylamine (entry <b>15</b>) was nearly equipotent to the <i>trans</i>-1,2-aminoalcohol (entry <b>11</b>), but the selectivity against the OPRM1 was 3.7-fold bigger. Introducing a methyl group at the 4-position of the cyclohexyl group (entry <b>16</b>) led to 4-fold loss of potency at the OPRK, 2-fold increase in selectivity against OPRM1 and ∼4-fold decrease in selectivity against the OPRD. Introducing an oxygen atom into the cyclohexyl ring (entry <b>17</b>) led to 4.5-fold loss in potency at the OPRK1, but the selectivity against OPRM1 was ∼11-fold higher than for compound <b>4</b>. Increasing (cyloheptyl, entry <b>18</b>) or decreasing (cyclopentyl, entry <b>19</b>) the ring size did not affect the potency at the OPRK1, whereas it increased the selectivity against the OPRM (4-5-fold) and decreased the selectivity against the OPRD1 (1.2-2.4-fold). Moving the cyclohexyl ring (entry <b>20</b>) or the cyclohexylamine (entry <b>21</b>) one methylene further from ring B led to 51-and 26-fold loss in potency, respectively. Methylation of the amino group (entry <b>22</b>) led to 20-fold loss in potency. When the cyclohexyl group was removed (entry <b>23</b>), the basicity of the amine reduced (entry <b>24</b>) or the nitrogen replaced by oxygen (entry <b>25</b>), the potency was completely lost. Taken together, it can be concluded that the basicity of the amine and its position in relation to the pyridine ring are fundamental for the activity at the OPRK1, while the installation of polar groups decreases the potency for the OPRK1, has small impact on the selectivity against OPRD1 but a greater impact against the OPRM1. A small lipophilic portion is important for the activity at the OPRK, however long lipophilic moieties lead to a decrease in potency on both OPRK1 and OPRM1.</p><p><i><u>Piperidine region (B)</u>:</i> When the cyclohexylamine was moved from 4- to 3-position (entry <b>26</b>) within the piperidine ring a complete loss of potency was observed. Interestingly, when a pyrrolidine system (entry <b>27</b>) was introduced the potency slightly decreased. Adding a methyl group on position 3 (entry <b>28</b>) led to small loss in potency. Interestingly, installing a hydroxyl group oriented in <i>trans</i> to the amino group (entry <b>29</b>) led to a substantial increase of potency at OPRK (8-14.5-fold) and the selectivity against the OPRM1 and OPRD1 were 20-35- and 219-382-fold, respectively.</p><p><i><u>Complementary SAR on compound <b>29</b></u>:</i> Removing the cyclohexyl group (entry <b>30</b>) led to a 368-644–fold loss in potency, as similarly observed for compound <b>23</b>. Removing the bromine from position 5 of the pyridine ring led to 137-240-fold loss in potency.</p></div><div id="ml350.s36"><h3>3.5. Cellular Activity</h3><p><a href="/pcsubstance/?term=ML350[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML350</a> has been evaluated in a series of cell-based assays (the OPRK1 primary and OPRD1, OPRM1, and S1P1 counterscreen assays, and a cytotoxicity assay) and shown to have activity in a cell-based system.</p></div><div id="ml350.s37"><h3>3.6. Profiling Assays</h3><p><a href="/pcsubstance/?term=ML350[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML350</a> was submitted to CEREP for broad panel screening of 52 protein targets (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/652083" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 652083</a>), including hERG (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/662075" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 662075</a>). The purpose of this panel of binding assays was to identify potential receptors, transporters, or ion channels for which <a href="/pcsubstance/?term=ML350[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML350</a> displays affinity. <a href="/pcsubstance/?term=ML350[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML350</a> exhibits 67% and 71% inhibition of the Na<sup>+</sup> channel (site 2) and the NOP receptor, respectively (<a class="figpopup" href="/books/NBK179827/table/ml350.t4/?report=objectonly" target="object" rid-figpopup="figml350t4" rid-ob="figobml350t4">Table 3</a>). The IC50s were determined to be 1.8 μM and 3 μM for these two targets, respectively. Thus, <a href="/pcsubstance/?term=ML350[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML350</a> is >100-fold selective for OPRK1 over the Na+ channel (site 2) and approximately 200-fold selective over the NOP receptor. These data suggest that <a href="/pcsubstance/?term=ML350[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML350</a> is generally inactive against a broad array of off targets and does not likely exert unwanted effects.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml350t4"><a href="/books/NBK179827/table/ml350.t4/?report=objectonly" target="object" title="Table 3" class="img_link icnblk_img figpopup" rid-figpopup="figml350t4" rid-ob="figobml350t4"><img class="small-thumb" src="/books/NBK179827/table/ml350.t4/?report=thumb" src-large="/books/NBK179827/table/ml350.t4/?report=previmg" alt="Table 3. Targets that exhibit ≥ 50% inhibition by ML350 in CEREP screen." /></a><div class="icnblk_cntnt"><h4 id="ml350.t4"><a href="/books/NBK179827/table/ml350.t4/?report=objectonly" target="object" rid-ob="figobml350t4">Table 3</a></h4><p class="float-caption no_bottom_margin">Targets that exhibit ≥ 50% inhibition by ML350 in CEREP screen. </p></div></div></div><div id="ml350.s38"><h3>3.7. Pharmacokinetic Assays</h3><p><i><u>Permeability</u>:</i>
