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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 activators for the M2 isoform of human pyruvate kinase" /><meta name="citation_publisher" content="National Center for Biotechnology Information (US)" /><meta name="citation_date" content="2010/08/06" /><meta name="citation_author" content="Douglas Auld" /><meta name="citation_author" content="Min Shen" /><meta name="citation_author" content="Amanda P Skoumbourdis" /><meta name="citation_author" content="Jian-kang Jiang" /><meta name="citation_author" content="Matthew Boxer" /><meta name="citation_author" content="Noel Southall" /><meta name="citation_author" content="James Inglese" /><meta name="citation_author" content="Craig Thomas" /><meta name="citation_pmid" content="21433354" /><meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK47338/" /><link rel="schema.DC" href="http://purl.org/DC/elements/1.0/" /><meta name="DC.Title" content="Identification of activators for the M2 isoform of human pyruvate kinase" /><meta name="DC.Type" content="Text" /><meta name="DC.Publisher" content="National Center for Biotechnology Information (US)" /><meta name="DC.Contributor" content="Douglas Auld" /><meta name="DC.Contributor" content="Min Shen" /><meta name="DC.Contributor" content="Amanda P Skoumbourdis" /><meta name="DC.Contributor" content="Jian-kang Jiang" /><meta name="DC.Contributor" content="Matthew Boxer" /><meta name="DC.Contributor" content="Noel Southall" /><meta name="DC.Contributor" content="James Inglese" /><meta name="DC.Contributor" content="Craig Thomas" /><meta name="DC.Date" content="2010/08/06" /><meta name="DC.Identifier" content="https://www.ncbi.nlm.nih.gov/books/NBK47338/" /><meta name="description" content="The expression of human pyruvate kinase M2 (hPK-M2) in cancer cells appears to be critical for tumor cell growth and proliferation in vivo. 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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="#__NBK47338_dtls__">Show details</a><div style="display:none" class="ui-widget" id="__NBK47338_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/ml084/" title="Previous page in this title">< Prev</a><a class="active page_link next" href="/books/n/mlprobe/ml081/" 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="_NBK47338_"><span class="title" itemprop="name">Identification of activators for the M2 isoform of human pyruvate kinase</span></h1><p class="contrib-group"><span itemprop="author">Douglas Auld</span>, <span itemprop="author">Min Shen</span>, <span itemprop="author">Amanda P Skoumbourdis</span>, <span itemprop="author">Jian-kang Jiang</span>, <span itemprop="author">Matthew Boxer</span>, <span itemprop="author">Noel Southall</span>, <span itemprop="author">James Inglese</span>, and <span itemprop="author">Craig Thomas</span>.</p><a data-jig="ncbitoggler" href="#__NBK47338_ai__" style="border:0;text-decoration:none">Author Information and Affiliations</a><div style="display:none" class="ui-widget" id="__NBK47338_ai__"><p class="contrib-group"><h4>Authors</h4><span itemprop="author">Douglas Auld</span>, <span itemprop="author">Min Shen</span>, <span itemprop="author">Amanda P Skoumbourdis</span>, <span itemprop="author">Jian-kang Jiang</span>, <span itemprop="author">Matthew Boxer</span>, <span itemprop="author">Noel Southall</span>, <span itemprop="author">James Inglese</span>, and <span itemprop="author">Craig Thomas</span>.</p><h4>Affiliations</h4><div class="affiliation"><sup>1</sup> NIH Chemical Genomics Center</div></div><p class="small">Received: <span itemprop="datePublished">April 16, 2009</span>; Last Update: <span itemprop="dateModified">August 6, 2010</span>.</p></div><div class="jig-ncbiinpagenav body-content whole_rhythm" data-jigconfig="allHeadingLevels: ['h2'],smoothScroll: false" itemprop="text"><div id="_abs_rndgid_" itemprop="description"><p> The expression of human pyruvate kinase M2 (hPK-M2) in cancer cells appears to be critical for tumor cell growth and proliferation in vivo. Because the PK-M2 isoform is expressed in all cancer cells studied, it represents a target for drug development that could potentially enable tumor cells to return to a normal state of metabolism. If this novel strategy for targeting malignancy were successful, it would be applicable to diverse types of cancer. The probes ML083 (CID-650361) and ML082 (CID-654376) are members of a series of highly specific allosteric activators for the tumor-specific isoform of human pyruvate kinase (M2 isoform). Both probes affect the cooperativity of phosphoenolpyruvate (PEP) binding with little effect on adenosine diphosphate (ADP) binding in a manner similar to FBP.</p></div><div class="h2"></div><p>
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<b>Assigned Assay Grant #:</b> 1 R03 MH085679-01 </p><p>
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<b>Screening Center Name & PI:</b> NIH Chemical Genomics Center,
|
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Christopher Austin </p><p>
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<b>Chemistry Center Name & PI:</b> NIH Chemical Genomics Center,
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Christopher Austin </p><p>
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<b>Assay Submitter & Institution:</b> Dr. Matthew George Vander Heiden,
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Harvard </p><p><b>PubChem Primary Bioassay Identifier (AID):</b>
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<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1631" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1631</a></p><div id="ml083.probestruct"><h2 id="_ml083_probestruct_">Probe Structure and Characteristics</h2><div id="ml083.tu1" class="table"><h3><span class="title">Probe 1 Structure & Characteristics</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK47338/table/ml083.tu1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml083.tu1_lrgtbl__"><table class="no_top_margin"><tbody><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">PubChem CID</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CID-650361</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">SID</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/847943" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-847943</a></td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Internal ID</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC-00030335</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Molecular Weight</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">454.52</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Molecular Formula</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">C<sub>19</sub>H<sub>22</sub>N<sub>2</sub>O<sub>7</sub>S<sub>2</sub></td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">XLogP</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">1.4</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">H-Bond Donor</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">H-Bond Acceptor</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">9</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Rotatable Bond Count</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">5</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Exact Mass</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">454.086842</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Topological Polar Surface Area</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">103</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Heavy Atom Count</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">30</td></tr></tbody></table></div></div><div id="ml083.fu1" class="figure"><div class="graphic"><img src="/books/NBK47338/bin/ml083fu1.jpg" alt="Image ml083fu1" /></div></div><div id="ml083.tu2" class="table"><h3><span class="title">PubChem Primary Bioassay Identifier (AID): 1631</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK47338/table/ml083.tu2/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml083.tu2_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml083.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CID/ML</th><th id="hd_h_ml083.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Target Name</th><th id="hd_h_ml083.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">IC50/EC 50 (nM) [SID,
|
||
AID]</th><th id="hd_h_ml083.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Anti-target Name(s)</th><th id="hd_h_ml083.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">IC50/EC50 (μM) [SID,
|
||
AID]</th><th id="hd_h_ml083.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Selectivity</th><th id="hd_h_ml083.tu2_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Secondary Assay(s) Name: IC50/EC50 (nM)
|
||
[SID, AID]</th></tr></thead><tbody><tr><td headers="hd_h_ml083.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">650361/<a href="/pcsubstance/?term=ML083[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML083</a></td><td headers="hd_h_ml083.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">hPK-M2</td><td headers="hd_h_ml083.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0.6 ± 0.3 [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/847943" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-847943</a>,
|
||
<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1540" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1540</a>]</td><td headers="hd_h_ml083.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">hPK-R</td><td headers="hd_h_ml083.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">30%@ 57 μM
|
||
[<a href="https://pubchem.ncbi.nlm.nih.gov/substance/847943" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-847943</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1543" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1543</a>]</td><td headers="hd_h_ml083.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">≥ 30-fold</td><td headers="hd_h_ml083.tu2_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">PK-LDH, [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/847943" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-847943</a>,
|
||
<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1543" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1543</a>]</td></tr><tr><td headers="hd_h_ml083.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"></td><td headers="hd_h_ml083.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"></td><td headers="hd_h_ml083.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"></td><td headers="hd_h_ml083.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">hPK-L</td><td headers="hd_h_ml083.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Inactive[<a href="https://pubchem.ncbi.nlm.nih.gov/substance/847943" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-847943</a>,
|
||
<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1541" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1541</a>]</td><td headers="hd_h_ml083.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">≥ 100-fold</td><td headers="hd_h_ml083.tu2_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">PK-LDH, [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/847943" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-847943</a>,
|
||
<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1541" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1541</a>]</td></tr><tr><td headers="hd_h_ml083.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"></td><td headers="hd_h_ml083.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"></td><td headers="hd_h_ml083.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"></td><td headers="hd_h_ml083.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">hPK-M1</td><td headers="hd_h_ml083.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Inactive[<a href="https://pubchem.ncbi.nlm.nih.gov/substance/847943" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-847943</a>,
|
||
<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1542" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1542</a>]</td><td headers="hd_h_ml083.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">≥ 100-fold</td><td headers="hd_h_ml083.tu2_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">PK-LDH, [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/847943" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-847943</a>,
|
||
