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<meta name="ncbi_db" content="books" /><meta name="ncbi_pdid" content="book-part" /><meta name="ncbi_acc" content="NBK153503" /><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="/NBK153503/" /><meta name="ncbi_pagename" content="Identification of selective inhibitors of cdc2-like kinases 1 and 4 (Clk1, Clk4) - 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 selective inhibitors of cdc2-like kinases 1 and 4 (Clk1, Clk4) - 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 selective inhibitors of cdc2-like kinases 1 and 4 (Clk1, Clk4)" /><meta name="citation_publisher" content="National Center for Biotechnology Information (US)" /><meta name="citation_date" content="2013/05/08" /><meta name="citation_author" content="Thomas C. Coombs" /><meta name="citation_author" content="Cordelle Tanega" /><meta name="citation_author" content="Min Shen" /><meta name="citation_author" content="Benjamin Neuenswander" /><meta name="citation_author" content="Patrick Porubsky" /><meta name="citation_author" content="Jenna L. Wang" /><meta name="citation_author" content="Tom Misteli" /><meta name="citation_author" content="Douglas S. Auld" /><meta name="citation_author" content="Frank Schoenen" /><meta name="citation_author" content="Craig J. Thomas" /><meta name="citation_author" content="Jeffrey Aubé" /><meta name="citation_pmid" content="23926621" /><meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK153503/" /><link rel="schema.DC" href="http://purl.org/DC/elements/1.0/" /><meta name="DC.Title" content="Identification of selective inhibitors of cdc2-like kinases 1 and 4 (Clk1, Clk4)" /><meta name="DC.Type" content="Text" /><meta name="DC.Publisher" content="National Center for Biotechnology Information (US)" /><meta name="DC.Contributor" content="Thomas C. Coombs" /><meta name="DC.Contributor" content="Cordelle Tanega" /><meta name="DC.Contributor" content="Min Shen" /><meta name="DC.Contributor" content="Benjamin Neuenswander" /><meta name="DC.Contributor" content="Patrick Porubsky" /><meta name="DC.Contributor" content="Jenna L. Wang" /><meta name="DC.Contributor" content="Tom Misteli" /><meta name="DC.Contributor" content="Douglas S. Auld" /><meta name="DC.Contributor" content="Frank Schoenen" /><meta name="DC.Contributor" content="Craig J. Thomas" /><meta name="DC.Contributor" content="Jeffrey Aubé" /><meta name="DC.Date" content="2013/05/08" /><meta name="DC.Identifier" content="https://www.ncbi.nlm.nih.gov/books/NBK153503/" /><meta name="description" content="The cdc2-like (Clk) kinases are critical elements of several important regulatory pathways involving pre-mRNA splicing. These kinases act by phosphorylating the serine- and arginine-rich (SR) proteins, which direct the early events of spliceosome assembly necessary for proper mRNA maturation. Mutations causing splicing defects have been linked to a number of disease processes, making the search for small-molecule regulators of enzymes associated with spliceosome function important for both the further study and potential treatment of these disorders. The dual-specificity tyrosine-regulated (Dyrk) kinases are associated with numerous cellular processes and disorders. In particular, overexpression of Dyrk1A may contribute to Down syndrome neurodevelopmental abnormalities, and Dyrk1B/Mirk has been implicated in the cellular repair of cancer cells impaired by chemotherapy. Developing inhibitors of the Dyrk kinases would likely have important biochemical and therapeutic uses. Continuing in the search for selective inhibitors of Clk and Dyrk kinases, we now report a series of amino-substituted pyrimidines with excellent potencies against several Clk and Dyrk isoforms. The probe compound and its analogs have been subjected to Clk and Dyrk isoform selectivity profiling by Ambit Biosciences and three compounds have also been sent for KINOMEscan analysis to assess their selectivity in the context of the greater kinome. The reported compounds are low-nanomolar Clk and Dyrk inhibitors, with the most potent compound having IC50s <10 nM against Clk1, Clk4, Dyrk1A and Dyrk1B. The probe molecule (ML315) is a substituted aminopyrimidine and structurally distinct from previous quinazoline probes. ML315 should provide biochemical investigators with a new tool to be used in parallel with the quinazoline probes in efforts to determine which Clk and/or Dyrk isoforms are associated with particular biochemical events. Until isoform-selective inhibitors of each Clk and Dyrk kinase can be developed, parallel use of the available panel of inhibitors should enable biological investigations with these kinase families to proceed through a “process of elimination” approach." /><meta name="og:title" content="Identification of selective inhibitors of cdc2-like kinases 1 and 4 (Clk1, Clk4)" /><meta name="og:type" content="book" /><meta name="og:description" content="The cdc2-like (Clk) kinases are critical elements of several important regulatory pathways involving pre-mRNA splicing. These kinases act by phosphorylating the serine- and arginine-rich (SR) proteins, which direct the early events of spliceosome assembly necessary for proper mRNA maturation. Mutations causing splicing defects have been linked to a number of disease processes, making the search for small-molecule regulators of enzymes associated with spliceosome function important for both the further study and potential treatment of these disorders. The dual-specificity tyrosine-regulated (Dyrk) kinases are associated with numerous cellular processes and disorders. In particular, overexpression of Dyrk1A may contribute to Down syndrome neurodevelopmental abnormalities, and Dyrk1B/Mirk has been implicated in the cellular repair of cancer cells impaired by chemotherapy. Developing inhibitors of the Dyrk kinases would likely have important biochemical and therapeutic uses. Continuing in the search for selective inhibitors of Clk and Dyrk kinases, we now report a series of amino-substituted pyrimidines with excellent potencies against several Clk and Dyrk isoforms. The probe compound and its analogs have been subjected to Clk and Dyrk isoform selectivity profiling by Ambit Biosciences and three compounds have also been sent for KINOMEscan analysis to assess their selectivity in the context of the greater kinome. The reported compounds are low-nanomolar Clk and Dyrk inhibitors, with the most potent compound having IC50s <10 nM against Clk1, Clk4, Dyrk1A and Dyrk1B. The probe molecule (ML315) is a substituted aminopyrimidine and structurally distinct from previous quinazoline probes. ML315 should provide biochemical investigators with a new tool to be used in parallel with the quinazoline probes in efforts to determine which Clk and/or Dyrk isoforms are associated with particular biochemical events. Until isoform-selective inhibitors of each Clk and Dyrk kinase can be developed, parallel use of the available panel of inhibitors should enable biological investigations with these kinase families to proceed through a “process of elimination” approach." /><meta name="og:url" content="https://www.ncbi.nlm.nih.gov/books/NBK153503/" /><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/ml315/" /><link rel="canonical" href="https://www.ncbi.nlm.nih.gov/books/NBK153503/" /><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="#__NBK153503_dtls__">Show details</a><div style="display:none" class="ui-widget" id="__NBK153503_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/ml316/" title="Previous page in this title">< Prev</a><a class="active page_link next" href="/books/n/mlprobe/ml314/" 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="_NBK153503_"><span class="title" itemprop="name">Identification of selective inhibitors of cdc2-like kinases 1 and 4 (Clk1, Clk4)</span></h1><p class="contrib-group"><span itemprop="author">Thomas C. Coombs</span>, <span itemprop="author">Cordelle Tanega</span>, <span itemprop="author">Min Shen</span>, <span itemprop="author">Benjamin Neuenswander</span>, <span itemprop="author">Patrick Porubsky</span>, <span itemprop="author">Jenna L. Wang</span>, <span itemprop="author">Tom Misteli</span>, <span itemprop="author">Douglas S. Auld</span>, <span itemprop="author">Frank Schoenen</span>, <span itemprop="author">Craig J. Thomas</span>, and <span itemprop="author">Jeffrey Aubé</span>.</p><a data-jig="ncbitoggler" href="#__NBK153503_ai__" style="border:0;text-decoration:none">Author Information and Affiliations</a><div style="display:none" class="ui-widget" id="__NBK153503_ai__"><p class="contrib-group"><h4>Authors</h4><span itemprop="author">Thomas C. Coombs</span>,<sup>a</sup> <span itemprop="author">Cordelle Tanega</span>,<sup>b</sup> <span itemprop="author">Min Shen</span>,<sup>b</sup> <span itemprop="author">Benjamin Neuenswander</span>,<sup>a</sup> <span itemprop="author">Patrick Porubsky</span>,<sup>a</sup> <span itemprop="author">Jenna L. Wang</span>,<sup>a</sup> <span itemprop="author">Tom Misteli</span>,<sup>c</sup> <span itemprop="author">Douglas S. Auld</span>,<sup>b</sup> <span itemprop="author">Frank Schoenen</span>,<sup>a</sup> <span itemprop="author">Craig J. Thomas</span>,<sup>b</sup><sup>,*</sup> and <span itemprop="author">Jeffrey Aubé</span><sup>d</sup><sup>,*</sup>.</p><h4>Affiliations</h4><div class="affiliation"><sup>a</sup>
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University of Kansas Specialized Chemistry Center, University of Kansas, KS 66047</div><div class="affiliation"><sup>b</sup>
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NIH Chemical Genomics Center, National Human Genome Research Institute, NIH 9800 Medical Center Drive, MSC 3370 Bethesda, MD 20892-3370 USA.</div><div class="affiliation"><sup>c</sup>
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Cell Biology of Genomes, National Cancer Institute, NIH, 41 Library Drive, Bethesda, MD 20892 USA.</div><div class="affiliation"><sup>d</sup>
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Department of Medicinal Chemistry, University of Kansas, Lawrence, KS, 66047</div><div class="affiliation">
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<sup>*</sup> To whom correspondence should be addressed: Email:
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<a href="mailto:dev@null" data-email="vog.hin.liam@tgiarc" class="oemail">vog.hin.liam@tgiarc</a> and
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<span class="before-email-separator">; </span><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="ude.uk@ebuaj" class="oemail">ude.uk@ebuaj</a></div></div><p class="small">Received: <span itemprop="datePublished">April 16, 2012</span>; Last Update: <span itemprop="dateModified">May 8, 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>The cdc2-like (Clk) kinases are critical elements of several important regulatory pathways involving pre-mRNA splicing. These kinases act by phosphorylating the serine- and arginine-rich (SR) proteins, which direct the early events of spliceosome assembly necessary for proper mRNA maturation. Mutations causing splicing defects have been linked to a number of disease processes, making the search for small-molecule regulators of enzymes associated with spliceosome function important for both the further study and potential treatment of these disorders. The dual-specificity tyrosine-regulated (Dyrk) kinases are associated with numerous cellular processes and disorders. In particular, overexpression of Dyrk1A may contribute to Down syndrome neurodevelopmental abnormalities, and Dyrk1B/Mirk has been implicated in the cellular repair of cancer cells impaired by chemotherapy. Developing inhibitors of the Dyrk kinases would likely have important biochemical and therapeutic uses. Continuing in the search for selective inhibitors of Clk and Dyrk kinases, we now report a series of amino-substituted pyrimidines with excellent potencies against several Clk and Dyrk isoforms. The probe compound and its analogs have been subjected to Clk and Dyrk isoform selectivity profiling by Ambit Biosciences and three compounds have also been sent for KINOMEscan analysis to assess their selectivity in the context of the greater kinome. The reported compounds are low-nanomolar Clk and Dyrk inhibitors, with the most potent compound having IC<sub>50</sub>s <10 nM against Clk1, Clk4, Dyrk1A and Dyrk1B. The probe molecule (<a href="/pcsubstance/?term=ML315[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML315</a>) is a substituted aminopyrimidine and structurally distinct from previous quinazoline probes. <a href="/pcsubstance/?term=ML315[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML315</a> should provide biochemical investigators with a new tool to be used in parallel with the quinazoline probes in efforts to determine which Clk and/or Dyrk isoforms are associated with particular biochemical events. Until isoform-selective inhibitors of each Clk and Dyrk kinase can be developed, parallel use of the available panel of inhibitors should enable biological investigations with these kinase families to proceed through a “process of elimination” approach.</p></div><div class="h2"></div><p><b>Screening Center Name & PI:</b> NIH Chemical Genomics Center, Christopher P. Austin</p><p><b>Chemistry Center Name & PI:</b> University of Kansas Specialized Chemistry Center; Jeffrey Aubé</p><p><b>Assay Submitter & Institution:</b> Tom Misteli, National Cancer Institute</p><p><b>PubChem Summary Bioassay Identifier (AID):</b>
