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<meta name="ncbi_db" content="books" /><meta name="ncbi_pdid" content="book-part" /><meta name="ncbi_acc" content="NBK63598" /><meta name="ncbi_domain" content="mlprobe" /><meta name="ncbi_report" content="record" /><meta name="ncbi_type" content="fulltext" /><meta name="ncbi_objectid" content="" /><meta name="ncbi_pcid" content="/NBK63598/" /><meta name="ncbi_pagename" content="HTS Assay for Discovery of Novel Metallo-Beta-lactamase (MBL) Inhibitors - Probe Reports from the NIH Molecular Libraries Program - NCBI Bookshelf" /><meta name="ncbi_bookparttype" content="chapter" /><meta name="ncbi_app" content="bookshelf" />
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<title>HTS Assay for Discovery of Novel Metallo-Beta-lactamase (MBL) Inhibitors - Probe Reports from the NIH Molecular Libraries Program - NCBI Bookshelf</title>
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<meta name="robots" content="INDEX,FOLLOW,NOARCHIVE" /><meta name="citation_inbook_title" content="Probe Reports from the NIH Molecular Libraries Program [Internet]" /><meta name="citation_title" content="HTS Assay for Discovery of Novel Metallo-Beta-lactamase (MBL) Inhibitors" /><meta name="citation_publisher" content="National Center for Biotechnology Information (US)" /><meta name="citation_date" content="2011/05/05" /><meta name="citation_author" content="D Minond" /><meta name="citation_author" content="SA Saldanha" /><meta name="citation_author" content="T Spicer" /><meta name="citation_author" content="Liu Qin" /><meta name="citation_author" content="BA Mercer" /><meta name="citation_author" content="WR Roush" /><meta name="citation_author" content="P Hodder" /><meta name="citation_pmid" content="21961119" /><meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK63598/" /><link rel="schema.DC" href="http://purl.org/DC/elements/1.0/" /><meta name="DC.Title" content="HTS Assay for Discovery of Novel Metallo-Beta-lactamase (MBL) Inhibitors" /><meta name="DC.Type" content="Text" /><meta name="DC.Publisher" content="National Center for Biotechnology Information (US)" /><meta name="DC.Contributor" content="D Minond" /><meta name="DC.Contributor" content="SA Saldanha" /><meta name="DC.Contributor" content="T Spicer" /><meta name="DC.Contributor" content="Liu Qin" /><meta name="DC.Contributor" content="BA Mercer" /><meta name="DC.Contributor" content="WR Roush" /><meta name="DC.Contributor" content="P Hodder" /><meta name="DC.Date" content="2011/05/05" /><meta name="DC.Identifier" content="https://www.ncbi.nlm.nih.gov/books/NBK63598/" /><meta name="description" content="VIM-2 is an Ambler class B metallo-β-lactamase (MBL) capable of hydrolyzing a broad-spectrum of β-lactam antibiotics. Although the discovery and development of MBL inhibitors continue to be an area of active research, an array of potent, small molecule inhibitors is yet to be fully characterized for VIM-2. This report claims CID4870494/SID 24790728 as a potent, selective and non-competitive VIM-2 probe (ML121). This compound was discovered via screening of more than 290,000 MLSMR compounds in a nitrocefin-based enzyme activity assay. Various secondary assays were run on cherry-picked MLSMR samples to determine its potency (VIM-2 IC50= 54 nM) and selectivity (it was inactive in IMP-1 and TEM-1 beta-lactamase assays). Other tests were run to verify it was not an optical artifact of the detection formats used for screening; this testing included assays with two different VIM-2 substrates (nitrocefin and CCF2). Analogs of CID4870494 were less active against VIM-2. A powder sample of CID4870494 was obtained for more detailed studies. Although the potency of the powder sample was slightly less (IC50= 0.223 ± 0.003 μM) than that of the MLSMR cherry-picked liquid stock, CID4870494 remained inactive in IMP-1 and VIM-2 assays. Kinetic analyses demonstrated that it behaves as a non-competitive inhibitor, with a submicromolar Ki (Ki = 148 ± 14 nM). This represents a 10-fold improvement in potency over the prior art probe, Mitoxantrone. It is also important to note that Mitoxantrone’s intense color limits its use in some assay detection formats; as described above, CID4870494 does not appear as an artifact in fluorescence- (CCF2) and absorbance-based (nitrocefin) assays. Further studies employing the TFA salt form of the probe (SID111978109/CID50909789) demonstrate that probe ML121 can potentiate the antibiotic activity of imipenem in VIM-2 transformed E. coli. Thus, these assays reveal the probe ML121 is a novel, selective, and potent VIM-2 inhibitor. This probe will serve as a valuable tool to elucidate the role of VIM-2 in nosocomial beta-lactam antibiotic resistance." /><meta