<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1542" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1542</a>]</td></tr></tbody></table></div></div><div id="ml083.tu3" class="table"><h3><span class="title">Probe 2 Structure & Characteristics</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK47338/table/ml083.tu3/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml083.tu3_lrgtbl__"><table class="no_top_margin"><tbody><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">PubChem CID</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CID-654376</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">SID</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/851783" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-851783</a></td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Internal ID</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC-00031955</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Molecular Weight</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">327.38</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Molecular Formula</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">C17H14FN3OS</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">XLogP</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">3.1</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">H-Bond Donor</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">H-Bond Acceptor</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">4</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Rotatable Bond Count</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">2</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Exact Mass</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">327.08</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Topological Polar Surface Area</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">37.6</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Heavy Atom Count</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">23</td></tr></tbody></table></div></div><div id="ml083.fu2" class="figure"><div class="graphic"><img src="/books/NBK47338/bin/ml083fu2.jpg" alt="Image ml083fu2" /></div></div><div id="ml083.tu4" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK47338/table/ml083.tu4/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml083.tu4_lrgtbl__"><table><thead><tr><th id="hd_h_ml083.tu4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">CID/ML</th><th id="hd_h_ml083.tu4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Target Name</th><th id="hd_h_ml083.tu4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">IC50/EC 50 (nM) [SID,
|
||
AID]</th><th id="hd_h_ml083.tu4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Anti-target Name(s)</th><th id="hd_h_ml083.tu4_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">IC50/EC50 (μM) [SID,
|
||
AID]</th><th id="hd_h_ml083.tu4_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Selectivity</th><th id="hd_h_ml083.tu4_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Secondary Assay(s) Name: IC50/EC50 (nM)
|
||
[SID, AID]</th></tr></thead><tbody><tr><td headers="hd_h_ml083.tu4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">CID-654376/<a href="/pcsubstance/?term=ML082[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML082</a></td><td headers="hd_h_ml083.tu4_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">hPK-M2</td><td headers="hd_h_ml083.tu4_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0.2 ± 0.1 [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/851783" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-851783</a>,
|
||
<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1540" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1540</a>]</td><td headers="hd_h_ml083.tu4_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">hPK-L</td><td headers="hd_h_ml083.tu4_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">4 [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/851783" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-851783</a>,
|
||
<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1541" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1541</a>]</td><td headers="hd_h_ml083.tu4_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">>10 fold</td><td headers="hd_h_ml083.tu4_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">PK-LDH, [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/851783" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-851783</a>,
|
||
<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1541" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1541</a>]</td></tr><tr><td headers="hd_h_ml083.tu4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"></td><td headers="hd_h_ml083.tu4_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"></td><td headers="hd_h_ml083.tu4_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"></td><td headers="hd_h_ml083.tu4_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">hPK-R</td><td headers="hd_h_ml083.tu4_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">4 [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/851783" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-851783</a>,
|
||
<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1543" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1543</a>]</td><td headers="hd_h_ml083.tu4_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">>10 fold</td><td headers="hd_h_ml083.tu4_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">PK-LDH, [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/851783" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-851783</a>,
|
||
<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1543" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1543</a>]</td></tr><tr><td headers="hd_h_ml083.tu4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"></td><td headers="hd_h_ml083.tu4_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"></td><td headers="hd_h_ml083.tu4_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"></td><td headers="hd_h_ml083.tu4_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">hPK-M1</td><td headers="hd_h_ml083.tu4_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Inactive [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/851783" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-851783</a>,
|
||
<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1542" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1542</a>]</td><td headers="hd_h_ml083.tu4_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">>100 fold</td><td headers="hd_h_ml083.tu4_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">PK-LDH, [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/851783" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-851783</a>,
|
||
<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1542" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1542</a>]</td></tr></tbody></table></div></div></div><div id="ml083.rationale"><h2 id="_ml083_rationale_">Recommendations for the scientific use of these probes</h2><div id="ml083.s5"><h3>Specific Aim</h3><p>We aimed to discover and optimize selective modulators of human pyruvate kinase
|
||
M2 (hPK-M2), a splice isoform of pyruvate kinase that is specifically expressed
|
||
in tumor cells. The research has the following specific aims: (1) To identify both activators and
|
||
inhibitors of human M2 PK using a quantitative high-throughput screening
|
||
approach against the MLSMR, containing 300,000 small molecules. (2) To confirm the potency of these
|
||
compounds in a panel of secondary assays, including selectivity assays for the
|
||
human R/L and M1 isoforms of pyruvate kinase which are expressed in most
|
||
differentiated tissues.</p></div><div id="ml083.s6"><h3>Significance</h3><p>Expression of hPK-M2 in cancer cells appears critical for tumor cell growth and
|
||
proliferation <i>in vivo</i>. In addition, the ability of PK-M2 to be
|
||
regulated by both FBP and tyrosine phosphorylated proteins is necessary for
|
||
cancer cell proliferation. Because PK-M2 is the sole pyruvate kinase isoform
|
||
expressed in all cancer cells studied, it represents a target for drug
|
||
development that would enable the inhibition of glucose metabolism in a
|
||
relatively tumor-specific manner. If this novel strategy for targeting
|
||
malignancy were successful, it would be applicable to diverse types of
|
||
cancer.</p></div><div id="ml083.s7"><h3>Rationale</h3><p>PK is a tetrameric enzyme composed of four identical monomers that form a dimer
|
||
of dimers in the final tetrameric structure. In humans, the M2, L, and R
|
||
isozymes are activated by fructose-1,6-bis phosphate (FBP) that binds to a
|
||
flexible loop region at the interface of the two dimers. Activation of PK shifts
|
||
the enzyme to a state showing high affinity for PEP and relieves the cooperative
|
||
kinetics of PEP binding found in the unactivated form (<a class="bk_pop" href="#ml083.r1">1</a>). In contrast, the M1 isoform is not regulated by
|
||
FBP and displays only high affinity PEP binding similar to the activated state
|
||
of PK. Importantly, the differentially spliced exons that distinguish PK-M1 from
|
||
PK-M2 are identical in size and encode a 56 amino acid stretch that covers the
|
||
activation loop that allows PK-M2, but not PK-M1, to be allosterically activated
|
||
by FBP (<a class="bk_pop" href="#ml083.r3">3</a>).</p><p>Tumor cells undergo a metabolic transformation that is required to supply the
|
||
biochemical precursors necessary for rapid cell growth and proliferation. Otto
|
||
Warburg was first to demonstrate in the 1920s that tumor cells show aberrant
|
||
energy metabolism and noted that cancer cells produce lactate even under aerobic
|
||
conditions (<a class="bk_pop" href="#ml083.r2" data-bk-pop-others="ml083.r3 ml083.r4">2–4</a>). The property of aerobic glycolysis in tumor cells
|
||
has been termed the “Warburg effect.” This work has
|
||
spawned generations of research on tumor metabolism and the high rate of glucose
|
||
used by tumors is exploited clinically today through the identification of tumor
|
||
cells using 18-fluorodeoxyglucose-PET scanning. This metabolic transformation of
|
||
tumor cells can be achieved by altering the expression of metabolic enzymes.
|
||
Current evidence suggests that expression of PK-M2 in tumor cells has a large
|
||
influence on this process, and that this is due to the unique allosteric
|
||
regulation of PK-M2 (<a class="bk_pop" href="#ml083.r2">2</a>,<a class="bk_pop" href="#ml083.r3">3</a>). In a recent study it was shown
|
||
that knock-down of PK-M2 and expression of PK-M1 in tumor cell lines was
|
||
sufficient to relieve the Warburg effect and rescue normal cellular respiration
|
||
(<a class="bk_pop" href="#ml083.r2">2</a>). In this study, tumor cells
|
||
engineered to express PK-M1 showed a reduced ability to form tumors in nude
|
||
mouse xenografts. As well, in related study, a link between PK-M2 and growth
|
||
factors that produce phosphotyrosine polypeptides in tumor cells was
|
||
demonstrated (<a class="bk_pop" href="#ml083.r3">3</a>). In that study it
|
||
was found that various phosphotyrosine peptides can bind to PK-M2 near the
|
||
activation loop, which results in the removal of FBP from the enzyme which
|
||
effectively down-regulates PK-M2 activity. This cascade leads to a building up
|
||
of glycolytic intermediates that can be diverted towards nucleotide and lipid
|
||
biosynthesis required for cell growth and proliferation.<sup>3</sup> These
|
||
studies suggest that growth factor signaling decreases PK-M2 activity, and that
|
||
this decrease in activity contributes to both the Warburg effect (<a class="bk_pop" href="#ml083.r4">4</a>) and the ability of tumor cells to
|
||
proliferate. Taken together, these studies imply a therapeutic strategy where
|
||
activation of PK-M2 should return normal cellular metabolism in a manner similar
|
||
to the experiments described above where tumor cells were rescued by the
|
||
expression of the constitutively active PK-M1 isozyme.</p><div id="ml083.fu3" class="figure bk_fig"><div class="graphic"><img src="/books/NBK47338/bin/ml083fu3.jpg" alt="A) Shown is normal respiration occurring in non-cancerous cell-types" /></div><div class="caption"><p><b>A)</b> Shown is normal respiration occurring in non-cancerous
|
||
cell-types. PK-M1 is constitutively active and acts primarily to drive
|
||
ATP formation. <b>B)</b> Shown is altered metabolism that occurs
|
||
in cancer cells. In this case, the activity of the PK-M2 isoform is
|
||
activated by phosphorylated sugars (e.g. FBP) and inhibited by
|
||
tyrosine-phosphorylated proteins derived from growth factor signaling.