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<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1997" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1997</a></p><div id="ml315.s1"><h2 id="_ml315_s1_">Probe Structure & Characteristics</h2><div id="ml315.fu1" class="figure bk_fig"><div class="graphic"><img src="/books/NBK153503/bin/ml315fu1.jpg" alt="ML315." /></div><h3><span class="title">ML315</span></h3></div><div id="ml315.tu1" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK153503/table/ml315.tu1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml315.tu1_lrgtbl__"><table><thead><tr><th id="hd_h_ml315.tu1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">CID/ML#</th><th id="hd_h_ml315.tu1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Target Name</th><th id="hd_h_ml315.tu1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">IC<sub>50</sub>/EC<sub>50</sub> (nM) [SID, AID</th><th id="hd_h_ml315.tu1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Anti-target Name(s)</th><th id="hd_h_ml315.tu1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">IC<sub>50</sub>/EC<sub>50</sub> (nM) [SID, AID]</th><th id="hd_h_ml315.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Fold Selective</th><th id="hd_h_ml315.tu1_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Secondary Assay(s) Name: IC<sub>50</sub>/EC<sub>50</sub> (nM) [SID, AID]</th></tr></thead><tbody><tr><td headers="hd_h_ml315.tu1_1_1_1_1" rowspan="5" colspan="1" style="text-align:left;vertical-align:top;">CID-46926514/<a href="/pcsubstance/?term=ML315[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML315</a></td><td headers="hd_h_ml315.tu1_1_1_1_2" rowspan="5" colspan="1" style="text-align:left;vertical-align:top;">Cdc2-like kinase 4 (Clk4)</td><td headers="hd_h_ml315.tu1_1_1_1_3" rowspan="5" colspan="1" style="text-align:left;vertical-align:top;">68 nM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/99431981" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 99431981</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/624034" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 624034</a>]</td><td headers="hd_h_ml315.tu1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Clk1</td><td headers="hd_h_ml315.tu1_1_1_1_5" rowspan="5" colspan="1" style="text-align:left;vertical-align:top;">68 nM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/99431981" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 99431981</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/624047" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 624047</a>]<br />231 nM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/99431981" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 99431981</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/624048" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 624048</a>]<br />>10,000 nM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/99431981" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 99431981</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/624049" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 624049</a>]<br />282 nM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/99431981" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 99431981</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/624045" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 624045</a>]<br />1156 nM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/99431981" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 99431981</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/624046" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 624046</a>]</td><td headers="hd_h_ml315.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">1 fold</td><td headers="hd_h_ml315.tu1_1_1_1_7" rowspan="5" colspan="1" style="text-align:left;vertical-align:top;">Kinase panel: Selectivity determined across the kinome via profiling within KinomeScan (DiscoveRx, <a href="http://www.kinomescan.com/" ref="pagearea=body&targetsite=external&targetcat=link&targettype=uri">http://www<wbr style="display:inline-block"></wbr>.kinomescan.com/</a>). Probe screened against 442 kinases.<br />Additional activity against CSNK1E, MAP3K1, PKNB, PRKCE.</td></tr><tr><td headers="hd_h_ml315.tu1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Clk2</td><td headers="hd_h_ml315.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">3.4 fold</td></tr><tr><td headers="hd_h_ml315.tu1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Clk3</td><td headers="hd_h_ml315.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">>147 fold</td></tr><tr><td headers="hd_h_ml315.tu1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Dyrk1A</td><td headers="hd_h_ml315.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">4.1 fold</td></tr><tr><td headers="hd_h_ml315.tu1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Dyrk1B</td><td headers="hd_h_ml315.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">17 fold</td></tr></tbody></table></div></div></div><div id="ml315.s2"><h2 id="_ml315_s2_">1. Recommendations for Scientific Use of the Probe</h2><p>Post-transcriptional and post-translational processes are required to expand the 20,000–25,000 genes into the estimated 50,000–500,000 human protein variants.<sup><a class="bk_pop" href="#ml315.r1">1</a></sup> Alternate gene splicing is one mechanism by which this rich diversity of protein isoforms is achieved, and novel tools to help researchers understand and control gene splicing outcomes continue to be of considerable interest. Since the report of the first Clk inhibitor probe from this project (CID 44968231/<a href="/pcsubstance/?term=ML167[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML167</a>), there have been a limited number of reports on additional inhibitors of the Clk kinases. Each has a distinctive signature and <a href="/pcsubstance/?term=ML315[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML315</a> represents a new chemotype with Clk activity and a unique structural and phamacological signature with which to interogate this complex process. Like the other inhibitors of this target, <a href="/pcsubstance/?term=ML315[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML315</a> will be used to interrogate the role that the Clk kinases play in altering SR protein phosphorylation and the resulting consequences on gene splicing.</p><p>Apart from Clk4 (inhibited by <a href="/pcsubstance/?term=ML167[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML167</a>), no selective inhibitors have been reported for the other Clk isoforms (Clk1-Clk3); published Clk inhibitors either potently inhibit more than one isoform or have not been tested against all four isoforms. Using the available panel of inhibitors, including <a href="/pcsubstance/?term=ML315[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML315</a>, in parallel would allow biological investigations involving these kinases to proceed until isoform-selective inhibitors become available. One approach to using the three probes in parallel is described here: Using previous probe <a href="/pcsubstance/?term=ML167[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML167</a> in Assay 1, one can inhibit Clk4 (136 nM) and observe the effect. Using previous probe <a href="/pcsubstance/?term=ML106[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML106</a> in Assay 2, one can inhibit Clk1, Clk4, and Dyrk1A (all <100 nM) and observe the effect. Phenotypic or biochemical differences between Assay 1 and Assay 2 could be attributed to the lack of Clk1 or Dyrk1A activity. Using the new probe (<a href="/pcsubstance/?term=ML315[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML315</a>) in Assay 3, one can potently inhibit Clk1 and Clk4 (<100 nM), while only moderately inhibiting Clk2 and Dyrk1A (>200 nM). Phenotypic or biochemical differences between Assay 2 and Assay 3 could be attributed to the lack of Clk1 activity, allowing the roles Clk1 and Dyrk1A to be further distinguished. The differences observed between Assay 2 and Assay 3 might also be the result of moderate Clk2 inhibition. The parallel use of these probes is further discussed in <a href="#ml315.s27">Section 4.1</a>. The previously reported probe (<a href="/pcsubstance/?term=ML167[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML167</a>) has been requested by numerous researchers and we plan to offer <a href="/pcsubstance/?term=ML315[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML315</a> to researchers alongside this agent. We have additionally made other Clk inhibitors from the literature and offer each to researchers in the hopes that off-target activities associated with the divergent polypharmacology of each chemotype will be mitigated by parallel assessment of the activity of multiple Clk inhibitors.</p></div><div id="ml315.s3"><h2 id="_ml315_s3_">2. Materials and Methods</h2><div id="ml315.s4"><h3>2.1. Assays</h3><div id="ml315.s5"><h4>Overall Assay Strategy</h4><p>Both the quinazoline and pyrimidine scaffolds were originally identified from a cell-based assay aimed at identifying modulators of Lamin A splicing (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1487" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1487</a>). Based upon these agents’ structures, we considered that kinase targets may be the mechanistic basis for the activity within this assay. From the literature it was determined that the cdc2-like kinases were likely targets and we followed up this phenotypic assay with an <i>in vitro</i> assay to determine these agents’ activity versus Clk4. In addition to this assay, we utilized commercially available assays from Reaction Biology (<a href="http://www.reactionbiology.com" ref="pagearea=body&targetsite=external&targetcat=link&targettype=uri">www.reactionbiology.com</a>) and DiscoveRx (<a href="http://www.kinomescan.com/" ref="pagearea=body&targetsite=external&targetcat=link&targettype=uri">www.kinomescan.com/</a>) to assess the selectivity of these agents.</p><div id="ml315.s6"><h5>Clk 4 qHTS Assays</h5><p>The application of bioluminescence to ATPase assays has relied on a substrate depletion format. In these assays, the ATP dependence of firefly luciferase is used to measure the remaining ATP concentration, where the luminescence signal is inversely proportional to kinase activity.<sup><a class="bk_pop" href="#ml315.r12">12</a>–<a class="bk_pop" href="#ml315.r16">16</a></sup> To provide a signal-to-background of approximately 2-fold, the substrate must be depleted by at least 50%. Operating enzyme assays under these high conversion conditions is not at all optimal for classical enzymological studies. However, this is acceptable for HTS, as shifts in potency are typically less than 2-fold with a percent conversion < 80%. Given that HTS assays typically show variability in potency determinations between ~2–3-fold, shifts due to high conversions in the range of 50–80% will not be easily discernible from the assay noise even if the assay is performed at lower conversions. Therefore, ATP depletion has become a popular choice to perform generic HTS assays for ATPases, particularly protein kinases.