name="og:title" content="HTS Assay for Discovery of Novel Metallo-Beta-lactamase (MBL) Inhibitors" /><meta name="og:type" content="book" /><meta name="og:description" content="VIM-2 is an Ambler class B metallo-β-lactamase (MBL) capable of hydrolyzing a broad-spectrum of β-lactam antibiotics. Although the discovery and development of MBL inhibitors continue to be an area of active research, an array of potent, small molecule inhibitors is yet to be fully characterized for VIM-2. This report claims CID4870494/SID 24790728 as a potent, selective and non-competitive VIM-2 probe (ML121). This compound was discovered via screening of more than 290,000 MLSMR compounds in a nitrocefin-based enzyme activity assay. Various secondary assays were run on cherry-picked MLSMR samples to determine its potency (VIM-2 IC50= 54 nM) and selectivity (it was inactive in IMP-1 and TEM-1 beta-lactamase assays). Other tests were run to verify it was not an optical artifact of the detection formats used for screening; this testing included assays with two different VIM-2 substrates (nitrocefin and CCF2). Analogs of CID4870494 were less active against VIM-2. A powder sample of CID4870494 was obtained for more detailed studies. Although the potency of the powder sample was slightly less (IC50= 0.223 ± 0.003 μM) than that of the MLSMR cherry-picked liquid stock, CID4870494 remained inactive in IMP-1 and VIM-2 assays. Kinetic analyses demonstrated that it behaves as a non-competitive inhibitor, with a submicromolar Ki (Ki = 148 ± 14 nM). This represents a 10-fold improvement in potency over the prior art probe, Mitoxantrone. It is also important to note that Mitoxantrone’s intense color limits its use in some assay detection formats; as described above, CID4870494 does not appear as an artifact in fluorescence- (CCF2) and absorbance-based (nitrocefin) assays. Further studies employing the TFA salt form of the probe (SID111978109/CID50909789) demonstrate that probe ML121 can potentiate the antibiotic activity of imipenem in VIM-2 transformed E. coli. Thus, these assays reveal the probe ML121 is a novel, selective, and potent VIM-2 inhibitor. This probe will serve as a valuable tool to elucidate the role of VIM-2 in nosocomial beta-lactam antibiotic resistance." /><meta name="og:url" content="https://www.ncbi.nlm.nih.gov/books/NBK63598/" /><meta name="og:site_name" content="NCBI Bookshelf" /><meta name="og:image" content="https://www.ncbi.nlm.nih.gov/corehtml/pmc/pmcgifs/bookshelf/thumbs/th-mlprobe-lrg.png" /><meta name="twitter:card" content="summary" /><meta name="twitter:site" content="@ncbibooks" /><meta name="bk-non-canon-loc" content="/books/n/mlprobe/ml121/" /><link rel="canonical" href="https://www.ncbi.nlm.nih.gov/books/NBK63598/" /><link rel="stylesheet" href="/corehtml/pmc/css/figpopup.css" type="text/css" media="screen" /><link rel="stylesheet" href="/corehtml/pmc/css/bookshelf/2.26/css/books.min.css" type="text/css" /><link rel="stylesheet" href="/corehtml/pmc/css/bookshelf/2.26/css/books_print.min.css" type="text/css" media="print" /><style type="text/css">p a.figpopup{display:inline !important} .bk_tt {font-family: monospace} .first-line-outdent .bk_ref {display: inline} .body-content h2, .body-content .h2 {border-bottom: 1px solid #97B0C8} .body-content h2.inline {border-bottom: none} a.page-toc-label , .jig-ncbismoothscroll a {text-decoration:none;border:0 !important} .temp-labeled-list .graphic {display:inline-block !important} .temp-labeled-list img{width:100%}</style><script type="text/javascript" src="/corehtml/pmc/js/jquery.hoverIntent.min.js"> </script><script type="text/javascript" src="/corehtml/pmc/js/common.min.js?_=3.18"> </script><script type="text/javascript" src="/corehtml/pmc/js/large-obj-scrollbars.min.js"> </script><script type="text/javascript">window.name="mainwindow";</script><script type="text/javascript" src="/corehtml/pmc/js/bookshelf/2.26/book-toc.min.js"> </script><script type="text/javascript" src="/corehtml/pmc/js/bookshelf/2.26/books.min.js"> </script><meta name="book-collection" content="NONE" />