|
||
The decrease in activity of PK-M2 in response to growth signals is
|
||
predicted to build-up intermediates of glycolysis and feed proliferation
|
||
of the cancer cells.</p></div></div></div></div><div id="ml083.s8"><h2 id="_ml083_s8_">Assay Implementation and Screening</h2><div id="ml083.s9"><h3>PubChem Bioassay Name</h3><p><i>Dmel</i> lipid storage</p></div><div id="ml083.s10"><h3>List of PubChem bioassay identifiers generated for this screening project
|
||
(AIDS)</h3><div id="ml083.tu5" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK47338/table/ml083.tu5/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml083.tu5_lrgtbl__"><table><thead><tr><th id="hd_h_ml083.tu5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">AID</th><th id="hd_h_ml083.tu5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Target</th><th id="hd_h_ml083.tu5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Concentration</th><th id="hd_h_ml083.tu5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Bioassay type</th></tr></thead><tbody><tr><td headers="hd_h_ml083.tu5_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1631" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1631</a></td><td headers="hd_h_ml083.tu5_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">hPKM2 Activator</td><td headers="hd_h_ml083.tu5_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">57 μM to 0.4 nM</td><td headers="hd_h_ml083.tu5_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Primary qHTS</td></tr><tr><td headers="hd_h_ml083.tu5_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1634" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1634</a></td><td headers="hd_h_ml083.tu5_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">hPKM2 Inhibitor</td><td headers="hd_h_ml083.tu5_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">57 μM to 0.4 nM</td><td headers="hd_h_ml083.tu5_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Primary qHTS</td></tr><tr><td headers="hd_h_ml083.tu5_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1540" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1540</a></td><td headers="hd_h_ml083.tu5_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">hPKM2</td><td headers="hd_h_ml083.tu5_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">57 μM to 0.4 nM</td><td headers="hd_h_ml083.tu5_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Confirmatory</td></tr><tr><td headers="hd_h_ml083.tu5_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1541" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1541</a></td><td headers="hd_h_ml083.tu5_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">hPKL</td><td headers="hd_h_ml083.tu5_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">57 μM to 0.4 nM</td><td headers="hd_h_ml083.tu5_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Selectivity</td></tr><tr><td headers="hd_h_ml083.tu5_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1542" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1542</a></td><td headers="hd_h_ml083.tu5_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">hPKM1</td><td headers="hd_h_ml083.tu5_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">57 μM to 0.4 nM</td><td headers="hd_h_ml083.tu5_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Selectivity</td></tr><tr><td headers="hd_h_ml083.tu5_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1543" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1543</a></td><td headers="hd_h_ml083.tu5_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">hPKR</td><td headers="hd_h_ml083.tu5_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">57 μM to 0.4 nM</td><td headers="hd_h_ml083.tu5_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Selectivity</td></tr></tbody></table></div></div></div><div id="ml083.s11"><h3>Primary Assay Description as defined in PubChem</h3><div id="ml083.s12"><h4>Overview</h4><p>To develop a robust assay for PK we took advantage of a well-utilized
|
||
luminescent assay detection system for protein kinases (<a class="bk_pop" href="#ml083.r5">5</a>,<a class="bk_pop" href="#ml083.r6">6</a>).
|
||
Typically, these assays use the ATP-dependent reaction catalyzed by firefly
|
||
luciferase to measure ATP depletion by protein kinases. In the present assay
|
||
we applied this assay to measure ATP production catalyzed by PK. This
|
||
provides for a robust increase in luminescent signal upon ATP product
|
||
formation (<a class="figpopup" href="/books/NBK47338/figure/ml083.f1/?report=objectonly" target="object" rid-figpopup="figml083f1" rid-ob="figobml083f1">Figure 2</a>) (<a class="bk_pop" href="#ml083.r7">7</a>). The primary high-throughput
|
||
screen was performed in 1536-well microtiter plates using 4 uL/well assay
|
||
volume with final concentrations of 0.1 nM human PK M2, 0.5 mM PEP, 0.1 mM
|
||
ADP in assay buffer that contained 50 mM Imidazole pH 7.2, 50 mM KCl, 7 mM
|
||
MgCl2, 0.01% Tween, 0.05% BSA. For compound transfer
|
||
we used a pintool equipped with a 1536 pins that each transferred 23 nL of a
|
||
DMSO solution containing compound. The enzymatic reaction was allowed to
|
||
proceed for 60 minutes and then ATP was detected using a bioluminescent
|
||
detection reagent containing D-luciferin and firefly luciferase (Kinase-Glo,
|
||
Promega) (<a class="bk_pop" href="#ml083.r8">8</a>). The detection
|
||
reagent also effectively stopped the PK reaction. Luminescence was then
|
||
measured on the Perkin Elmer Viewlux using an exposure of 2 secs (with 2X
|
||
binning). The final concentration of DMSO in the enzyme assay is
|
||
0.5% and was found not to affect the assay signal.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml083f1" co-legend-rid="figlgndml083f1"><a href="/books/NBK47338/figure/ml083.f1/?report=objectonly" target="object" title="Figure 2" class="img_link icnblk_img figpopup" rid-figpopup="figml083f1" rid-ob="figobml083f1"><img class="small-thumb" src="/books/NBK47338/bin/ml083f1.gif" src-large="/books/NBK47338/bin/ml083f1.jpg" alt="Figure 2. Pyruvate kinase-luciferase coupled assay." /></a><div class="icnblk_cntnt" id="figlgndml083f1"><h4 id="ml083.f1"><a href="/books/NBK47338/figure/ml083.f1/?report=objectonly" target="object" rid-ob="figobml083f1">Figure 2</a></h4><p class="float-caption no_bottom_margin">Pyruvate kinase-luciferase coupled assay. </p></div></div><p>All compounds were screened using a qHTS approach, where compounds are
|
||
assayed using at least seven concentrations to generate
|
||
concentration-response curves for every compounds (<a class="bk_pop" href="#ml083.r7">7</a>). 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
|
||
(<a class="bk_pop" href="#ml083.r9">9</a>). 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 successive 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></div><div id="ml083.s13"><h3>Summary of the Primary Screen</h3><div id="ml083.s14"><h4>Assay protocol</h4><p>The optimized 1536-well protocol is given in <a class="figpopup" href="/books/NBK47338/table/ml083.t1/?report=objectonly" target="object" rid-figpopup="figml083t1" rid-ob="figobml083t1">Table 1</a>.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml083t1"><a href="/books/NBK47338/table/ml083.t1/?report=objectonly" target="object" title="Table 1" class="img_link icnblk_img figpopup" rid-figpopup="figml083t1" rid-ob="figobml083t1"><img class="small-thumb" src="/books/NBK47338/table/ml083.t1/?report=thumb" src-large="/books/NBK47338/table/ml083.t1/?report=previmg" alt="Table 1. Final 1536-well assay protocol." /></a><div class="icnblk_cntnt"><h4 id="ml083.t1"><a href="/books/NBK47338/table/ml083.t1/?report=objectonly" target="object" rid-ob="figobml083t1">Table 1</a></h4><p class="float-caption no_bottom_margin">Final 1536-well assay protocol. </p></div></div><div id="ml083.f2" class="figure bk_fig"><div class="graphic"><img src="/books/NBK47338/bin/ml083f2.jpg" alt="Figure 2. qHTS Performance Summary." /></div><h3><span class="label">Figure 2</span><span class="title">qHTS Performance Summary</span></h3></div><div id="ml083.s15"><h5>Identification of hPK M2 Activators</h5><p>Following the qHTS the CRC data was subjected to a classification scheme
|
||
to rank the quality of the CRCs as described by Inglese and coworkers
|
||
(<a class="bk_pop" href="#ml083.r7">7</a>) (see <a class="figpopup" href="/books/NBK47338/figure/ml083.f3/?report=objectonly" target="object" rid-figpopup="figml083f3" rid-ob="figobml083f3">scheme 1</a>). Briefly, CRCs are
|
||
placed into four classes. Class 1 contains complete CRCs showing both
|
||
upper and lower asymptotes and r<sup>2</sup> values > 0.9. Class
|
||
2 contains incomplete CRCs lacking the lower asymptote and shows
|
||
r<sup>2</sup> values greater than 0.9. Class 3 curves are of the
|
||
lowest confidence because they are defined by a single concentration