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<a href="/pcsubstance/?term=ML350[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML350</a> has high passive membrane permeability as measured in a PAMPA assay (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/652113" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 652113</a>) as compared to standard compounds (<a class="figpopup" href="/books/NBK179827/figure/ml350.f9/?report=objectonly" target="object" rid-figpopup="figml350f9" rid-ob="figobml350f9">Figure 7</a>).</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml350f9" co-legend-rid="figlgndml350f9"><a href="/books/NBK179827/figure/ml350.f9/?report=objectonly" target="object" title="Figure 7" class="img_link icnblk_img figpopup" rid-figpopup="figml350f9" rid-ob="figobml350f9"><img class="small-thumb" src="/books/NBK179827/bin/ml350f9.gif" src-large="/books/NBK179827/bin/ml350f9.jpg" alt="Figure 7. ML350 has high passive membrane permeability, similar to positive controls propanolol and antipirine." /></a><div class="icnblk_cntnt" id="figlgndml350f9"><h4 id="ml350.f9"><a href="/books/NBK179827/figure/ml350.f9/?report=objectonly" target="object" rid-ob="figobml350f9">Figure 7</a></h4><p class="float-caption no_bottom_margin">ML350 has high passive membrane permeability, similar to positive controls propanolol and antipirine. </p></div></div><p><i><u>Brain penetration</u>:</i>
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<a href="/pcsubstance/?term=ML350[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML350</a> exhibited good brain penetration when administered by intraperitoneal (IP) injection in mice (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/662085" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 662085</a>). Thus, dosed at 10 mg/kg in mice, <a href="/pcsubstance/?term=ML350[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML350</a> (1 mg/mL in 10/10/80 DMSO/Tween/Water), reached brain at levels 3 times higher than plasma (<a class="figpopup" href="/books/NBK179827/table/ml350.t5/?report=objectonly" target="object" rid-figpopup="figml350t5" rid-ob="figobml350t5">Table 4</a>).</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml350t5"><a href="/books/NBK179827/table/ml350.t5/?report=objectonly" target="object" title="Table 4" class="img_link icnblk_img figpopup" rid-figpopup="figml350t5" rid-ob="figobml350t5"><img class="small-thumb" src="/books/NBK179827/table/ml350.t5/?report=thumb" src-large="/books/NBK179827/table/ml350.t5/?report=previmg" alt="Table 4. Levels of ML350 in plasma and brain of mice 30 and 120 minutes after IP administration." /></a><div class="icnblk_cntnt"><h4 id="ml350.t5"><a href="/books/NBK179827/table/ml350.t5/?report=objectonly" target="object" rid-ob="figobml350t5">Table 4</a></h4><p class="float-caption no_bottom_margin">Levels of ML350 in plasma and brain of mice 30 and 120 minutes after IP administration. </p></div></div><p><i><u>Cytochrome P450 inhibition</u>:</i>
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<a href="/pcsubstance/?term=ML350[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML350</a> had no significant activity at 3 of 4 human cytochrome P450 subtypes (<a class="figpopup" href="/books/NBK179827/table/ml350.t6/?report=objectonly" target="object" rid-figpopup="figml350t6" rid-ob="figobml350t6">Table 5</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/652076" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 652076</a>). <a href="/pcsubstance/?term=ML350[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML350</a> inhibited CYP2D6 by 89% at 10 µM.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml350t6"><a href="/books/NBK179827/table/ml350.t6/?report=objectonly" target="object" title="Table 5" class="img_link icnblk_img figpopup" rid-figpopup="figml350t6" rid-ob="figobml350t6"><img class="small-thumb" src="/books/NBK179827/table/ml350.t6/?report=thumb" src-large="/books/NBK179827/table/ml350.t6/?report=previmg" alt="Table 5. Inhibition of human cytochrome P450 subtypes 1A2, 2C9, 2D6, and 3A4 by ML350." /></a><div class="icnblk_cntnt"><h4 id="ml350.t6"><a href="/books/NBK179827/table/ml350.t6/?report=objectonly" target="object" rid-ob="figobml350t6">Table 5</a></h4><p class="float-caption no_bottom_margin">Inhibition of human cytochrome P450 subtypes 1A2, 2C9, 2D6, and 3A4 by ML350. Furafylline, sulfaphenazole, quinidine, and ketoconazone were the positive controls used for CYP1A2, CYP2C9, CYP2D6, and CYP3A4, respectively. </p></div></div><p><i><u>Plasma protein binding</u>:</i>
