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<meta name="ncbi_db" content="books" /><meta name="ncbi_pdid" content="book-part" /><meta name="ncbi_acc" content="NBK50685" /><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="/NBK50685/" /><meta name="ncbi_pagename" content="Discovery of a small molecule inhibitor of ROMK and Kir7.1 - 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>Discovery of a small molecule inhibitor of ROMK and Kir7.1 - 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="Discovery of a small molecule inhibitor of ROMK and Kir7.1" /><meta name="citation_publisher" content="National Center for Biotechnology Information (US)" /><meta name="citation_date" content="2010/10/20" /><meta name="citation_author" content="Jerod S Denton" /><meta name="citation_author" content="C David Weaver" /><meta name="citation_author" content="L Michelle Lewis" /><meta name="citation_author" content="Brian A Chauder" /><meta name="citation_author" content="Craig W Lindsley" /><meta name="citation_pmid" content="21433378" /><meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK50685/" /><link rel="schema.DC" href="http://purl.org/DC/elements/1.0/" /><meta name="DC.Title" content="Discovery of a small molecule inhibitor of ROMK and Kir7.1" /><meta name="DC.Type" content="Text" /><meta name="DC.Publisher" content="National Center for Biotechnology Information (US)" /><meta name="DC.Contributor" content="Jerod S Denton" /><meta name="DC.Contributor" content="C David Weaver" /><meta name="DC.Contributor" content="L Michelle Lewis" /><meta name="DC.Contributor" content="Brian A Chauder" /><meta name="DC.Contributor" content="Craig W Lindsley" /><meta name="DC.Date" content="2010/10/20" /><meta name="DC.Identifier" content="https://www.ncbi.nlm.nih.gov/books/NBK50685/" /><meta name="description" content="The specific aim of this project is to identify small molecule inhibitors of Renal Outer Medullary Potassium Channel (ROMK, Kir1.1, KCNJ1), a potassium channel located in the renal tubule, where it critically regulates sodium and potassium balance. However, due to the lack of selective small molecule inhibitors of ROMK, understanding of the molecular pharmacology of ROMK, and indeed that of the entire inward rectifier K+ channel family (kir), is undeveloped, precluding assessment of ROMK's potential as a diuretic target. Furthermore, at least 6 other members (Kir2.3, Kir4.1, Kir4.2, Kir5.1, Kir7.1) of the Kir channel family are expressed in the nephron, but their physiological functions are not well understood. This project aimed to develop ROMK inhibitors with submicromolar potency, cell penetrance and greater than 10-fold selectivity versus other members of the Kir channel family (Kir2.3, Kir4.1, Kir4.2, Kir5.1 and Kir7.1). The currently identified probe, ML111 (CID-4536383) can be used for in vitro and electrophysiology studies to probe the role of ROMK inhibition without inhibition of closely related channels Kir2.1 and Kir4.1. Moreover, ML111 is the first small molecule known to inhibit Kir7.1; thus, the probe also can be used to explore the role of Kir7.1. A second probe from this screen, ML112 (CID-44123657) was a potent ROMK inhibitor with complete selectivity versus Kir2.1, Kir4.1, Kir7.1, Kir2.3 or hERG." /><meta name="og:title" content="Discovery of a small molecule inhibitor of ROMK and Kir7.1" /><meta name="og:type" content="book" /><meta name="og:description" content="The specific aim of this project is to identify small molecule inhibitors of Renal Outer Medullary Potassium Channel (ROMK, Kir1.1, KCNJ1), a potassium channel located in the renal tubule, where it critically regulates sodium and potassium balance. However, due to the lack of selective small molecule inhibitors of ROMK, understanding of the molecular pharmacology of ROMK, and indeed that of the entire inward rectifier K+ channel family (kir), is undeveloped, precluding assessment of ROMK's potential as a diuretic target. Furthermore, at least 6 other members (Kir2.3, Kir4.1, Kir4.2, Kir5.1, Kir7.1) of the Kir channel family are expressed in the nephron, but their physiological functions are not well understood. This project aimed to develop ROMK inhibitors with submicromolar potency, cell penetrance and greater than 10-fold selectivity versus other members of the Kir channel family (Kir2.3, Kir4.1, Kir4.2, Kir5.1 and Kir7.1). The currently identified probe, ML111 (CID-4536383) can be used for in vitro and electrophysiology studies to probe the role of ROMK inhibition without inhibition of closely related channels Kir2.1 and Kir4.1. Moreover, ML111 is the first small molecule known to inhibit Kir7.1; thus, the probe also can be used to explore the role of Kir7.1. A second probe from this screen, ML112 (CID-44123657) was a potent ROMK inhibitor with complete selectivity versus Kir2.1, Kir4.1, Kir7.1, Kir2.3 or hERG." /><meta name="og:url" content="https://www.ncbi.nlm.nih.gov/books/NBK50685/" /><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/ml111/" /><link rel="canonical" href="https://www.ncbi.nlm.nih.gov/books/NBK50685/" /><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="#__NBK50685_dtls__">Show details</a><div style="display:none" class="ui-widget" id="__NBK50685_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/ml112/" title="Previous page in this title">< Prev</a><a class="active page_link next" href="/books/n/mlprobe/ml109/" 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="_NBK50685_"><span class="title" itemprop="name">Discovery of a small molecule inhibitor of ROMK and Kir7.1</span></h1><p class="contrib-group"><span itemprop="author">Jerod S Denton</span>, <span itemprop="author">C David Weaver</span>, <span itemprop="author">L Michelle Lewis</span>, <span itemprop="author">Brian A Chauder</span>, and <span itemprop="author">Craig W Lindsley</span>.</p><a data-jig="ncbitoggler" href="#__NBK50685_ai__" style="border:0;text-decoration:none">Author Information and Affiliations</a><div style="display:none" class="ui-widget" id="__NBK50685_ai__"><p class="contrib-group"><h4>Authors</h4><span itemprop="author">Jerod S Denton</span>, <span itemprop="author">C David Weaver</span>, <span itemprop="author">L Michelle Lewis</span>, <span itemprop="author">Brian A Chauder</span>, and <span itemprop="author">Craig W Lindsley</span><sup>1</sup>.