|
||
point where the minimal acceptable activity is set at 3 SD of the mean
|
||
activity calculated from the lowest tested concentration. Finally, class
|
||
4 contains compounds that do not show any CRCs and are therefore
|
||
classified as inactive.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml083f3" co-legend-rid="figlgndml083f3"><a href="/books/NBK47338/figure/ml083.f3/?report=objectonly" target="object" title="Scheme 1" class="img_link icnblk_img figpopup" rid-figpopup="figml083f3" rid-ob="figobml083f3"><img class="small-thumb" src="/books/NBK47338/bin/ml083f3.gif" src-large="/books/NBK47338/bin/ml083f3.jpg" alt="Scheme 1. Example qHTS data and classification scheme for assignment of resulting curve-fit data into classes." /></a><div class="icnblk_cntnt" id="figlgndml083f3"><h4 id="ml083.f3"><a href="/books/NBK47338/figure/ml083.f3/?report=objectonly" target="object" rid-ob="figobml083f3">Scheme 1</a></h4><p class="float-caption no_bottom_margin">Example qHTS data and classification scheme for assignment of
|
||
resulting curve-fit data into classes. <i>Top</i>, qHTS curve-fit data from AID-361 binned into
|
||
curve classifications 1–4 based classification
|
||
criteria. <i>Below</i>, Examples of curves fitting the
|
||
following <a href="/books/NBK47338/figure/ml083.f3/?report=objectonly" target="object" rid-ob="figobml083f3">(more...)</a></p></div></div><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml083f4" co-legend-rid="figlgndml083f4"><a href="/books/NBK47338/figure/ml083.f4/?report=objectonly" target="object" title="Figure 3" class="img_link icnblk_img figpopup" rid-figpopup="figml083f4" rid-ob="figobml083f4"><img class="small-thumb" src="/books/NBK47338/bin/ml083f4.gif" src-large="/books/NBK47338/bin/ml083f4.jpg" alt="Figure 3. Summary of activity from the qHTS." /></a><div class="icnblk_cntnt" id="figlgndml083f4"><h4 id="ml083.f4"><a href="/books/NBK47338/figure/ml083.f4/?report=objectonly" target="object" rid-ob="figobml083f4">Figure 3</a></h4><p class="float-caption no_bottom_margin">Summary of activity from the qHTS. The table summarizes the number actives associated with CRC
|
||
classes described in Scheme
|
||
1(highlighted in red are the percentages for the high
|
||
quality activating CRCs. The flow chart below summarizes the how
|
||
this information <a href="/books/NBK47338/figure/ml083.f4/?report=objectonly" target="object" rid-ob="figobml083f4">(more...)</a></p></div></div></div></div></div><div id="ml083.s16"><h3>Probe Characterization</h3><div id="ml083.fu4" class="figure bk_fig"><div class="graphic"><img src="/books/NBK47338/bin/ml083fu4.jpg" alt="Mode of action." /></div><h3><span class="title">Mode of action</span></h3><div class="caption"><p>Both CID-650361 (<b>A</b>) and CID-654376 (<b>B</b>) affect
|
||
the cooperativity of PEP binding with little affect on ADP binding in a
|
||
manner similar to FBP but with lower efficacy. Kinetics of substrate
|
||
binding in the presence (open circles) or absence (filled squares) of
|
||
activator (10 μM was used). Vo, initial rate in pmol/min as
|
||
determined in the PK-LDH coupled assay</p></div></div><div id="ml083.s18"><h4>Synthesis of analogs CID-650361</h4><div id="ml083.s19"><h5>General procedure for the synthesis of analogues</h5><p>The bis-sulfonamide was elaborated upon via the synthetic strategy
|
||
outline in <a class="figpopup" href="/books/NBK47338/figure/ml083.f5/?report=objectonly" target="object" rid-figpopup="figml083f5" rid-ob="figobml083f5">Scheme 1</a>. A Boc
|
||
protected piperizine (alternate ring systems were additionally explored;
|
||
for instance, 1,4-diazepane,
|
||
2,6-diazabicyclo[3.2.1]octane,
|
||
2,5-diazabicyclo[2.2.1]heptane,
|
||
2-methylpiperazine and piperazin-2-one) was treated with selected
|
||
sulfonyl chlorides in basic methylene chloride. Boc removal was
|
||
accomplished by treatment with trifluoroacetic acid, and a subsequent
|
||
treatment with selected sulfonyl chlorides in basic methylene chloride
|
||
provided numerous analogues.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml083f5" co-legend-rid="figlgndml083f5"><a href="/books/NBK47338/figure/ml083.f5/?report=objectonly" target="object" title="Scheme 1" class="img_link icnblk_img figpopup" rid-figpopup="figml083f5" rid-ob="figobml083f5"><img class="small-thumb" src="/books/NBK47338/bin/ml083f5.gif" src-large="/books/NBK47338/bin/ml083f5.jpg" alt="Scheme 1" /></a><div class="icnblk_cntnt" id="figlgndml083f5"><h4 id="ml083.f5"><a href="/books/NBK47338/figure/ml083.f5/?report=objectonly" target="object" rid-ob="figobml083f5">Scheme 1</a></h4></div></div><p>Additionally, numerous derivatives were synthesized that incorporated
|
||
selected amide functions in place of sulfonamides. The synthesis of
|
||
these analogues followed similar methods and is outlined in <a class="figpopup" href="/books/NBK47338/figure/ml083.f6/?report=objectonly" target="object" rid-figpopup="figml083f6" rid-ob="figobml083f6">Scheme 2</a>.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml083f6" co-legend-rid="figlgndml083f6"><a href="/books/NBK47338/figure/ml083.f6/?report=objectonly" target="object" title="Scheme 2" class="img_link icnblk_img figpopup" rid-figpopup="figml083f6" rid-ob="figobml083f6"><img class="small-thumb" src="/books/NBK47338/bin/ml083f6.gif" src-large="/books/NBK47338/bin/ml083f6.jpg" alt="Scheme 2" /></a><div class="icnblk_cntnt" id="figlgndml083f6"><h4 id="ml083.f6"><a href="/books/NBK47338/figure/ml083.f6/?report=objectonly" target="object" rid-ob="figobml083f6">Scheme 2</a></h4></div></div><p>It was also of interest to explore selected sulfone moieties in place of
|
||
the sulfonamides. To achieve these derivatives, we first displaced the
|
||
halogen on 4-bromopiperidine (alternate ring systems were additionally
|
||
explored; for instance 4-bromoazepane) via attach by selected aromatic
|
||
thiols in basic <i>N,N</i>-dimethylformamide (<a class="figpopup" href="/books/NBK47338/figure/ml083.f7/?report=objectonly" target="object" rid-figpopup="figml083f7" rid-ob="figobml083f7">Scheme 3</a>). Oxidation with
|
||
meta-chloroperbenzoic acid provided the sulfones and the remainder of
|
||
the synthesis followed the strategy outline in <a class="figpopup" href="/books/NBK47338/figure/ml083.f5/?report=objectonly" target="object" rid-figpopup="figml083f5" rid-ob="figobml083f5">Scheme 1</a>.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml083f7" co-legend-rid="figlgndml083f7"><a href="/books/NBK47338/figure/ml083.f7/?report=objectonly" target="object" title="Scheme 3" class="img_link icnblk_img figpopup" rid-figpopup="figml083f7" rid-ob="figobml083f7"><img class="small-thumb" src="/books/NBK47338/bin/ml083f7.gif" src-large="/books/NBK47338/bin/ml083f7.jpg" alt="Scheme 3" /></a><div class="icnblk_cntnt" id="figlgndml083f7"><h4 id="ml083.f7"><a href="/books/NBK47338/figure/ml083.f7/?report=objectonly" target="object" rid-ob="figobml083f7">Scheme 3</a></h4></div></div><p>Additional efforts surrounded the incorporation of selected functionality
|
||
directly on the piperizine ring system (alternate ring systems were
|
||
additionally explored; for instance, 1,4-diazepane, 2-methylpiperazine
|
||
and piperazin-2-one). In these efforts, the utility of appropriately
|
||
substituted piperazin-2-ones were treated with lithium
|
||
hexamethyldisilazide followed by Selectfluor® to accomplish
|
||
the fluorination of the ring system. These analogues were explored
|
||
further by direct reduction of the carbonyl with borane (<a class="figpopup" href="/books/NBK47338/figure/ml083.f8/?report=objectonly" target="object" rid-figpopup="figml083f8" rid-ob="figobml083f8">Scheme 4</a>).</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml083f8" co-legend-rid="figlgndml083f8"><a href="/books/NBK47338/figure/ml083.f8/?report=objectonly" target="object" title="Scheme 4" class="img_link icnblk_img figpopup" rid-figpopup="figml083f8" rid-ob="figobml083f8"><img class="small-thumb" src="/books/NBK47338/bin/ml083f8.gif" src-large="/books/NBK47338/bin/ml083f8.jpg" alt="Scheme 4" /></a><div class="icnblk_cntnt" id="figlgndml083f8"><h4 id="ml083.f8"><a href="/books/NBK47338/figure/ml083.f8/?report=objectonly" target="object" rid-ob="figobml083f8">Scheme 4</a></h4></div></div></div><div id="ml083.s20"><h5>SAR of CID650361 analogs</h5><p>The SAR of the bis-sulfonamide chemotype was explored on multiple
|
||
fronts. The central ring system was found to be necessary as was the
|
||
presence of the sulfonamides (amides were not active). Further, both
|
||
aryl derivatives were altered to gain an appreciation of their
|
||
effect on activity. The lone phenyl ring was explored with standard
|
||
substitutions (F, Me, OMe, Cl, CF3) at all positions (ortho, meta,
|
||
para) and several di-substitutions were explored (see table).