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<a href="/pcsubstance/?term=ML350[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML350</a> (1uM) had modest plasma protein binding in human (73%), but was higher in rodent, 96% in mouse and 99% in rat (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/652078" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 652078</a>, <a class="figpopup" href="/books/NBK179827/table/ml350.t7/?report=objectonly" target="object" rid-figpopup="figml350t7" rid-ob="figobml350t7">Table 6</a>).</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml350t7"><a href="/books/NBK179827/table/ml350.t7/?report=objectonly" target="object" title="Table 6" class="img_link icnblk_img figpopup" rid-figpopup="figml350t7" rid-ob="figobml350t7"><img class="small-thumb" src="/books/NBK179827/table/ml350.t7/?report=thumb" src-large="/books/NBK179827/table/ml350.t7/?report=previmg" alt="Table 6. Binding of ML350 to human, mouse, and rat plasma proteins." /></a><div class="icnblk_cntnt"><h4 id="ml350.t7"><a href="/books/NBK179827/table/ml350.t7/?report=objectonly" target="object" rid-ob="figobml350t7">Table 6</a></h4><p class="float-caption no_bottom_margin">Binding of ML350 to human, mouse, and rat plasma proteins. </p></div></div><p><i><u>Hepatic microsome stability</u>:</i> In vitro hepatic microsomal stability of <a href="/pcsubstance/?term=ML350[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML350</a> was measured (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/652086" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 652086</a>, <a class="figpopup" href="/books/NBK179827/table/ml350.t8/?report=objectonly" target="object" rid-figpopup="figml350t8" rid-ob="figobml350t8">Table 7</a>). The half life in 0.2 mg/mL hepatic microsomes was >120, 20, 5, 13 and >120 minutes in human, rat, mouse, monkey and dog hepatic microsomes, respectively.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml350t8"><a href="/books/NBK179827/table/ml350.t8/?report=objectonly" target="object" title="Table 7" class="img_link icnblk_img figpopup" rid-figpopup="figml350t8" rid-ob="figobml350t8"><img class="small-thumb" src="/books/NBK179827/table/ml350.t8/?report=thumb" src-large="/books/NBK179827/table/ml350.t8/?report=previmg" alt="Table 7. Hepatic microsome stability of ML350." /></a><div class="icnblk_cntnt"><h4 id="ml350.t8"><a href="/books/NBK179827/table/ml350.t8/?report=objectonly" target="object" rid-ob="figobml350t8">Table 7</a></h4><p class="float-caption no_bottom_margin">Hepatic microsome stability of ML350. </p></div></div><p><i><u>Rat pharmacokinetics</u></i>: <a href="/pcsubstance/?term=ML350[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML350</a> has an encouraging <i>in vivo</i> pharmacokinetic profile in rats (<a class="figpopup" href="/books/NBK179827/table/ml350.t9/?report=objectonly" target="object" rid-figpopup="figml350t9" rid-ob="figobml350t9">Table 8</a>). The oral results are particularly exciting with, concentrations in plasma 10 times the cell-based IC50 after the relatively modest dose of 2 mg/kg.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml350t9"><a href="/books/NBK179827/table/ml350.t9/?report=objectonly" target="object" title="Table 8" class="img_link icnblk_img figpopup" rid-figpopup="figml350t9" rid-ob="figobml350t9"><img class="small-thumb" src="/books/NBK179827/table/ml350.t9/?report=thumb" src-large="/books/NBK179827/table/ml350.t9/?report=previmg" alt="Table 8. In vivo pharmacokinetic profile of ML350 in rats." /></a><div class="icnblk_cntnt"><h4 id="ml350.t9"><a href="/books/NBK179827/table/ml350.t9/?report=objectonly" target="object" rid-ob="figobml350t9">Table 8</a></h4><p class="float-caption no_bottom_margin"><i>In vivo</i> pharmacokinetic profile of ML350 in rats. ML350 was administered by IV at 1 mg/kg, or by mouth at 2 mg/kg </p></div></div></div><div id="ml350.s39"><h3>3.8. Reversibility of Analgesic Effects in Mice</h3><p>In order to assess the reversibility of analgesic effects of <a href="/pcsubstance/?term=ML350[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML350</a> in mice, a tail flick assay was used (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/652108" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 652108</a>, <a class="figpopup" href="/books/NBK179827/figure/ml350.f10/?report=objectonly" target="object" rid-figpopup="figml350f10" rid-ob="figobml350f10">Figure 8</a>). This is a pain receptive assay in which a mouse is placed within a restraining tube with its tail protruding. The tail is placed on a level surface, radiant heat is applied to the tail and the latency of the mouse to remove its tail from the heat is recorded. This latency is used as a measure to indicate neurological pathology. In this assay, the mice are administered <a href="/pcsubstance/?term=ML350[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML350</a>, vehicle, or the OPRK1 antagonist Nor-BNI, and subsequently challenged with OPRK agonist U-69,593 at one hour, 24 hours, and 1 week post pre-treatment. The ability of <a href="/pcsubstance/?term=ML350[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML350</a> to block the analgesic effect of the agonist compound is measured. After each agonist challenge, each moue was tested by application of a heat source and the latency time of the mouse to remove its tail from the heat is measured and reported in seconds. The ability of <a href="/pcsubstance/?term=ML350[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML350</a> to block the analgesic effect of the agonist is gone after 24 hours, whereas Nor-BNI is still efficacious after 1 week.