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<meta name="ncbi_db" content="books" /><meta name="ncbi_pdid" content="book-part" /><meta name="ncbi_acc" content="NBK143190" /><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="/NBK143190/" /><meta name="ncbi_pagename" content="Identification of Small Molecule Inhibitors of Wee1 Degradation and Mitotic Entry - 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>Identification of Small Molecule Inhibitors of Wee1 Degradation and Mitotic Entry - 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="Identification of Small Molecule Inhibitors of Wee1 Degradation and Mitotic Entry" /><meta name="citation_publisher" content="National Center for Biotechnology Information (US)" /><meta name="citation_date" content="2013/03/22" /><meta name="citation_author" content="Scott Simanski" /><meta name="citation_author" content="Franck Madoux" /><meta name="citation_author" content="Ronald J. Rahaim" /><meta name="citation_author" content="Peter Chase" /><meta name="citation_author" content="Stephan Schurer" /><meta name="citation_author" content="Michael Cameron" /><meta name="citation_author" content="Becky A. Mercer" /><meta name="citation_author" content="Peter Hodder" /><meta name="citation_author" content="William R. Roush" /><meta name="citation_author" content="Nagi G. Ayad" /><meta name="citation_pmid" content="23762957" /><meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK143190/" /><link rel="schema.DC" href="http://purl.org/DC/elements/1.0/" /><meta name="DC.Title" content="Identification of Small Molecule Inhibitors of Wee1 Degradation and Mitotic Entry" /><meta name="DC.Type" content="Text" /><meta name="DC.Publisher" content="National Center for Biotechnology Information (US)" /><meta name="DC.Contributor" content="Scott Simanski" /><meta name="DC.Contributor" content="Franck Madoux" /><meta name="DC.Contributor" content="Ronald J. Rahaim" /><meta name="DC.Contributor" content="Peter Chase" /><meta name="DC.Contributor" content="Stephan Schurer" /><meta name="DC.Contributor" content="Michael Cameron" /><meta name="DC.Contributor" content="Becky A. Mercer" /><meta name="DC.Contributor" content="Peter Hodder" /><meta name="DC.Contributor" content="William R. Roush" /><meta name="DC.Contributor" content="Nagi G. Ayad" /><meta name="DC.Date" content="2013/03/22" /><meta name="DC.Identifier" content="https://www.ncbi.nlm.nih.gov/books/NBK143190/" /><meta name="description" content="Cell cycle progression and entry into mitosis are regulated by a highly conserved cellular process known as checkpoint signaling. The Wee1 nuclear tyrosine kinase functions in this process by regulating the cdc2/cyclinB protein complex. Specifically, Wee1 mediates inhibitory phosphorylation of cdc2, leading to delayed mitosis and cell cycle arrest in cells with DNA damage so that DNA repair and replication can occur. Wee1 activity is inhibited during mitosis by its phosphorylation and ubiquitination by E3 ligases, and its subsequent degradation by the proteasome. Studies showing that Wee1 expression is reduced in colon carcinoma cells and that Wee1 overexpression can block cell division, suggest that Wee1 may act as a tumor suppressor. Thus, the identification of probes that selectively increase levels of Wee1 may provide useful insights into the roles of Wee1 in cell cycle control and tumor pathogenesis. The Scripps Research Institute Molecular Screening Center (SRIMSC), part of the Molecular Libraries Probe Production Centers Network (MLPCN), utilized a chemical biology approach to uncover kinases controlling Wee1 proteolysis by identifying small molecule inhibitors stabilizing a reporter of Wee1 degradation, K328M-Wee1-luciferase. High-throughput screening followed by medicinal chemistry optimization driven by secondary assays yielded a selective stabilizing probe, ML177, which is 200 times more potent than our previously described probe, ML118. ML177 exhibits a Wee1 stabilization EC50 value of 40 nM. The probe has been tested against several cell-cycle related targets and anti-targets, including Wee1, cyclin B, p21, p27, and ability to induce HeLa (cancer) cell apoptosis. The identified probe exhibited significantly improved selectivity and potency in these assays compared to the previous probe. These results, combined with additional kinase profiling by the assay provider, confirm the value of this probe and its usefulness in biologically relevant, cell-based assays." /><meta name="og:title" content="Identification of Small Molecule Inhibitors of Wee1 Degradation and Mitotic Entry" /><meta name="og:type" content="book" /><meta name="og:description" content="Cell cycle progression and entry into mitosis are regulated by a highly conserved cellular process known as checkpoint signaling. The Wee1 nuclear tyrosine kinase functions in this process by regulating the cdc2/cyclinB protein complex. Specifically, Wee1 mediates inhibitory phosphorylation of cdc2, leading to delayed mitosis and cell cycle arrest in cells with DNA damage so that DNA repair and replication can occur. Wee1 activity is inhibited during mitosis by its phosphorylation and ubiquitination by E3 ligases, and its subsequent degradation by the proteasome. Studies showing that Wee1 expression is reduced in colon carcinoma cells and that Wee1 overexpression can block cell division, suggest that Wee1 may act as a tumor suppressor. Thus, the identification of probes that selectively increase levels of Wee1 may provide useful insights into the roles of Wee1 in cell cycle control and tumor pathogenesis. The Scripps Research Institute Molecular Screening Center (SRIMSC), part of the Molecular Libraries Probe Production Centers Network (MLPCN), utilized a chemical biology approach to uncover kinases controlling Wee1 proteolysis by identifying small molecule inhibitors stabilizing a reporter of Wee1 degradation, K328M-Wee1-luciferase. High-throughput screening followed by medicinal chemistry optimization driven by secondary assays yielded a selective stabilizing probe, ML177, which is 200 times more potent than our previously described probe, ML118. ML177 exhibits a Wee1 stabilization EC50 value of 40 nM. The probe has been tested against several cell-cycle related targets and anti-targets, including Wee1, cyclin B, p21, p27, and ability to induce HeLa (cancer) cell apoptosis. The identified probe exhibited significantly improved selectivity and potency in these assays compared to the previous probe. These results, combined with additional kinase profiling by the assay provider, confirm the value of this probe and its usefulness in biologically relevant, cell-based assays." /><meta name="og:url" content="https://www.ncbi.nlm.nih.gov/books/NBK143190/" /><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/ml177/" /><link rel="canonical" href="https://www.ncbi.nlm.nih.gov/books/NBK143190/" /><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="#__NBK143190_dtls__">Show details</a><div style="display:none" class="ui-widget" id="__NBK143190_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/ml178/" title="Previous page in this title">< Prev</a><a class="active page_link next" href="/books/n/mlprobe/ml176/" 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="_NBK143190_"><span class="title" itemprop="name">Identification of Small Molecule Inhibitors of Wee1 Degradation and Mitotic Entry</span></h1><p class="contrib-group"><span itemprop="author">Scott Simanski</span>, <span itemprop="author">Franck Madoux</span>, <span itemprop="author">Ronald J. Rahaim</span>, <span itemprop="author">Peter Chase</span>, <span itemprop="author">Stephan Schurer</span>, <span itemprop="author">Michael Cameron</span>, <span itemprop="author">Becky A. Mercer</span>, <span itemprop="author">Peter Hodder</span>, <span itemprop="author">William R. Roush</span>, and <span itemprop="author">Nagi G. Ayad</span>.</p><a data-jig="ncbitoggler" href="#__NBK143190_ai__" style="border:0;text-decoration:none">Author Information and Affiliations</a><div style="display:none" class="ui-widget" id="__NBK143190_ai__"><p class="contrib-group"><h4>Authors</h4><span itemprop="author">Scott Simanski</span>,<sup>1</sup><sup>,<a href="#ml177.fn2" class="bk_pop">6</a></sup> <span itemprop="author">Franck Madoux</span>,<sup>2</sup><sup>,<a href="#ml177.fn2" class="bk_pop">6</a></sup> <span itemprop="author">Ronald J. Rahaim</span>,<sup>3</sup> <span itemprop="author">Peter Chase</span>,<sup>2</sup> <span itemprop="author">Stephan Schurer</span>,<sup>4</sup> <span itemprop="author">Michael Cameron</span>,<sup>5</sup> <span itemprop="author">Becky A. Mercer</span>,<sup>2</sup> <span itemprop="author">Peter Hodder</span>,<sup>2</sup> <span itemprop="author">William R. Roush</span>,<sup>3</sup> and <span itemprop="author">Nagi G. Ayad</span><sup>1,4</sup><sup>,5</sup>.</p><h4>Affiliations</h4><div class="affiliation"><sup>1</sup>
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Department of Cancer Biology, Scripps Florida, C130 Scripps Way, Jupiter, Fl, 33458</div><div class="affiliation"><sup>2</sup>
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Lead Identification Division, Translational Research Institute, Scripps Florida, C130 Scripps Way, Jupiter, Fl, 33458</div><div class="affiliation"><sup>3</sup>
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Department of Chemistry, Scripps Florida, C130 Scripps Way, Jupiter, Fl, 33458</div><div class="affiliation"><sup>4</sup>
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Department of Psychiatry and Behavioral Sciences, University of Miami Miller School of Medicine, 1501 NW 10<sup>th</sup> Ave, Miami, FL. 33136</div><div class="affiliation">