</p><h4>Contact</h4><div class="affiliation"><sup>1</sup> <span class="email-label">Email: </span><a href="mailto:dev@null" data-email="ude.tlibrednav@yelsdnil.giarc" class="oemail">ude.tlibrednav@yelsdnil.giarc</a></div></div><p class="small">Received: <span itemprop="datePublished">September 1, 2009</span>; Last Update: <span itemprop="dateModified">October 20, 2010</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 specific aim of this project is to identify small molecule inhibitors of Renal Outer Medullary Potassium Channel (ROMK, Kir1.1, KCNJ1), a potassium channel located in the renal tubule, where it critically regulates sodium and potassium balance. However, due to the lack of selective small molecule inhibitors of ROMK, understanding of the molecular pharmacology of ROMK, and indeed that of the entire inward rectifier K+ channel family (kir), is undeveloped, precluding assessment of ROMK's potential as a diuretic target. Furthermore, at least 6 other members (Kir2.3, Kir4.1, Kir4.2, Kir5.1, Kir7.1) of the Kir channel family are expressed in the nephron, but their physiological functions are not well understood. This project aimed to develop ROMK inhibitors with submicromolar potency, cell penetrance and greater than 10-fold selectivity versus other members of the Kir channel family (Kir2.3, Kir4.1, Kir4.2, Kir5.1 and Kir7.1). The currently identified probe, ML111 (CID-4536383) can be used for in vitro and electrophysiology studies to probe the role of ROMK inhibition without inhibition of closely related channels Kir2.1 and Kir4.1. Moreover, ML111 is the first small molecule known to inhibit Kir7.1; thus, the probe also can be used to explore the role of Kir7.1. A second probe from this screen, ML112 (CID-44123657) was a potent ROMK inhibitor with complete selectivity versus Kir2.1, Kir4.1, Kir7.1, Kir2.3 or hERG.</p></div><div class="h2"></div><p><b>Assigned Assay Grant #:</b> NS057041-01</p><p><b>Screening Center Name & PI:</b> Vanderbilt Screening Center for GPCRs, Ion Channels and Transporters, C. David Weaver</p><p><b>Chemistry Center Name & PI:</b> Vanderbilt Specialized Chemistry Center for Accelerated Probe Development, Craig W. Lindsley</p><p><b>Assay Submitter & Institution:</b> Jerod S. Denton, Vanderbilt University</p><p><b>PubChem Summary Bioassay Identifier (AID):</b>
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<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2436" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-2436</a></p><div id="ml111.s2"><h2 id="_ml111_s2_">Probe Structure & Characteristics</h2><p>7,13-bis(4-nitorbenzyl)-1,4,10-trioxa-7,13-diazacyclopentadecane</p><p>MW = 488.5, ClogP = 3.06</p><div id="ml111.fu1" class="figure"><div class="graphic"><img src="/books/NBK50685/bin/ml111fu1.jpg" alt="Image ml111fu1" /></div></div><div id="ml111.tu1" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK50685/table/ml111.tu1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml111.tu1_lrgtbl__"><table><thead><tr><th id="hd_h_ml111.tu1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CID/ML#</th><th id="hd_h_ml111.tu1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Target Name</th><th id="hd_h_ml111.tu1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">IC50/E C50 (nM) [SID, AID]</th><th id="hd_h_ml111.tu1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Anti-target Name(s)</th><th id="hd_h_ml111.tu1_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">IC50/EC50 (μM) [SID, AID]</th><th id="hd_h_ml111.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Selectivity</th><th id="hd_h_ml111.tu1_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Secondary Assay(s) Name: IC50/EC50 (nM) [SID, AID]</th></tr></thead><tbody><tr><td headers="hd_h_ml111.tu1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CID 4536383 <a href="/pcsubstance/?term=ML111[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML111</a></td><td headers="hd_h_ml111.tu1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">ROMK</td><td headers="hd_h_ml111.tu1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">294 [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/84975334" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-84975334</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1917" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1917</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1918" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1918</a>]</td><td headers="hd_h_ml111.tu1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Kir2.1, Kir4.1,</td><td headers="hd_h_ml111.tu1_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">>10 μM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/84975334" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-84975334</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1916" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1916</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1924" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1924</a>]</td><td headers="hd_h_ml111.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">>30</td><td headers="hd_h_ml111.tu1_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Electrophysiology [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/84975334" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-84975334</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1922" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1922</a>]</td></tr><tr><td headers="hd_h_ml111.tu1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CID 4536383 <a href="/pcsubstance/?term=ML111[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML111</a></td><td headers="hd_h_ml111.tu1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">ROMK</td><td headers="hd_h_ml111.tu1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml111.tu1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Kir7.1</td><td headers="hd_h_ml111.tu1_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">70% @ 10 μM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/84975334" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-84975334</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1916" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1916</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1924" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1924</a></td><td headers="hd_h_ml111.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml111.tu1_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td></tr></tbody></table></div></div></div><div id="ml111.s3"><h2 id="_ml111_s3_">Recommendations for the scientific use of this probe</h2><p>This probe (CID 4536383) can be used for <i>in vitro</i> and electrophysiology studies to probe the role of ROMK inhibition without inhibition of closely related channels Kir2.1 and Kir4.1. Moreover CID 4536383 is the first small molecule known to inhibit Kir7.1; thus, CID 4536383 can also be used to explore the role of Kir7.1. A second probe from this screen, CID 44123657 was a potent ROMK inhibitor with complete selectivity versus Kir2.1, Kir4.1, Kir7.1, Kir2.3 or hERG.</p><div id="ml111.s4"><h3>Specific AIM</h3><p>To identify small molecule inhibitors of Renal Outer Medullary Potassium Channel (ROMK, Kir1.1, KCNJ1), a potassium channel located in the renal tubule where it critically regulates sodium and potassium balance. There are no selective small molecule inhibitors of ROMK. We aimed to develop ROMK inhibitors with submicromolar potency, cell penetrance and greater than 10-fold selectivity versus other members of the Kir channel family (Kir2.3, Kir4.1, Kir4.2, Kir5.1 and Kir7.1).</p></div><div id="ml111.s5"><h3>Significance</h3><p>The <u>R</u>enal <u>O</u>uter <u>M</u>edullary