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml350f10" co-legend-rid="figlgndml350f10"><a href="/books/NBK179827/figure/ml350.f10/?report=objectonly" target="object" title="Figure 8" class="img_link icnblk_img figpopup" rid-figpopup="figml350f10" rid-ob="figobml350f10"><img class="small-thumb" src="/books/NBK179827/bin/ml350f10.gif" src-large="/books/NBK179827/bin/ml350f10.jpg" alt="Figure 8. Results of Tail Flick assay to determine the reversibility of the analgesic effect of ML350." /></a><div class="icnblk_cntnt" id="figlgndml350f10"><h4 id="ml350.f10"><a href="/books/NBK179827/figure/ml350.f10/?report=objectonly" target="object" rid-ob="figobml350f10">Figure 8</a></h4><p class="float-caption no_bottom_margin">Results of Tail Flick assay to determine the reversibility of the analgesic effect of ML350. Mice received ML350 (5 mg/kg) administered IP, Nor-BNI (10 mg/kg) administered SC, or vehicle/. Mice were then challenged with agonist U-69,593 (2mg/kg) administered <a href="/books/NBK179827/figure/ml350.f10/?report=objectonly" target="object" rid-ob="figobml350f10">(more...)</a></p></div></div></div></div><div id="ml350.s40"><h2 id="_ml350_s40_">4. Discussion</h2><p><a href="/pcsubstance/?term=ML350[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML350</a> was identified by high-throughput screening using a cell-based Tango™-format assay. A set of pharmacokinetic analyses show that <a href="/pcsubstance/?term=ML350[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML350</a> has high passive membrane permeability, good brain penetration, no significant activity at three of four human cytochrome P450 subtypes, high binding for rodent plasma protein and modest binding for human plasma protein, and an encouraging <i>in vivo</i> pharmacokinetic profile in rats. <a href="/pcsubstance/?term=ML350[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML350</a> was submitted to CEREP for broad panel screening against a panel of receptors, transporters, and ion channels; the data suggest that <a href="/pcsubstance/?term=ML350[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML350</a> is generally inactive against a broad array of off targets and does not likely exert unwanted effects. Importantly, <a href="/pcsubstance/?term=ML350[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML350</a> was shown to have a reversible analgesic effect when challenged with an OPRK1 agonist in a tail flick assay in mice.</p><div id="ml350.s41"><h3>4.1. Comparison to existing art and how the new probe is an improvement</h3><p>Several OPRK1 antagonists have been described in the literature. Norbinaltorphimine (nor-BNI) [<a class="bk_pop" href="#ml350.r89">89</a>-<a class="bk_pop" href="#ml350.r90">90</a>], 5′-guanidinonaltrindole (GNTI) [<a class="bk_pop" href="#ml350.r89">89</a>, <a class="bk_pop" href="#ml350.r91">91</a>], and JDTic [<a class="bk_pop" href="#ml350.r89">89</a>, <a class="bk_pop" href="#ml350.r92">92</a>] all exhibit a delay in the onset of action of approximately 24 hours [<a class="bk_pop" href="#ml350.r89">89</a>, <a class="bk_pop" href="#ml350.r93">93</a>-<a class="bk_pop" href="#ml350.r95">95</a>], and have very long lasting <i>in vivo</i> effects of up to 56 days [<a class="bk_pop" href="#ml350.r89">89</a>, <a class="bk_pop" href="#ml350.r95">95</a>-<a class="bk_pop" href="#ml350.r96">96</a>]. Due to their pharmacodynamics/pharmacokinetics and poor unbound brain/plasma ratio, morphine-like derivatives, nor-BNI and GNTI, are only used as pharmacological tools. The non-opioid compound JDTic exhibited a poor brain exposure but had an extraordinary persistence in brain (mean brain concentration declined by only 56% over 24 hours, and the drug was still detectable at 1 week in mice) despite its P-glycoprotein-mediated efflux, and its moderate lipophilicity and low affinity for cell homogenates. The presence of two basic nitrogens and entrapment in cellular compartments such as lysosomes have been proposed as a possible mechanism of this persistence [<a class="bk_pop" href="#ml350.r89">89</a>, <a class="bk_pop" href="#ml350.r97">97</a>]. A Phase 1 clinical trial of JDTic for cocaine dependence was terminated in 2012 due to adverse events. Several short-acting compounds from distinct chemotypes have been developed with greater CNS exposure compared to the prototypic ligands (nor-BNI, GNTI, JDTic). AstraZeneca disclosed a series of 8-azabicyclo[3.2.1]octan-3-yloxy-benzamides. AZ-MTAB was efficacious in animal models of mood disorders, but was associated with a significant hERG liability (IC50 of 260 nM) [<a class="bk_pop" href="#ml350.r97">97</a>-<a class="bk_pop" href="#ml350.r98">98</a>]. Another OPRK1 antagonist PF-04455242 [<a class="bk_pop" href="#ml350.r99">99</a>] was developed by Pfizer. However, on January 6, 2010 the phase 1 study for dipolar disorder and depression was terminated due to toxicology findings in animals exposed to PF-04455242 for three months. Eli Lilly identified a subnanomolar OPRK1 antagonist with selectivity of 21 and 135 over OPRM1 and OPRD1 respectively [<a class="bk_pop" href="#ml350.r100">100</a>]. In 2010 LY2456302 was advanced to phase I clinical trial for the oral treatment of alcohol dependence. The clinical study was to assess the brain OPRK1 occupancy after single oral doses of LY2456302 as measured by positron emission tomography with radioligand LY2879788 (<sup>11</sup>C PKAB) in healthy subjects. The last update of the study was May 5, 2011, and no new studies for the compound are listed in <a href="http://ClinicalTrials.gov" ref="pagearea=body&targetsite=external&targetcat=link&targettype=uri">ClinicalTrials.gov</a>.</p><p>Even though a few drug-like OPRK1 antagonists with more favorable pharmacokinetics than nor-BNI, GNTI, JDTic have been developed, new OPRK1 antagonists possessing novel scaffolds and improved selectivity are still needed both as pharmacological tools to better understand the OPRK1-dynorphin system and as potential pharmacotherapies.