</p><p>We used two bioluminescent assays for Clk4 qHTS (<a class="figpopup" href="/books/NBK153503/figure/ml315.f1/?report=objectonly" target="object" rid-figpopup="figml315f1" rid-ob="figobml315f1">Figure 1</a>). Measurement of ATP depletion was assessed using the Kinase-Glo™ assay system (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1770" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1770</a>), where a firefly luciferase detection reagent containing D-luciferin and buffer components are added to detect the remaining ATP following the Clk4 kinase assay (<a class="figpopup" href="/books/NBK153503/figure/ml315.f1/?report=objectonly" target="object" rid-figpopup="figml315f1" rid-ob="figobml315f1">Figure 1a</a>). The second system, ADP-Glo<sup>®</sup> measures kinase activity by quantifying the amount of ADP formed after the kinase reaction (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1771" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1771</a>). Bioluminescent detection of ADP levels is achieved through the addition of two different detection reagents (<a class="figpopup" href="/books/NBK153503/figure/ml315.f1/?report=objectonly" target="object" rid-figpopup="figml315f1" rid-ob="figobml315f1">Figure 1b</a>). First, a reagent that stops the protein kinase reaction and depletes the remaining ATP is added. Then, a second reagent is added to stop ATP degradation. In addition, the second reagent also contains an enzyme such as pyruvate kinase that efficiently converts the ADP to ATP, and the same firefly luciferase/D-luciferin components present in Kinase-Glo, which generate a luminescent signal proportional to the ADP concentration produced. Therefore, the two assay formats show opposite luminescent signal changes in response to protein kinase inhibitors.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml315f1" co-legend-rid="figlgndml315f1"><a href="/books/NBK153503/figure/ml315.f1/?report=objectonly" target="object" title="Figure 1" class="img_link icnblk_img figpopup" rid-figpopup="figml315f1" rid-ob="figobml315f1"><img class="small-thumb" src="/books/NBK153503/bin/ml315f1.gif" src-large="/books/NBK153503/bin/ml315f1.jpg" alt="Figure1" /></a><div class="icnblk_cntnt" id="figlgndml315f1"><h4 id="ml315.f1"><a href="/books/NBK153503/figure/ml315.f1/?report=objectonly" target="object" rid-ob="figobml315f1">Figure 1</a></h4><p class="float-caption no_bottom_margin">Bioluminescent assays used for Clk4 qHTS. <i>a</i>. Bioluminescent measurement of ATP depletion using Kinase-Glo. <i>b</i>. Bioluminescent measurement of ADP formation using ADP-Glo. </p></div></div><p>All compounds were screened using a qHTS approach,<sup><a class="bk_pop" href="#ml315.r15">15</a></sup> in which compounds were assayed using at least seven concentrations to generate concentration-response curves for each compound tested. The methodology for creating a concentration-titration series between successive copies of library plates for the purpose of large-scale titration-based screening has been described. Briefly, qHTS uses an inter-plate dilution method where the first plate contains the highest concentration of a set of compounds in DMSO, while subsequent plates contain the same compounds in the same well locations, but at successively lower concentrations. Using the protocol outlined above, we calculated a plate throughput of 18 plates/hr, or approximately 7 samples/sec, on the Kalypsys robotic system, which means that a 7 point CRC was obtained every second on the robotic system.</p><div id="ml315.t1" class="table"><h3><span class="label">Table 1</span><span class="title">Assay protocol: The optimized 1536-well protocol</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK153503/table/ml315.t1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml315.t1_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml315.t1_1_1_1_1" colspan="4" rowspan="1" style="text-align:center;vertical-align:top;">Kinase-Glo</th><th id="hd_h_ml315.t1_1_1_1_2" colspan="3" rowspan="1" style="text-align:center;vertical-align:top;">ADP-Glo</th></tr><tr><th headers="hd_h_ml315.t1_1_1_1_1 hd_h_ml315.t1_1_1_1_2" id="hd_h_ml315.t1_1_1_2_1" colspan="7" rowspan="1" style="text-align:left;vertical-align:top;">
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<span class="hr"></span></th></tr><tr><th headers="hd_h_ml315.t1_1_1_1_1 hd_h_ml315.t1_1_1_2_1" id="hd_h_ml315.t1_1_1_3_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Step</th><th headers="hd_h_ml315.t1_1_1_1_1 hd_h_ml315.t1_1_1_2_1" id="hd_h_ml315.t1_1_1_3_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Parameter</th><th headers="hd_h_ml315.t1_1_1_1_1 hd_h_ml315.t1_1_1_2_1" id="hd_h_ml315.t1_1_1_3_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Value</th><th headers="hd_h_ml315.t1_1_1_1_1 hd_h_ml315.t1_1_1_2_1" id="hd_h_ml315.t1_1_1_3_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Description</th><th headers="hd_h_ml315.t1_1_1_1_2 hd_h_ml315.t1_1_1_2_1" id="hd_h_ml315.t1_1_1_3_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Parameter</th><th headers="hd_h_ml315.t1_1_1_1_2 hd_h_ml315.t1_1_1_2_1" id="hd_h_ml315.t1_1_1_3_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Value</th><th headers="hd_h_ml315.t1_1_1_1_2 hd_h_ml315.t1_1_1_2_1" id="hd_h_ml315.t1_1_1_3_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Description</th></tr></thead><tbody><tr><td headers="hd_h_ml315.t1_1_1_1_1 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><b>1</b></td><td headers="hd_h_ml315.t1_1_1_1_1 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Reagent</td><td headers="hd_h_ml315.t1_1_1_1_1 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">2 μL</td><td headers="hd_h_ml315.t1_1_1_1_1 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">ATP/peptide</td><td headers="hd_h_ml315.t1_1_1_1_2 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Reagent</td><td headers="hd_h_ml315.t1_1_1_1_2 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">2 μL</td><td headers="hd_h_ml315.t1_1_1_1_2 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">ATP/peptide</td></tr><tr><td headers="hd_h_ml315.t1_1_1_1_1 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><b>2</b></td><td headers="hd_h_ml315.t1_1_1_1_1 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Library</td><td headers="hd_h_ml315.t1_1_1_1_1 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">23 nL</td><td headers="hd_h_ml315.t1_1_1_1_1 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0.5 nM- 46 μM</td><td headers="hd_h_ml315.t1_1_1_1_2 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Library</td><td headers="hd_h_ml315.t1_1_1_1_2 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">23 nL</td><td headers="hd_h_ml315.t1_1_1_1_2 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0.6 nM- 55.2 μM</td></tr><tr><td headers="hd_h_ml315.t1_1_1_1_1 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><b>3</b></td><td headers="hd_h_ml315.t1_1_1_1_1 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Controls</td><td headers="hd_h_ml315.t1_1_1_1_1 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">23 nL</td><td headers="hd_h_ml315.t1_1_1_1_1 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">TG003</td><td headers="hd_h_ml315.t1_1_1_1_2 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Controls</td><td headers="hd_h_ml315.t1_1_1_1_2 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">23 nL</td><td headers="hd_h_ml315.t1_1_1_1_2 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">TG003</td></tr><tr><td headers="hd_h_ml315.t1_1_1_1_1 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><b>4</b></td><td headers="hd_h_ml315.t1_1_1_1_1 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Reagent</td><td headers="hd_h_ml315.t1_1_1_1_1 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">1 μL</td><td headers="hd_h_ml315.t1_1_1_1_1 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Clk4</td><td headers="hd_h_ml315.t1_1_1_1_2 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Reagent</td><td headers="hd_h_ml315.t1_1_1_1_2 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0.5 μL</td><td headers="hd_h_ml315.t1_1_1_1_2 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Clk4</td></tr><tr><td headers="hd_h_ml315.t1_1_1_1_1 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><b>5</b></td><td headers="hd_h_ml315.t1_1_1_1_1 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Time</td><td headers="hd_h_ml315.t1_1_1_1_1 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">4.5 hrs</td><td headers="hd_h_ml315.t1_1_1_1_1 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">r.t. incubation</td><td headers="hd_h_ml315.t1_1_1_1_2 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Time</td><td headers="hd_h_ml315.t1_1_1_1_2 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">1 hr</td><td headers="hd_h_ml315.t1_1_1_1_2 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">r.t. incubation</td></tr><tr><td headers="hd_h_ml315.t1_1_1_1_1 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><b>6</b></td><td headers="hd_h_ml315.t1_1_1_1_1 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Reagent</td><td headers="hd_h_ml315.t1_1_1_1_1 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">3 μL</td><td headers="hd_h_ml315.t1_1_1_1_1 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Kinase-Glo</td><td headers="hd_h_ml315.t1_1_1_1_2 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Reagent</td><td headers="hd_h_ml315.t1_1_1_1_2 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">2.5 μL</td><td headers="hd_h_ml315.t1_1_1_1_2 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Deplete ATP</td></tr><tr><td headers="hd_h_ml315.t1_1_1_1_1 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><b>7</b></td><td headers="hd_h_ml315.t1_1_1_1_1 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Read</td><td headers="hd_h_ml315.t1_1_1_1_1 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">2 sec</td><td headers="hd_h_ml315.t1_1_1_1_1 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">ViewLux</td><td headers="hd_h_ml315.t1_1_1_1_2 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Time</td><td headers="hd_h_ml315.t1_1_1_1_2 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">45 min</td><td headers="hd_h_ml315.t1_1_1_1_2 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">r.t. incubation</td></tr><tr><td headers="hd_h_ml315.t1_1_1_1_1 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><b>8</b></td><td headers="hd_h_ml315.t1_1_1_1_1 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_2 hd_h_ml315.t1_1_1_3_3 hd_h_ml315.t1_1_1_3_4" colspan="3" rowspan="3" style="text-align:left;vertical-align:top;"></td><td headers="hd_h_ml315.t1_1_1_1_2 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Reagent</td><td headers="hd_h_ml315.t1_1_1_1_2 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">5 μL</td><td headers="hd_h_ml315.t1_1_1_1_2 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">ADP→ATP/Luc</td></tr><tr><td headers="hd_h_ml315.t1_1_1_1_1 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><b>9</b></td><td headers="hd_h_ml315.t1_1_1_1_2 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Time</td><td headers="hd_h_ml315.t1_1_1_1_2 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">30 min</td><td headers="hd_h_ml315.t1_1_1_1_2 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">r.t. incubation</td></tr><tr><td headers="hd_h_ml315.t1_1_1_1_1 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><b>10</b></td><td headers="hd_h_ml315.t1_1_1_1_2 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Read</td><td headers="hd_h_ml315.t1_1_1_1_2 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">2 sec</td><td headers="hd_h_ml315.t1_1_1_1_2 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">ViewLux</td></tr><tr><td headers="hd_h_ml315.t1_1_1_1_1 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_1 hd_h_ml315.t1_1_1_3_2 hd_h_ml315.t1_1_1_3_3 hd_h_ml315.t1_1_1_3_4 hd_h_ml315.t1_1_1_1_2 hd_h_ml315.t1_1_1_3_5 hd_h_ml315.t1_1_1_3_6 hd_h_ml315.t1_1_1_3_7" colspan="7" rowspan="1" style="text-align:left;vertical-align:top;">