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potassium (<u>K</u>) channel (ROMK, Kir1.1, KCNJ1) is expressed in the kidney tubule
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where it plays key roles in regulating fluid and electrolyte homeostasis. (<a class="bk_pop" href="#ml111.r1">1</a>,<a class="bk_pop" href="#ml111.r2">2</a>) In the thick ascending limb
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of Henle, luminal K recycling by ROMK supports NaCl reabsorption by the Na-K-2Cl co-transporter and
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loop diuretic (e.g. furosemide) target NKCC2, which in turn promotes osmotic water reabsorption in
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the distal nephron. (<a class="bk_pop" href="#ml111.r3" data-bk-pop-others="ml111.r4 ml111.r5">3–5</a>) In the
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connecting tubule, distal convoluted tubule and collecting duct, ROMK mediates the final step in K
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secretion and functions to match dietary K intake with urinary K excretion. (<a class="bk_pop" href="#ml111.r6">6</a>,<a class="bk_pop" href="#ml111.r7">7</a>) A growing body of genetic evidence (<a class="bk_pop" href="#ml111.r8" data-bk-pop-others="ml111.r9 ml111.r10">8–10</a>) suggests that pharmacological antagonists of ROMK could have potent diuretic effects while minimizing potentially dangerous urinary K loss caused by furosemide. (<a class="bk_pop" href="#ml111.r11">11</a>,<a class="bk_pop" href="#ml111.r12">12</a>) However, the molecular pharmacology of ROMK, and indeed that of the entire inward rectifier K<sup>+</sup> channel family, is virtually undeveloped, precluding assessment of ROMK’s potential as a diuretic target.</p><p>At least 6 other members (Kir2.3, Kir4.1, Kir4.2, Kir5.1, Kir7.1) of the Kir channel family are
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expressed in the nephron, but their physiological functions are not well understood. (<a class="bk_pop" href="#ml111.r2">2</a>,<a class="bk_pop" href="#ml111.r13" data-bk-pop-others="ml111.r14 ml111.r15 ml111.r16">13–16</a>) The newest family member, Kir7.1 (KCNJ13), is expressed in
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several nephron segments. In principal cells of the collecting duct, Kir7.1 is believed to
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contribute to basolateral K recycling necessary for Na-K-ATPase-dependent K secretion. (<a class="bk_pop" href="#ml111.r15">15</a>) However, there is no direct evidence that Kir7.1 forms
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functional ion channels in the nephron. Kir7.1 has an unusually low unitary conductance (~50 fS),
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(<a class="bk_pop" href="#ml111.r17">17</a>) making it difficult to identify in single-channel recordings, and there have been no pharmacological tools available with which to discriminate Kir7.1 from other channels in whole-cell recordings. The identification of pharmacological probes would be helpful in defining the functions of Kir7.1 in the nephron and other tissues. (<a class="bk_pop" href="#ml111.r17">17</a>,<a class="bk_pop" href="#ml111.r25">25</a>) To date, there are no potent and or selective ROMK inhibitors; however, known K<sub>ATP</sub> blockers have been shown to exhibit low potency toward ROMK together with undesirable cardiovascular and metabolic side effects mediated by K<sub>ATP</sub> channels. (<a class="bk_pop" href="#ml111.r37">37</a>)</p></div><div id="ml111.s6"><h3>Rationale</h3><p>In the present study, we developed and implemented a fluorescence-based assay for high-throughput
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screening (HTS) of chemical libraries for novel modulators of ROMK function, and developed the
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appropriate counter screens within the Kir family and electrophysiology assays to rapidly confirm
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and optimize putative ROMK inhibitor hits into selective ROMK inhibitors. (<a class="bk_pop" href="#ml111.r18" data-bk-pop-others="ml111.r19 ml111.r20 ml111.r21 ml111.r22 ml111.r23 ml111.r24 ml111.r25">18–25</a>)</p></div></div><div id="ml111.s7"><h2 id="_ml111_s7_">Assay Implementation and Screening</h2><div id="ml111.s8"><h3>PubChem Bioassay Name</h3><p>Identification of Novel Modulators of ROMK K+ Channel Activity</p></div><div id="ml111.s9"><h3>List of PubChem bioassay identifiers generated for this screening project (AIDs)</h3><p><b><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1916" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1916</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1917" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1917</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1918" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1918</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1922" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1922</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1924" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1924</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2436" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-2436</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2753" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-2753</a></b></p></div><div id="ml111.s10"><h3>PubChem Primary Assay Description</h3><p>We used C1 cells to develop a fluorescence-based assay of ROMK function for high-throughput
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screening (HTS). The assay reports flux of the K+ congener thallium (Tl+) through