</p><div id="ml350.t10" class="table"><h3><span class="label">Table 9</span><span class="title">Prior Art OPRK Antagonist Compounds</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK179827/table/ml350.t10/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml350.t10_lrgtbl__"><table class="no_margin"><thead><tr><th id="hd_h_ml350.t10_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Compound Name</th><th id="hd_h_ml350.t10_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Structure</th><th id="hd_h_ml350.t10_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">OPRK<br />IC50 (nM)</th><th id="hd_h_ml350.t10_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Selectivity (nM)</th><th id="hd_h_ml350.t10_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">B/P<sup><a class="bk_pop" href="#ml350.tfn5">*</a></sup></th><th id="hd_h_ml350.t10_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Ref(s)</th></tr></thead><tbody><tr><td headers="hd_h_ml350.t10_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Norbinaltorphimine (nor-BNI)</td><td headers="hd_h_ml350.t10_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK179827/bin/ml350fu95.jpg" alt="Image ml350fu95.jpg" /></div></td><td headers="hd_h_ml350.t10_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.07 ± 0.03</td><td headers="hd_h_ml350.t10_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">OPRM: IC50 15.8 ± 5.7; OPRD: IC50 12.1 ± 3.1</td><td headers="hd_h_ml350.t10_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">< 0.05</td><td headers="hd_h_ml350.t10_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">[<a class="bk_pop" href="#ml350.r92">92</a>, <a class="bk_pop" href="#ml350.r97">97</a>]</td></tr><tr><td headers="hd_h_ml350.t10_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">5′-guanidinonaltrindole (GNTI)</td><td headers="hd_h_ml350.t10_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK179827/bin/ml350fu96.jpg" alt="Image ml350fu96.jpg" /></div></td><td headers="hd_h_ml350.t10_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.04</td><td headers="hd_h_ml350.t10_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">OPRM: IC50 3.2; OPRD: IC50 15.5</td><td headers="hd_h_ml350.t10_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">< 0.0007</td><td headers="hd_h_ml350.t10_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">[<a class="bk_pop" href="#ml350.r97">97</a>, <a class="bk_pop" href="#ml350.r102">102</a>]</td></tr><tr><td headers="hd_h_ml350.t10_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">JDTic</td><td headers="hd_h_ml350.t10_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
|
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<div class="graphic"><img src="/books/NBK179827/bin/ml350fu97.jpg" alt="Image ml350fu97.jpg" /></div></td><td headers="hd_h_ml350.t10_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.006 ± 0.001</td><td headers="hd_h_ml350.t10_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">OPRM: IC50 3.42 ± 0.83; OPRD: IC50 > 100</td><td headers="hd_h_ml350.t10_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">< 0.05</td><td headers="hd_h_ml350.t10_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">[<a class="bk_pop" href="#ml350.r92">92</a>, <a class="bk_pop" href="#ml350.r97">97</a>]</td></tr><tr><td headers="hd_h_ml350.t10_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">AZ-MTAB</td><td headers="hd_h_ml350.t10_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
|
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<div class="graphic"><img src="/books/NBK179827/bin/ml350fu98.jpg" alt="Image ml350fu98.jpg" /></div></td><td headers="hd_h_ml350.t10_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">20 ± 3</td><td headers="hd_h_ml350.t10_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">OPRM: IC50 722 ± 47; OPRD: IC50 8306 ± 1635; (hERG: IC50 260 nM)</td><td headers="hd_h_ml350.t10_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">∼ 1</td><td headers="hd_h_ml350.t10_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">[<a class="bk_pop" href="#ml350.r97">97</a>-<a class="bk_pop" href="#ml350.r98">98</a>]</td></tr><tr><td headers="hd_h_ml350.t10_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">PF-04455242</td><td headers="hd_h_ml350.t10_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
|
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<div class="graphic"><img src="/books/NBK179827/bin/ml350fu99.jpg" alt="Image ml350fu99.jpg" /></div></td><td headers="hd_h_ml350.t10_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1.23</td><td headers="hd_h_ml350.t10_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">OPRM: IC50 10 nM; OPRD: Ki > 4000</td><td headers="hd_h_ml350.t10_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">∼ 1</td><td headers="hd_h_ml350.t10_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">[<a class="bk_pop" href="#ml350.r99">99</a>, <a class="bk_pop" href="#ml350.r103">103</a>]</td></tr><tr><td headers="hd_h_ml350.t10_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">LY2456302</td><td headers="hd_h_ml350.t10_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
|
||