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<sup>5</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.imaim.dem@dayan" class="oemail">ude.imaim.dem@dayan</a>;</div></div><p class="small">Received: <span itemprop="datePublished">September 1, 2010</span>; Last Update: <span itemprop="dateModified">March 22, 2013</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>Cell cycle progression and entry into mitosis are regulated by a highly conserved cellular process known as checkpoint signaling. The Wee1 nuclear tyrosine kinase functions in this process by regulating the cdc2/cyclinB protein complex. Specifically, Wee1 mediates inhibitory phosphorylation of cdc2, leading to delayed mitosis and cell cycle arrest in cells with DNA damage so that DNA repair and replication can occur. Wee1 activity is inhibited during mitosis by its phosphorylation and ubiquitination by E3 ligases, and its subsequent degradation by the proteasome. Studies showing that Wee1 expression is reduced in colon carcinoma cells and that Wee1 overexpression can block cell division, suggest that Wee1 may act as a tumor suppressor. Thus, the identification of probes that selectively increase levels of Wee1 may provide useful insights into the roles of Wee1 in cell cycle control and tumor pathogenesis. The Scripps Research Institute Molecular Screening Center (SRIMSC), part of the Molecular Libraries Probe Production Centers Network (MLPCN), utilized a chemical biology approach to uncover kinases controlling Wee1 proteolysis by identifying small molecule inhibitors stabilizing a reporter of Wee1 degradation, K328M-Wee1-luciferase. High-throughput screening followed by medicinal chemistry optimization driven by secondary assays yielded a selective stabilizing probe, <a href="/pcsubstance/?term=ML177[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML177</a>, which is 200 times more potent than our previously described probe, <a href="/pcsubstance/?term=ML118[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML118</a>. <a href="/pcsubstance/?term=ML177[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML177</a> exhibits a Wee1 stabilization EC50 value of 40 nM. The probe has been tested against several cell-cycle related targets and anti-targets, including Wee1, cyclin B, p21, p27, and ability to induce HeLa (cancer) cell apoptosis. The identified probe exhibited significantly improved selectivity and potency in these assays compared to the previous probe. These results, combined with additional kinase profiling by the assay provider, confirm the value of this probe and its usefulness in biologically relevant, cell-based assays.</p></div><div class="h2"></div><p><b>Assigned Assay Grant #:</b> 1R21NS056991-01</p><p><b>Screening Center Name & PI:</b> 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> Nagi Ayad, The Scripps Research Institute (TSRI), University of Miami</p><p><b>Summary Bioassay Identifier (AID):</b>
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<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1807" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1807</a></p><div id="ml177.s1"><h2 id="_ml177_s1_">Probe Structure & Characteristics</h2><div id="ml177.fu1" class="figure bk_fig"><div class="graphic"><img src="/books/NBK143190/bin/ml177fu1.jpg" alt="ML177." /></div><h3><span class="title">ML177</span></h3></div><div id="ml177.tu1" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK143190/table/ml177.tu1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml177.tu1_lrgtbl__"><table><thead><tr><th id="hd_h_ml177.tu1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CID/ML#</th><th id="hd_h_ml177.tu1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Target Name</th><th id="hd_h_ml177.tu1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">EC50 (nM) [SID, AID]</th><th id="hd_h_ml177.tu1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Anti-target</th><th id="hd_h_ml177.tu1_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">EC50 (μM) [SID, AID]</th><th id="hd_h_ml177.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Fold Selective</th><th id="hd_h_ml177.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_ml177.tu1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">CID 45100431/<a href="/pcsubstance/?term=ML177[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML177</a></td><td headers="hd_h_ml177.tu1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Wee1 Degradation Inhibition</td><td headers="hd_h_ml177.tu1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">40nM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/92092801" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 92092801</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463080" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 463080</a>] Active<br /><br />23.3-fold activation, [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/92092801" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 92092801</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463169" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 463169</a>] Active</td><td headers="hd_h_ml177.tu1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Cyclin B</td><td headers="hd_h_ml177.tu1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">2.6-fold activation [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/92092801" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 92092801</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463186" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 463186</a>] Inactive</td><td headers="hd_h_ml177.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">23.3/2.6 = 8.9-fold</td><td headers="hd_h_ml177.tu1_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;"><b>CK1d IC50 Assay</b>: 49 nM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/92092801" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 92092801</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463077" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 463077</a>] Active<br /><b>FLT3 IC50 Assay</b>: 0.305 nM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/92092801" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 92092801</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463076" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 463076</a>] Active<br /><b>p21 Stabilization Assay</b>: 3.41-fold change [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/92092801" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 92092801</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463170" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 463170</a>] Inactive<br /><b>p27 Stabilization Assay</b>: 4.61-fold change [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/92092801" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 92092801</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463171" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 463171</a>] Inactive<br /><b>HeLa Apoptosis Assay</b>: Active [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/92092801" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 92092801</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463177" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 463177</a>]<br /><b>Amyloid Assay</b>: 60% signal; Active [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/92092801" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 92092801</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504929" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504929</a>]<br /><b>GCP (Granule Cell Proliferation) Assay</b>: IC50 = 0.46; Active [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/92092801" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 92092801</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504930" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504930</a>]</td></tr></tbody></table></div></div></div><div id="ml177.s2"><h2 id="_ml177_s2_">1. Recommendations for Scientific Use of the Probe</h2><div id="ml177.s3"><h3>What limitations in current state of the art is the probe addressing?</h3><p>Although selective inhibitors of Wee1 kinase activity have been reported, none have been shown to inhibit Wee1 turnover [<a class="bk_pop" href="#ml177.r1">1</a>–<a class="bk_pop" href="#ml177.r2">2</a>]. For example, inhibition of Wee1 kinase activity by compounds that abrogate the G2/M checkpoint improve the cytotoxic effects of DNA damaging agents on p53-negative cells [<a class="bk_pop" href="#ml177.r3">3</a>–<a class="bk_pop" href="#ml177.r6">6</a>]. However, these compounds are dual Src/Wee1 kinase inhibitors (i.e. they are not inhibitors of Wee1 degradation). In addition, the assay provider screened LOPAC and other libraries that contain Src inhibitors and identified no hits; thus, Src activity is unrelated to inhibition of Wee1 degradation. As a result, the current state of the art lacks potent inhibitors that selectively inhibit degradation of Wee1. <i>Non-selective</i> inhibitors of protein degradation exist but these compounds are not useful for elucidating the specific roles of Wee1 in cell cycle progression, cancer development, and other cellular events. For example, MG132 is not selective as it inhibits general protein degradation via proteasome inhibition, as measured by similar EC50 values (~5 μM) in Wee1 and cyclin B assays.</p><p>One other small molecule has been reported in the literature to stabilize Wee1 in HeLa cells: the dihydropteridinone BI 2536 [<a class="bk_pop" href="#ml177.r2">2</a>]. The mechanism by which BI 2536 leads to Wee1 stabilization is unknown. BI 2536 is an inhibitor of Plk-1, which phosphorylates many proteins associated with cell cycle progression and mitotic entry. A sample of BI 2536 was obtained from commercial sources [Selleck Chemicals, catalog number S1109] and was determined by the assay provider to be inactive in the Wee1 degradation inhibition assay. Thus, dissecting the role of BI 2536 on Wee1 biology relative to its multiple other cellular roles is an exceedingly challenging process. A second commercially available Plk-1 inhibitor, [5-(5,6-dimethoxybenzimidazol-1-yl)-3-(4-methanesulfonyl-benzyloxy)-thiophene-2-carboxamide, purchased from EMD Chemicals, catalog number 528282-5MG] was determined by the assay provider to be inactive in the Wee1 degradation inhibition assay. Moreover, probe compound <a href="https://pubchem.ncbi.nlm.nih.gov/substance/4243143" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 4243143</a>/CID 3237904/powder <a href="https://pubchem.ncbi.nlm.nih.gov/substance/87235992" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 87235992</a> does not inhibit Plk-1, so its mechanism of action for Wee1 stabilization is distinct from that of BI 2536.