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<div class="pre-content"><div><div class="bk_prnt"><p class="small">NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health.</p><p>Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-. </p></div><div class="iconblock clearfix whole_rhythm no_top_margin bk_noprnt"><a class="img_link icnblk_img" title="Table of Contents Page" href="/books/n/mlprobe/"><img class="source-thumb" src="/corehtml/pmc/pmcgifs/bookshelf/thumbs/th-mlprobe-lrg.png" alt="Cover of Probe Reports from the NIH Molecular Libraries Program" height="100px" width="80px" /></a><div class="icnblk_cntnt eight_col"><h2>Probe Reports from the NIH Molecular Libraries Program [Internet].</h2><a data-jig="ncbitoggler" href="#__NBK63598_dtls__">Show details</a><div style="display:none" class="ui-widget" id="__NBK63598_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/ml123/" title="Previous page in this title">< Prev</a><a class="active page_link next" href="/books/n/mlprobe/ml120/" 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="_NBK63598_"><span class="title" itemprop="name">HTS Assay for Discovery of Novel Metallo-Beta-lactamase (MBL) Inhibitors</span></h1><p class="contrib-group"><span itemprop="author">D Minond</span>, <span itemprop="author">SA Saldanha</span>, <span itemprop="author">T Spicer</span>, <span itemprop="author">Liu Qin</span>, <span itemprop="author">BA Mercer</span>, <span itemprop="author">WR Roush</span>, and <span itemprop="author">P Hodder</span>.</p><a data-jig="ncbitoggler" href="#__NBK63598_ai__" style="border:0;text-decoration:none">Author Information and Affiliations</a><div style="display:none" class="ui-widget" id="__NBK63598_ai__"><p class="contrib-group"><h4>Authors</h4><span itemprop="author">D Minond</span>,<sup>1</sup> <span itemprop="author">SA Saldanha</span>,<sup>1</sup> <span itemprop="author">T Spicer</span>,<sup>1</sup> <span itemprop="author">Liu Qin</span>,<sup>2</sup> <span itemprop="author">BA Mercer</span>,<sup>1</sup> <span itemprop="author">WR Roush</span>,<sup>2</sup> and <span itemprop="author">P Hodder</span><sup>1,3</sup><sup>,4</sup>.</p><h4>Affiliations</h4><div class="affiliation"><sup>1</sup>
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Lead Identification Division, Translational Research Institute, Scripps Florida, 130 Scripps Way, Jupiter, Fl, 33458</div><div class="affiliation"><sup>2</sup>
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Department of Chemistry, Scripps Florida, 130 Scripps Way, Jupiter, Fl, 33458</div><div class="affiliation"><sup>3</sup>
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Department of Molecular Therapeutics</div><div class="affiliation">
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<sup>4</sup> To whom correspondence should be addressed,
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<span class="before-email-separator"></span><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="ude.sppircs@preddoh" class="oemail">ude.sppircs@preddoh</a></div></div><p class="small">Received: <span itemprop="datePublished">November 13, 2009</span>; Last Update: <span itemprop="dateModified">May 5, 2011</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>VIM-2 is an Ambler class B metallo-β-lactamase (MBL) capable of hydrolyzing a broad-spectrum of β-lactam antibiotics. Although the discovery and development of MBL inhibitors continue to be an area of active research, an array of potent, small molecule inhibitors is yet to be fully characterized for VIM-2. This report claims <b>CID4870494/<a href="https://pubchem.ncbi.nlm.nih.gov/substance/24790728" ref="pagearea=abstract&targetsite=entrez&targetcat=link&targettype=pubchem">SID 24790728</a></b> as a potent, selective and non-competitive VIM-2 probe (<a href="/pcsubstance/?term=ML121[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML121</a>). This compound was discovered via screening of more than 290,000 MLSMR compounds in a nitrocefin-based enzyme activity assay. Various secondary assays were run on cherry-picked MLSMR samples to determine its potency (VIM-2 IC<sub>50</sub>= 54 nM) and selectivity (it was inactive in IMP-1 and TEM-1 beta-lactamase assays). Other tests were run to verify it was not an optical artifact of the detection formats used for screening; this testing included assays with two different VIM-2 substrates (nitrocefin and CCF2). Analogs of CID4870494 were less active against VIM-2. A powder sample of CID4870494 was obtained for more detailed studies. Although the potency of the powder sample was slightly less (IC<sub>50</sub>= 0.223 ± 0.003 μM) than that of the MLSMR cherry-picked liquid stock<b>, CID4870494</b> remained inactive in IMP-1 and VIM-2 assays. Kinetic analyses demonstrated that it behaves as a non-competitive inhibitor, with a submicromolar K<sub>i</sub> (K<sub>i</sub> = 148 ± 14 nM). This represents a 10-fold improvement in potency over the prior art probe, Mitoxantrone. It is also important to note that Mitoxantrone’s intense color limits its use in some assay detection formats; as described above, CID4870494 does not appear as an artifact in fluorescence- (CCF2) and absorbance-based (nitrocefin) assays. Further studies employing the TFA salt form of the probe (<a href="https://pubchem.ncbi.nlm.nih.gov/substance/111978109" ref="pagearea=abstract&targetsite=entrez&targetcat=link&targettype=pubchem">SID111978109</a>/CID50909789) demonstrate that probe <a href="/pcsubstance/?term=ML121[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML121</a> can potentiate the antibiotic activity of imipenem in VIM-2 transformed <i>E. coli</i>. Thus, these assays reveal the probe <a href="/pcsubstance/?term=ML121[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML121</a> is a novel, selective, and potent VIM-2 inhibitor. This probe will serve as a valuable tool to elucidate the role of VIM-2 in nosocomial beta-lactam antibiotic resistance.