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ROMK channels using the fluorescent dye Fluozin-2. (<a class="bk_pop" href="#ml111.r22" data-bk-pop-others="ml111.r23 ml111.r24">22–24</a>) <a class="figpopup" href="/books/NBK50685/figure/ml111.f1/?report=objectonly" target="object" rid-figpopup="figml111f1" rid-ob="figobml111f1">Figure 1A</a> shows representative fluorescence traces recorded from individual wells of a 384-well plate containing uninduced (−Tet) or Tet-induced cells bathed in control (+Tet) or TPNQ-containing assay buffer (Tet + TPNQ). Tl+ evoked a rapid fluorescence increase in induced cells, but not in uninduced cells or induced cells pre-treated with the ROMK blocker TPNQ. The slope of the Fluozin-2 fluorescence increase between 7 and 12 seconds after Tl+ addition was used as a measure of Tl+ flux. TPNQ dose-dependently decreased Tl+ flux in induced cells with an IC<sub>50</sub> of 5.2 nM (<a class="figpopup" href="/books/NBK50685/figure/ml111.f1/?report=objectonly" target="object" rid-figpopup="figml111f1" rid-ob="figobml111f1">Fig. 1C</a>), indicating the assay is sensitive and capable of reporting graded changes in ROMK activity. To assess the suitability of the assay for HTS, every other well of a 384-well plate was pre-treated with control assay buffer or TPNQ-containing buffer before Tl+ addition. The rate of Tl+ flux varied little within treatments, but was dramatically different between control- and TPNQ-treated wells (<a class="figpopup" href="/books/NBK50685/figure/ml111.f1/?report=objectonly" target="object" rid-figpopup="figml111f1" rid-ob="figobml111f1">Fig. 1B</a>). For this plate the calculated Z′ value, a statistical indicator of the assay’s ability to correctly identify hits in a screen, was 0.68. Assays with Z′ values between 0.5 and 1.0 are considered suitable for HTS. (<a class="bk_pop" href="#ml111.r26">26</a>)</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml111f1" co-legend-rid="figlgndml111f1"><a href="/books/NBK50685/figure/ml111.f1/?report=objectonly" target="object" title="Figure 1" class="img_link icnblk_img figpopup" rid-figpopup="figml111f1" rid-ob="figobml111f1"><img class="small-thumb" src="/books/NBK50685/bin/ml111f1.gif" src-large="/books/NBK50685/bin/ml111f1.jpg" alt="Figure 1. High-throughput assay of ROMK function." /></a><div class="icnblk_cntnt" id="figlgndml111f1"><h4 id="ml111.f1"><a href="/books/NBK50685/figure/ml111.f1/?report=objectonly" target="object" rid-ob="figobml111f1">Figure 1</a></h4><p class="float-caption no_bottom_margin">High-throughput assay of ROMK function. (A) Fluozin-2 fluorescence traces recorded
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fram C1 cells before and after addition of extracellular Tl+ (shaded area); (B) Plot of the slope of Tl+-induced fluorescence increase recorded from individual wells of <a href="/books/NBK50685/figure/ml111.f1/?report=objectonly" target="object" rid-ob="figobml111f1">(more...)</a></p></div></div></div><div id="ml111.s11"><h3>Summary of Screen</h3><p>We developed a robust HTS assay for small-molecule ROMK modulators, which enabled the
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identification of a novel blocker of ROMK and Kir7.1 channels. The assay extends previous work using
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Tl+ flux to detect potassium channel and transporter activities (<a class="bk_pop" href="#ml111.r22" data-bk-pop-others="ml111.r23 ml111.r24 ml111.r25">22–25</a>) and overcomes ROMK-specific
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technical hurdles associated with reportedly poor channel expression in mammalian cells (<a class="bk_pop" href="#ml111.r27" data-bk-pop-others="ml111.r28 ml111.r29 ml111.r30 ml111.r31 ml111.r32 ml111.r33">27–33</a>) by using an inducible expression system and point mutation (S44D) that promotes cell surface expression. (<a class="bk_pop" href="#ml111.r21" data-bk-pop-others="ml111.r22 ml111.r23 ml111.r24 ml111.r25 ml111.r26 ml111.r27 ml111.r28 ml111.r29 ml111.r30 ml111.r31 ml111.r32 ml111.r33 ml111.r34 ml111.r35">21–35</a>) The identification by HTS of numerous ROMK antagonists, some of which preferentially block other Kir channels, is a significant step toward developing the molecular pharmacology of the inward rectifier channel family. Using the Tl+ flux assay we screened 126,009 compounds in 384-well plates at a single dose of 10 μM. DMSO at a final concentration 0.1% was used as the compound vehicle. During assay development, we established that Tl+ flux through ROMK was insensitive to DMSO concentrations up to 10% (data not shown). Four rows in each plate were filled with control or TPNQ-containing buffer for determination of Z′ (e.g. <a class="figpopup" href="/books/NBK50685/figure/ml111.f3/?report=objectonly" target="object" rid-figpopup="figml111f3" rid-ob="figobml111f3">Fig. 3C</a>). Plates with Z′ values less than 0.45 were not included in the data analysis. The mean ± SD Z′ of plates passing quality control was 0.72 ± 0.08. The processed primary data resulted in 1,758 compounds designated as inhibitors. After re-orders from ChemDiv and confirmation screening in electrophysiology experiments, we were left with three hits (<a class="figpopup" href="/books/NBK50685/figure/ml111.f2/?report=objectonly" target="object" rid-figpopup="figml111f2" rid-ob="figobml111f2">Figure 2</a>): <a href="https://pubchem.ncbi.nlm.nih.gov/substance/17510129" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-17510129</a> (IC<sub>50</sub> = 3 μM), <a href="https://pubchem.ncbi.nlm.nih.gov/substance/24802482" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-24802482</a> (80% block @ 10 μM) and <a href="https://pubchem.ncbi.nlm.nih.gov/substance/17409040" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-17409040</a> (30% block @ 10 μM).</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml111f3" co-legend-rid="figlgndml111f3"><a href="/books/NBK50685/figure/ml111.f3/?report=objectonly" target="object" title="Figure 3" class="img_link icnblk_img figpopup" rid-figpopup="figml111f3" rid-ob="figobml111f3"><img class="small-thumb" src="/books/NBK50685/bin/ml111f3.gif" src-large="/books/NBK50685/bin/ml111f3.jpg" alt="Figure 3. Lead Optimization of SID-17510129." /></a><div class="icnblk_cntnt" id="figlgndml111f3"><h4 id="ml111.f3"><a href="/books/NBK50685/figure/ml111.f3/?report=objectonly" target="object" rid-ob="figobml111f3">Figure 3</a></h4><p class="float-caption no_bottom_margin">Lead Optimization of SID-17510129. </p></div></div><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml111f2" co-legend-rid="figlgndml111f2"><a href="/books/NBK50685/figure/ml111.f2/?report=objectonly" target="object" title="Figure 2" class="img_link icnblk_img figpopup" rid-figpopup="figml111f2" rid-ob="figobml111f2"><img class="small-thumb" src="/books/NBK50685/bin/ml111f2.gif" src-large="/books/NBK50685/bin/ml111f2.jpg" alt="Figure 2. Confirmed ROMK HTS hits." /></a><div class="icnblk_cntnt" id="figlgndml111f2"><h4 id="ml111.f2"><a href="/books/NBK50685/figure/ml111.f2/?report=objectonly" target="object" rid-ob="figobml111f2">Figure 2</a></h4><p class="float-caption no_bottom_margin">Confirmed ROMK HTS hits. </p></div></div></div><div id="ml111.s12"><h3>Chemical Probe Lead Optimization</h3><p>Based on the initial activity at inhibiting ROMK and the fact that PubChem data indicated that it