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<span class="hr"></span></td></tr><tr><th headers="hd_h_ml315.t1_1_1_1_1 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_1" id="hd_b_ml315.t1_1_1_12_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Step</th><th headers="hd_h_ml315.t1_1_1_1_1 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_2 hd_h_ml315.t1_1_1_3_3 hd_h_ml315.t1_1_1_3_4" id="hd_b_ml315.t1_1_1_12_2" colspan="3" rowspan="1" style="text-align:left;vertical-align:top;">Notes</th><th headers="hd_h_ml315.t1_1_1_1_2 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_5 hd_h_ml315.t1_1_1_3_6 hd_h_ml315.t1_1_1_3_7" id="hd_b_ml315.t1_1_1_12_3" colspan="3" rowspan="1" style="text-align:left;vertical-align:top;">Notes</th></tr><tr><td headers="hd_h_ml315.t1_1_1_1_1 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_1 hd_b_ml315.t1_1_1_12_1 hd_h_ml315.t1_1_1_3_2 hd_b_ml315.t1_1_1_12_2 hd_h_ml315.t1_1_1_3_3 hd_h_ml315.t1_1_1_3_4 hd_h_ml315.t1_1_1_1_2 hd_h_ml315.t1_1_1_3_5 hd_b_ml315.t1_1_1_12_3 hd_h_ml315.t1_1_1_3_6 hd_h_ml315.t1_1_1_3_7" colspan="7" rowspan="1" style="text-align:left;vertical-align:top;">
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<span class="hr"></span></td></tr><tr><td headers="hd_h_ml315.t1_1_1_1_1 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_1 hd_b_ml315.t1_1_1_12_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><b>1</b></td><td headers="hd_h_ml315.t1_1_1_1_1 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_2 hd_b_ml315.t1_1_1_12_2 hd_h_ml315.t1_1_1_3_3 hd_h_ml315.t1_1_1_3_4" colspan="3" rowspan="1" style="text-align:left;vertical-align:top;">100 μM RS peptide, 1 μM ATP (final) concentration in buffer; FRD dispense</td><td headers="hd_h_ml315.t1_1_1_1_2 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_5 hd_b_ml315.t1_1_1_12_3 hd_h_ml315.t1_1_1_3_6 hd_h_ml315.t1_1_1_3_7" colspan="3" rowspan="1" style="text-align:left;vertical-align:top;">100 μM RS peptide, 1 μM ATP (final) concentration in buffer; FRD dispense</td></tr><tr><td headers="hd_h_ml315.t1_1_1_1_1 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_1 hd_b_ml315.t1_1_1_12_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><b>2</b></td><td headers="hd_h_ml315.t1_1_1_1_1 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_2 hd_b_ml315.t1_1_1_12_2 hd_h_ml315.t1_1_1_3_3 hd_h_ml315.t1_1_1_3_4" colspan="3" rowspan="1" style="text-align:left;vertical-align:top;">Pin-tool transfer compound library for a (final) range of 46 μM to 0.5 nM</td><td headers="hd_h_ml315.t1_1_1_1_2 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_5 hd_b_ml315.t1_1_1_12_3 hd_h_ml315.t1_1_1_3_6 hd_h_ml315.t1_1_1_3_7" colspan="3" rowspan="1" style="text-align:left;vertical-align:top;">Pin-tool transfer compound library for a (final) range of 55.2 μM to 0.6 nM</td></tr><tr><td headers="hd_h_ml315.t1_1_1_1_1 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_1 hd_b_ml315.t1_1_1_12_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><b>3</b></td><td headers="hd_h_ml315.t1_1_1_1_1 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_2 hd_b_ml315.t1_1_1_12_2 hd_h_ml315.t1_1_1_3_3 hd_h_ml315.t1_1_1_3_4" colspan="3" rowspan="1" style="text-align:left;vertical-align:top;"></td><td headers="hd_h_ml315.t1_1_1_1_2 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_5 hd_b_ml315.t1_1_1_12_3 hd_h_ml315.t1_1_1_3_6 hd_h_ml315.t1_1_1_3_7" colspan="3" rowspan="1" style="text-align:left;vertical-align:top;"></td></tr><tr><td headers="hd_h_ml315.t1_1_1_1_1 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_1 hd_b_ml315.t1_1_1_12_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><b>4</b></td><td headers="hd_h_ml315.t1_1_1_1_1 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_2 hd_b_ml315.t1_1_1_12_2 hd_h_ml315.t1_1_1_3_3 hd_h_ml315.t1_1_1_3_4" colspan="3" rowspan="1" style="text-align:left;vertical-align:top;">Clk4 at 25 nM final, FRD dispense</td><td headers="hd_h_ml315.t1_1_1_1_2 hd_h_ml315.t1_1_1_2_1 hd_h_ml315.t1_1_1_3_5 hd_b_ml315.t1_1_1_12_3 hd_h_ml315.t1_1_1_3_6 hd_h_ml315.t1_1_1_3_7" colspan="3" rowspan="1" style="text-align:left;vertical-align:top;">Clk4 at 25 nM final, FRD dispense</td></tr></tbody></table></div></div><p>The assay showed excellent performance (the signal-to-background ratio was 3.2 +/− 0.07, the average Z′ screening factor associated with each plate was 0.86 +/− 0.02 and the CV was 7.2 +/− 1.9, indicating a robust performance of the screen).</p></div><div id="ml315.s7"><h5>Clk4 confirmation assay</h5><p>Kinase-Glo™ assay to measure ATP depletion was used in the confirmation of Clk4 inhibition. All compounds were tested in 12-pt concentration response from 77 μM to 0.4 nM.</p></div><div id="ml315.s8"><h5>Dyrk1A selectivity assay</h5><p>Among the members of the CMGC group, the Clks and the Dyrks are very closely related. Therefore to determine the activity and selectivity of the Clk4 pyrimidine analogs, a bioluminescent counter-screen against Dyrk1A was developed. Dyrk1A (Invitrogen, cat # PV3785) was assayed using ATP and DYRKtide (AnaSpec, cat# 62698) as substrates. Promega Kinase-Glo (cat# V6712) technology was used to detect the residual ATP following kinetic reaction. The Kinase-Glo Plus contains Ultra-Glo luciferase and D-luciferin which generates a bioluminescence signal from the remaining ATP. No enzyme and harmine (Sigma, cat # 286044-1G), a compound shown to inhibit Dyrks, were used as positive controls; DMSO treatment was used as a negative control. All compounds were tested in 12-pt concentration response from 77μM to 0.4nM.</p><p>Dyrk1A assay protocol summary: Two μL/well of substrate solution (8 μM Dyrktide, 2 μM ATP, 25 mM Tris-HCl pH 7.5, 10 mM MgCl2, 0.5 mM EGTA, 5 mM cystein, 0.01% Triton X-100 final concentration) was dispensed into 1536-well assay plates (Greiner, solid white, medium-binding plates) with Aurora Discovery BioRAPTR Flying Reagent Dispenser (FRD, Beckton-Dickenson). Compound solution (23 nL) was transferred to the assay plate using a Kalypsys pin tool equipped with a 1536-pin tool. One μL/well of enzyme solution (5 nM Dyrk1A, 2 5mM Tris-HCl pH7.5, 10 mM MgCl2, 0.5 mM EGTA, 5 mM cystein, 0.01% Triton X-100 final concentration) was then added using FRD, yielding a total kinase reaction volume of 3 μL/well. After 2 hours of room temperature incubation, 3 μL of Kinase-Glo detection reagent was added for a final assay volume of 6 μL/well. The plates were spun for 30 seconds (1000 rpm) and incubated at ambient temperature for 2 minutes, followed by a luminescence read using Perkin Elmer ViewLux plate reader at 3 seconds exposure time and 2× binning.</p></div><div id="ml315.s9"><h5>Clk1-4, Dyrk1A, Dyrk1B Assay</h5><p>Reaction Biology (<a href="http://www.reactionbiology.com" ref="pagearea=body&targetsite=external&targetcat=link&targettype=uri">www.reactionbiology.com</a>) offers <i>in vitro</i> assays for multiple kinases including all Clk isoforms, Dyrk1A and Dyrk1B. The assay format relies upon a <sup>33</sup>P-γ-ATP radiometric based filtration binding assay. Please refer to BioAssay <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/624047" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 624047</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/624048" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 624048</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/624049" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 624049</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/624034" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 624034</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/624045" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 624045</a> and <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/624046" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 624046</a>.</p></div><div id="ml315.s10"><h5>KinomeScan Assay</h5><p>The KinomeScan assay [originally developed by Ambit Biosciences and currently offered by DiscoverX (<a href="http://www.kinomescan.com/" ref="pagearea=body&targetsite=external&targetcat=link&targettype=uri">www.kinomescan.com/</a>)] is based upon a competition binding assay of kinases fused to a proprietary tag.</p></div></div></div><div id="ml315.s11"><h3>2.2. Probe Chemical Characterization</h3><div id="ml315.s12"><h4>Probe Chemical Structure, Physical parameters, and Properties</h4><div id="ml315.fu2" class="figure"><div class="graphic"><img src="/books/NBK153503/bin/ml315fu2.jpg" alt="Image ml315fu2" /></div></div></div><div id="ml315.s13"><h4>Structure Verification and Purity: <sup>1</sup>H NMR, <sup>13</sup>C NMR, RP HPLC/UV/HRMS Data</h4><p><b>Proton and carbon NMR data for <a href="/pcsubstance/?term=ML315[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML315</a>:</b> Detailed analytical methods and associated instrumentation are described in <a href="#ml315.s18">section 2.3</a>, entitled “Probe Preparation”, under general experimental and analytical details. The numerical experimental proton and carbon NMR data are presented.</p><p><b>Proton NMR Data for <a href="/pcsubstance/?term=ML315[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML315</a>:</b><sup>1</sup>H NMR (500 MHz, DMSO-d<sub>6</sub>): δ 8.41 (s, 1H), 7.96 (s, 1H), 7.41 (t, <i>J</i> = 2.0 Hz, 1H), 7.35-7.32 (m, 3H), 7.05 (d, <i>J</i> = 8.0 Hz, 1H), 7.02 (d, <i>J</i> = 1.5 Hz, 1H), 6.90 (dd, <i>J</i> = 8.0 Hz, 1.5 Hz, 1H), 6.09 (s, 2H), 4.54 (d, <i>J</i> = 6.5 Hz, 2H).</p><p><b>Carbon NMR Data for <a href="/pcsubstance/?term=ML315[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML315</a>:</b><sup>13</sup>C NMR (125 MHz, DMSO-d<sub>6</sub>): δ 158.9, 156.9, 153.4, 147.8, 147.3, 144.7, 133.8 (2), 127.6, 126.2, 125.9 (2), 122.6, 119.1, 109.4, 109.0, 101.3, 42.8.</p><p><b>RP HPLC/UV/HRMS Data for <a href="/pcsubstance/?term=ML315[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML315</a>:</b> Detailed analytical methods and associated instrumentation are described in <a href="#ml315.s18">section 2.3</a>, entitled “Probe Preparation”, under general experimental and analytical details. Purity assessment by RP HPLC/UV/HRMS at 214 nm for <b><a href="/pcsubstance/?term=ML315[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML315</a></b> revealed purity of 97.6% (retention time = 3.37 minutes). HRMS (m/z): calcd for C<sub>18</sub>H<sub>14</sub>Cl<sub>2</sub>N<sub>3</sub>O<sub>2</sub> (MH<sup>+</sup>) 374.0465; found 374.0486.</p></div><div id="ml315.s14"><h4>Solubility</h4><p>Aqueous solubility for the probe was assessed in phosphate-buffered saline (PBS) at room temperature. PBS by definition is 137 mM NaCl, 2.7 mM KCl, 10 mM sodium phosphate dibasic, 2 mM potassium phosphate monobasic at a pH of 7.4 (ref to SBMRI). Under these conditions, the solubility of <a href="/pcsubstance/?term=ML315[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML315</a> was determined to be 1.15 μM. Solubility was also assessed in the assay media of the primary assay (25 mM Tris pH7.5, 10 mM MgCl<sub>2</sub>, 0.5 mM EGTA, 2.5 mM DTT, 0.01% Triton X-100). Under these conditions, the solubility of probe <a href="/pcsubstance/?term=ML315[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML315</a> was determined to be 1.71 μM. Though the solubility of <a href="/pcsubstance/?term=ML315[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML315</a> is limited, the compound concentration was 17-fold greater in PBS than the measured IC<sub>50</sub> as determined in the AMBIT panel, and in the assay media, the compound concentration was 25-fold greater than the measured IC<sub>50</sub>, allaying concerns about solubility-limited potency and SAR.</p></div><div id="ml315.s15"><h4>Stability</h4><p>Aqueous stability for the probe was assessed using two solvent systems (100% aqueous PBS,
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||
and 50:50 aqueous PBS:acetonitrile). The probe’s stability was measured in aqueous PBS (no
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||
antioxidants or other protectants, DMSO concentration below 1%, room temperature) and the
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||
results are reported as circles in the graph in <a class="figpopup" href="/books/NBK153503/figure/ml315.f2/?report=objectonly" target="object" rid-figpopup="figml315f2" rid-ob="figobml315f2">Figure 2</a>.