</p><p>Recent improvements have been made to the field, as demonstrated by a previous <a href="/books/n/mlprobe/ml118/">probe report</a> submitted by the SRIMSC and assay provider Dr. Nagi Ayad. In this prior <a href="/books/n/mlprobe/ml118/">report</a>, the authors describe the identification of a first generation selective inhibitor of Wee1 degradation [<a class="bk_pop" href="#ml177.r7">7</a>]. This prior probe, (CID 3237904/<a href="https://pubchem.ncbi.nlm.nih.gov/substance/4243143" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 4243143</a>/powder <a href="https://pubchem.ncbi.nlm.nih.gov/substance/87235992" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 87235992</a>/<a href="/pcsubstance/?term=ML118[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML118</a>) exhibits a Wee1 EC50 value of ~8 μM and a Cyclin B EC50 of >50 μM, demonstrating a >6.3-fold selectivity towards Wee1 over cyclin B.</p><p>In an effort to improve upon this current art by improving the potency and selectivity for Wee1, the SRIMSC and Dr. Ayad extended their chemistry and biology efforts, resulting in a more potent and selective probe (CID 45100431/<a href="https://pubchem.ncbi.nlm.nih.gov/substance/92092801" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 92092801</a>/<a href="/pcsubstance/?term=ML177[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML177</a>). <b>This new probe exhibits a Wee1 stabilization EC50 of 40nM</b>, and thus is clearly an improvement over the prior art compounds. It exhibits an 8-fold selectivity for Wee1 over cyclin B. In summary, this new probe <a href="/pcsubstance/?term=ML177[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML177</a>/CID 45100431 addresses the limitations of the current art by specifically inhibiting turnover of Wee1 with a <b>greater potency and selectivity</b> than other available chemical tools such as the prior probe and the prior art compound MG132, without acting as a general proteasome inhibitor. <b>Thus, the new probe <a href="/pcsubstance/?term=ML177[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML177</a>/CID 45100431 represents a 200-fold improvement in potency over prior probe <a href="/pcsubstance/?term=ML118[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML118</a>.</b></p></div><div id="ml177.s4"><h3>What will the probe be used for?</h3><p>The probe can be used to elucidate the specific role of Wee1 in cell cycle-related tumorigenesis and other cellular events. The importance of this probe is underscored by recent reports suggesting that Wee1 is shuttled from the nucleus to the cytoplasm, and thus there may be different pools of Wee1 that are located in different intracellular compartments and turned over via distinct mechanisms [<a class="bk_pop" href="#ml177.r8">8</a>]. In this regard, the probe can also be used to elucidate Wee1 turnover in different intracellular compartments. This is not possible with siRNA mediated knockdown of intracellular components that generally inhibit turnover of all proteins since such treatments are often toxic and nonspecific.</p></div><div id="ml177.s5"><h3>Who in the research community will use the probe?</h3><p>The probe can be used by academic researchers studying cell biology, molecular biology, and tumor biology. In addition, this probe was recently applied to studies of central nervous system neurogenesis. Thus it is conceivable that scientists in diverse fields will be able to apply this powerful chemical probe to elucidate the role of Wee1 in disease pathways.</p></div><div id="ml177.s6"><h3>What is the relevant biology to which the probe can be applied?</h3><p>Cell cycle progression and entry into mitosis are regulated by a highly conserved cellular process known as checkpoint signaling [<a class="bk_pop" href="#ml177.r8">8</a>–<a class="bk_pop" href="#ml177.r9">9</a>]. The Wee1 nuclear tyrosine kinase functions in this process by regulating the cdc2/cyclin B protein complex. Specifically, Wee1 mediates inhibitory phosphorylation of cdc2, leading to delayed mitosis and cell cycle arrest in cells with DNA damage so that DNA repair and replication can occur [<a class="bk_pop" href="#ml177.r3">3</a>, <a class="bk_pop" href="#ml177.r8">8</a>–<a class="bk_pop" href="#ml177.r9">9</a>]. Wee1 activity is inhibited during mitosis by its phosphorylation and ubiquitination by E3 ligases, and its subsequent degradation by the proteasome [<a class="bk_pop" href="#ml177.r4">4</a>, <a class="bk_pop" href="#ml177.r6">6</a>]. Studies showing that Wee1 expression is reduced in colon carcinoma cells suggest that Wee1 may act as a tumor suppressor [<a class="bk_pop" href="#ml177.r10">10</a>]. Thus, the identification of probes that selectively increase levels of Wee1 may provide useful insights into the roles of Wee1 in cell cycle control and tumor pathogenesis. The assay provider hypothesizes that compounds that inhibit Wee1 degradation will provide information about the role of Wee1 in mitotic entry and cell cycle progression. As a result, the current probe can be applied to the characterization of Wee1 in cellular events, and may also be useful in identifying proteins that bind to, and/or modify the post-translational state of Wee1.</p></div></div><div id="ml177.s7"><h2 id="_ml177_s7_">2. Materials and Methods</h2><p>Following a successful HTS campaign, which resulted in the identification of a Wee1 degradation inhibitor probe (CID 3237904/<a href="https://pubchem.ncbi.nlm.nih.gov/substance/4243143" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 4243143</a>/powder <a href="https://pubchem.ncbi.nlm.nih.gov/substance/87235992" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 87235992</a>/<a href="/pcsubstance/?term=ML118[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML118</a>), a Center-based initiative was implemented to further characterize the mechanisms of Wee1 stabilization and its role in cell cycle regulation. <b>The approach taken was to uncover kinases controlling Wee1 proteolysis by identifying small molecule kinase inhibitors stabilizing a reporter of Wee1 degradation, K328M-Wee1-luciferase</b>. We screened a proprietary 16,000 compound kinase inhibitor library at Scripps Florida with K328M-Wee1-luciferase expressing cells and identified several structurally related members of a purine scaffold that increased the steady-state levels of K328M-Wee1-luciferase. In order to further examine the specific kinases involved the assay provider tested several leads against fms-related tyrosine kinase 3 (FLT3) and casein kinase 1 delta (CK1d). Furthermore, since Wee1, p21, and p27 are targeted by the same class of ubiquitin ligases (the SCF ubiquitin ligases), the assay provider performed counter-screens against p21 and p27, which served to identify and eliminate non-selective inhibitors of SCF ligases (compounds active against p21 and p27 were not pursued). Counter-screening against cyclin B was also performed to eliminate non-selective inhibitors. Finally, to characterize the mechanism of action of probe candidates, studies were performed to determine the cytotoxicity of probe candidates against neuroblastoma and HeLa cancer cells. </p><div id="ml177.s8"><h3>2.1. Assays</h3><p>The specific assays are listed in <a class="figpopup" href="/books/NBK143190/table/ml177.t1/?report=objectonly" target="object" rid-figpopup="figml177t1" rid-ob="figobml177t1">Table 1</a>.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml177t1"><a href="/books/NBK143190/table/ml177.t1/?report=objectonly" target="object" title="Table 1" class="img_link icnblk_img figpopup" rid-figpopup="figml177t1" rid-ob="figobml177t1"><img class="small-thumb" src="/books/NBK143190/table/ml177.t1/?report=thumb" src-large="/books/NBK143190/table/ml177.t1/?report=previmg" alt="Table 1. Related PubChem Bioassays." /></a><div class="icnblk_cntnt"><h4 id="ml177.t1"><a href="/books/NBK143190/table/ml177.t1/?report=objectonly" target="object" rid-ob="figobml177t1">Table 1</a></h4><p class="float-caption no_bottom_margin">Related PubChem Bioassays. </p></div></div><div id="ml177.s9"><h4>Descriptions and protocols for each assay</h4><div id="ml177.s10"><h5>AID 434972, AID 463080, AID 463169 (Wee1 Degradation Inhibitor Assays)</h5><div id="ml177.s11"><h5>Late stage results from the probe development effort to identify inhibitors of Wee1 degradation: luminescence-based cell-based assay to determine inhibition of Wee1 degradation by kinase inhibitors</h5><p>The purpose of this assay is to identify whether powder samples of compounds identified as possible WEE1 probe candidates can act as inhibitors of WEE1 degradation. The assay uses HeLa cells transfected with a kinase negative mutant of WEE1 (WEE1K328M) fused to a luciferase reporter gene. As designed, compounds that increase WEE1K328M-luciferase stability and/or prevent its degradation will lead to increased well luminescence compared to untreated wells. Specifically, compounds that increase luminescence are considered WEE1 degradation inhibitors. Compounds were tested in quadruplicate at a final nominal concentration of 250 nM.</p><p>HeLa cells were routinely grown in T75 tissue culture flasks in growth media composed of Dulbecco’s Modified Eagle’s Media (DMEM) supplemented with 10% v/v fetal bovine serum (FBS) and 1% pen-strep-neo antibiotic mixture at 37 degrees C in an atmosphere of 10% CO2 and 95% relative humidity (RH). Cells were transiently transfected in flasks by mixing 6 million cells with 29 μg of the WEE1K328M-luciferase plasmid complexed with 87 μL of TransIT-LT1 reagent in a final volume of 24 mL of a 1:1 mix of OptiMEM and 2X supplemented DMEM, according to the manufacturer’s protocol. Cells were then returned to the incubator for 48 hours. Next, the transfected cells were trypsinized and resuspended at a concentration of 850,000 cells per mL in growth media. Then, 50 μL of cells were added in quadruplicate to wells of a 96 well plate. Compounds were made in DMSO at 2,000 times the required concentration and diluted in media 1:1000. 50 μL of each concentration was added to 50 μL of cells in the appropriate wells resulting in a 1:1000 final dilution. The plates were incubated for 20 hours at 37 degrees Celsius (10% CO2, 95% RH). The assay was stopped by adding 100 μL of Brite-lite Reagent to each well and incubating at room temperature for 1 minute. Well luminescence was measured on the Perkin Elmer Envision Plate Reader. K328M-WEE1-luciferase levels (RLUs) were divided by luciferase only levels (RLU) for RJR159, analog, or DMSO. The ratio for each of the compounds or controls was then divided by the ratio for RJR159 to yield %RJR159 levels.