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had been tested in 181 bioassays, and only active in the ROMK screen, we focused on optimization of
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<a href="https://pubchem.ncbi.nlm.nih.gov/substance/17510129" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-17510129</a>, a bis-benzylated kryptofix analog. Under our standard paradigm, (<a class="bk_pop" href="#ml111.r36">36</a>) we resynthesized <a href="https://pubchem.ncbi.nlm.nih.gov/substance/17510129" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-17510129</a> in a library formpat with 21 other analogs wherein we varied the aryl moiety (<a class="figpopup" href="/books/NBK50685/figure/ml111.f3/?report=objectonly" target="object" rid-figpopup="figml111f3" rid-ob="figobml111f3">Figure 3A</a>) utilizing solution phase parallel synthesis (<a class="figpopup" href="/books/NBK50685/figure/ml111.f3/?report=objectonly" target="object" rid-figpopup="figml111f3" rid-ob="figobml111f3">Figure 3B</a>). In the event, kryptofix <b>1</b> underwent a bis-reductive amination with 22 different aldehydes <b>2</b> employing MP-B(OAc)<sub>3</sub>H to deliver a total of 21 analogs <b>3</b> and analytically pure, fresh stock of <a href="https://pubchem.ncbi.nlm.nih.gov/substance/17510129" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-17510129</a> (CID 4536383). Upon testing fresh powder of analytically pure CID 4536383, we were pleased to find that the IC<sub>50</sub> was 294 nM, a potency meeting MLPCN probe criteria. Moving the NO<sub>2</sub> group from the 4-position to the 3-position (CID 44123652) led to a significant decrease in potency (IC<sub>50</sub> = 14.2 μM) and further migration to the 2-position led a complete loss of activity (IC<sub>50</sub> = >100 μM). SAR proved to be quite shallow (<a class="figpopup" href="/books/NBK50685/figure/ml111.f3/?report=objectonly" target="object" rid-figpopup="figml111f3" rid-ob="figobml111f3">Figure 3B</a>), with any other substituents on the aryl ring leading to ROMK inhibitors in the micromolar range (IC<sub>50</sub>s from 6.6 M to >100 μM). We then synthesized three analogs (<a class="figpopup" href="/books/NBK50685/figure/ml111.f3/?report=objectonly" target="object" rid-figpopup="figml111f3" rid-ob="figobml111f3">Figure 3C</a>) in which we maintained the 4-nitrophenyl moiety, but attenuated the basicity of the nitrogen atoms by capping as an amide <b>4</b>, a urea <b>5</b> or a sulfonamide <b>6</b>; however, all were inactive (IC<sub>50</sub> = >100 μM), suggesting the basic nitrogen moieties are required for ROMK activity. Since CID 4536383 possessed the potency requirements for an MLPCN probe (IC<sub>50</sub> = 294 nM) in both Tl+ flux assay (<a class="figpopup" href="/books/NBK50685/figure/ml111.f4/?report=objectonly" target="object" rid-figpopup="figml111f4" rid-ob="figobml111f4">Figure 4B</a>) and the in confirmatory electrophysiology assay (<a class="figpopup" href="/books/NBK50685/figure/ml111.f4/?report=objectonly" target="object" rid-figpopup="figml111f4" rid-ob="figobml111f4">Figure 4C</a>), we next evaluated Kir selectivity. We were pleased to find that CID 4536383 was selective versus the closely related Kir2.1 and Kir4.1 potassium channels. Interestingly, low micromolar concentrations of CID 4536383 inhibit Kir7.1 (70% @ 10 μM). Other than non-selective cationic pore blockers, CID 4536383 is the first small molecule inhibitor of Kir7.1, the newest Kir family member. Kir7.1 (KCNJ13), is expressed in several nephron segments. In principal cells of the collecting duct, Kir7.1 is believed to contribute to basolateral K+ recycling necessary for Na-K-ATPase-dependent K secretion. However, there is no direct evidence that Kir7.1 forms functional ion channels in the nephron. Kir7.1 has an unusually low unitary conductance (~50 fS), making it difficult to identify in single-channel recordings, and there have been no pharmacological tools available with which to discriminate Kir7.1 from other channels in whole-cell recordings. The identification of CID 4536383 as the first pharmacological probe of Kir7.1 will be helpful in defining the functions of Kir7.1 in the nephron and other tissues.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml111f4" co-legend-rid="figlgndml111f4"><a href="/books/NBK50685/figure/ml111.f4/?report=objectonly" target="object" title="Figure 4" class="img_link icnblk_img figpopup" rid-figpopup="figml111f4" rid-ob="figobml111f4"><img class="small-thumb" src="/books/NBK50685/bin/ml111f4.gif" src-large="/books/NBK50685/bin/ml111f4.jpg" alt="Figure 4. (A) CID 4536838; (B) CRC in Tl+ Flux assay; (C) Electrophysiology." /></a><div class="icnblk_cntnt" id="figlgndml111f4"><h4 id="ml111.f4"><a href="/books/NBK50685/figure/ml111.f4/?report=objectonly" target="object" rid-ob="figobml111f4">Figure 4</a></h4><p class="float-caption no_bottom_margin">(A) CID 4536838; (B) CRC in Tl+ Flux assay; (C) Electrophysiology. </p></div></div><div id="ml111.f5" class="figure bk_fig"><div class="graphic"><img src="/books/NBK50685/bin/ml111f5.jpg" alt="Figure 5. CID 4536838 selectivity versus Kir members." /></div><h3><span class="label">Figure 5</span><span class="title">CID 4536838 selectivity versus Kir members</span></h3></div><p>This intriguing Kir selectivity profile led to additional experiments to determine a mode of
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action for ROMK inhibition. The rate of ROMK inhibition by 10 M CID 4536383 was slow (time constant
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= 40 ± 0.5 sec; n = 8), and washout was incomplete, suggesting the compound
|
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might first cross the plasma membrane to reach an intracellular binding site. Furthermore, we noted
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a large hyperpolarizing shift in the rectification potential during the onset of inhibition. Because