|
||
Additionally, the
|
||
2,3-dihydrobenzo[b][1,4]dioxine
|
||
heterocycle was altered. Related heterocycles and substituted phenyl
|
||
rings were explored. Symmetric versions of this chemotype were also
|
||
explored and found to be active.</p><div id="ml083.tu6" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK47338/table/ml083.tu6/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml083.tu6_lrgtbl__"><table><thead><tr><th id="hd_h_ml083.tu6_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;"></th><th id="hd_h_ml083.tu6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">R<sub>1</sub></th><th id="hd_h_ml083.tu6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">R<sub>2</sub></th><th id="hd_h_ml083.tu6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">R<sub>3</sub></th><th id="hd_h_ml083.tu6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">R<sub>4</sub></th><th id="hd_h_ml083.tu6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">R<sub>5</sub></th><th id="hd_h_ml083.tu6_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">
|
||
<i>IC</i>
|
||
<i>
|
||
<sub>50</sub>
|
||
</i>
|
||
<i>(μM)</i>
|
||
</th><th id="hd_h_ml083.tu6_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">
|
||
<i>Max Responce</i>
|
||
</th></tr></thead><tbody><tr><td headers="hd_h_ml083.tu6_1_1_1_1" rowspan="17" colspan="1" style="text-align:left;vertical-align:top;">
|
||
<div class="graphic"><img src="/books/NBK47338/bin/ml083fu5.jpg" alt="Image ml083fu5.jpg" /></div>
|
||
</td><td headers="hd_h_ml083.tu6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu6_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.398</td><td headers="hd_h_ml083.tu6_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">310</td></tr><tr><td headers="hd_h_ml083.tu6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">OMe</td><td headers="hd_h_ml083.tu6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu6_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.562</td><td headers="hd_h_ml083.tu6_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">195</td></tr><tr><td headers="hd_h_ml083.tu6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu6_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.708</td><td headers="hd_h_ml083.tu6_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">162</td></tr><tr><td headers="hd_h_ml083.tu6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">OCF3</td><td headers="hd_h_ml083.tu6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu6_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">1.00</td><td headers="hd_h_ml083.tu6_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">21</td></tr><tr><td headers="hd_h_ml083.tu6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu6_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.708</td><td headers="hd_h_ml083.tu6_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">369</td></tr><tr><td headers="hd_h_ml083.tu6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Br</td><td headers="hd_h_ml083.tu6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu6_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">4.47</td><td headers="hd_h_ml083.tu6_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">310</td></tr><tr><td headers="hd_h_ml083.tu6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">COOH</td><td headers="hd_h_ml083.tu6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu6_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">15.8</td><td headers="hd_h_ml083.tu6_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">164</td></tr><tr><td headers="hd_h_ml083.tu6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CH<sub>2</sub>COOH</td><td headers="hd_h_ml083.tu6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu6_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">17.8</td><td headers="hd_h_ml083.tu6_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">276</td></tr><tr><td headers="hd_h_ml083.tu6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><i>n</i>Pr</td><td headers="hd_h_ml083.tu6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu6_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">31.6</td><td headers="hd_h_ml083.tu6_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">234</td></tr><tr><td headers="hd_h_ml083.tu6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">COOMe</td><td headers="hd_h_ml083.tu6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu6_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.224</td><td headers="hd_h_ml083.tu6_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">306</td></tr><tr><td headers="hd_h_ml083.tu6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">COOH</td><td headers="hd_h_ml083.tu6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu6_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.355</td><td headers="hd_h_ml083.tu6_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">12.5</td></tr><tr><td headers="hd_h_ml083.tu6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">OMe</td><td headers="hd_h_ml083.tu6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu6_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.447</td><td headers="hd_h_ml083.tu6_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">103</td></tr><tr><td headers="hd_h_ml083.tu6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">OH</td><td headers="hd_h_ml083.tu6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu6_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.562</td><td headers="hd_h_ml083.tu6_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">227</td></tr><tr><td headers="hd_h_ml083.tu6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">OMe</td><td headers="hd_h_ml083.tu6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu6_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">1.00</td><td headers="hd_h_ml083.tu6_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">232</td></tr><tr><td headers="hd_h_ml083.tu6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu6_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">1.21</td><td headers="hd_h_ml083.tu6_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">199</td></tr><tr><td headers="hd_h_ml083.tu6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CF<sub>3</sub></td><td headers="hd_h_ml083.tu6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu6_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">1.26</td><td headers="hd_h_ml083.tu6_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">193</td></tr><tr><td headers="hd_h_ml083.tu6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu6_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">1.58</td><td headers="hd_h_ml083.tu6_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">229</td></tr></tbody></table></div></div></div></div><div id="ml083.s21"><h4>Synthesis of analogs CID-654376</h4><p>The 2-(benzyl)pyridazin-3(2H)-one based heterocycle was expanded via the
|
||
synthetic strategy outline in <a class="figpopup" href="/books/NBK47338/figure/ml083.f9/?report=objectonly" target="object" rid-figpopup="figml083f9" rid-ob="figobml083f9">Scheme
|
||
5</a>. Mixing a 5-substituted thiophene-2-carbaldehyde (substitutions
|
||
included alkyl groups and halogens) and ethyl 2-azidoacetate in basic
|
||
ethanol provided the (Z)-ethyl
|
||
2-azido-3-(5-substituted-thiophen-2-yl)acrylates, which can be directly
|
||
cyclized to ethyl
|
||
2-substituted-4H-thieno[3,2-b]pyrrole-5-carboxylates
|
||
by refluxing in O-xylene. Treatment of this heterocycle with piperidine and
|
||
formaldehyde in glacial acetic acid produced the ethyl
|
||
2-methyl-6-(piperidin-1-ylmethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylates
|
||
that can be methylated (the quarternary amine is preferred) and converted to
|
||
the aldehyde. Treatment of the ethyl
|
||
6-formyl-2-substituted-4H-thieno[3,2-b]pyrrole-5-carboxylates
|
||
with methyl iodide in basic conditions (K<sub>2</sub>CO<sub>3</sub>, DMF)
|
||
directly methylates the pyrole nitrogen [NOTE: it is essential
|
||
to alkylate the pyrole nitrogen at this synthetic step to avoid competition
|
||
with the pyridazin-3(2H)-one nitrogens]. Treatment with
|
||
hydrazine forms the pyridazin-3(2H)-one ring system and alkylation with
|
||
selected benzyl bromides provides the final structures.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml083f9" co-legend-rid="figlgndml083f9"><a href="/books/NBK47338/figure/ml083.f9/?report=objectonly" target="object" title="Scheme 5" class="img_link icnblk_img figpopup" rid-figpopup="figml083f9" rid-ob="figobml083f9"><img class="small-thumb" src="/books/NBK47338/bin/ml083f9.gif" src-large="/books/NBK47338/bin/ml083f9.jpg" alt="Scheme 5" /></a><div class="icnblk_cntnt" id="figlgndml083f9"><h4 id="ml083.f9"><a href="/books/NBK47338/figure/ml083.f9/?report=objectonly" target="object" rid-ob="figobml083f9">Scheme 5</a></h4></div></div><div id="ml083.s22"><h5>General procedure for the synthesis of analogues</h5><div id="ml083.fu5" class="figure"><div class="graphic"><img src="/books/NBK47338/bin/ml083fu6.jpg" alt="Image ml083fu6" /></div></div><p>Synthesis of (Z)-ethyl 2-azido-3-(5-substituted-thiophen-2-yl)acrylates.
|
||
A solution of sodium (2.76 g, 120 mmol) in absolute EtOH (120 ml) was
|
||
cooled in an ice-bath and a mixture of 5-substituted-2-formylthiophene
|
||
(5.73 g, 30 mmol) and ethyl azidoacetate (15.49 g, 120 mmol) was added
|
||
dropwise during 30 min period. The bath was removed and the reaction
|
||
mixture was stirred at room temperature for another 30 min. A cold
|
||
solution of saturated aqueous NH<sub>4</sub>Cl solution (100 ml) was
|
||
added and the resulting solution was extracted with diethyl ether (3x100
|
||
ml) and the combined organic layers were washed with brine (200 ml),
|
||
dried over Na<sub>2</sub>SO<sub>4</sub>. After removing diethyl ether
|
||
under reduced pressure, the crude product was purified by column
|
||
chromatography (EtOAc/Hexane 1/50) to give substituted acrylates as
|
||
light yellow solids</p><div id="ml083.fu6" class="figure"><div class="graphic"><img src="/books/NBK47338/bin/ml083fu7.jpg" alt="Image ml083fu7" /></div></div><p>Synthesis of ethyl
|
||
2-substituted-4H-thieno[3,2-b]pyrrole-5-carboxylate.</p><p>A solution of substituted acrylates (3.81 g, 12.6 mmol) in
|
||
<i>o</i>-xylene was refluxed for 20 min. After removing
|
||
the <i>o</i>-xylene, the crude product was purified by column
|
||
chromatography (EtOAc/Hexane 1/10) to give substituted
|
||
thieno[3,2-<i>b</i>]pyrroles as
|
||
white solids.</p><div id="ml083.fu7" class="figure"><div class="graphic"><img src="/books/NBK47338/bin/ml083fu8.jpg" alt="Image ml083fu8" /></div></div><p>Synthesis of ethyl
|
||
2-substituted-6-(piperidin-1-ylmethyl)-4H-thieno[3,2-b]pyrrole-5-carboxylate.</p><p>Piperidine (1.02 ml, 10.2 mmol), 37% aqueous formaldehyde
|
||
(0.80 ml, 10.2 mmol) and substituted
|
||
thieno[3,2-<i>b</i>]pyrrole (2.80
|
||
g, 10.2 mmol) were added to glacial acetic acid (3 ml) with ice-bath
|
||
cooling. The mixture was heated to 95 °C for 30 min and
|
||
allowed to react at room temperature overnight. After adding
|
||
H<sub>2</sub>O (10 ml), the mixture was adjusted to pH 8 by slow
|
||
addition of saturated aqueous K<sub>2</sub>CO<sub>3</sub> solution and
|
||
resulting solution was extracted with diethyl ether (3x15 ml). The
|
||
combined organic layers were washed with brine, dried over
|
||
Na<sub>2</sub>SO<sub>4</sub>. After removing the organic solvent
|
||
under reduced pressure, the residue was purified by column
|
||
chromatography (EtOAc/Hexane 1/2) to give the desired substituted
|
||
Mannich bases as white solids</p><div id="ml083.fu8" class="figure"><div class="graphic"><img src="/books/NBK47338/bin/ml083fu9.jpg" alt="Image ml083fu9" /></div></div><p>Synthesis of
|
||
1-((5-(ethoxycarbonyl)-2-substituted-4H-thieno[3,2-b]pyrrol-6-yl)methyl)-1-methylpiperidinium
|
||
iodides.</p><p>A solution of substituted Mannich bases (2.70 g, 7.30 mmol) in diethyl
|
||
ether (60 ml) was added dropwise to iodomethane (15 ml) with stirring in
|
||
30 min. The resulting mixture was refluxed for 2 hr. After cooling to
|
||
room temperature, the mixture was refrigerated at −20
|
||
°C overnight. The solid was filtered and washed with diethyl
|
||
ether to give the desired iodo salts as light yellow solids.</p><div id="ml083.fu9" class="figure"><div class="graphic"><img src="/books/NBK47338/bin/ml083fu10.jpg" alt="Image ml083fu10" /></div></div><p>Synthesis of ethyl
|
||
6-formyl-2-substituted-4H-thieno[3,2-b]pyrrole-5-carboxylates.</p><p>To a flask containing glacial acetic acid (5 ml) was added
|
||
1-((5-(ethoxycarbonyl)-2-substituted-4H-thieno[3,2-b]pyrrol-6-yl)methyl)-1-methylpiperidinium
|
||
iodides (2.30 g, 4.48 mmol) and the mixture was heated to 115
|
||
°C (oil bath), and hexamethylenetetramine (4.40 g, 31.4
|
||
mmol) was added as one portion and the temperature was kept at 115
|
||
°C for 20 min. After cooling to room temperature,
|
||
H<sub>2</sub>O (20 ml) was added and the resulting mixture was
|
||
extracted with EtOAc (3x25 ml) and the combined organic layers were
|
||
washed with saturated aqueous NaHCO<sub>3</sub> solution and brine.