</p><p>For dose response assays, a ratio of luminescence signals was then calculated and plotted against the concentration to generate the EC50s. Compounds with an EC50 greater than 1 μM were considered inactive. Compounds with an EC50 equal to or less than 1 μM were considered active.</p><div id="ml177.s12"><h5>List of Reagents</h5><ul><li class="half_rhythm"><div>Dulbecco’s Modified Eagle’s Media (Invitrogen, part 11965-092)</div></li><li class="half_rhythm"><div>Fetal Bovine Serum (Hyclone, part SH 30088.03)</div></li><li class="half_rhythm"><div>Penicillin-Streptomycin-Neomycin antibiotic mix (Invitrogen, part 15640-055)</div></li><li class="half_rhythm"><div>SteadyLite HTS luciferase substrate (PerkinElmer, part 6016989)</div></li><li class="half_rhythm"><div>Reference agonist MG132 (American Peptide, part 81-5-15)</div></li><li class="half_rhythm"><div>TransIT-LT1 (Mirus, part MIR2306)</div></li><li class="half_rhythm"><div>T75 flasks (Corning, part 430641)</div></li><li class="half_rhythm"><div>1536-well plates (Greiner, part 789173)</div></li></ul></div></div></div><div id="ml177.s13"><h5>AID 463076</h5><div id="ml177.s14"><h5>Late stage assay provider results from the probe development effort to identify inhibitors of casein kinase 1 delta (CK1d): radioactivity-based in vitro biochemical kinase assay for inhibitors of fms-related tyrosine kinase 3 (FLT3)</h5><p>The purpose of this assay is to determine whether powder samples of probe candidates can inhibit the activity of FLT3. This in vitro kinase assay was performed by Reaction Biology Corporation (RBC). For FLT3, 20 μM final Abltide was used, sequence: [EAIYAAPFAKKK] in a standard kinase assay with 33P-ATP and purified kinase. Incorporation of 33P-ATP into the peptide was measured after a filter-binding assay. Compounds were assayed in a 10-point 1:3 dilution series starting at a nominal concentration of 50 μM.</p><p>Kinase reactions were performed using the “Hotspot” kinase profiling service of Reaction Biology Corporation.</p><ol><li class="half_rhythm"><div>Prepare indicated substrate in freshly prepared Base Reaction Buffer.</div></li><li class="half_rhythm"><div>Deliver any required cofactors to the substrate solution above.</div></li><li class="half_rhythm"><div>Deliver indicated kinase into the substrate solution and gently mix.</div></li><li class="half_rhythm"><div>Deliver compounds in DMSO into the kinase reaction mixture.</div></li><li class="half_rhythm"><div>Deliver 33P-ATP (specific activity 500 μCi/μl) into the reaction mixture to initiate the reaction.</div></li><li class="half_rhythm"><div>Incubate kinase reaction for 120 min. at room temperature.</div></li></ol><p>A ratio of radioactivity signals was then calculated and plotted against the concentration to generate the IC50s. Compounds with an IC50 greater than 1 μM were considered inactive. Compounds with an IC50 equal to or less than 1 μM were considered active.</p><div id="ml177.s15"><h5>List of Reagents</h5><ul><li class="half_rhythm"><div>Base Reaction buffer (20 mM Hepes (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO)</div></li></ul><p>Additional details can be found at <a href="http://www.reactionbiology.com/" ref="pagearea=body&targetsite=external&targetcat=link&targettype=uri">http://www.reactionbiology.com/</a></p></div></div></div><div id="ml177.s16"><h5>AID 463077</h5><div id="ml177.s17"><h5>Late stage assay provider results from the probe development effort to identify inhibitors of casein kinase 1 delta (CK1d): radioactivity-based in vitro biochemical kinase assay to identify CK1d inhibitors</h5><p>The purpose of this assay is to determine whether powder samples of probe candidates can inhibit the activity of CK1d. This in vitro kinase assay was performed by Reaction Biology Corporation (RBC). For CK1, 20 μM final CK1tide was used, sequence: [KRRRAL[pS]VASLPGL] in a standard kinase assay with 33P-ATP and purified kinase. Incorporation of 33P-ATP into the peptide was measured after a filter-binding assay. Compounds were assayed in a 10-point 1:3 dilution series starting at a nominal concentration of 50 μM.</p><p>Kinase reactions were performed using the “Hotspot” kinase profiling service of Reaction Biology Corporation.</p><ol><li class="half_rhythm"><div>Prepare indicated substrate in freshly prepared Base Reaction Buffer.</div></li><li class="half_rhythm"><div>Deliver any required cofactors to the substrate solution above.</div></li><li class="half_rhythm"><div>Deliver indicated kinase into the substrate solution and gently mix.</div></li><li class="half_rhythm"><div>Deliver compounds in DMSO into the kinase reaction mixture.</div></li><li class="half_rhythm"><div>Deliver 33P-ATP (specific activity 500 μCi/μl) into the reaction mixture to initiate the reaction.</div></li><li class="half_rhythm"><div>Incubate kinase reaction for 120 min. at room temperature.</div></li></ol><p>A ratio of radioactivity signals was then calculated and plotted against the concentration to generate the IC50s. Compounds with an IC50 greater than 1 μM were considered inactive. Compounds with an IC50 equal to or less than 1 μM were considered active.</p><div id="ml177.s18"><h5>List of Reagents</h5><ul><li class="half_rhythm"><div>Base Reaction buffer (20 mM Hepes (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO)</div></li></ul><p>Additional details can be found at <a href="http://www.reactionbiology.com/" ref="pagearea=body&targetsite=external&targetcat=link&targettype=uri">http://www.reactionbiology.com/</a></p></div></div></div><div id="ml177.s19"><h5>AID 434972</h5><div id="ml177.s20"><h5>Late stage assay provider results from the probe development effort to identify inhibitors of Wee1 degradation: luminescence-based cell-based assay to identify inhibitors of Wee1 degradation</h5><p>The purpose of this assay is to determine whether powder samples of compounds identified as possible WEE1 degradation inhibitor probe candidates can induce stabilization of a Wee1 luciferase fusion construct. This assay uses HeLa cells transfected with a plasmid that encodes a Wee1-luciferase fusion protein to monitor Wee1 levels. The Wee1-luciferase complex is rapidly turned over in these cells. As designed, compounds that inhibit Wee1 degradation will increase Wee1-luciferase stability, leading to increased well luminescence. Compounds were tested in quadruplicate at a nominal concentration of 100 nanomolar (nM).</p><div id="ml177.s21"><h5>Protocol Summary</h5><p>HeLa cells were routinely grown in T75 tissue culture flasks in growth media composed of Dulbecco’s Modified Eagle’s Media (DMEM) supplemented with 10% v/v fetal bovine serum (FBS) and 1% pen-strep-neo antibiotic mixture at 37 degrees C in an atmosphere of 10% CO2 and 95% relative humidity (RH). Cells were transiently transfected in flasks by mixing 6 million cells with 29 micrograms of the Wee1-luciferase plasmid complexed with 87 microliters of TransIT-LT1 reagent in a final volume of 24 mL of a 1:1 mix of OptiMEM and 2X supplemented DMEM, according to the manufacturer’s protocol. Cells were then returned to the incubator for 48 hours. Next, the transfected cells were trypsinized and re-suspended at a concentration of 850,000 cells per mL in growth media. Then, 50 microliters of cells were added in quadruplicate to wells of a 96 well plate. Compounds were prepared in DMSO at 2,000 times the required concentration and diluted in media 1:1000. Fifty microliters of compound was added to 50 microliters of cells in the appropriate wells resulting in a 1:1000 final dilution. The plates were incubated for 20 hours at 37°C (10% CO2, 95% RH). The assay was stopped by adding 100 microliters of Brite-lite Reagent to each well and incubating at room temperature for 1 minute. Well luminescence was measured on the Perkin Elmer Envision Plate Reader. Wee1-luciferase levels (RLUs) were divided by luciferase only levels (RLU) for DMSO.</p><p>The fold-change inhibition for each compound was calculated as follows:</p><p>Cells_treated_with_Test_Compound/Cells_treated_with_Vehicle (DMSO).</p><p>The average fold-change of each compound tested was calculated.</p><p>PubChem Activity Outcome and Score:</p><p>Compounds that induced a fold-change less than 10 were considered inactive. Compounds that induced a fold-change equal to or less than 10 were considered active.</p><p>Activity score was ranked by the potency of the compounds, with the most potent compounds assigned the highest activity scores.</p></div></div></div><div id="ml177.s22"><h5>AID 463170</h5><div id="ml177.s23"><h5>Late stage assay provider counter-screen results from the probe development effort to identify inhibitors of Wee1 degradation: luminescence-based cell-based assay to identify inhibitors of p21 (CDKN1A) degradation</h5><p>The purpose of this assay is to determine whether powder samples of compounds identified as possible WEE1 degradation inhibitor probe candidates are non-selective inhibitors of protein turnover by the proteasome. Furthermore, since Wee1 and p21 are targeted by the same class of ubiquitin ligases (the SCF ubiquitin ligases), this p21 assay serves to identify non-selective inhibitors of SCF ligases. This assay uses HeLa cells transfected with a plasmid that encodes a p21-luciferase fusion protein to monitor p21 levels and its turnover. The p21-luciferase complex is rapidly turned over in these cells. As designed, compounds that inhibit p21 degradation will increase p21-luciferase stability, leading to increased well luminescence. Compounds were tested in quadruplicate at a nominal concentration of 100 nanomolar (nM).