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rectification in Kir channels is caused by block of outward current by intracellular solutes, this
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observation raised the possibility that CID 4536383 binds in the cytoplasmic pore. (<a class="bk_pop" href="#ml111.r28" data-bk-pop-others="ml111.r29 ml111.r30 ml111.r31">28–31</a>) We therefore tested if CID 4536383 could be displaced from the cytoplasmic pore with inwardly directed K<sup>+</sup> ions through a mechanism known as “knock-off”. (<a class="bk_pop" href="#ml111.r32">32</a>,<a class="bk_pop" href="#ml111.r33">33</a>) Experiments were performed by pre-blocking ROMK with 10 μM CID 4536383 and voltage clamping the cell to potentials more negative than the K<sup>+</sup> equilibrium potential (E<sub>K</sub>) to drive K+ ions intracellularly through the ROMK channel pore. In the presence of 5 mM extracellular K<sup>+</sup> and 135 mM intracellular K<sup>+</sup> (E<sub>K</sub> = −83 mV), block by CID 4536383 was virtually insensitive to repetitive pulses to −120 mV. However, increasingly stronger pulses led to a knock-off of CID 4536383 that was both time- and voltage-dependent (Fig. 7B′). To confirm that CID 4536383 knock-off is caused by permeant ions and not some voltage-dependent conformational change in the channel, we raised extracellular K<sup>+</sup> concentration to 50 mM (E<sub>K</sub> = −25 mV) and repeated the -120 mV step protocol. CID 4536383 was readily displaced from the pore under these conditions. The most parsimonious interpretation of these data is that CID 4536383 is an intracellular pore blocker of ROMK. (<a class="bk_pop" href="#ml111.r25">25</a>)</p></div><div id="ml111.s13"><h3>Solubility</h3><p>~ 100 M in DMSO. Homogeneous/microsuspension at 10 mg/mL in : 20% β-cyclodextrin, 10% cremaphor, 40% PEG-400/H<sub>2</sub>O. As bis-HCl salt, soluble in saline at 10 mg/mL.</p></div><div id="ml111.s14"><h3>Synthetic procedure and Spectral data for CID 4536383</h3><div id="ml111.fu2" class="figure"><div class="graphic"><img src="/books/NBK50685/bin/ml111fu2.jpg" alt="Image ml111fu2" /></div></div><div id="ml111.s15"><h4>7,13-bis(4-nitrobenzyl)-1,4,10-trioxa-7,13-diazacyclopentadecane(CID 4536383) [<a href="/pcsubstance/?term=ML111[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML111</a>]</h4><p>To an 8 mL vial was added 4-nitrobenzaldehyde (70 mg, 0.46 mmol), Kryptofix (50 mg, 0.23 mmol) and MP-BH(OAc)<sub>3</sub> (500 mg) in DCM (3 mL). The mixture was rotated for 12 h, filtered and concentrated. Purification of the crude oil by ISCO flash chromatography [10% MeOH/DCM containing 1% NH<sub>4</sub>OH (12CV)] provided the title compound <b>CID 4536383</b> as an orange oil (39 mg, 35%). <sup>1</sup>H-NMR (400 MHz, CDCl<sub>3</sub>) δ 8.18 (d, <i>J</i>=8.8 Hz, 4H), 7.60 (d, <i>J</i>=8.8 Hz, 4H), 3.79 (s, 4H), 3.65–3.62 (m, 12H), 2.86–2.79 (m, 8H); HRMS (calculated for C<sub>24</sub>H<sub>32</sub>N<sub>4</sub>O<sub>7</sub>+H) 489.2349; Found 489.2349.</p></div></div><div id="ml111.s16"><h3>MLS#s</h3><p>002474506 (CID 4536383, 500 mg), 002474503, 002474504, 002474505, 002765789,
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002765790</p></div></div><div id="ml111.rl1"><h2 id="_ml111_rl1_">Bibliography</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="ml111.r1">Wang W, Hebert SC, Giebisch G. <span><span class="ref-journal">Annual. 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[<a href="/pmc/articles/PMC2774996/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2774996</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/19706730" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19706730</span></a>] [<a href="http://dx.crossref.org/10.1124/mol.109.059840" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>26.</dt><dd><div class="bk_ref" id="ml111.r26">Zhang JH, Chung TD, Oldenburg KR. <span><span class="ref-journal">J Biomol Screen. </span>1999;<span class="ref-vol">4</span>:67–73.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/10838414" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 10838414</span></a>]</div></dd><dt>27.</dt><dd><div class="bk_ref" id="ml111.r27">Schulte U, Hahn H, Konrad M, Jeck N, Derst C, Wild K, Weidemann S, Ruppersberg JP, Fakler B, Ludwig J. <span><span class="ref-journal">Proc Natl Acad Sci U S A. </span>1999;<span class="ref-vol">96</span>:15298–15303.</span> [<a href="/pmc/articles/PMC24814/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC24814</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/10611379" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 10611379</span></a>]</div></dd><dt>28.</dt><dd><div class="bk_ref" id="ml111.r28">Fakler B, Brandle U, Glowatzki E, Weidemann S, Zenner HP, Ruppersberg JP. <span><span class="ref-journal">Cell. </span>1995;<span class="ref-vol">80</span>:149–154.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/7813010" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 7813010</span></a>]</div></dd><dt>29.</dt><dd><div class="bk_ref" id="ml111.r29">Lopatin AN, Makhina EN, Nichols CG. <span><span class="ref-journal">Nature. </span>1994;<span class="ref-vol">372</span>:366–369.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/7969496" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 7969496</span></a>]</div></dd><dt>30.</dt><dd><div class="bk_ref" id="ml111.r30">Lu Z, MacKinnon R. <span><span class="ref-journal">Nature. </span>1994;<span class="ref-vol">371</span>:243–246.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/7915826" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 7915826</span></a>]</div></dd><dt>31.</dt><dd><div class="bk_ref" id="ml111.r31">Wible BA, Taglialatela M, Ficker E, Brown AM. <span><span class="ref-journal">Nature. </span>1994;<span class="ref-vol">371</span>:246–249.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/8078584" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 8078584</span></a>]</div></dd><dt>32.</dt><dd><div class="bk_ref" id="ml111.r32">Sali A, Blundell TL. <span><span class="ref-journal">J Mol Biol. </span>1993;<span class="ref-vol">234</span>:779–815.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/8254673" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 8254673</span></a>]</div></dd><dt>33.</dt><dd><div class="bk_ref" id="ml111.r33">Shin HG, Lu Z. <span><span class="ref-journal">J Gen Physiol. </span>2005;<span class="ref-vol">125</span>:413–426.</span> [<a href="/pmc/articles/PMC2217510/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2217510</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/15795311" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 15795311</span></a>]</div></dd><dt>34.