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The probe’s stability was also measured in 50:50 aqueous PBS and acetonitrile and the results are reported as triangles in the graph in <a class="figpopup" href="/books/NBK153503/figure/ml315.f2/?report=objectonly" target="object" rid-figpopup="figml315f2" rid-ob="figobml315f2">Figure 2</a>. Stability data in each case is depicted as the loss of compound with time over 48 hours with a minimum of six time points and providing the percent compound remaining after 48 hours. The apparent compound instability in aqueous PBS is much more likely attributed to compound insolubility in that medium, because 100% of the compound was detected after 48 hours in PBS with 50% acetonitrile (50% ACN).</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml315f2" co-legend-rid="figlgndml315f2"><a href="/books/NBK153503/figure/ml315.f2/?report=objectonly" target="object" title="Figure 2" class="img_link icnblk_img figpopup" rid-figpopup="figml315f2" rid-ob="figobml315f2"><img class="small-thumb" src="/books/NBK153503/bin/ml315f2.gif" src-large="/books/NBK153503/bin/ml315f2.jpg" alt="Figure 2. Stability studies for ML315 in aqueous PBS (circles) and 50:50 aqueous PBS:acetonitrile (triangles)." /></a><div class="icnblk_cntnt" id="figlgndml315f2"><h4 id="ml315.f2"><a href="/books/NBK153503/figure/ml315.f2/?report=objectonly" target="object" rid-ob="figobml315f2">Figure 2</a></h4><p class="float-caption no_bottom_margin">Stability studies for <i>ML315</i> in aqueous PBS (circles) and 50:50 aqueous PBS:acetonitrile (triangles). </p></div></div></div><div id="ml315.s16"><h4>Synthesis Route</h4><p>The probe compound <a href="/pcsubstance/?term=ML315[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML315</a> was synthesized according to <a class="figpopup" href="/books/NBK153503/figure/ml315.f4/?report=objectonly" target="object" rid-figpopup="figml315f4" rid-ob="figobml315f4">Scheme 1</a>. Bromination of 4-aminopyrimidine afforded 5-bromo-4-amino pyrimidine, which was subjected to Suzuki cross-coupling conditions with 3,4-(methylenedioxy)phenylboronic acid in a microwave reactor to produce <b>S-1</b>. Reductive amination of <b>S-1</b> with 3,5-dichlorobenzaldehyde delivered <a href="/pcsubstance/?term=ML315[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML315</a>, which was purified by preparative reverse-phase HPLC.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml315f4" co-legend-rid="figlgndml315f4"><a href="/books/NBK153503/figure/ml315.f4/?report=objectonly" target="object" title="Scheme 1" class="img_link icnblk_img figpopup" rid-figpopup="figml315f4" rid-ob="figobml315f4"><img class="small-thumb" src="/books/NBK153503/bin/ml315f4.gif" src-large="/books/NBK153503/bin/ml315f4.jpg" alt="Scheme 1. Synthetic route to ML315." /></a><div class="icnblk_cntnt" id="figlgndml315f4"><h4 id="ml315.f4"><a href="/books/NBK153503/figure/ml315.f4/?report=objectonly" target="object" rid-ob="figobml315f4">Scheme 1</a></h4><p class="float-caption no_bottom_margin">Synthetic route to <i>ML315</i>. </p></div></div><p>Benzodioxole and benzodioxane analogs were synthesized via the sequence shown in <a class="figpopup" href="/books/NBK153503/figure/ml315.f5/?report=objectonly" target="object" rid-figpopup="figml315f5" rid-ob="figobml315f5">Scheme 2</a>. Suzuki cross-coupling of 2-amino-4-chloropyrimidine with either 3,4-(methylenedioxy)phenylboronic acid or 1,4-benzodioxane-6-boronic acid in a microwave reactor afforded <b>S-2</b> or <b>S-3</b>, respectively. Subjecting the cross-coupling products to reductive amination conditions with the appropriate aldehyde in each case produced the desired analogs, which were purified by preparative reverse-phase HPLC.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml315f5" co-legend-rid="figlgndml315f5"><a href="/books/NBK153503/figure/ml315.f5/?report=objectonly" target="object" title="Scheme 2" class="img_link icnblk_img figpopup" rid-figpopup="figml315f5" rid-ob="figobml315f5"><img class="small-thumb" src="/books/NBK153503/bin/ml315f5.gif" src-large="/books/NBK153503/bin/ml315f5.jpg" alt="Scheme 2. Synthetic route to oxygenated analogues." /></a><div class="icnblk_cntnt" id="figlgndml315f5"><h4 id="ml315.f5"><a href="/books/NBK153503/figure/ml315.f5/?report=objectonly" target="object" rid-ob="figobml315f5">Scheme 2</a></h4><p class="float-caption no_bottom_margin">Synthetic route to oxygenated analogues. </p></div></div><p>Indazole analogs were synthesized according to the sequence shown in <a class="figpopup" href="/books/NBK153503/figure/ml315.f6/?report=objectonly" target="object" rid-figpopup="figml315f6" rid-ob="figobml315f6">Scheme 3</a>. 2,4-Dichloropyrimidine was reacted with 3,4-dichlorobenzylamine via S<sub>N</sub>Ar reaction in a microwave reactor at 110 °C, producing <b>S-4</b> and <b>S-5</b> in a ratio of 72:28. The isomeric mixture was readily separated using silica gel column chromatography. The individual isomers <b>S-4</b> and <b>S-5</b> were then used in microwave-assisted Suzuki cross-coupling reactions with the appropriate indazolylboronic acids to afford the desired indazole analogs, which were purified by preparative reverse-phase HPLC. These compounds were also synthesized via Suzuki cross-coupling/reductive amination sequences starting from either 2-amino-4-chloropyrimidine or 4-amino-2-chloropyrimidine to confirm the identities of the desired analogs.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml315f6" co-legend-rid="figlgndml315f6"><a href="/books/NBK153503/figure/ml315.f6/?report=objectonly" target="object" title="Scheme 3" class="img_link icnblk_img figpopup" rid-figpopup="figml315f6" rid-ob="figobml315f6"><img class="small-thumb" src="/books/NBK153503/bin/ml315f6.gif" src-large="/books/NBK153503/bin/ml315f6.jpg" alt="Scheme 3. Synthetic route to indazole analogues." /></a><div class="icnblk_cntnt" id="figlgndml315f6"><h4 id="ml315.f6"><a href="/books/NBK153503/figure/ml315.f6/?report=objectonly" target="object" rid-ob="figobml315f6">Scheme 3</a></h4><p class="float-caption no_bottom_margin">Synthetic route to indazole analogues. </p></div></div></div><div id="ml315.s17"><h4>Submission of Probe and Five Supporting Analogues to the MLSMR</h4><p>Samples of the probe and five analogs were prepared, analytically characterized, and shipped to the MLSMR. The structures for the five supporting analogues are shown in <a class="figpopup" href="/books/NBK153503/table/ml315.t2/?report=objectonly" target="object" rid-figpopup="figml315t2" rid-ob="figobml315t2">Table 2</a>. The screening results from NCGC in-house bioluminescent luciferase-based assays are shown in parentheses, while the remaining data (IC<sub>50</sub> values, nM) was generated using a commercial vendor for AMBIT subtype selectivity profiling against the Clk and Dyrk kinases.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml315t2"><a href="/books/NBK153503/table/ml315.t2/?report=objectonly" target="object" title="Table 2" class="img_link icnblk_img figpopup" rid-figpopup="figml315t2" rid-ob="figobml315t2"><img class="small-thumb" src="/books/NBK153503/table/ml315.t2/?report=thumb" src-large="/books/NBK153503/table/ml315.t2/?report=previmg" alt="Table 2. Supporting analogs with IC50 values (nM) against Clk and Dyrk kinases." /></a><div class="icnblk_cntnt"><h4 id="ml315.t2"><a href="/books/NBK153503/table/ml315.t2/?report=objectonly" target="object" rid-ob="figobml315t2">Table 2</a></h4><p class="float-caption no_bottom_margin">Supporting analogs with IC<sub>50</sub> values (nM) against Clk and Dyrk kinases. </p></div></div></div></div><div id="ml315.s18"><h3>2.3. Probe Preparation</h3><div id="ml315.s19"><h4>General experimental and analytical details</h4><p>All reagents were used as received from commercial suppliers. Microwave reactions were carried out using a Biotage Initiator. The <sup>1</sup>H and <sup>13</sup>C spectra were recorded on Bruker Avance 400 MHz or 500 MHz spectrometers. Chemical shifts are reported in parts per million and referenced to the center line of residual solvent signals: CDCl<sub>3</sub> (7.26 ppm for <sup>1</sup>H and 77.23 ppm for <sup>13</sup>C), d<sub>3</sub>-MeCN (1.94 ppm for <sup>1</sup>H and 1.39 ppm for <sup>13</sup>C), and d<sub>6</sub>-DMSO (2.50 ppm for <sup>1</sup>H and 39.51 ppm for <sup>13</sup>C). Flash column chromatography separations were performed using Sorbent Technologies standard grade silica gel (40–63 μm particle size, 230 × 400 mesh) with compressed nitrogen as a source of positive pressure or the Teledyne Isco CombiFlash <i>R</i><i><sub>F</sub></i> using RediSep <i>R</i><i><sub>F</sub></i> silica gel columns. TLC was performed on Analtech UNIPLATE silica gel GHLF plates (gypsum inorganic hard layer with fluorescence). TLC plates were developed using UV light or aqueous KMnO<sub>4</sub>. HPLC/MS analysis was carried out with gradient elution (5% CH<sub>3</sub>CN to 100% CH<sub>3</sub>CN) on an Agilent 1200 RRLC with a photodiode array UV detector and an Agilent 6224 TOF mass spectrometer (also used to produce high resolution mass spectra). Purification was carried out by mass-directed fractionation with gradient elution (a narrow CH<sub>3</sub>CN gradient was chosen based on the retention time of the target from LCMS analysis of the crude sample) on an Agilent 1200 instrument with photodiode array detector, an Agilent 6120 quadrupole mass spectrometer, and a HTPAL LEAP autosampler. Fractions were triggered using an MS and UV threshold determined by HPLC/MS analysis of the crude sample. The conditions for HPLC analysis included the following: Waters BEH C-18, 1.7 μm, 2.1 × 50mm column; 0.6 ml/min flow rate; and pH 9.8 NH<sub>4</sub>OH aqueous mobile phase. The conditions for purification included: Waters XBridge C18 5μm, 19 × 150mm column; 20 ml/min flowrate pH 9.8 NH<sub>4</sub>OH aqueous mobile phase.