</p><p>HeLa cells were routinely grown in T75 tissue culture flasks in growth media composed of Dulbecco’s Modified Eagle’s Media (DMEM) supplemented with 10% v/v fetal bovine serum (FBS) and 1% pen-strep-neo antibiotic mixture at 37 degrees C in an atmosphere of 10% CO2 and 95% relative humidity (RH). Cells were transiently transfected in flasks by mixing 6 million cells with 29 micrograms of the p21-luciferase plasmid complexed with 87 microliters of TransIT-LT1 reagent in a final volume of 24 mL of a 1:1 mix of OptiMEM and 2X supplemented DMEM, according to the manufacturer’s protocol. Cells were then returned to the incubator for 48 hours. Next, the transfected cells were trypsinized and re-suspended at a concentration of 850,000 cells per mL in growth media. Then, 50 microliters of cells were added in quadruplicate to wells of a 96 well plate. Compounds were prepared in DMSO at 2,000 times the required concentration and diluted in media 1:1000. Fifty microliters of compound was added to 50 microliters of cells in the appropriate wells resulting in a 1:1000 final dilution. The plates were incubated for 20 hours at 37°C (10% CO2, 95% RH). The assay was stopped by adding 100 microliters of Brite-lite Reagent to each well and incubating at room temperature for 1 minute. Well luminescence was measured on the Perkin Elmer Envision Plate Reader. P21-luciferase levels (RLUs) were divided by luciferase only levels (RLU) for DMSO.</p><p>The fold-change inhibition for each compound was calculated as follows:</p><p>Cells_treated_with_Test_Compound/Cells_treated_with_Vehicle (DMSO).</p><p>The average fold-change of each compound tested was calculated.</p><p>PubChem Activity Outcome and Score:</p><p>Compounds that induced a fold-change less than 10 were considered inactive. Compounds that induced a fold-change equal to or less than 10 were considered active.</p><p>Activity score was ranked by the potency of the compounds, with the most potent compounds assigned the highest activity scores.</p></div></div><div id="ml177.s24"><h5>AID 463171</h5><div id="ml177.s25"><h5>Late stage assay provider counter-screen results from the probe development effort to identify inhibitors of Wee1 degradation: luminescence-based cell-based assay to identify p27 (CDKN1B) degradation</h5><p>The purpose of this assay is to determine whether powder samples of compounds identified as possible WEE1 degradation inhibitor probe candidates are non-selective inhibitors of protein turnover by the proteasome. Furthermore, since Wee1 and p27 are targeted by the same class of ubiquitin ligases (the SCF ubiquitin ligases), this p27 assay serves to identify non-selective inhibitors of SCF ligases. This assay uses HeLa cells transfected with a plasmid that encodes a p27-luciferase fusion protein to monitor p27 levels and its turnover. The p27-luciferase complex is rapidly turned over in these cells. As designed, compounds that inhibit p27 degradation will increase p27-luciferase stability, leading to increased well luminescence. Compounds were tested in quadruplicate at a nominal concentration of 100 nanomolar (nM).</p><div id="ml177.s26"><h5>Protocol Summary</h5><p>HeLa cells were routinely grown in T75 tissue culture flasks in growth media composed of Dulbecco’s Modified Eagle’s Media (DMEM) supplemented with 10% v/v fetal bovine serum (FBS) and 1% pen-strep-neo antibiotic mixture at 37 degrees C in an atmosphere of 10% CO2 and 95% relative humidity (RH). Cells were transiently transfected in flasks by mixing 6 million cells with 29 micrograms of the p27-luciferase plasmid complexed with 87 microliters of TransIT-LT1 reagent in a final volume of 24 mL of a 1:1 mix of OptiMEM and 2X supplemented DMEM, according to the manufacturer’s protocol. Cells were then returned to the incubator for 48 hours. Next, the transfected cells were trypsinized and re-suspended at a concentration of 850,000 cells per mL in growth media. Then, 50 microliters of cells were added in quadruplicate to wells of a 96 well plate. Compounds were prepared in DMSO at 2,000 times the required concentration and diluted in media 1:1000. Fifty microliters of compound was added to 50 microliters of cells in the appropriate wells resulting in a 1:1000 final dilution. The plates were incubated for 20 hours at 37°C (10% CO2, 95% RH). The assay was stopped by adding 100 microliters of Brite-lite Reagent to each well and incubating at room temperature for 1 minute. Well luminescence was measured on the Perkin Elmer Envision Plate Reader. p27-luciferase levels (RLUs) were divided by luciferase only levels (RLU) for DMSO.</p><p>The fold-change inhibition for each compound was calculated as follows:</p><p>Cells_treated_with_Test_Compound/Cells_treated_with_Vehicle (DMSO).</p><p>The percentage of cells in each stage of the cell cycle was determined directly from FACS.</p><p>PubChem Activity Outcome and Score: Compounds that induce >20% of the HeLa cell population to enter the “subG1” phase of the cell cycle at concentrations greater than100nM are considered Active. Compounds that induce <20% of the HeLa cell population to enter the “subG1” phase of the cell cycle at all concentrations are considered inactive. Both compounds were active in this assay.</p><p>Activity score was ranked by the potency of the compounds, with the most potent compounds assigned the highest activity scores.</p></div></div></div><div id="ml177.s27"><h5>AID 463177</h5><div id="ml177.s28"><h5>Late stage assay provider counter-screen results from the probe development effort to identify inhibitors of Wee1 degradation: fluorescence activated cell sorting (FACS)-based cell-based assay to identify inducers of Hela cell apoptosis</h5><p>The purpose of this assay is to determine whether powder samples of compounds identified as possible WEE1 degradation inhibitor probe candidates can induce apoptosis in parental HeLa cells, as measured by the percentage of cells in the subG1 phase of the cell cycle (9). The prediction is that a small-molecule inhibitor of Wee1 degradation would inhibit cell cycle progression because overexpression of non-degradable Wee1 induces a G2-M arrest. In this assay, compounds were tested using a 7-point dose response starting at a nominal high concentration of 5 micromolar, followed by determination of the percentage of cells in subG1, G1, S, and G2 phases of the cell cycle.</p><div id="ml177.s29"><h5>Protocol Summary</h5><p>HeLa cells were routinely grown in T75 tissue culture flasks in growth media composed of Dulbecco’s Modified Eagle’s Media (DMEM) supplemented with 10% v/v fetal bovine serum (FBS) and 1% pen-strep-neo antibiotic mixture at 37 degrees C in an atmosphere of 10% CO2 and 95% relative humidity (RH). HeLa cells were treated for 20 hours with different concentrations of test compounds. Next, compound-treated HeLa cells were harvested by collecting culture media, washing once in phosphate-buffered saline (PBS), and trypsinizing. Cells were centrifuged at 400 g for 5 min at 4°C. Cells were then washed with cold PBS, centrifuged at 400 g for 5 min at 4°C, and rapidly resuspended with 7.5 mL 70% ethanol and incubated at −20°C overnight. Cells were then centrifuged at 400 g for 10 min at 4°C and washed with 10 mL cold PBS. The supernatant was removed and the cell pellet re-suspended in 38 mM sodium citrate containing 69 mM of propidium iodide and 19 mg/mL of Rnase A. Fluorescence-activated cell sorting (FACS) analysis was performed on a BD Biosciences (San Jose, CA) LSR II system and analyzed using Flowjo 8.7.3 software.</p><p>The fold-change inhibition for each compound was calculated as follows:</p><p>Cells_treated_with_Test_Compound/Cells_treated_with_Vehicle (DMSO).</p><p>The percentage of cells in each stage of the cell cycle was determined directly from FACS. Compounds that induce >20% of the HeLa cell population to enter the “subG1” phase of the cell cycle at concentrations greater than100nM are considered Active. Compounds that induce <20% of the HeLa cell population to enter the “subG1” phase of the cell cycle at all concentrations are considered inactive. Both compounds were active in this assay.</p></div></div></div><div id="ml177.s30"><h5>AID 463186</h5><div id="ml177.s31"><h5>Late stage assay provider results from the probe development effort to identify inhibitors of Wee1 degradation: luminescence-based dose response cell-based assay to identify inhibitors of cyclin B degradation</h5><p>The purpose of this assay is to determine whether powder samples of compounds identified as possible WEE1 degradation inhibitor probe candidates are non-selective due to stabilization of a cyclin B luciferase fusion construct. This assay uses HeLa cells transfected with a plasmid that encodes a cyclin B-luciferase fusion protein to monitor cyclin B levels. The cyclin B-luciferase complex is rapidly turned over in these cells. As designed, compounds that inhibit cyclin B degradation will increase cyclin B-luciferase stability, leading to increased well luminescence. Compounds were tested in quadruplicate at a nominal concentration of 100 nanomolar (nM).</p><div id="ml177.s32"><h5>Protocol Summary</h5><p>HeLa cells were routinely grown in T75 tissue culture flasks in growth media composed of Dulbecco’s Modified Eagle’s Media (DMEM) supplemented with 10% v/v fetal bovine serum (FBS) and 1% pen-strep-neo antibiotic mixture at 37 degrees C in an atmosphere of 10% CO2 and 95% relative humidity (RH). Cells were transiently transfected in flasks by mixing 6 million cells with 29 micrograms of the Cyclin B-luciferase plasmid complexed with 87 microliters of TransIT-LT1 reagent in a final volume of 24 mL of a 1:1 mix of OptiMEM and 2X supplemented DMEM, according to the manufacturer’s protocol. Cells were then returned to the incubator for 48 hours. Next, the transfected cells were trypsinized and re-suspended at a concentration of 850,000 cells per mL in growth media. Then, 50 microliters of cells were added in quadruplicate to wells of a 96 well plate. Compounds were prepared in DMSO at 2,000 times the required concentration and diluted in media 1:1000. Fifty microliters of compound was added to 50 microliters of cells in the appropriate wells resulting in a 1:1000 final dilution. The plates were incubated for 20 hours at 37°C (10% CO2, 95% RH). The assay was stopped by adding 100 microliters of Brite-lite Reagent to each well and incubating at room temperature for 1 minute. Well luminescence was measured on the Perkin Elmer Envision Plate Reader. Cyclin B-luciferase levels (RLUs) were divided by luciferase only levels (RLU) for DMSO.</p><p>The fold-change inhibition for each compound was calculated as follows:</p><p>Cells_treated_with_Test_Compound/Cells_treated_with_Vehicle (DMSO).</p><p>The average fold-change of each compound tested was calculated.