|
||
After drying over Na<sub>2</sub>SO<sub>4</sub>, the organic solvent was
|
||
removed under reduced pressure and the residue was purified by column
|
||
chromatography (EtOAc/Hexane 1/6) to give the desired ethyl
|
||
6-formyl-2-substituted-4H-thieno[3,2-b]pyrrole-5-carboxylates
|
||
(302 mg, 40%) as white solids.</p><div id="ml083.fu10" class="figure"><div class="graphic"><img src="/books/NBK47338/bin/ml083fu11.jpg" alt="Image ml083fu11" /></div></div><p>Synthesis of ethyl
|
||
6-formyl-2,4-disubstituted-4H-thieno[3,2-b]pyrrole-5-carboxylates.</p><p>To a solution of ethyl
|
||
6-formyl-2-substituted-4H-thieno[3,2-b]pyrrole-5-carboxylates
|
||
(515 mg, 1.70 mmol) in DMF (5 ml) was added potassium carbonate (707 mg,
|
||
5.12 mmol) and iodomethane (0.27 ml, 3.40 mmol), and the resulting
|
||
mixture was stirred at room temperature for 2 hr. To the mixture was
|
||
added H<sub>2</sub>O (30 ml) and EtOAc (50 ml) and the organic layer was
|
||
separated and washed with brine, dried over
|
||
Na<sub>2</sub>SO<sub>4</sub>. After removing the organic solvent under
|
||
reduced pressure, the residue was purified by column chromatography
|
||
(EtOAc/Hexane 1/8) to give the desired <i>N</i>-methylated
|
||
ethyl
|
||
6-formyl-2,4-disubstituted-4H-thieno[3,2-b]pyrrole-5-carboxylates
|
||
(440 mg, 82%) as a white solid.</p><div id="ml083.fu11" class="figure"><div class="graphic"><img src="/books/NBK47338/bin/ml083fu12.jpg" alt="Image ml083fu12" /></div></div><p>Synthesis of substituted pyridazin-3(2H)-ones.</p><p>The
|
||
6-formyl-2,4-disubstituted-4H-thieno[3,2-b]pyrrole-5-carboxylates
|
||
(420 mg, 1.33 mmol) was dissolved in warm 2-ethoxyethanol (26 ml). To a
|
||
refluxed solution of hydrazine monohydrate (1 ml, 31.9 mmol) and
|
||
2-ethoxyethanol (5 ml) under nitrogen was added prepared
|
||
<i>N</i>-methylated ethyl
|
||
6-formyl-2,4-disubstituted-4H-thieno[3,2-b]pyrrole-5-carboxylates
|
||
solution dropwise over a 2 hr period, and the solution continued to
|
||
reflux for additional 1 hr. After cooling to room temperature, about
|
||
half of the 2-ethoxyethanol was removed under reduced pressure and the
|
||
remaining solution was refrigerated at −20 °C
|
||
overnight. The precipitate was filtered and washed with 2-ethoxyethanol
|
||
to give desired substituted pyridazin-3(2H)-ones (354 mg,
|
||
94%) as white solids.</p><div id="ml083.fu12" class="figure"><div class="graphic"><img src="/books/NBK47338/bin/ml083fu13.jpg" alt="Image ml083fu13" /></div></div><p>Synthesis of substituted 2-benzylpyridazin-3(2H)-ones.</p><p>To a solution of substituted pyridazin-3(2H)-ones (354 mg, 1.25 mmol) in
|
||
DMF (5 ml) was added potassium carbonate (1.02 g, 7.35 mmol) and
|
||
2-fluorobenzyl bromide (0.94, 4.98), and the resulting mixture was
|
||
heated at 60 °C for 2 hr. After cooling to room temperature,
|
||
to the mixture H<sub>2</sub>O (20 ml) and EtOAc (50 ml) was added and
|
||
the organic layer was separated and washed with brine, dried over
|
||
Na<sub>2</sub>SO<sub>4</sub>. After removing the solvent under
|
||
reduced pressure, the residue was purified by column chromatography
|
||
(EtOAc/Hexane 1/4) to give substituted 2-benzylpyridazin-3(2H)-ones (371
|
||
mg, 76%) as white solids</p><div id="ml083.fu13" class="figure"><div class="graphic"><img src="/books/NBK47338/bin/ml083fu14.jpg" alt="Image ml083fu14" /></div></div><p><b>2,4-dimethyl-4H-thieno[3,2-b]pyrrole-2-(2-fluorobenzyl)pyridazin-3(2H)-one.</b>
|
||
<sup>1</sup>H NMR (400 MHz, CDCl<sub>3</sub>) δ 2.64 (d, 3H,
|
||
<i>J</i>=1.2 Hz), 4.27 (s, 3H), 5.53 (s, 2H),
|
||
6.92 (q, 1H, <i>J</i>=1.2 Hz),
|
||
7.02–7.09 (m, 2H), 7.19–7.26 (m, 2H), 8.20 (s,
|
||
1H). HRMS; Calculated for
|
||
C<sub>17</sub>H<sub>15</sub>FN<sub>3</sub>OS+
|
||
(M+H): 328.0920, found: 328.0925.</p></div><div id="ml083.s23"><h5>SAR of CID-654376 analogs</h5><p>The SAR of the substituted 2-benzylpyridazin-3(2H)-one chemotype was
|
||
explored on multiple fronts. The central 6:5:5 ring system was found to
|
||
be necessary as was the presence of the alkyl group of off the central
|
||
pyrole ring (methyl>ethyl>isoproply). Further, alkyl
|
||
groups at the 2 position of the thiophene ring were limited by size
|
||
(again, methyl>ethyl>isoproply). The lone phenyl ring was
|
||
explored with standard substitutions (F, Me, OMe, Cl, CF3) at all
|
||
positions (ortho, meta, para) and several di-substitutions were explored
|
||
(see table). Additionally, the 2-position of the thiophene ring was
|
||
explored in terms of heteroatomic substitutions (OMe, SMe, S(O)Me,
|
||
S(O<sub>2</sub>)Me, NO<sub>2</sub>) with varying results.</p><div id="ml083.tu7" class="table"><h3><span class="title">SAR of CID-654376 analogs</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK47338/table/ml083.tu7/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml083.tu7_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml083.tu7_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;"></th><th id="hd_h_ml083.tu7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">R<sub>1</sub></th><th id="hd_h_ml083.tu7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">R<sub>2</sub></th><th id="hd_h_ml083.tu7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">R<sub>3</sub></th><th id="hd_h_ml083.tu7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">R<sub>4</sub></th><th id="hd_h_ml083.tu7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">R<sub>5</sub></th><th id="hd_h_ml083.tu7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">
|
||
<i>IC</i>
|
||
<i>
|
||
<sub>50</sub>
|
||
</i>
|
||
<i>( μM)</i>
|
||
</th><th id="hd_h_ml083.tu7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">
|
||
<i>Max Responce</i>
|
||
</th></tr></thead><tbody><tr><td headers="hd_h_ml083.tu7_1_1_1_1" rowspan="45" colspan="1" style="text-align:left;vertical-align:top;">
|
||
<div class="graphic"><img src="/books/NBK47338/bin/ml083fu15.jpg" alt="Image ml083fu15.jpg" /></div>
|
||
</td><td headers="hd_h_ml083.tu7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.200</td><td headers="hd_h_ml083.tu7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">354</td></tr><tr><td headers="hd_h_ml083.tu7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.251</td><td headers="hd_h_ml083.tu7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">330</td></tr><tr><td headers="hd_h_ml083.tu7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.282</td><td headers="hd_h_ml083.tu7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">331</td></tr><tr><td headers="hd_h_ml083.tu7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">OMe</td><td headers="hd_h_ml083.tu7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.282</td><td headers="hd_h_ml083.tu7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">353</td></tr><tr><td headers="hd_h_ml083.tu7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Cl</td><td headers="hd_h_ml083.tu7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.316</td><td headers="hd_h_ml083.tu7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">302</td></tr><tr><td headers="hd_h_ml083.tu7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.316</td><td headers="hd_h_ml083.tu7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">389</td></tr><tr><td headers="hd_h_ml083.tu7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Cl</td><td headers="hd_h_ml083.tu7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CF<sub>3</sub></td><td headers="hd_h_ml083.tu7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.316</td><td headers="hd_h_ml083.tu7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">316</td></tr><tr><td headers="hd_h_ml083.tu7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.355</td><td headers="hd_h_ml083.tu7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">365</td></tr><tr><td headers="hd_h_ml083.tu7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Cl</td><td headers="hd_h_ml083.tu7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.398</td><td headers="hd_h_ml083.tu7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">322</td></tr><tr><td headers="hd_h_ml083.tu7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.398</td><td headers="hd_h_ml083.tu7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">389</td></tr><tr><td headers="hd_h_ml083.tu7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Cl</td><td headers="hd_h_ml083.tu7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.447</td><td headers="hd_h_ml083.tu7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">313</td></tr><tr><td headers="hd_h_ml083.tu7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">OMe</td><td headers="hd_h_ml083.tu7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.501</td><td headers="hd_h_ml083.tu7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">409</td></tr><tr><td headers="hd_h_ml083.tu7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.501</td><td headers="hd_h_ml083.tu7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">322</td></tr><tr><td headers="hd_h_ml083.tu7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Me</td><td headers="hd_h_ml083.tu7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.562</td><td headers="hd_h_ml083.tu7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">293</td></tr><tr><td headers="hd_h_ml083.tu7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Me</td><td headers="hd_h_ml083.tu7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.562</td><td headers="hd_h_ml083.tu7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">335</td></tr><tr><td headers="hd_h_ml083.tu7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.631</td><td headers="hd_h_ml083.tu7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">324</td></tr><tr><td headers="hd_h_ml083.tu7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Cl</td><td headers="hd_h_ml083.tu7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.631</td><td headers="hd_h_ml083.tu7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">382</td></tr><tr><td headers="hd_h_ml083.tu7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Br</td><td headers="hd_h_ml083.tu7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.631</td><td