</dt><dd><div class="bk_ref" id="ml111.r34">Brejon M, Le Maout S, Welling PA, Merot J. <span><span class="ref-journal">Biochem Biophys Res Commun. </span>1999;<span class="ref-vol">261</span>:364–371.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/10425191" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 10425191</span></a>]</div></dd><dt>35.</dt><dd><div class="bk_ref" id="ml111.r35">Yoo D, Kim BY, Campo C, Nance L, King A, Maouyo D, Welling PA. <span><span class="ref-journal">J Biol Chem. </span>2003;<span class="ref-vol">278</span>:23066–23075.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/12684516" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 12684516</span></a>]</div></dd><dt>36.</dt><dd><div class="bk_ref" id="ml111.r36">Kennedy JP, Williams L, Bridges TM, Daniels RN, Weaver D, Lindsley CW. <span><span class="ref-journal">J Comb Chem 10. </span>2008:345–356.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/18220367" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18220367</span></a>]</div></dd><dt>37.</dt><dd><div class="bk_ref" id="ml111.r37">Clark MA, Humphrey SJ, Smith MP, Ludens JH. <span><span class="ref-journal">J Pharmacol Exp Ther. </span>1993;<span class="ref-vol">265</span>:933–937.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/8496833" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 8496833</span></a>]</div></dd></dl></div><div id="ml111.app1"><h2 id="_ml111_app1_">APPENDIX I. Solubility, Stability and Reactivity data as determined by Absorption Systems</h2><div id="ml111.s17"><h3>Solubility</h3><p>Solubility in PBS (at pH = 7.4) for <a href="/pcsubstance/?term=ML111[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML111</a> was 58.2 μM.</p></div><div id="ml111.s18"><h3>Stability</h3><p>Stability (at room temperature = 23 °C) for <a href="/pcsubstance/?term=ML111[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML111</a> in PBS (no antioxidants or other protectorants and DMSO concentration below 0.1%) is shown in the table below. After 48 hours, the percent of parent compound remaining was 91%, but the assay variability over the course of the experiment ranged from a low of 91% (at 48 hours) to a high of 107% (at 30 minutes).</p><div id="ml111.tu2" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK50685/table/ml111.tu2/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml111.tu2_lrgtbl__"><table><thead><tr><th id="hd_h_ml111.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"></th><th id="hd_h_ml111.tu2_1_1_1_2" colspan="7" rowspan="1" style="text-align:center;vertical-align:bottom;">Percent Remaining (%)</th></tr><tr><th headers="hd_h_ml111.tu2_1_1_1_1" id="hd_h_ml111.tu2_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Compound</th><th headers="hd_h_ml111.tu2_1_1_1_2" id="hd_h_ml111.tu2_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">0 Min</th><th headers="hd_h_ml111.tu2_1_1_1_2" id="hd_h_ml111.tu2_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">15 Min</th><th headers="hd_h_ml111.tu2_1_1_1_2" id="hd_h_ml111.tu2_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">30 Min</th><th headers="hd_h_ml111.tu2_1_1_1_2" id="hd_h_ml111.tu2_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">1 Hour</th><th headers="hd_h_ml111.tu2_1_1_1_2" id="hd_h_ml111.tu2_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">2 Hour</th><th headers="hd_h_ml111.tu2_1_1_1_2" id="hd_h_ml111.tu2_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">24 Hour</th><th headers="hd_h_ml111.tu2_1_1_1_2" id="hd_h_ml111.tu2_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">48 Hour</th></tr></thead><tbody><tr><td headers="hd_h_ml111.tu2_1_1_1_1 hd_h_ml111.tu2_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="/pcsubstance/?term=ML111[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML111</a></td><td headers="hd_h_ml111.tu2_1_1_1_2 hd_h_ml111.tu2_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">---</td><td headers="hd_h_ml111.tu2_1_1_1_2 hd_h_ml111.tu2_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">100</td><td headers="hd_h_ml111.tu2_1_1_1_2 hd_h_ml111.tu2_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">107</td><td headers="hd_h_ml111.tu2_1_1_1_2 hd_h_ml111.tu2_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">99</td><td headers="hd_h_ml111.tu2_1_1_1_2 hd_h_ml111.tu2_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">97</td><td headers="hd_h_ml111.tu2_1_1_1_2 hd_h_ml111.tu2_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">95</td><td headers="hd_h_ml111.tu2_1_1_1_2 hd_h_ml111.tu2_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">91</td></tr></tbody></table></div></div></div><div id="ml111.s19"><h3>Reactivity</h3><p>As assessed through a glutathione (GSH) trapping experiment in phosphate buffered saline (with a substrate concentration of typically 5–50 μM and a GSH concentration of 5 mM, at t = 60 minutes) <a href="/pcsubstance/?term=ML111[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML111</a> was found to not form any detectable GSH adducts.<sup><a class="bk_pop" href="#ml111.fn1">*</a></sup></p></div></div><h2 id="NBK50685_footnotes">Footnotes</h2><dl class="temp-labeled-list small"><dt>*</dt><dd><div id="ml111.fn1"><p class="no_top_margin">Solubility (PBS at pH = 7.4), Stability and Reactivity experiments were conducted at Absorption Systems. For additional information see: <a href="https://www.absorption.com/site" ref="pagearea=body&targetsite=external&targetcat=link&targettype=uri">https://www<wbr style="display:inline-block"></wbr>.absorption.com/site</a></p></div></dd></dl><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/NBK50685/?report=reader">PubReader</a></li><li><a href="/books/NBK50685/?report=printable">Print View</a></li><li><a data-jig="ncbidialog" href="#_ncbi_dlg_citbx_NBK50685" data-jigconfig="width:400,modal:true">Cite this Page</a><div id="_ncbi_dlg_citbx_NBK50685" style="display:none" title="Cite this Page"><div class="bk_tt">Denton JS, Weaver CD, Lewis LM, et al. Discovery of a small molecule inhibitor of ROMK and Kir7.1. 2009 Sep 1 [Updated 2010 Oct 20]. 