</p><p>PubChem Activity Outcome and Score: Compounds that induced a fold-change less than 10 were considered inactive. Compounds that induced a fold-change equal to or less than 10 were considered active. Activity score was ranked by the potency of the compounds, with the most potent compounds assigned the highest activity scores.</p></div><div id="ml177.s33"><h5>List of Reagents</h5><ul><li class="half_rhythm"><div>Dulbecco’s Modified Eagle’s Media (Invitrogen, part 11965-092)</div></li><li class="half_rhythm"><div>Fetal Bovine Serum (Hyclone, part SH 30088.03)</div></li><li class="half_rhythm"><div>Penicillin-Streptomycin-Neomycin antibiotic mix (Invitrogen, part 15640-055)</div></li><li class="half_rhythm"><div>Brite-lite reagent (PerkinElmer, part 6016769)</div></li><li class="half_rhythm"><div>TransIT-LT1 (Mirus, part MIR2306)</div></li><li class="half_rhythm"><div>T75 flasks (BD Falcon 353136)</div></li><li class="half_rhythm"><div>96-well plates (Corning part 3917)</div></li></ul></div></div></div></div></div><div id="ml177.s34"><h3>2.2. Probe Chemical Characterization</h3><p>Graphical representations of the stability of the new and prior probe compounds are shown in <a class="figpopup" href="/books/NBK143190/figure/ml177.f1/?report=objectonly" target="object" rid-figpopup="figml177f1" rid-ob="figobml177f1">Figure 1</a>. The new probe (<a href="/pcsubstance/?term=ML177[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML177</a>; CID 45100431) has been tested in numerous cell-based assays, demonstrating the solubility and stability of the probe.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml177f1" co-legend-rid="figlgndml177f1"><a href="/books/NBK143190/figure/ml177.f1/?report=objectonly" target="object" title="Figure 1" class="img_link icnblk_img figpopup" rid-figpopup="figml177f1" rid-ob="figobml177f1"><img class="small-thumb" src="/books/NBK143190/bin/ml177f1.gif" src-large="/books/NBK143190/bin/ml177f1.jpg" alt="Figure 1. Stability of Probes ML177 and ML118." /></a><div class="icnblk_cntnt" id="figlgndml177f1"><h4 id="ml177.f1"><a href="/books/NBK143190/figure/ml177.f1/?report=objectonly" target="object" rid-ob="figobml177f1">Figure 1</a></h4><p class="float-caption no_bottom_margin">Stability of Probes ML177 and ML118. </p></div></div><p>The results summarized in <a class="figpopup" href="/books/NBK143190/table/ml177.t2/?report=objectonly" target="object" rid-figpopup="figml177t2" rid-ob="figobml177t2">Table 2</a>, indicating that <a href="/pcsubstance/?term=ML177[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML177</a> is unstable in PBS solution is very surprising and highly suspect, given that the SRIMSC medicinal chemistry team have observed absolutely no issues of chemical instability of this compound. Indeed, in one test, a 5 mg sample of <a href="/pcsubstance/?term=ML177[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML177</a> was dissolved in 0.5 mL of d<sub>6</sub>-DMSO and monitored by 1H NMR spectroscopy. After one week at ambient temperature, the 1H NMR spectrum of this sample was identical to that of the freshly prepared sample at the beginning of the experiment. This experiment verifies the chemical stability of the probe. However, <a href="/pcsubstance/?term=ML177[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML177</a> has poor solubility in PBS buffer and aqueous solution. Indeed, when a 10μM solution of <a href="/pcsubstance/?term=ML177[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML177</a> in DMSO was titrated with water, <a href="/pcsubstance/?term=ML177[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML177</a> began to precipitate from solution after 5% water had been added. Therefore, the “apparent” instability of <a href="/pcsubstance/?term=ML177[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML177</a> suggested by the graph in <a class="figpopup" href="/books/NBK143190/figure/ml177.f1/?report=objectonly" target="object" rid-figpopup="figml177f1" rid-ob="figobml177f1">Figure 1</a>, is attributed to solubility problems of the probe.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml177t2"><a href="/books/NBK143190/table/ml177.t2/?report=objectonly" target="object" title="Table 2" class="img_link icnblk_img figpopup" rid-figpopup="figml177t2" rid-ob="figobml177t2"><img class="small-thumb" src="/books/NBK143190/table/ml177.t2/?report=thumb" src-large="/books/NBK143190/table/ml177.t2/?report=previmg" alt="Table 2. Summary of Results." /></a><div class="icnblk_cntnt"><h4 id="ml177.t2"><a href="/books/NBK143190/table/ml177.t2/?report=objectonly" target="object" rid-ob="figobml177t2">Table 2</a></h4><p class="float-caption no_bottom_margin">Summary of Results. </p></div></div><p>Preliminary efforts have been made to improve the solubility of <a href="/pcsubstance/?term=ML177[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML177</a> by generation of salts. However, the several salts that we have attempted to generate (by concentrating a solution of <a href="/pcsubstance/?term=ML177[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML177</a> in the presence of the indicated acids) are even less soluble than the parent <a href="/pcsubstance/?term=ML177[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML177</a>. Therefore, solubility of the probe is an issue that will be addressed in further optimization of <a href="/pcsubstance/?term=ML177[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML177</a> for in vivo applications.</p><div id="ml177.tu2" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK143190/table/ml177.tu2/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml177.tu2_lrgtbl__"><table><thead><tr><th id="hd_h_ml177.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="/pcsubstance/?term=ML177[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML177</a> Salts</th><th id="hd_h_ml177.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><i>Solubility (μM)</i></th></tr></thead><tbody><tr><td headers="hd_h_ml177.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="/pcsubstance/?term=ML177[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML177</a>·AcOH</td><td headers="hd_h_ml177.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0.6</td></tr><tr><td headers="hd_h_ml177.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="/pcsubstance/?term=ML177[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML177</a>·HCl</td><td headers="hd_h_ml177.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">1.3</td></tr><tr><td headers="hd_h_ml177.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="/pcsubstance/?term=ML177[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML177</a>·TFA</td><td headers="hd_h_ml177.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0.7</td></tr><tr><td headers="hd_h_ml177.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="/pcsubstance/?term=ML177[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML177</a>·Sulfuric Acid</td><td headers="hd_h_ml177.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">5.6</td></tr></tbody></table></div></div></div><div id="ml177.s35"><h3>2.3. Probe Preparation</h3><div id="ml177.f2" class="figure bk_fig"><div class="graphic"><img src="/books/NBK143190/bin/ml177f2.jpg" alt="Figure 2. Synthesis of Wee1 Degradation Inhibitor Probe (ML177/CID 45100431)." /></div><h3><span class="label">Figure 2</span><span class="title">Synthesis of Wee1 Degradation Inhibitor Probe (ML177/CID 45100431)</span></h3></div><p><b>Methods</b>: All reactions were magnetically stirred, and monitored by thin-layer chromatography using 0.25-mm precoated silica gel Kieselgel 60 F<sub>254</sub> plates, unless otherwise noted. Visualization of all TLC plates was performed by UV. Product purifications were performed by silica gel flash chromatography (Kieselgel 60; 230–400 mesh) packed in glass columns and eluted with CH<sub>2</sub>Cl<sub>2</sub>/MeOH, or with EtOAc/MeOH buffered with 1% NH<sub>4</sub>OH.</p><p><b>Materials</b>: Commercial grade reagents and solvents were used without further purification unless otherwise specified. DMF was dried by passing through a column of activated molecular sieves [<a class="bk_pop" href="#ml177.r11">11</a>]. <i>N,N</i>-Diisopropylethylamine was distilled under argon from calcium hydride.</p><p><b>Instrumentation</b>: <sup>1</sup>H and <sup>13</sup>C NMR spectra were recorded on a commercial NMR spectrometer (400 MHz for <sup>1</sup>H, respectively, and 100 MHz for <sup>13</sup>C, respectively), with chemical shifts reported relative to the residue peaks of solvent DMSO (δ 2.50 for <sup>1</sup>H and 39.5 for <sup>13</sup>C). IR spectra were obtained on a FT-IR spectrophotometer and are given in cm<sup>−1</sup>. Low resolution mass spectra (LRMS) and high resolution mass spectra were obtained on commercial instruments.</p><p><b>Synthesis of RJR-159 (probe <a href="/pcsubstance/?term=ML177[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML177</a>)</b>[<a class="bk_pop" href="#ml177.r15">15</a>]: A 2–5 mL Biotage microwave vial was charged with 2,6-dichloropurine-9-(3-thiophene)-purine (<b>2</b>) (0.136 g, 0.5 mmol), 4-nitro-2-(aminomethyl)-benzimidazole (<b>5</b>) (0.105 g, 0.55 mmol), freshly distilled <i>N,N-</i>diisopropylethylamine (0.435 mL, 2.5 mmol), and isopropanol (2 mL). The vial was sealed with a microwave cap, and the reaction mixture was heated to 90 °C for 30 minutes in a Biotage microwave unit. The cooled vial was then placed on a rotary evaporator and the reaction mixture was concentrated. Morpholine (2.5 mL) was added to the vial containing the concentrated crude mixture. The vial was resealed and the reaction was heated to 130 °C for 30 minutes in the microwave reactor. The cooled reaction mixture was concentrated in vacuo, and the crude product was subjected to flash chromatographic purification (98/2 (500mL) then 95/5 (500mL) [CH<sub>2</sub>Cl<sub>2</sub>/MeOH]) to give 0.156 g (65%) of RJR-159 as a tan-orange powder; m.p. = decomposed at 290 °C; <sup>1</sup>H NMR (400 MHz, d6-DMSO) δ 13.1 (s, 1H), 8.39 (s, 1H), 8.15 (bs, 1H), 8.09 (d, <i>J</i> = 8.05 Hz, 1H), 8.07 (dd, <i>J</i> = 1.51 & 3.03 Hz, 1H), 8.03 (d, <i>J</i> = 7.32 Hz, 1H), 7.78 (dd, <i>J</i> = 1.51 & 5.30 Hz, 1H), 7.73 (dd, <i>J</i> = 3.28 & 5.30 Hz, 1H), 7.36 (t, <i>J</i> = 8.08 Hz, 1H), 4.95 (bs, 2H), 3.60 (bs, 4H), 3.53 (bs, 4H); <sup>13</sup>C NMR (100 MHz, d<sub>6</sub>-DMSO) δ 158.6, 156.8, 154.0, 153.9, 150.0, 136.4, 133.6, 126.7, 121.0, 120.8, 118.1, 113.6, 112.8, 65.8, 48.5, 44.4; IR (neat) 1605, 1484, 1411, 1325, 1268, 1238, 1115, 1014, 901, 864, 783, 733 cm<sup>-1</sup>; LRMS-ESI (M+1) <i>m/z</i>: 478, 398, 279, 201, 171, 139.</p></div></div><div id="ml177.s36"><h2 id="_ml177_s36_">3. Results</h2><div id="ml177.s37"><h3>3.1. Dose Response Curves for Probe</h3><p>The new probe RJR-159 (<a href="/pcsubstance/?term=ML177[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML177</a>/CID 45100431) is a more potent inhibitor of GCP (Granule cell