<div class="graphic"><img src="/books/NBK179827/bin/ml350fu100.jpg" alt="Image ml350fu100.jpg" /></div></td><td headers="hd_h_ml350.t10_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.813 ± 0.285</td><td headers="hd_h_ml350.t10_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">OPRM: IC50 17.4 ± 6.33; OPRD: IC50 110 ± 33.6</td><td headers="hd_h_ml350.t10_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">∼ 1</td><td headers="hd_h_ml350.t10_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">[<a class="bk_pop" href="#ml350.r97">97</a>, <a class="bk_pop" href="#ml350.r100">100</a>]</td></tr></tbody></table></div><div><div><dl class="temp-labeled-list small"><dt>*</dt><dd><div id="ml350.tfn5"><p class="no_margin">Unbound brain/plasma ratio</p></div></dd></dl></div></div></div></div><div id="ml350.s42"><h3>4.2. Mechanism of Action Studies</h3><p>Planned future studies (see below) will help elucidate the mechanism of action of <a href="/pcsubstance/?term=ML350[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML350</a> and future improved analogs. New OPRK1 antagonists possessing novel scaffolds and improved selectivity will serve as useful pharmacological tools to better understand the OPRK-dynorphin system.</p></div><div id="ml350.s43"><h3>4.3. Planned Future Studies</h3><p>Additional medicinal chemistry to improve potency, selectivity, and pharmacokinetic properties is in progress. Working with collaborators, we plan to test <a href="/pcsubstance/?term=ML350[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML350</a> and improved analogs in: 1) a stress-induced migraine model assay that measures cutaneous (tactile) allodynia in rats as a measurable translational endpoint for headache-related pain [<a class="bk_pop" href="#ml350.r5">5</a>], 2) an alcohol withdrawal assay that measures working memory performance and anxiety-like behavior in rats after withdrawal from alcohol [<a class="bk_pop" href="#ml350.r6">6</a>], and 3) a cocaine reward assay that measures the effect on drug-seeking behavior in a cocaine addiction model. A CNS-penetrant single oral daily dosed picomolar OPRK antagonist with 3 logs selectivity of OPRM1 and OPRD1 for titration of OPRK1 tone in depression and psychosis is the desired goal.</p></div></div><div id="ml350.s44"><h2 id="_ml350_s44_">5. References</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="ml350.r1">Kuzmin AV, Gerrits MA, Van Ree JM. Kappa-opioid receptor blockade with nor-binaltorphimine modulates cocaine self-administration in drug-naive rats. <span><span class="ref-journal">Eur J Pharmacol. </span>1998;<span class="ref-vol">358</span>(3):197–202.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/9822884" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 9822884</span></a>]</div></dd><dt>2.</dt><dd><div class="bk_ref" id="ml350.r2">Mague SD, et al. Antidepressant-like effects of kappa-opioid receptor antagonists in the forced swim test in rats. <span><span class="ref-journal">J Pharmacol Exp Ther. </span>2003;<span class="ref-vol">305</span>(1):323–30.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/12649385" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 12649385</span></a>]</div></dd><dt>3.</dt><dd><div class="bk_ref" id="ml350.r3">Jewett DC, et al. The kappa-opioid antagonist GNTI reduces U50,488-, DAMGO-, and deprivation-induced feeding, but not butorphanol- and neuropeptide Y-induced feeding in rats. <span><span class="ref-journal">Brain Res. </span>2001;<span class="ref-vol">909</span>(1-2):75–80.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/11478923" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 11478923</span></a>]</div></dd><dt>4.</dt><dd><div class="bk_ref" id="ml350.r4">Roth BL, et al. Salvinorin A: a potent naturally occurring nonnitrogenous kappa opioid selective agonist. <span><span class="ref-journal">Proc Natl Acad Sci U S A. </span>2002;<span class="ref-vol">99</span>(18):11934–9.</span> [<a href="/pmc/articles/PMC129372/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC129372</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/12192085" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 12192085</span></a>]</div></dd><dt>5.</dt><dd><div class="bk_ref" id="ml350.r5">De Felice M, et al. Triptan-induced enhancement of neuronal nitric oxide synthase in trigeminal ganglion dural afferents underlies increased responsiveness to potential migraine triggers. <span><span class="ref-journal">Brain. </span>2010;<span class="ref-vol">133</span>(Pt 8):2475–88.</span> [<a href="/pmc/articles/PMC3139937/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3139937</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/20627971" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 20627971</span></a>]</div></dd><dt>6.</dt><dd><div class="bk_ref" id="ml350.r6">George O, et al. Recruitment of medial prefrontal cortex neurons during alcohol withdrawal predicts cognitive impairment and excessive alcohol drinking. <span><span class="ref-journal">Proc Natl Acad Sci U S A. </span>2012;<span class="ref-vol">109</span>(44):18156–61.</span> [<a href="/pmc/articles/PMC3497825/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3497825</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/23071333" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 23071333</span></a>]</div></dd><dt>7.