</p></div><div id="ml315.s20"><h4>The probe was prepared using the following protocols</h4><div id="ml315.fu3" class="figure"><div class="graphic"><img src="/books/NBK153503/bin/ml315fu9.jpg" alt="Image ml315fu9" /></div></div><p><b>5-Bromopyrimidin-4-amine</b>. This compound was synthesized according to the published procedure.<sup><a class="bk_pop" href="#ml315.r17">17</a></sup> 4-Aminopyrimidine (1.00 g, 10.5 mmol, 1.0 equiv) and CaCO<sub>3</sub> (263 mg, 2.63 mmol, 0.25 equiv) were combined in H<sub>2</sub>O (20 mL) and Br<sub>2</sub> (1.06 mL, 20.5 mmol, 2.0 equiv) was added dropwise. The reaction mixture was stirred at 60 °C for 45 minutes, cooled to room temperature, and then poured into CH<sub>2</sub>Cl<sub>2</sub> (20 mL). The layers were separated and the aqueous layer was extracted with CH<sub>2</sub>Cl<sub>2</sub> (2 × 20 mL). The combined organic layers were set aside. The aqueous layer was treated with 20% aqueous K<sub>2</sub>CO<sub>3</sub> until pH 9–10 was achieved, and the product precipitated. The precipitate was removed via filtration and dried, affording the product (1.08 g, 6.22 mmol, 59%) as a light tan solid. <sup>1</sup>H NMR (500 MHz, DMSO-d<sub>6</sub>): δ 8.32 (s, 2H), 7.87–6.62 (v br s, 2H). <sup>13</sup>C NMR (125 MHz, DMSO-d<sub>6</sub>): δ 160.1, 156.8<sub>,</sub> 156.0, 102.5.</p><div id="ml315.fu4" class="figure"><div class="graphic"><img src="/books/NBK153503/bin/ml315fu10.jpg" alt="Image ml315fu10" /></div></div><p><b>5-(Benzo[</b><b><i>d</i></b><b>][1,3]dioxol-5-yl)pyrimidin-4-amine</b>. 5-Bromopyrimidin-4-amine (400 mg, 2.30 mmol, 1.0 equiv) was dissolved in dioxane (7 mL) in a 20-mL microwave vial and to it was added 3,4-(methylenedioxy)-phenylboronic acid (763 mg, 4.60 mmol, 2.0 equiv), aqueous Na<sub>2</sub>CO<sub>3</sub> (2.0 M, 3.45 mL, 6.90 mmol, 3.0 equiv), and Pd(PPh<sub>3</sub>)<sub>4</sub> (266 mg, 0.23 mmol, 0.10 equiv). The sealed vial was placed in a microwave reactor and heated at 110 °C for 30 minutes. The crude reaction mixture was partitioned between CH<sub>2</sub>Cl<sub>2</sub> (20 mL)/H<sub>2</sub>O (20 mL) and the layers were separated. The aqueous layer was extracted with CH<sub>2</sub>Cl<sub>2</sub> (2 × 20 mL), and the combined organic layers were dried over Na<sub>2</sub>SO<sub>4</sub> and concentrated under reduced pressure. The crude reaction mixture was purified via silica gel column chromatography using MeOH/CH<sub>2</sub>Cl<sub>2</sub> (1:19), affording the product (220 mg, 1.02 mmol, 44%) as a light tan solid. <sup>1</sup>H NMR (500 MHz, DMSO-d<sub>6</sub>): δ 8.33 (s, 1H), 7.95 (s, 1H), 7.00 (d, <i>J</i> = 8.0 Hz, 1H), 6.97 (d, <i>J</i> = 1.5 Hz, 1H), 6.87 (dd, <i>J</i> = 8.0 Hz, 1.5 Hz, 1H), 6.60 (br s, 2H), 6.06 (s, 2H) <sup>13</sup>C NMR (125 MHz, DMSO-d<sub>6</sub>): δ 160.8, 157.0, 154.0, 147.7, 147.0, 128.3, 122.2, 117.7, 109.0, 108.9, 101.2.</p><div id="ml315.fu5" class="figure"><div class="graphic"><img src="/books/NBK153503/bin/ml315fu11.jpg" alt="Image ml315fu11" /></div></div><p><b>5-(Benzo[</b><b><i>d</i></b><b>][1,3]dioxol-5-yl)-</b><b><i>N</i></b><b>-(3,5-dichlorobenzyl)pyrimidin-4-amine</b>. 5-(Benzo[<i>d</i>][1,3]dioxol-5-yl)pyrimidin-4-amine (150 mg, 0.70 mmol, 1.0 equiv) was suspended in MeCN (15 mL), and then 3,5-dichlorobenzaldehyde (610 mg, 3.49 mmol, 5.0 equiv), ClTi(O<i>i</i>-Pr)<sub>3</sub> (95%, 1.05 mL, 4.20 mmol, 6.0 equiv) and AcOH (5 drops) were added. The reaction mixture was stirred for 5 minutes at room temperature and then Na(OAc)<sub>3</sub>BH (95%, 779 mg, 3.49 mmol, 5.0 equiv) was added. The mixture was stirred for two hours and then aqueous NH<sub>4</sub>OH (15%, 15 mL) was added. The mixture was stirred for a further one hour and then solids were filtered off and washed with CH<sub>2</sub>Cl<sub>2</sub> (20 mL) and H<sub>2</sub>O (20 mL). The layers of the biphasic mixture were separated and the aqueous layer was extracted with CH<sub>2</sub>Cl<sub>2</sub> (2 × 20 mL). The combined organic layers were dried over Na<sub>2</sub>SO<sub>4</sub> and then concentrated under reduced pressure. The crude reaction mixture was submitted for preparative reverse-phase HPLC purification, affording the product as a white solid (86.1 mg, 0.23 mmol, 33%). <sup>1</sup>H NMR (500 MHz, DMSO-d<sub>6</sub>): δ 8.41 (s, 1H), 7.96 (s, 1H), 7.41 (t, <i>J</i> = 2.0 Hz, 1H), 7.35-7.32 (m, 3H), 7.05 (d, <i>J</i> = 8.0 Hz, 1H), 7.02 (d, <i>J</i> = 1.5 Hz, 1H), 6.90 (dd, <i>J</i> = 8.0 Hz, 1.5 Hz, 1H), 6.09 (s, 2H), 4.54 (d, <i>J</i> = 6.5 Hz, 2H). <sup>13</sup>C NMR (125 MHz, DMSO-d<sub>6</sub>): δ 158.9, 156.9, 153.4, 147.8, 147.3, 144.7, 133.8 (2), 127.6, 126.2, 125.9 (2), 122.6, 119.1, 109.4, 109.0, 101.3, 42.8. HRMS (ESI/APCI) Calcd for C<sub>18</sub>H<sub>14</sub>Cl<sub>2</sub>N<sub>3</sub>O<sub>2</sub> ([M+H]<sup>+</sup>): 374.0465. Found: 374.0486.</p></div></div></div><div id="ml315.s21"><h2 id="_ml315_s21_">3. Results</h2><div id="ml315.s22"><h3>3.1. Dose Response Curves for Probe</h3><div id="ml315.f3" class="figure bk_fig"><div class="graphic"><img src="/books/NBK153503/bin/ml315f3.jpg" alt="Figure 3. Dose response curve for ML315." /></div><h3><span class="label">Figure 3</span><span class="title">Dose response curve for <b>ML315</b></span></h3></div></div><div id="ml315.s23"><h3>3.2. Cellular Activity</h3><p>We are currently conducting studies with <a href="/pcsubstance/?term=ML315[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML315</a> (alongside <a href="/pcsubstance/?term=ML106[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML106</a> and <a href="/pcsubstance/?term=ML167[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML167</a>) to assess all of these agents’ ability to modulate the splicing of VEGF isoforms (namely VEGFa and VEGFb) via a cell-based RT-PCR assay. Preliminary results suggest that these agents are capable of modulating VEGF splice variants.</p></div><div id="ml315.s24"><h3>3.3. Profiling Assays</h3><div id="ml315.s25"><h4><i>In vitro</i> pharmacokinetics profiling</h4><p>The <i>in vitro</i> pharmacokinetic (PK) properties of the probe (<a href="/pcsubstance/?term=ML315[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML315</a>) were profiled
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||
using a standard panel of assays (<a class="figpopup" href="/books/NBK153503/table/ml315.t3/?report=objectonly" target="object" rid-figpopup="figml315t3" rid-ob="figobml315t3">Table 3</a>). The results from this profile suggested that <a href="/pcsubstance/?term=ML315[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML315</a> has modest aqueous and assay media solubility, good permeability across an artificial membrane (PAMPA), good plasma stability but low microsomal stability.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml315t3"><a href="/books/NBK153503/table/ml315.t3/?report=objectonly" target="object" title="Table 3" class="img_link icnblk_img figpopup" rid-figpopup="figml315t3" rid-ob="figobml315t3"><img class="small-thumb" src="/books/NBK153503/table/ml315.t3/?report=thumb" src-large="/books/NBK153503/table/ml315.t3/?report=previmg" alt="Table 3. Summary of in vitro ADME properties of ML315." /></a><div class="icnblk_cntnt"><h4 id="ml315.t3"><a href="/books/NBK153503/table/ml315.t3/?report=objectonly" target="object" rid-ob="figobml315t3">Table 3</a></h4><p class="float-caption no_bottom_margin">Summary of <i>in vitro</i> ADME properties of <i>ML315</i>. </p></div></div><p><b>Broad-spectrum target profiling:</b>
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<a href="/pcsubstance/?term=ML315[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML315</a> was submitted for assessing off-target
|
||
pharmacology using a Ricerca LeadProfiling® screen made up of 67 assays. The probe was
|
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assayed in duplicate at a concentration of 10 μM for all targets, and the following
|
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responses were noted as ≥ 50% inhibition or stimulation for biochemical assays (see
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<a class="figpopup" href="/books/NBK153503/table/ml315.t4/?report=objectonly" target="object" rid-figpopup="figml315t4" rid-ob="figobml315t4">Table 4</a>).</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml315t4"><a href="/books/NBK153503/table/ml315.t4/?report=objectonly" target="object" title="Table 4" class="img_link icnblk_img figpopup" rid-figpopup="figml315t4" rid-ob="figobml315t4"><img class="small-thumb" src="/books/NBK153503/table/ml315.t4/?report=thumb" src-large="/books/NBK153503/table/ml315.t4/?report=previmg" alt="Table 4. Off-target pharmacology data for ML315 highlighting targets with ≥ 50% inhibition or stimulation." /></a><div class="icnblk_cntnt"><h4 id="ml315.t4"><a href="/books/NBK153503/table/ml315.t4/?report=objectonly" target="object" rid-ob="figobml315t4">Table 4</a></h4><p class="float-caption no_bottom_margin">Off-target pharmacology data for ML315 highlighting targets with ≥ 50% inhibition or stimulation. </p></div></div></div></div></div><div id="ml315.s26"><h2 id="_ml315_s26_">4. Discussion</h2><div id="ml315.s27"><h3>4.1. Comparison to Existing Art and How the New Probe is an Improvement</h3><p>The new probe (<a href="/pcsubstance/?term=ML315[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML315</a><b>,</b>