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progenitor) cell proliferation than FKL135. <sup>3</sup>H-thymidine incorporation of GCPs treated
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with DMSO, FKL-135 (analog), or probe RJR-159 was measured after 24 hours. Well-established methods
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for purifying GCPs were developed by the Hatten laboratory [<a class="bk_pop" href="#ml177.r16">16</a>]. In brief, to purify immature granule cells, cerebella from
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postnatal mice are isolated and triturated, and a mixed cell suspension containing GCPs is applied
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to a Percoll gradient (35%/65%), followed by three sequential panning steps to remove glia and fibroblasts from meninges and enrich GCPs. By electron microscopy (EM) and immunocytochemistry, the final suspension is 99% pure GCPs. 40 million GCPs or more are purified from a single litter of C57Bl6J mice. <sup>3</sup>H-thymidine uptake has been extensively utilized to measure GCP proliferation [<a class="bk_pop" href="#ml177.r16">16</a>]. The extent of <sup>3</sup>H-thymidine incorporation was normalized to DMSO control and plotted. Moreover, these cell-based studies support the solubility and stability of the probe. These assays are available in PubChem as <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504930" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504930</a>.</p><div id="ml177.f3" class="figure bk_fig"><div class="graphic"><img src="/books/NBK143190/bin/ml177f3.jpg" alt="Figure 3. Dose Response Curve." /></div><h3><span class="label">Figure 3</span><span class="title">Dose Response Curve</span></h3></div></div><div id="ml177.s38"><h3>3.2. Cellular Activity</h3><p>The results of a Cell Titer Glo-based cytotoxicity assay of the probe RJR-159 (<a href="/pcsubstance/?term=ML177[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML177</a>/CID 45100431) performed using neuroblastoma cell lines are provided in <a class="figpopup" href="/books/NBK143190/figure/ml177.f4/?report=objectonly" target="object" rid-figpopup="figml177f4" rid-ob="figobml177f4">Figure 4</a>. The cell toxicity observed in this assay is presumed to be related to the ability of probe RJR-159 (<a href="/pcsubstance/?term=ML177[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML177</a>/CID 45100431) to inhibit kinases associated with cell-cycle progression.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml177f4" co-legend-rid="figlgndml177f4"><a href="/books/NBK143190/figure/ml177.f4/?report=objectonly" target="object" title="Figure 4" class="img_link icnblk_img figpopup" rid-figpopup="figml177f4" rid-ob="figobml177f4"><img class="small-thumb" src="/books/NBK143190/bin/ml177f4.gif" src-large="/books/NBK143190/bin/ml177f4.jpg" alt="Figure 4. Results of a Cell Titer Glo-based cytotoxicity assay." /></a><div class="icnblk_cntnt" id="figlgndml177f4"><h4 id="ml177.f4"><a href="/books/NBK143190/figure/ml177.f4/?report=objectonly" target="object" rid-ob="figobml177f4">Figure 4</a></h4><p class="float-caption no_bottom_margin">Results of a Cell Titer Glo-based cytotoxicity assay. </p></div></div></div><div id="ml177.s39"><h3>3.3. Profiling Assays</h3><p>The probe has been tested against several cell-cycle related targets and anti-targets, including Wee1, cyclin B, p21, p27, and ability to induce HeLa (cancer) cell apoptosis. The objective is to identify a compound that could stabilize (inhibit the degradation of) Wee1, without acting as a general protein stabilizer. For this reason, the assay provider selected cyclin B stabilization as the counter-screen target. These studies employed a luciferase-fusion protein to monitor protein degradation. Further studies were performed using p21 (CDKN1A) and p27 (CDKN1B) fusion constructs. These latter assays were performed by the assay provider. The identified probe exhibited significantly improved selectivity and potency in these AIDs, compared to the previous probe. These results, combined with additional kinase profiling by the assay provider, confirm the value of this probe and its usefulness in biologically-relevant, cell-based assays. These results are available in PubChem as <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504930" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504930</a> and <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504930" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504930</a> (GCP). The data for kinase profiling of probes against the casein kinases (Ck1delta, epsilon) and FLT3 have been reported in PubChem <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463077" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 463077</a> and <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463076" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 463076</a>, respectively.</p><p>The probe has not been tested in other PubChem assays, as it is a newly synthesized molecule. The probe and its analog FKL-135 have been profiled against a set of 296 kinases (performed by Reaction Corporation Biology). These data, summarized in <a class="figpopup" href="/books/NBK143190/figure/ml177.f5/?report=objectonly" target="object" rid-figpopup="figml177f5" rid-ob="figobml177f5">Figure 5</a> as an activity heat map for assays performed in a 1 μM inhibitor, demonstrate that the probe, CID 45100431/<a href="/pcsubstance/?term=ML177[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML177</a> (e.g., RJR-159), is a highly selective kinase inhibitor—much more so than the initial hit FLK-135.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml177f5" co-legend-rid="figlgndml177f5"><a href="/books/NBK143190/figure/ml177.f5/?report=objectonly" target="object" title="Figure 5" class="img_link icnblk_img figpopup" rid-figpopup="figml177f5" rid-ob="figobml177f5"><img class="small-thumb" src="/books/NBK143190/bin/ml177f5.gif" src-large="/books/NBK143190/bin/ml177f5.jpg" alt="Figure 5. Summary of profile between FKL-135 and a set of 296 kinases." /></a><div class="icnblk_cntnt" id="figlgndml177f5"><h4 id="ml177.f5"><a href="/books/NBK143190/figure/ml177.f5/?report=objectonly" target="object" rid-ob="figobml177f5">Figure 5</a></h4><p class="float-caption no_bottom_margin">Summary of profile between FKL-135 and a set of 296 kinases. </p></div></div></div></div><div id="ml177.s40"><h2 id="_ml177_s40_">4. Discussion</h2><div id="ml177.s41"><h3>4.1. Comparison to Existing Art and How the New Probe is an Improvement</h3><p>As summarized in <a class="figpopup" href="/books/NBK143190/table/ml177.t3/?report=objectonly" target="object" rid-figpopup="figml177t3" rid-ob="figobml177t3">Table 3</a>, the current probe <a href="/pcsubstance/?term=ML177[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML177</a>/CID 45100431 is significantly more potent and selective than the previous probe and the prior art compound MG132.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml177t3"><a href="/books/NBK143190/table/ml177.t3/?report=objectonly" target="object" title="Table 3" class="img_link icnblk_img figpopup" rid-figpopup="figml177t3" rid-ob="figobml177t3"><img class="small-thumb" src="/books/NBK143190/table/ml177.t3/?report=thumb" src-large="/books/NBK143190/table/ml177.t3/?report=previmg" alt="Table 3. Wee1 Degradation Inhibitor Probes and Prior Art." /></a><div class="icnblk_cntnt"><h4 id="ml177.t3"><a href="/books/NBK143190/table/ml177.t3/?report=objectonly" target="object" rid-ob="figobml177t3">Table 3</a></h4><p class="float-caption no_bottom_margin">Wee1 Degradation Inhibitor Probes and Prior Art. </p></div></div></div></div><div id="ml177.s42"><h2 id="_ml177_s42_">5. References</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="ml177.r1">Hashimoto O, et al. Cell cycle regulation by the Wee1 inhibitor PD0166285, pyrido [2,3-d] pyimidine, in the B16 mouse melanoma cell line. <span><span class="ref-journal">BMC Cancer. </span>2006;<span class="ref-vol">6</span>:292.</span> [<a href="/pmc/articles/PMC1770931/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC1770931</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/17177986" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17177986</span></a>]</div></dd><dt>2.</dt><dd><div class="bk_ref" id="ml177.r2">Steegmaier M, et al. BI 2536, a potent and selective inhibitor of polo-like kinase 1, inhibits tumor growth in vivo. <span><span class="ref-journal">Curr Biol. </span>2007;<span class="ref-vol">17</span>(4):316–22.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/17291758" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17291758</span></a>]</div></dd><dt>3.</dt><dd><div class="bk_ref" id="ml177.r3">Coleman TR, Dunphy WG. Cdc2 regulatory factors. <span><span class="ref-journal">Curr Opin Cell Biol. </span>1994;<span class="ref-vol">6</span>(6):877–82.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/7880537" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 7880537</span></a>]</div></dd><dt>4.</dt><dd><div class="bk_ref" id="ml177.r4">Kellogg DR. Wee1-dependent mechanisms required for coordination of cell growth and cell division. <span><span class="ref-journal">J Cell Sci. </span>2003;<span class="ref-vol">116</span>(Pt 24):4883–90.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/14625382" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 14625382</span></a>]</div></dd><dt>5.</dt><dd><div class="bk_ref" id="ml177.r5">Smith A, et al. Redundant ubiquitin ligase activities regulate wee1 degradation and mitotic entry. <span><span class="ref-journal">Cell Cycle. </span>2007;<span class="ref-vol">6</span>(22):2795–9.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/18032919" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18032919</span></a>]</div></dd><dt>6.