</dt><dd><div class="bk_ref" id="ml350.r7">Belcheva MM, et al. Mu and kappa opioid receptors activate ERK/MAPK via different protein kinase C isoforms and secondary messengers in astrocytes. <span><span class="ref-journal">J Biol Chem. </span>2005;<span class="ref-vol">280</span>(30):27662–9.</span> [<a href="/pmc/articles/PMC1400585/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC1400585</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/15944153" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 15944153</span></a>]</div></dd><dt>8.</dt><dd><div class="bk_ref" id="ml350.r8">Bruchas MR, Chavkin C. Kinase cascades and ligand-directed signaling at the kappa opioid receptor. <span><span class="ref-journal">Psychopharmacology (Berl). </span>2010;<span class="ref-vol">210</span>(2):137–47.</span> [<a href="/pmc/articles/PMC3671863/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3671863</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/20401607" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 20401607</span></a>]</div></dd><dt>9.</dt><dd><div class="bk_ref" id="ml350.r9">Bruchas MR, et al. Stress-induced p38 mitogen-activated protein kinase activation mediates kappa-opioid-dependent dysphoria. <span><span class="ref-journal">J Neurosci. </span>2007;<span class="ref-vol">27</span>(43):11614–23.</span> [<a href="/pmc/articles/PMC2481272/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2481272</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/17959804" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17959804</span></a>]</div></dd><dt>10.</dt><dd><div class="bk_ref" id="ml350.r10">Bruchas MR, et al. Kappa opioid receptor activation of p38 MAPK is GRK3- and arrestin-dependent in neurons and astrocytes. <span><span class="ref-journal">J Biol Chem. </span>2006;<span class="ref-vol">281</span>(26):18081–9.</span> [<a href="/pmc/articles/PMC2096730/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2096730</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/16648139" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 16648139</span></a>]</div></dd><dt>11.</dt><dd><div class="bk_ref" id="ml350.r11">Bruchas MR, et al. Selective p38alpha MAPK deletion in serotonergic neurons produces stress resilience in models of depression and addiction. <span><span class="ref-journal">Neuron. </span>2011;<span class="ref-vol">71</span>(3):498–511.</span> [<a href="/pmc/articles/PMC3155685/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3155685</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/21835346" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 21835346</span></a>]</div></dd><dt>12.</dt><dd><div class="bk_ref" id="ml350.r12">Childers SR, et al. Opiate receptor binding affected differentially by opiates and opioid peptides. <span><span class="ref-journal">Eur J Pharmacol. </span>1979;<span class="ref-vol">55</span>(1):11–8.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/220062" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 220062</span></a>]</div></dd><dt>13.</dt><dd><div class="bk_ref" id="ml350.r13">Hahn JW, et al. Mu and kappa opioids modulate mouse embryonic stem cell-derived neural progenitor differentiation via MAP kinases. <span><span class="ref-journal">J Neurochem. </span>2010;<span class="ref-vol">112</span>(6):1431–41.</span> [<a href="/pmc/articles/PMC2856797/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2856797</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/19895666" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19895666</span></a>]</div></dd><dt>14.</dt><dd><div class="bk_ref" id="ml350.r14">Walwyn WM, Miotto KA, Evans CJ. 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Nor-binaltorphimine: a potent and selective kappa-opioid receptor antagonist with long-lasting activity in vivo. <span><span class="ref-journal">Arch Int Pharmacodyn Ther. </span>1992;<span class="ref-vol">316</span>:30–42.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/1326932" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 1326932</span></a>]</div></dd><dt>94.</dt><dd><div class="bk_ref" id="ml350.r94">Horan P, et al. Extremely long-lasting antagonistic actions of nor-binaltorphimine (nor-BNI) in the mouse tail-flick test. <span><span class="ref-journal">J Pharmacol Exp Ther. </span>1992;<span class="ref-vol">260</span>(3):1237–43.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/1312164" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 1312164</span></a>]</div></dd><dt>95.</dt><dd><div class="bk_ref" id="ml350.r95">Metcalf MD, Coop A. Kappa opioid antagonists: past successes and future prospects. <span><span class="ref-journal">AAPS J. </span>2005;<span class="ref-vol">7</span>(3):E704–22.</span> [<a href="/pmc/articles/PMC2751273/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2751273</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/16353947" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 16353947</span></a>]</div></dd><dt>96.</dt><dd><div class="bk_ref" id="ml350.r96">Beguin C, Cohen BM. Medicinal Chemistry of Kappa Opioid Receptor Antagonists. In: Dean RL, Bilsky EJ, Negus SS, editors. <span class="ref-journal">Opiate Receptors and Antagonists: from Bench to Clinic.</span> Humana Press; New York: 2009. pp. 99–118.</div></dd><dt>97.</dt><dd><div class="bk_ref" id="ml350.r97">Peters MF, et al. Identification of short-acting kappa-opioid receptor antagonists with anxiolytic-like activity. <span><span class="ref-journal">Eur J Pharmacol. </span>2011;<span class="ref-vol">661</span>(1-3):27–34.