</p></div><div class="h2"></div><p><b>Assigned Assay Grant #:</b> 1 R21 NS059451-01 Fast Track</p><p><b>Screening Center Name & PI:</b> Scripps Research Institute Molecular Screening Center (SRIMSC); Hugh Rosen</p><p><b>Chemistry Center Name & PI:</b> SRIMSC; Hugh Rosen</p><p><b>Assay Submitter & Institution:</b> Peter Hodder, TSRI</p><p><b>PubChem Summary Bioassay Identifier (AID):</b>
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<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1854" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1854</a></p><div id="ml121.fu1" class="figure bk_fig"><div class="graphic"><img src="/books/NBK63598/bin/ml121fu1.jpg" alt="Selective VIM-2 Inhibitor Probe ML121." /></div><h3><span class="title">Selective VIM-2 Inhibitor Probe <a href="/pcsubstance/?term=ML121[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML121</a></span></h3><div class="caption"><p>MLS 000680027</p><p>VIM-2 K<sub>i</sub> = 148 ± 14 nM (non-competitive inhibitor)</p><p>IMP-1 IC<sub>50</sub> > 60 μM (Not Active)</p><p>TEM-1 IC<sub>50</sub> > 60 μM (Not Active)</p></div></div><div id="ml121.tu1" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK63598/table/ml121.tu1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml121.tu1_lrgtbl__"><table><thead><tr><th id="hd_h_ml121.tu1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CID/ML#</th><th id="hd_h_ml121.tu1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Target Name</th><th id="hd_h_ml121.tu1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">IC50 (nM) [SID, AID]</th><th id="hd_h_ml121.tu1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Anti-target Name</th><th id="hd_h_ml121.tu1_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">IC50 (μM) [SID, AID]</th><th id="hd_h_ml121.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Fold Selective</th><th id="hd_h_ml121.tu1_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Secondary Assay(s) Name: IC50 (nM) [SID, AID]</th></tr></thead><tbody><tr><td headers="hd_h_ml121.tu1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CID 4870494/<a href="/pcsubstance/?term=ML121[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML121</a></td><td headers="hd_h_ml121.tu1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">VIM-2</td><td headers="hd_h_ml121.tu1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">54 nm (liquid)<br /><br />223 nM powder [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/24790728" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID24790728</a>
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<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2128" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 2128</a>]</td><td headers="hd_h_ml121.tu1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">IMP-1</td><td headers="hd_h_ml121.tu1_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>60 μM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/24790728" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID24790728</a>
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<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2128" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 2128</a>]</td><td headers="hd_h_ml121.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>269</td><td headers="hd_h_ml121.tu1_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">TEM-1 Assay: >60 μM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/24790728" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 24790728</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2128" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 2128</a>]<br /><br />IM-2 Ki Assay = 148 nM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/85856282" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID85856282</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2128" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 2128</a>]<br /><br />Imipenem Synergy Assay: Active. Reduces the MIC of imipenem by four fold at 12.5μM [TFA Salt <a href="https://pubchem.ncbi.nlm.nih.gov/substance/111978109" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID111978109</a>/CID50909789 <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504620" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504620</a>]</td></tr></tbody></table></div></div><div id="ml121.s1"><h2 id="_ml121_s1_">Recommendations for scientific use of the probe (<a href="/pcsubstance/?term=ML121[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML121</a>)</h2><p><i>Limitations in state of the art.