</dt><dd><div class="bk_ref" id="ml177.r6">Watanabe N, et al. M-phase kinases induce phospho-dependent ubiquitination of somatic Wee1 by SCFbeta-TrCP. <span><span class="ref-journal">Proc Natl Acad Sci U S A. </span>2004;<span class="ref-vol">101</span>(13):4419–24.</span> [<a href="/pmc/articles/PMC384762/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC384762</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/15070733" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 15070733</span></a>]</div></dd><dt>7.</dt><dd><div class="bk_ref" id="ml177.r7">Madoux F, et al. An Ultra-High Throughput Cell-Based Screen for Wee1 Degradation Inhibitors. <span><span class="ref-journal">J Biomol Screen. </span>2010</span> [<a href="/pmc/articles/PMC3082437/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3082437</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/20660794" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 20660794</span></a>]</div></dd><dt>8.</dt><dd><div class="bk_ref" id="ml177.r8">Lee MH, Yang HY. Negative regulators of cyclin-dependent kinases and their roles in cancers. <span><span class="ref-journal">Cell Mol Life Sci. </span>2001;<span class="ref-vol">58</span>(12–13):1907–22.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/11766887" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 11766887</span></a>]</div></dd><dt>9.</dt><dd><div class="bk_ref" id="ml177.r9">Heald R, McLoughlin M, McKeon F. Human wee1 maintains mitotic timing by protecting the nucleus from cytoplasmically activated Cdc2 kinase. <span><span class="ref-journal">Cell. </span>1993;<span class="ref-vol">74</span>(3):463–74.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/8348613" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 8348613</span></a>]</div></dd><dt>10.</dt><dd><div class="bk_ref" id="ml177.r10">Backert S, et al. Differential gene expression in colon carcinoma cells and tissues detected with a cDNA array. <span><span class="ref-journal">Int J Cancer. </span>1999;<span class="ref-vol">82</span>(6):868–74.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/10446455" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 10446455</span></a>]</div></dd><dt>11.</dt><dd><div class="bk_ref" id="ml177.r11">Pangborn ABG, MA, Grubbs RH, Rosen RK, Timmers FJ. Safe and Convenient Procedure for Solvent Purification. <span><span class="ref-journal">Organometallics. </span>1996;<span class="ref-vol">15</span>(5):1518–1520.</span></div></dd><dt>12.</dt><dd><div class="bk_ref" id="ml177.r12">Behrend L, et al. IC261, a specific inhibitor of the protein kinases casein kinase 1-delta and -epsilon, triggers the mitotic checkpoint and induces p53-dependent postmitotic effects. <span><span class="ref-journal">Oncogene. </span>2000;<span class="ref-vol">19</span>(47):5303–13.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/11103931" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 11103931</span></a>]</div></dd><dt>13.</dt><dd><div class="bk_ref" id="ml177.r13">Rena G, et al. D4476, a cell-permeant inhibitor of CK1, suppresses the site-specific phosphorylation and nuclear exclusion of FOXO1a. <span><span class="ref-journal">EMBO Rep. </span>2004;<span class="ref-vol">5</span>(1):60–5.</span> [<a href="/pmc/articles/PMC1298959/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC1298959</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/14710188" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 14710188</span></a>]</div></dd><dt>14.</dt><dd><div class="bk_ref" id="ml177.r14">Walton KM, et al. Selective inhibition of casein kinase 1 epsilon minimally alters circadian clock period. <span><span class="ref-journal">J Pharmacol Exp Ther. </span>2009;<span class="ref-vol">330</span>(2):430–9.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/19458106" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19458106</span></a>]</div></dd><dt>15.</dt><dd><div class="bk_ref" id="ml177.r15">Ding S, Gray NS, Ding Q, Schultz PG. A concise and traceless linker strategy toward combinatorial libraries of 2,6,9-substituted purines. <span><span class="ref-journal">J Org
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Chem. </span>2001;<span class="ref-vol">66</span>(24):8273–6.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/11722241" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 11722241</span></a>]</div></dd><dt>16.</dt><dd><div class="bk_ref" id="ml177.r16">Hatten ME. Neuronal regulation of astroglial morphology
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and proliferation in vitro. <span><span class="ref-journal">J Cell
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Biol. </span>1985;<span class="ref-vol">100</span>(2):384–96.</span> [<a href="/pmc/articles/PMC2113456/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2113456</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/3881455" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 3881455</span></a>]</div></dd></dl></div><div><dl class="temp-labeled-list small"><dt>6</dt><dd><div id="ml177.fn2"><p class="no_top_margin">Equal contribution.</p></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/NBK143190/?report=reader">PubReader</a></li><li><a href="/books/NBK143190/?report=printable">Print View</a></li><li><a data-jig="ncbidialog" href="#_ncbi_dlg_citbx_NBK143190" data-jigconfig="width:400,modal:true">Cite this Page</a><div id="_ncbi_dlg_citbx_NBK143190" style="display:none" title="Cite this Page"><div class="bk_tt">Simanski S, Madoux F, Rahaim RJ, et al. Identification of Small Molecule Inhibitors of Wee1 Degradation and Mitotic Entry. 2010 Sep 1 [Updated 2013 Mar 22]. In: Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-. <span class="bk_cite_avail"></span></div></div></li></ul></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>In this Page</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="page-toc" id="Shutter"></a></div><div class="portlet_content"><ul xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="simple-list"><li><a href="#ml177.s1" ref="log$=inpage&link_id=inpage">Probe Structure & Characteristics</a></li><li><a href="#ml177.s2" ref="log$=inpage&link_id=inpage">Recommendations for Scientific Use of the Probe</a></li><li><a href="#ml177.s7" ref="log$=inpage&link_id=inpage">Materials and Methods</a></li><li><a href="#ml177.s36" ref="log$=inpage&link_id=inpage">Results</a></li><li><a href="#ml177.s40" ref="log$=inpage&link_id=inpage">Discussion</a></li><li><a href="#ml177.s42" ref="log$=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=3034134" ref="log$=recordlinks">PMC</a><div class="brieflinkpop offscreen_noflow">PubMed Central citations</div></li><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pcassay&DbFrom=books&Cmd=Link&LinkName=books_pcassay_probe&IdsFromResult=3034134" ref="log$=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=3034134" ref="log$=recordlinks">PubChem Substance</a><div class="brieflinkpop offscreen_noflow">Related PubChem Substances</div></li><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pubmed&DbFrom=books&Cmd=Link&LinkName=books_pubmed_refs&IdsFromResult=3034134" ref="log$=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/21735595" ref="ordinalpos=1&linkpos=1&log$=relatedreviews&logdbfrom=pubmed"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> Small Molecule Inhibitors of Wee1 Degradation and Mitotic Entry.</a><span class="source">[Probe Reports from the NIH Mol...]</span><div class="brieflinkpop offscreen_noflow"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> Small Molecule Inhibitors of Wee1 Degradation and Mitotic Entry.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Madoux F, Mishra J, Mercer BA, Ayad N, Roush W, Hodder P, Rosen HR. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Probe Reports from the NIH Molecular Libraries Program. 2010</em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/20660794" ref="ordinalpos=1&linkpos=2&log$=relatedarticles&logdbfrom=pubmed">An ultra-high throughput cell-based screen for wee1 degradation inhibitors.</a><span class="source">[J Biomol Screen. 2010]</span><div class="brieflinkpop offscreen_noflow">An ultra-high throughput cell-based screen for wee1 degradation inhibitors.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Madoux F, Simanski S, Chase P, Mishra JK, Roush WR, Ayad NG, Hodder P. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">J Biomol Screen. 2010 Sep; 15(8):907-17. Epub 2010 Jul 26.</em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/24817118" ref="ordinalpos=1&linkpos=3&log$=relatedarticles&logdbfrom=pubmed">Casein kinase 1δ-dependent Wee1 protein degradation.</a><span class="source">[J Biol Chem. 2014]</span><div class="brieflinkpop offscreen_noflow">Casein kinase 1δ-dependent Wee1 protein degradation.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Penas C, Ramachandran V, Simanski S, Lee C, Madoux F, Rahaim RJ, Chauhan R, Barnaby O, Schurer S, Hodder P, et al. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">J Biol Chem. 2014 Jul 4; 289(27):18893-903. Epub 2014 May 9.</em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/8515817" ref="ordinalpos=1&linkpos=4&log$=relatedarticles&logdbfrom=pubmed">Phosphorylation and inactivation of the mitotic inhibitor Wee1 by the nim1/cdr1 kinase.</a><span class="source">[Nature. 1993]</span><div class="brieflinkpop offscreen_noflow">Phosphorylation and inactivation of the mitotic inhibitor Wee1 by the nim1/cdr1 kinase.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Parker LL, Walter SA, Young PG, Piwnica-Worms H. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Nature. 1993 Jun 24; 363(6431):736-8. </em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/35251998" 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> Targeting the DNA Damage Response for Cancer Therapy by Inhibiting the Kinase Wee1.</a><span class="source">[Front Oncol. 2022]</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> Targeting the DNA Damage Response for Cancer Therapy by Inhibiting the Kinase Wee1.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Bukhari AB, Chan GK, Gamper AM. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Front Oncol. 2022; 12:828684. 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