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/21539838" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 21539838</span></a>]</div></dd><dt>98.</dt><dd><div class="bk_ref" id="ml350.r98">Brugel TA, et al. Discovery of 8-azabicyclo[3.2.1]octan-3-yloxy-benzamides as selective antagonists of the kappa opioid receptor. Part 1. <span><span class="ref-journal">Bioorg Med Chem Lett. </span>2010;<span class="ref-vol">20</span>(19):5847–52.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/20727752" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 20727752</span></a>]</div></dd><dt>99.</dt><dd><div class="bk_ref" id="ml350.r99">Verhoest PR, et al. Design and discovery of a selective small molecule kappa opioid antagonist (2-methyl-N-((2′-(pyrrolidin-1-ylsulfonyl)biphenyl-4-yl)methyl)propan-1-amine, PF-4455242). <span><span class="ref-journal">J Med Chem. </span>2011;<span class="ref-vol">54</span>(16):5868–77.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/21744827" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 21744827</span></a>]</div></dd><dt>100.</dt><dd><div class="bk_ref" id="ml350.r100">Mitch CH, et al. Discovery of aminobenzyloxyarylamides as kappa opioid receptor selective antagonists: application to preclinical development of a kappa opioid receptor antagonist receptor occupancy tracer. <span><span class="ref-journal">J Med Chem. </span>2011;<span class="ref-vol">54</span>(23):8000–12.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/21958337" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 21958337</span></a>]</div></dd><dt>101.</dt><dd><div class="bk_ref" id="ml350.r101">Li X, et al. Characterization of dasatinib and its structural analogs as CYP3A4 mechanism-based inactivators and the proposed bioactivation pathways. <span><span class="ref-journal">Drug Metab Dispos. </span>2009;<span class="ref-vol">37</span>(6):1242–50.</span> [<a href="/pmc/articles/PMC3202349/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3202349</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/19282395" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19282395</span></a>]</div></dd><dt>102.</dt><dd><div class="bk_ref" id="ml350.r102">Jones RM, Portoghese PS. 5′-Guanidinonaltrindole, a highly selective and potent kappa-opioid receptor antagonist. <span><span class="ref-journal">Eur J Pharmacol. </span>2000;<span class="ref-vol">396</span>(1):49–52.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/10822054" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 10822054</span></a>]</div></dd><dt>103.</dt><dd><div class="bk_ref" id="ml350.r103">Grimwood S, et al. Pharmacological characterization of 2-methyl-N-((2′-(pyrrolidin-1-ylsulfonyl)biphenyl-4-yl)methyl)propan-1-amine (PF-04455242), a high-affinity antagonist selective for kappa-opioid receptors. <span><span class="ref-journal">J Pharmacol Exp Ther. </span>2011;<span class="ref-vol">339</span>(2):555–66.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/21821697" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 21821697</span></a>]</div></dd></dl></div><div id="bk_toc_contnr"></div></div></div>
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<div xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance"></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Views</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="PDF_download" id="Shutter"></a></div><div class="portlet_content"><ul xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="simple-list"><li><a href="/books/NBK179827/?report=reader">PubReader</a></li><li><a href="/books/NBK179827/?report=printable">Print View</a></li><li><a data-jig="ncbidialog" href="#_ncbi_dlg_citbx_NBK179827" data-jigconfig="width:400,modal:true">Cite this Page</a><div id="_ncbi_dlg_citbx_NBK179827" style="display:none" title="Cite this Page"><div class="bk_tt">Guerrero M, Urbano M, Brown SJ, et al. Optimization and characterization of an opioid kappa receptor (OPRK1) antagonist. 2013 Apr 15 [Updated 2014 Sep 18]. 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/NBK179827/pdf/Bookshelf_NBK179827.pdf">PDF version of this page</a> (936K)</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="#ml350.s1" ref="log$=inpage&link_id=inpage">Probe Structure & Characteristics</a></li><li><a href="#ml350.s2" ref="log$=inpage&link_id=inpage">Recommendations for scientific use of the probe</a></li><li><a href="#ml350.s3" ref="log$=inpage&link_id=inpage">Introduction</a></li><li><a href="#ml350.s4" ref="log$=inpage&link_id=inpage">Materials and Methods</a></li><li><a href="#ml350.s31" ref="log$=inpage&link_id=inpage">Results</a></li><li><a href="#ml350.s40" ref="log$=inpage&link_id=inpage">Discussion</a></li><li><a href="#ml350.s44" ref="log$=inpage&link_id=inpage">References</a></li></ul></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Related information</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="discovery_db_links" id="Shutter"></a></div><div class="portlet_content"><ul><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pmc&DbFrom=books&Cmd=Link&LinkName=books_pmc_refs&IdsFromResult=3070012" ref="log$=recordlinks">PMC</a><div class="brieflinkpop offscreen_noflow">PubMed Central citations</div></li><li class="brieflinkpopper"><a class="brieflinkpopperctrl" 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