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/NBK50685/pdf/Bookshelf_NBK50685.pdf">PDF version of this page</a> (640K)</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="#ml111.s2" ref="log$=inpage&link_id=inpage">Probe Structure & Characteristics</a></li><li><a href="#ml111.s3" ref="log$=inpage&link_id=inpage">Recommendations for the scientific use of this probe</a></li><li><a href="#ml111.s7" ref="log$=inpage&link_id=inpage">Assay Implementation and Screening</a></li><li><a href="#ml111.rl1" ref="log$=inpage&link_id=inpage">Bibliography</a></li><li><a href="#ml111.app1" ref="log$=inpage&link_id=inpage">Solubility, Stability and Reactivity data as determined by Absorption Systems</a></li></ul></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Related information</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="discovery_db_links" id="Shutter"></a></div><div class="portlet_content"><ul><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pmc&DbFrom=books&Cmd=Link&LinkName=books_pmc_refs&IdsFromResult=2376246" ref="log$=recordlinks">PMC</a><div class="brieflinkpop offscreen_noflow">PubMed Central citations</div></li><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pcassay&DbFrom=books&Cmd=Link&LinkName=books_pcassay_probe&IdsFromResult=2376246" ref="log$=recordlinks">PubChem BioAssay for Chemical Probe</a><div class="brieflinkpop offscreen_noflow">PubChem BioAssay records reporting screening data for the development of the chemical probe(s) described in this book chapter</div></li><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pcsubstance&DbFrom=books&Cmd=Link&LinkName=books_pcsubstance&IdsFromResult=2376246" ref="log$=recordlinks">PubChem Substance</a><div class="brieflinkpop offscreen_noflow">Related PubChem Substances</div></li><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pubmed&DbFrom=books&Cmd=Link&LinkName=books_pubmed_refs&IdsFromResult=2376246" ref="log$=recordlinks">PubMed</a><div class="brieflinkpop offscreen_noflow">Links to PubMed</div></li></ul></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Similar articles in PubMed</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="PBooksDiscovery_RA" id="Shutter"></a></div><div class="portlet_content"><ul><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/21433391" ref="ordinalpos=1&linkpos=1&log$=relatedreviews&logdbfrom=pubmed"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> Discovery of a small molecule inhibitor of ROMK with unprecedented selectivity.</a><span class="source">[Probe Reports from the NIH Mol...]</span><div class="brieflinkpop offscreen_noflow"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> Discovery of a small molecule inhibitor of ROMK with unprecedented selectivity.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Denton JS, Weaver CD, Chauder BA, Lindsley CW. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Probe Reports from the NIH Molecular Libraries Program. 2010</em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/19706730" ref="ordinalpos=1&linkpos=2&log$=relatedarticles&logdbfrom=pubmed">High-throughput screening reveals a small-molecule inhibitor of the renal outer medullary potassium channel and Kir7.1.</a><span class="source">[Mol Pharmacol. 2009]</span><div class="brieflinkpop offscreen_noflow">High-throughput screening reveals a small-molecule inhibitor of the renal outer medullary potassium channel and Kir7.1.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Lewis LM, Bhave G, Chauder BA, Banerjee S, Lornsen KA, Redha R, Fallen K, Lindsley CW, Weaver CD, Denton JS. </em><em 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Epub 2009 Aug 25.</em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/29895592" ref="ordinalpos=1&linkpos=3&log$=relatedarticles&logdbfrom=pubmed">Discovery, Characterization, and Effects on Renal Fluid and Electrolyte Excretion of the Kir4.1 Potassium Channel Pore Blocker, VU0134992.</a><span class="source">[Mol Pharmacol. 2018]</span><div class="brieflinkpop offscreen_noflow">Discovery, Characterization, and Effects on Renal Fluid and Electrolyte Excretion of the Kir4.1 Potassium Channel Pore Blocker, VU0134992.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Kharade SV, Kurata H, Bender AM, Blobaum AL, Figueroa EE, Duran A, Kramer M, Days E, Vinson P, Flores D, et al. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Mol Pharmacol. 2018 Aug; 94(2):926-937. Epub 2018 Jun 12.</em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/36439248" ref="ordinalpos=1&linkpos=4&log$=relatedarticles&logdbfrom=pubmed">The effect of high-dietary K(+) (HK) on Kir4.1/Kir5.1 and ROMK in the distal convoluted tubule (DCT) is not affected by gender and Cl(-) content of the diet.</a><span class="source">[Front Physiol. 2022]</span><div class="brieflinkpop offscreen_noflow">The effect of high-dietary K(+) (HK) on Kir4.1/Kir5.1 and ROMK in the distal convoluted tubule (DCT) is not affected by gender and Cl(-) content of the diet.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Meng XX, Zhang H, Meng GL, Jiang SP, Duan XP, Wang WH, Wang MX. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Front Physiol. 2022; 13:1039029. Epub 2022 Nov 9.</em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/21433384" ref="ordinalpos=1&linkpos=5&log$=relatedreviews&logdbfrom=pubmed"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> A potent and selective small molecule Kir2.1 inhibitor.</a><span class="source">[Probe Reports from the NIH Mol...]</span><div class="brieflinkpop offscreen_noflow"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> A potent and selective small molecule Kir2.1 inhibitor.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Wu M, Wang HR, Yu H, Makhina E, Xu J, Dawson ES, Hopkins CR, Lindsley CW, McManus OB, Li M. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Probe Reports from the NIH Molecular Libraries Program. 2010</em></div></div></li></ul><a class="seemore" href="/sites/entrez?db=pubmed&cmd=link&linkname=pubmed_pubmed_reviews&uid=21433378" ref="ordinalpos=1&log$=relatedreviews_seeall&logdbfrom=pubmed">See reviews...</a><a class="seemore" href="/sites/entrez?db=pubmed&cmd=link&linkname=pubmed_pubmed&uid=21433378" ref="ordinalpos=1&log$=relatedarticles_seeall&logdbfrom=pubmed">See all...</a></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Recent Activity</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="recent_activity" id="Shutter"></a></div><div class="portlet_content"><div xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" id="HTDisplay" class=""><div class="action"><a href="javascript:historyDisplayState('ClearHT')">Clear</a><a href="javascript:historyDisplayState('HTOff')" class="HTOn">Turn Off</a><a href="javascript:historyDisplayState('HTOn')" class="HTOff">Turn On</a></div><ul id="activity"><li class="ra_rcd ralinkpopper two_line"><a class="htb ralinkpopperctrl" ref="log$=activity&linkpos=1" href="/portal/utils/pageresolver.fcgi?recordid=67d66c1d2f30673f7bf8fdd0">Discovery of a small molecule inhibitor of ROMK and Kir7.1 - 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