headers="hd_h_ml083.tu7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">384</td></tr><tr><td headers="hd_h_ml083.tu7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Me</td><td headers="hd_h_ml083.tu7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.631</td><td headers="hd_h_ml083.tu7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">326</td></tr><tr><td headers="hd_h_ml083.tu7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Br</td><td headers="hd_h_ml083.tu7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.708</td><td headers="hd_h_ml083.tu7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">318</td></tr><tr><td headers="hd_h_ml083.tu7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Cl</td><td headers="hd_h_ml083.tu7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.708</td><td headers="hd_h_ml083.tu7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">308</td></tr><tr><td headers="hd_h_ml083.tu7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Me</td><td headers="hd_h_ml083.tu7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.708</td><td headers="hd_h_ml083.tu7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">309</td></tr><tr><td headers="hd_h_ml083.tu7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Cl</td><td headers="hd_h_ml083.tu7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.708</td><td headers="hd_h_ml083.tu7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">332</td></tr><tr><td headers="hd_h_ml083.tu7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.708</td><td headers="hd_h_ml083.tu7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">323</td></tr><tr><td headers="hd_h_ml083.tu7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Cl</td><td headers="hd_h_ml083.tu7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.794</td><td headers="hd_h_ml083.tu7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">355</td></tr><tr><td headers="hd_h_ml083.tu7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.794</td><td headers="hd_h_ml083.tu7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">348</td></tr><tr><td headers="hd_h_ml083.tu7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Cl</td><td headers="hd_h_ml083.tu7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.794</td><td headers="hd_h_ml083.tu7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">303</td></tr><tr><td headers="hd_h_ml083.tu7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Me</td><td headers="hd_h_ml083.tu7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.794</td><td headers="hd_h_ml083.tu7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">285</td></tr><tr><td headers="hd_h_ml083.tu7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.891</td><td headers="hd_h_ml083.tu7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">309</td></tr><tr><td headers="hd_h_ml083.tu7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Me</td><td headers="hd_h_ml083.tu7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.891</td><td headers="hd_h_ml083.tu7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">351</td></tr><tr><td headers="hd_h_ml083.tu7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Me</td><td headers="hd_h_ml083.tu7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Me</td><td headers="hd_h_ml083.tu7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.891</td><td headers="hd_h_ml083.tu7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">62</td></tr><tr><td headers="hd_h_ml083.tu7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Me</td><td headers="hd_h_ml083.tu7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">1.00</td><td headers="hd_h_ml083.tu7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">380</td></tr><tr><td headers="hd_h_ml083.tu7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Me</td><td headers="hd_h_ml083.tu7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">1.00</td><td headers="hd_h_ml083.tu7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">321</td></tr><tr><td headers="hd_h_ml083.tu7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Me</td><td headers="hd_h_ml083.tu7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Cl</td><td headers="hd_h_ml083.tu7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">1.12</td><td headers="hd_h_ml083.tu7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">323</td></tr><tr><td headers="hd_h_ml083.tu7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Me</td><td headers="hd_h_ml083.tu7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Me</td><td headers="hd_h_ml083.tu7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">1.12</td><td headers="hd_h_ml083.tu7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">343</td></tr><tr><td headers="hd_h_ml083.tu7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">OMe</td><td headers="hd_h_ml083.tu7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">1.41</td><td headers="hd_h_ml083.tu7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">297</td></tr><tr><td headers="hd_h_ml083.tu7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Me</td><td headers="hd_h_ml083.tu7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Cl</td><td headers="hd_h_ml083.tu7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">1.41</td><td headers="hd_h_ml083.tu7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">307</td></tr><tr><td headers="hd_h_ml083.tu7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Me</td><td headers="hd_h_ml083.tu7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">1.59</td><td headers="hd_h_ml083.tu7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">237</td></tr><tr><td headers="hd_h_ml083.tu7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Cl</td><td headers="hd_h_ml083.tu7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Cl</td><td headers="hd_h_ml083.tu7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">1.59</td><td headers="hd_h_ml083.tu7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">272</td></tr><tr><td headers="hd_h_ml083.tu7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Br</td><td headers="hd_h_ml083.tu7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">1.59</td><td headers="hd_h_ml083.tu7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">250</td></tr><tr><td headers="hd_h_ml083.tu7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CF<sub>3</sub></td><td headers="hd_h_ml083.tu7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">1.78</td><td headers="hd_h_ml083.tu7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">292</td></tr><tr><td headers="hd_h_ml083.tu7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Cl</td><td headers="hd_h_ml083.tu7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">1.78</td><td headers="hd_h_ml083.tu7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">335</td></tr><tr><td headers="hd_h_ml083.tu7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Me</td><td headers="hd_h_ml083.tu7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">2.00</td><td headers="hd_h_ml083.tu7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">381</td></tr><tr><td headers="hd_h_ml083.tu7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Cl</td><td headers="hd_h_ml083.tu7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">2.00</td><td headers="hd_h_ml083.tu7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">347</td></tr><tr><td headers="hd_h_ml083.tu7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">F</td><td headers="hd_h_ml083.tu7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H</td><td headers="hd_h_ml083.tu7_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">2.00</td><td headers="hd_h_ml083.tu7_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">266</td></tr></tbody></table></div></div><p>These compounds have been provided to the MLSMR: </p><p><b>MLS-00030964 (CID-650361)</b></p><p>
|
||
<i>Canonical SMILES</i>:
|
||
COC1=CC=C(C=C1)S(=O)(=O)N2CCN(CC2)S(=O)(=O)C3=CC4=C(C=C3)OCCO4</p><p>
|
||
<i>InChI</i>: InChI
|
||
=1S/C19H22N2O7S2/c1-26-15-2-4-16(5-3-15)29(22,
|
||
23)20-8-10-21(11-9-20)30(24,25)17-6-7-18-19(14–17)28-13-12-27-18/h2-7,
|
||
14H,8-13H2,1H3 </p><p><b>MLS-000076148 (CID-654376)</b></p><p>
|
||
<i>Canonical SMILES:</i>
|
||
CC1=CC2=C(S1)C3=C(N2C)C(=O)N(N=C3)CC4=CC=CC=C4F </p><p>
|
||
<i>InChI</i>:
|
||
InChI=1S/C17H14FN3OS/c1-10-7-14-16(23-10)12-8-19-21(17(22)15(12)20(14)2)
|
||
9-11-5-3-4-6-13(11)18/h3-8H,9H2,1-2H3 </p></div></div><div id="ml083.s25"><h4>Description of secondary assays used in probe characterization</h4><div id="ml083.s26"><h5>PK-LDH coupled</h5><p>A second assay was used to evaluate compounds. This assay couples the
|
||
formation of pyruvate from PK using lactate dehydrogenase (LDH) and
|
||
NADH. The depletion of NADH is followed in a fluorescent-based kinetic
|
||
assay that follows the formation of pyruvate to lactate, as catalyzed
|
||
LDH (described in ref [10]). This format can be
|
||
used to determine kinetic parameters and mechanism of action for
|
||
activators/inhibitors. This kinetic assay used 1 nM human PK-M2, and
|
||
initial rates are determined by measuring the fluorescence signal every
|
||
30 secs for 10 minutes. The same assay was used for isoform
|
||
selectivity.</p></div><div id="ml083.s27"><h5>Compound preparation</h5><p>Compound is prepared in DMSO at 10 mM stock concentration. Assays
|
||
described above have 0.6% DMSO final concentration in
|
||
buffer.</p></div></div></div></div><div id="ml083.s28"><h2 id="_ml083_s28_">Known probe properties</h2><p>Summary of probe properties (solubility, absorbance/fluorescence, reactivity,
|
||
toxicity, etc.) and recommendations for the scientific use of probe as research
|
||
tool:</p><div id="ml083.s29"><h3>1. Probe</h3><div id="ml083.s30"><h4>a. Chemical name</h4><p>
|
||
1-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)-4-(4-methoxyphenylsulfonyl)piperazine
|
||
[<a href="/pcsubstance/?term=ML083[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML083</a>]
|
||
</p></div><div id="ml083.s31"><h4>b. Probe chemical structure</h4><div id="ml083.fu14" class="figure"><div class="graphic"><img src="/books/NBK47338/bin/ml083fu16.jpg" alt="Image ml083fu16" /></div></div></div><div id="ml083.s32"><h4>c. Structural Verification Information of probe SID</h4><p><sup>1</sup>H NMR (400 MHz, DMSO-<i>d</i>6) δ: 2.94 (s,