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<a href="https://pubchem.ncbi.nlm.nih.gov/substance/99431981" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 99431981</a>, CID 46926514, <b>91</b>) has a pyrimidine core, which is a chemotype that is structurally distinct from prior probes (<a href="/pcsubstance/?term=ML106[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML106</a> and <a href="/pcsubstance/?term=ML167[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML167</a>) having quinazoline cores. <a href="/pcsubstance/?term=ML315[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML315</a> exhibits a 1,2-relationship between the substituents appended to the pyrimidine ring, while <a href="/pcsubstance/?term=ML106[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML106</a> and <a href="/pcsubstance/?term=ML167[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML167</a> display appended substituents on different rings of the fused bicyclic quinazoline. Though benzodioxolane substituents are found in both <a href="/pcsubstance/?term=ML315[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML315</a> and <a href="/pcsubstance/?term=ML106[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML106</a>, the benzodioxolane is directly attached to the pyrimidine in <a href="/pcsubstance/?term=ML315[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML315</a>, while it is bound to the benzene ring in <a href="/pcsubstance/?term=ML106[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML106</a>, providing these inhibitors with different molecular topologies. For instance, comparing the distances between the NH groups and oxygen atoms of the benzodioxolanes in each inhibitor, it is clear that these hydrogen-bonding groups are spread much further apart in <a href="/pcsubstance/?term=ML106[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML106</a> than in <a href="/pcsubstance/?term=ML315[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML315</a>. The divergent topological features of <a href="/pcsubstance/?term=ML315[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML315</a> and <a href="/pcsubstance/?term=ML106[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML106</a>, including the differences in arrangement of hydrogen-bond-capable functional groups, is expected to distinguish these probes from one another during binding events in both the Clk/Dyrk families and throughout the greater kinome, as each will fit differently into the binding pocket of a given enzyme. Furthermore, the effect of the benzodioxolane’s electron density on the basicity of the benzylamine is also expected to differ between the pyrimidine and quinazoline chemotypes. In the quinazoline (<a href="/pcsubstance/?term=ML106[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML106</a>), electron density is spread over the six additional atoms of the benzene ring, diminishing the effect on the benzylamine appended to the fused pyrimidine. However, in the pyrimidine (<a href="/pcsubstance/?term=ML315[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML315</a>), the electron-rich benzodioxolane and the benzylamine are both bound to the same heterocycle.</p><p>Although <a href="/pcsubstance/?term=ML167[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML167</a> (Entry 1 in <a class="figpopup" href="/books/NBK153503/table/ml315.t5/?report=objectonly" target="object" rid-figpopup="figml315t5" rid-ob="figobml315t5">Table 5</a>) selectively inhibits Clk4 (136 nM), none of the remaining Clk inhibitors appear to have single isoform selectivity (> 10-fold activity) against Clk1-Clk3; published Clk inhibitors either potently inhibit more than one isoform or have not been tested against all four isoforms. Therefore, until single-isoform-selective inhibitors can be developed, the parallel use of available inhibitors can still be used to gather biological data involving these kinases. The selectivity profile for the new probe differs from the profiles of previous probes in interesting and potentially useful ways. As mentioned above, <a href="/pcsubstance/?term=ML167[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML167</a> is an isoform-selective Clk4 inhibitor. Comparing entries 2 and 3, it may be seen that while this prior quinazoline probe <a href="/pcsubstance/?term=ML106[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML106</a> is a potent inhibitor of Clk1, Clk4, and Dyrk1A and not very potent against Clk2, the new pyrimidine probe <a href="/pcsubstance/?term=ML315[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML315</a> is a potent inhibitor of Clk1 and Clk4 and a moderate inhibitor of Clk2 and Dyrk 1A. Using <a href="/pcsubstance/?term=ML315[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML315</a> in parallel with previous probes <a href="/pcsubstance/?term=ML106[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML106</a> and <a href="/pcsubstance/?term=ML167[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML167</a> should allow researchers to address questions that would not be possible with the previous probes alone. For instance, employing <a href="/pcsubstance/?term=ML167[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML167</a> in a biochemical assay (Assay 1) would allow one to determine the effect of inhibiting Clk4 (136 nM). Administering <a href="/pcsubstance/?term=ML106[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML106</a> (Assay 2) would then allow one to inhibit Clk1, Clk4, and Dyrk1A (59 nM, 39 nM, and 62 nM, respectively). Differences between the results of Assay 1 and Assay 2 might be attributed to the lack of Clk1 and Dyrk1A activity, thereby helping to define the functions of these two kinases. Running the assay a third time (Assay 3) using <a href="/pcsubstance/?term=ML315[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML315</a> would further allow one to inhibit Clk1 (68 nM) and Clk4 (68 nM), while only moderately inhibiting Dyrk1A (282 nM) and Clk2 (231 nM). Differences between the results of Assay 2 and Assay 3 might in this case be attributed to the loss of Clk1 activity, allowing the functions of Clk1 and Dyrk1A to be further separated and understood. These differences might also result from the moderate inhibition of Clk2. Thus, by running the same assay with each of the three probes, researchers should be able to exclude isoforms from consideration and begin to hone in on which Clk or Dyrk kinase is responsible for particular biochemical observations. Furthermore, in advanced pharmacological studies, compounds with similar activity profiles could have distinctly different liabilities (metabolic instability, toxicity, etc.), making the availability of multiple chemotypes with different chemical structures advantageous.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml315t5"><a href="/books/NBK153503/table/ml315.t5/?report=objectonly" target="object" title="Table 5" class="img_link icnblk_img figpopup" rid-figpopup="figml315t5" rid-ob="figobml315t5"><img class="small-thumb" src="/books/NBK153503/table/ml315.t5/?report=thumb" src-large="/books/NBK153503/table/ml315.t5/?report=previmg" alt="Table 5. IC50 values (nM) against Clk and Dyrk kinases for quinazoline and pyrimidine chemotypes." /></a><div class="icnblk_cntnt"><h4 id="ml315.t5"><a href="/books/NBK153503/table/ml315.t5/?report=objectonly" target="object" rid-ob="figobml315t5">Table 5</a></h4><p class="float-caption no_bottom_margin">IC<sub>50</sub> values (nM) against Clk and Dyrk kinases for quinazoline and pyrimidine chemotypes. </p></div></div></div></div><div id="ml315.s28"><h2 id="_ml315_s28_">5. References</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="ml315.r1">Uhlen M, Ponten F. Antibody-based Proteomics for Human Tissue Profiling. <span><span class="ref-journal">Molecular & Cellular Proteomics. </span>2005;<span class="ref-vol">4</span>(4):384–393.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/15695805" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 15695805</span></a>]</div></dd><dt>2.</dt><dd><div class="bk_ref" id="ml315.r2">Prasad J, Manley JL. <span><span class="ref-journal">Mol Cell Bio. </span>2003;<span class="ref-vol">23</span>:4139–4149.</span> [<a href="/pmc/articles/PMC156123/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC156123</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/12773558" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 12773558</span></a>]</div></dd><dt>3.</dt><dd><div class="bk_ref" id="ml315.r3">Mott BT, Tanega C, Shen M, Maloney DJ, Shinn P, Leister W, Marugan JJ, Inglese J, Austin CP, Mistelli T, Auld DS, Thomas CJ. <span><span class="ref-journal">Bioorg Med Chem Lett. </span>2009;<span class="ref-vol">19</span>:6700–6705.</span> [<a href="/pmc/articles/PMC2807730/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2807730</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/19837585" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19837585</span></a>]</div></dd><dt>4.</dt><dd><div class="bk_ref" id="ml315.r4">Rosenthal AS, Tanega C, Shen M, Mott BT, Bougie JM, Nguyen D.-T, Misteli T, Auld DS, Maloney DJ, Thomas CJ. <span><span class="ref-journal">Bioorg Med Chem Lett. </span>2011;<span class="ref-vol">21</span>:3152–3158.</span> [<a href="/pmc/articles/PMC3085634/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3085634</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/21450467" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 21450467</span></a>]</div></dd><dt>5.</dt><dd><div class="bk_ref" id="ml315.r5">Muraki M, Ohkawara B, Hosoya T, Onogi H, Koizumi J, Koizumi T, Sumi K, Yomoda J-i, Murray MV, Kimura H, Furuichi K, Shibuya H, Drainer AR, Suzuki M, Hagiwara M. <span><span class="ref-journal">J Biol Chem. </span>2004;<span class="ref-vol">279</span>:24246–24254.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/15010457" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 15010457</span></a>]</div></dd><dt>6.</dt><dd><div class="bk_ref" id="ml315.r6">Hekimi S, McBride K, Hihi AK, Kianicka I, Wang Y, Hayes SL, Guimond M.-P, Sevigny G, Dumas D, Smith J. WO 2008014602. <span></span></div></dd><dt>7.</dt><dd><div class="bk_ref" id="ml315.r7">Fedorov O, Huber K, Eisenreich A, Filippakopoulos P, King O, Bullock AN, Szklarczyk D, Jensen LJ, Fabbro D, Trappe J, Rauch U, Bracher F, Knapp S. <span><span class="ref-journal">Chem Biol. </span>2011;<span class="ref-vol">18</span>:67–76.</span> [<a href="/pmc/articles/PMC3145970/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3145970</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/21276940" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 21276940</span></a>]</div></dd><dt>8.</dt><dd><div class="bk_ref" id="ml315.r8">Debdad M, Carreaux F, Renault S, Soundararajan M, Fedorov O, Filippakopoulos P, Lozach O, Babault L, Tahtouh T, Baratte B, Ogawa Y, Hagiwara M, Eisenreich A, Rauch U, Knapp S, Meijer L, Bazureau J.-P. <span><span class="ref-journal">J Med Chem. </span>2011;<span class="ref-vol">54</span>:4172–4186.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/21615147" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 21615147</span></a>]</div></dd><dt>9.</dt><dd><div class="bk_ref" id="ml315.r9">Gockler N, Jofre G, Papadopoulos C, Soppa U, Tejedor FJ, Becker W. <span><span class="ref-journal">FEBS Journal. </span>2009;<span class="ref-vol">276</span>:6324–6337.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/19796173" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19796173</span></a>]</div></dd><dt>10.</dt><dd><div class="bk_ref" id="ml315.r10">Wang S, Wood G, Duncan KW, Meades C, Gibson D, McLachlan JC, Perry A, Blake D, Zheleva DI, Fischer P. <span class="ref-journal">PCT Int. Appl.</span> 2005. p. 105. CODEN: PIXXD2 WO 2005042525 A1 20050512 CA 142:463748 AN 2005:409513 CAPLUS.</div></dd><dt>11.</dt><dd><div class="bk_ref" id="ml315.r11">Aubé J, Thomas C, Schoenen FJ, Auld D, Coombs TC, Shen M, Tanega CUS. Provisional Application entitled “CLK and DYRK Inhibitors and Methods of Use Thereof” Serial No : 61/584,935. Filing Date: January 10, 2012.</div></dd><dt>12.</dt><dd><div class="bk_ref" id="ml315.r12">Fan F, Wood KV. <span><span class="ref-journal">Assay Drug Dev Technol. </span>2007;<span class="ref-vol">5</span>:127–36.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/17355205" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17355205</span></a>]</div></dd><dt>13.</dt><dd><div class="bk_ref" id="ml315.r13">Schroter T, Minond D, Weiser A, Dao C, Habel J, Spicer T, Chase P, Baillargeon P, Scampavia L, Schurer S, Chung C, Mader C, Southern M, Tsinoremas N, LoGrasso P, Hodder P. <span><span class="ref-journal">J Biomol Screen. </span>2008;<span class="ref-vol">13</span>:17–28.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/18227223" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18227223</span></a>]</div></dd><dt>14.</dt><dd><div class="bk_ref" id="ml315.r14">Singh P, Harden BJ, Lillywhite BJ, Broad PM. <span><span class="ref-journal">Assay Drug Dev Technol. </span>2004;<span class="ref-vol">2</span>:161–9.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/15165512" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 15165512</span></a>]</div></dd><dt>15.</dt><dd><div class="bk_ref" id="ml315.r15">Inglese J, Auld DS, Jadhav A, Johnson RL, Simeonov A, Yasgar A, Zheng W, Austin CP. <span><span class="ref-journal">Proc Natl Acad Sci USA. </span>2006;<span class="ref-vol">103</span>:11473–8.</span> [<a href="/pmc/articles/PMC1518803/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC1518803</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/16864780" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 16864780</span></a>]</div></dd><dt>16.</dt><dd><div class="bk_ref" id="ml315.r16">Tanega C, Shen M, Mott BT, Thomas CJ, MacArthur R, Inglese J, Auld DS. <span><span class="ref-journal">Assay & Drug Dev Tech. </span>2010;<span class="ref-vol">7</span>:606–614.</span> [<a href="/pmc/articles/PMC3096547/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3096547</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/20059377" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 20059377</span></a>]</div></dd><dt>17.</dt><dd><div class="bk_ref" id="ml315.r17">Reigan P, Gbaj A, Stratford IJ, Bryce RA, Freeman S. <span><span class="ref-journal">Eur J Med Chem. </span>2008;<span class="ref-vol">43</span>:1248–1260.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/17870212" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17870212</span></a>]</div></dd><dt>18.</dt><dd><div class="bk_ref" id="ml315.r18">Woolard J, Wang W.-Y, Bevan HS, Qiu Y, Morbidelli L, Pritchard-Jones RO, Cui T.-G, Sugiono M, Waine E, Perrin R, Foster R, Digby-Bell J, Shields JD, Whittles CE, Mushens RE, Gillatt DA, Ziche M, Harper SJ, Bates DO. <span><span class="ref-journal">Cancer Res. </span>2004;<span class="ref-vol">64</span>:7822–7835.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/15520188" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 15520188</span></a>]</div></dd></dl></div><div id="bk_toc_contnr"></div></div></div>
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<div xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance"></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Views</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="PDF_download" id="Shutter"></a></div><div class="portlet_content"><ul xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="simple-list"><li><a href="/books/NBK153503/?report=reader">PubReader</a></li><li><a href="/books/NBK153503/?report=printable">Print View</a></li><li><a data-jig="ncbidialog" href="#_ncbi_dlg_citbx_NBK153503" data-jigconfig="width:400,modal:true">Cite this Page</a><div id="_ncbi_dlg_citbx_NBK153503" style="display:none" title="Cite this Page"><div class="bk_tt">Coombs TC, Tanega C, Shen M, et al. Identification of selective inhibitors of cdc2-like kinases 1 and 4 (Clk1, Clk4) 2012 Apr 16 [Updated 2013 May 8]. 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="#ml315.s1" ref="log$=inpage&link_id=inpage">Probe Structure & Characteristics</a></li><li><a href="#ml315.s2" ref="log$=inpage&link_id=inpage">Recommendations for Scientific Use of the Probe</a></li><li><a href="#ml315.s3" ref="log$=inpage&link_id=inpage">Materials and Methods</a></li><li><a href="#ml315.s21" ref="log$=inpage&link_id=inpage">Results</a></li><li><a href="#ml315.s26" ref="log$=inpage&link_id=inpage">Discussion</a></li><li><a href="#ml315.s28" ref="log$=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=3047408" ref="log$=recordlinks">PMC</a><div class="brieflinkpop offscreen_noflow">PubMed Central citations</div></li><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pcsubstance&DbFrom=books&Cmd=Link&LinkName=books_pcsubstance&IdsFromResult=3047408" ref="log$=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=3047408" ref="log$=recordlinks">PubMed</a><div class="brieflinkpop offscreen_noflow">Links to PubMed</div></li></ul></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Similar articles in PubMed</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="PBooksDiscovery_RA" id="Shutter"></a></div><div class="portlet_content"><ul><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/23642479" ref="ordinalpos=1&linkpos=1&log$=relatedarticles&logdbfrom=pubmed">Small-molecule pyrimidine inhibitors of the cdc2-like (Clk) and dual specificity tyrosine phosphorylation-regulated (Dyrk) kinases: development of chemical probe ML315.</a><span class="source">[Bioorg Med Chem Lett. 2013]</span><div class="brieflinkpop offscreen_noflow">Small-molecule pyrimidine inhibitors of the cdc2-like (Clk) and dual specificity tyrosine phosphorylation-regulated (Dyrk) kinases: development of chemical probe ML315.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Coombs TC, Tanega C, Shen M, Wang JL, Auld DS, Gerritz SW, Schoenen FJ, Thomas CJ, Aubé J. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Bioorg Med Chem Lett. 2013 Jun 15; 23(12):3654-61. 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Epub 2024 Feb 19.</em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/21433360" ref="ordinalpos=1&linkpos=5&log$=relatedreviews&logdbfrom=pubmed"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> A high-throughput screen for pre-mRNA splicing modulators.</a><span class="source">[Probe Reports from the NIH Mol...]</span><div class="brieflinkpop offscreen_noflow"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> A high-throughput screen for pre-mRNA splicing modulators.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Auld D, Shen M, Thomas C. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Probe Reports from the NIH Molecular Libraries Program. 2010</em></div></div></li></ul><a class="seemore" href="/sites/entrez?db=pubmed&cmd=link&linkname=pubmed_pubmed_reviews&uid=23926621" ref="ordinalpos=1&log$=relatedreviews_seeall&logdbfrom=pubmed">See reviews...</a><a class="seemore" href="/sites/entrez?db=pubmed&cmd=link&linkname=pubmed_pubmed&uid=23926621" ref="ordinalpos=1&log$=relatedarticles_seeall&logdbfrom=pubmed">See all...</a></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Recent Activity</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="recent_activity" id="Shutter"></a></div><div class="portlet_content"><div xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" id="HTDisplay" class=""><div class="action"><a href="javascript:historyDisplayState('ClearHT')">Clear</a><a href="javascript:historyDisplayState('HTOff')" class="HTOn">Turn Off</a><a href="javascript:historyDisplayState('HTOn')" class="HTOff">Turn On</a></div><ul id="activity"><li class="ra_rcd ralinkpopper two_line"><a class="htb ralinkpopperctrl" ref="log$=activity&linkpos=1" href="/portal/utils/pageresolver.fcgi?recordid=67d66b6c2f30673f7bf651e6">Identification of selective inhibitors of cdc2-like kinases 1 and 4 (Clk1, Clk4)...</a><div class="ralinkpop offscreen_noflow">Identification of selective inhibitors of cdc2-like kinases 1 and 4 (Clk1, Clk4) - 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