|
||
8H), 3.86 (s, 3H), 4.23–4.45 (m, 4H), 7.01–7.08 (m,
|
||
1H), 7.08–7.19 (m, 4H), 7.57–7.67 (m, 2H). HRMS;
|
||
Calculated for
|
||
C<sub>19</sub>H<sub>22</sub>N<sub>2</sub>O<sub>7</sub>S<sub>2</sub>
|
||
(M+): 454.0868, found: 454.0865.</p></div><div id="ml083.s33"><h4>d. PubChem CID (corresponding to the SID)</h4><p>CID-650361</p></div><div id="ml083.s34"><h4>e. Availability from a vendor</h4><p>Aliquots of MLS-00030964-01 (CID-650361) is available from the NCGC upon
|
||
request.</p></div><div id="ml083.s35"><h4>f. Mode of action for biological activity of probe</h4><p>The probe is a member of a series of highly specific allosteric activators
|
||
for the tumor-specific isoform of human pyruvate kinase (M2 isoform). The
|
||
activation occurs in a manner that is kinetically similar to the natural
|
||
activator (FBP) by decreasing the Km for PEP and reducing the cooperative
|
||
binding of PEP. Preliminary X-ray data obtained for co-crystals of
|
||
NCGC00030335 bound to human PK M2 (see PDB:3GQY) suggests this occurs by
|
||
binding at interface that spans the dimer-dimer interaction region of the
|
||
tetramer, which we infer stabilizes the high affinity R-state of the
|
||
tetramer.</p></div><div id="ml083.s36"><h4>g. Detailed synthetic pathway for making probe</h4><p>See schemes above.</p></div><div id="ml083.s37"><h4>h. Summary of probe properties</h4><p>Compound is soluble at 10 mM in DMSO. The compound is not fluorescent with
|
||
blue excitation wavelengths (~340 nm). Solubility in buffer has not been
|
||
determined.</p></div></div><div id="ml083.s38"><h3>2. Probe</h3><div id="ml083.s39"><h4>a. Chemical name</h4><p>
|
||
2,4-dimethyl-4H-thieno[3,2-b]pyrrole-2-(2-fluorobenzyl)pyridazin-3(2H)-one
|
||
[<a href="/pcsubstance/?term=ML082[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML082</a>]
|
||
</p></div><div id="ml083.s40"><h4>b. Probe chemical structure</h4><div id="ml083.fu15" class="figure"><div class="graphic"><img src="/books/NBK47338/bin/ml083fu17.jpg" alt="Image ml083fu17" /></div></div></div><div id="ml083.s41"><h4>c. Structural Verification Information of probe SID</h4><p><sup>1</sup>H NMR (400 MHz, CDCl<sub>3</sub>) δ 2.64 (d, 3H,
|
||
<i>J</i>=1.2 Hz), 4.27 (s, 3H), 5.53 (s, 2H), 6.92
|
||
(q, 1H, <i>J</i>=1.2 Hz), 7.02–7.09 (m,
|
||
2H), 7.19–7.26 (m, 2H), 8.20 (s, 1H). HRMS; Calculated for
|
||
C<sub>17</sub>H<sub>15</sub>FN<sub>3</sub>OS+
|
||
(M+H): 328.0920, found: 328.0925.</p></div><div id="ml083.s42"><h4>d. PubChem CID (corresponding to the SID)</h4><p>CID-654376</p></div><div id="ml083.s43"><h4>e. Availability from a vendor</h4><p>Aliquots of MLS-000076148 (CID-654376) are available from the NCGC upon
|
||
request.</p></div><div id="ml083.s44"><h4>f. Mode of action for biological activity of probe</h4><p>The probe is a member of a series of highly specific allosteric activators
|
||
for the tumor-specific isoform of human pyruvate kinase (M2 isoform). The
|
||
activation occurs in a manner that is kinetically similar to the natural
|
||
activator (FBP) by decreasing the Km for PEP and reducing the cooperative
|
||
binding of PEP.</p></div><div id="ml083.s45"><h4>g. Detailed synthetic pathway for making probe</h4><p>See Scheme above.</p></div><div id="ml083.s46"><h4>h. Summary of probe properties</h4><p>Compound is soluble at 10 mM in DMSO. The compound is not fluorescent with
|
||
blue excitation wavelengths (~340 nm). Solubility in buffer has not been
|
||
determined.</p></div></div></div><div id="ml083.bib"><h2 id="_ml083_bib_">Bibliography</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="ml083.r1">Dombrauckas JD, Santarsiero BD, Mesecar AD. Structural basis for tumor pyruvate kinase M2 allosteric
|
||
regulation and catalysis. <span><span class="ref-journal">Biochemistry. </span>2005;<span class="ref-vol">44</span>:9417–29.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/15996096" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 15996096</span></a>]</div></dd><dt>2.</dt><dd><div class="bk_ref" id="ml083.r2">Christofk HR, Vander Heiden MG, Harris MH, Ramanathan A, Gerszten RE, Wei R, Fleming MD, Schreiber SL, Cantley LC. The M2 splice isoform of pyruvate kinase is important for
|
||
cancer metabolism and tumour growth. <span><span class="ref-journal">Nature. </span>2008;<span class="ref-vol">452</span>:230–3.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/18337823" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18337823</span></a>]</div></dd><dt>3.</dt><dd><div class="bk_ref" id="ml083.r3">Christofk HR, Vander Heiden MG, Wu N, Asara JM, Cantley LC. Pyruvate kinase M2 is a phosphotyrosine-binding
|
||
protein. <span><span class="ref-journal">Nature. </span>2008;<span class="ref-vol">452</span>:181–6.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/18337815" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18337815</span></a>]</div></dd><dt>4.</dt><dd><div class="bk_ref" id="ml083.r4">Warburg O. On the origin of cancer cells. <span><span class="ref-journal">Science. </span>1956;<span class="ref-vol">123</span>:309–14.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/13298683" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 13298683</span></a>]</div></dd><dt>5.</dt><dd><div class="bk_ref" id="ml083.r5">Fan F, Wood KV. Bioluminescent assays for high-throughput
|
||
screening. <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>6.</dt><dd><div class="bk_ref" id="ml083.r6">Singh P, Harden BJ, Lillywhite BJ, Broad PM. Identification of kinase inhibitors by an ATP depletion
|
||
method. <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>7.</dt><dd><div class="bk_ref" id="ml083.r7">Inglese J, Auld DS, Jadhav A, Johnson RL, Simeonov A, Yasgar A, Zheng W, Austin CP. Quantitative high-throughput screening: A titration-based
|
||
approach that efficiently identifies biological activities in large
|
||
chemical libraries. <span><span class="ref-journal">Proc Natl Acad Sci U S A. </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>8.</dt><dd><div class="bk_ref" id="ml083.r8">Auld DS, Zhang YQ, Southall NT, Rai G, Landsman M, Maclure J, Langevin D, Thomas CJ, Austin CP, Inglese J. A Basis for Reduced Chemical Library Inhibition of Firefly
|
||
Luciferase Obtained from Directed Evolution. <span><span class="ref-journal">J Med Chem. </span>2009;<span class="ref-vol">52</span>:1450–1458.</span> [<a href="/pmc/articles/PMC3430137/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3430137</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/19215089" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19215089</span></a>]</div></dd><dt>9.</dt><dd><div class="bk_ref" id="ml083.r9">Yasgar A, Shinn P, Jadhav A, Auld DS, Michael S, Zheng W, Austin CP, Inglese J, Simeonov A. Compound Management for Quantitative High-Throughput
|
||
Screening. <span><span class="ref-journal">J Assoc Lab Automation. </span>2008;<span class="ref-vol">13</span>:79–89.</span> [<a href="/pmc/articles/PMC2390859/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2390859</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/18496600" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18496600</span></a>]</div></dd><dt>10.</dt><dd><div class="bk_ref" id="ml083.r10">Hannaert V, Yernaux C, Rigden DJ, Fothergill-Gilmore LA, Opperdoes FR, Michels PA. The putative effector-binding site of Leishmania mexicana
|
||
pyruvate kinase studied by site-directed mutagenesis. <span><span class="ref-journal">FEBS Lett. </span>2002;<span class="ref-vol">514</span>:255–9.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/11943161" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 11943161</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/NBK47338/?report=reader">PubReader</a></li><li><a href="/books/NBK47338/?report=printable">Print View</a></li><li><a data-jig="ncbidialog" href="#_ncbi_dlg_citbx_NBK47338" data-jigconfig="width:400,modal:true">Cite this Page</a><div id="_ncbi_dlg_citbx_NBK47338" style="display:none" title="Cite this Page"><div class="bk_tt">Auld D, Shen M, Skoumbourdis AP, et al. Identification of activators for the M2 isoform of human pyruvate kinase. 2009 Apr 16 [Updated 2010 Aug 6]. In: Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-. <span class="bk_cite_avail"></span></div></div></li><li><a href="/books/NBK47338/pdf/Bookshelf_NBK47338.pdf">PDF version of this page</a> (555K)</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="#ml083.probestruct" ref="log$=inpage&link_id=inpage">Probe Structure and Characteristics</a></li><li><a href="#ml083.rationale" ref="log$=inpage&link_id=inpage">Recommendations for the scientific use of these probes</a></li><li><a href="#ml083.s8" ref="log$=inpage&link_id=inpage">Assay Implementation and Screening</a></li><li><a href="#ml083.s28" ref="log$=inpage&link_id=inpage">Known probe properties</a></li><li><a href="#ml083.bib" ref="log$=inpage&link_id=inpage">Bibliography</a></li></ul></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Related information</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="discovery_db_links" id="Shutter"></a></div><div class="portlet_content"><ul><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pmc&DbFrom=books&Cmd=Link&LinkName=books_pmc_refs&IdsFromResult=2358220" ref="log$=recordlinks">PMC</a><div class="brieflinkpop offscreen_noflow">PubMed Central citations</div></li><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pcassay&DbFrom=books&Cmd=Link&LinkName=books_pcassay_probe&IdsFromResult=2358220" ref="log$=recordlinks">PubChem BioAssay for Chemical Probe</a><div class="brieflinkpop offscreen_noflow">PubChem BioAssay records reporting screening data for the development of the chemical probe(s) described in this book chapter</div></li><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pcsubstance&DbFrom=books&Cmd=Link&LinkName=books_pcsubstance&IdsFromResult=2358220" ref="log$=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=2358220" 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" 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