</i> Unfortunately, only one selective, non-competitive VIM-2 inhibitor, <b>mitoxantrone (1,4-dihydroxy-5,8-bis([2-([2-hydroxyethyl]amino)ethyl]amino)-9,10-anthracenedione; CID 4212)</b>, has been reported. Mitoxantrone is a type II topoisomerase inhibitor that disrupts DNA synthesis and DNA repair in both healthy cells and cancer cells [<a class="bk_pop" href="#ml121.r1">1</a>]. Its use to examine VIM-2 function is hampered by its moderate potency (K<sub>i</sub>= 1.5 ± 0.2 μM). The probe reported here exhibits a 10-fold improvement in potency over mitoxantrone. Mitoxantrone’s intense color limits its use in some assay detection formats. In addition, <b>4-chloromercuribenzoic acid</b> (<b><i>p</i>CMB; CID 1730)</b>, a slowly reversible/irreversible VIM-2 inhibitor, has been shown to have a synergistic effect with β-lactam antibacterials in VIM-2-expressing bacteria.[<a class="bk_pop" href="#ml121.r2">2</a>] However, this cysteine-reactive reagent is known to have several off-target activities, lessening its value for mechanistic studies.</p><p><i>Probe Applications.</i> Here we demonstrate that the probe <a href="/pcsubstance/?term=ML121[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML121</a> (CID4870494/<a href="https://pubchem.ncbi.nlm.nih.gov/substance/24790728" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 24790728</a>) modulates the activities of VIM-2 in a variety of biochemical assays, but is inactive against other metallo- and serine-beta-lactamases, IMP-1 and TEM-1. The probe binds to VIM-2 and inhibits its metallo-beta-lactamase enzymatic activity. This finding has significant implications for studies that probe the enzymology of VIM-2, especially mechanistic studies to better understand VIM-2’s notorious broad-spectrum activity against various beta-lactam based antibiotics. Further, the selectivity of this probe can be useful for experiments that aim to inhibit VIM-2 activity, without inhibiting IMP-1 or TEM-1 activity. In a broader role, the selectivity, potency, and non-competitive action of this compound will enable further investigations into the biological and biochemical roles of metallo-beta-lactamase enzymes, and may be useful in the design of inhibitors to inhibit this clinically relevant enzyme.</p><p><i>Expected end-users of the probe in the research community</i>. The probe can be used by researchers studying microbiology, antibiotic chemistry, lactamase enzymology, and bacterial cell biology. Thus it is conceivable that scientists in diverse fields will be able to apply this powerful chemical probe to elucidate the role of VIM-2 in cellular pathways and investigate mechanisms of VIM-2 inhibition in biochemical and microbiology-based assays.</p><p><i>Relevant biology of the probe.</i> The metallo-beta-lactamase VIM-2 is a zinc-dependent, Ambler Class “B” beta-lactamase that hydrolyzes beta-lactam based antibiotics (e.g. penicillins, carbapenems), rendering them ineffective. No VIM-2 inhibitors yet exist for clinical use, and all VIM-2 inhibitors reported to date have been designed to bind zinc or modify cysteine found in the enzyme’s active site. Therefore, selective, non-competitive VIM-2 inhibitors are desired to probe VIM-2 function exclusive of active site inhibition.</p></div><div id="ml121.s2"><h2 id="_ml121_s2_">1. Introduction</h2><p>The emergence of gram-negative bacteria that exhibit multi-drug resistance, combined with the paucity of new antibiotics, poses a public health challenge [<a class="bk_pop" href="#ml121.r3">3</a>]. The production of bacterial beta-lactamase enzymes, in particular, is a common mechanism of drug resistance [<a class="bk_pop" href="#ml121.r4" data-bk-pop-others="ml121.r5 ml121.r6">4–6</a>]. The beta-lactamases evolved from bacteria with resistance to naturally-occurring beta-lactams or penams [<a class="bk_pop" href="#ml121.r7">7</a>], agents which inhibit the transpeptidase involved in cell wall biosynthesis [<a class="bk_pop" href="#ml121.r8">8</a>]. Human medicine adapted these agents into synthetic antibiotics such as penicillins, cephalosporins, carbapenems, and monobactams that contain a 2-azetidone ring [<a class="bk_pop" href="#ml121.r7">7</a>, <a class="bk_pop" href="#ml121.r9">9</a>]. The metallo-beta-lactamases (MBL) are zinc-dependent class B beta-lactamases that hydrolyze the beta-lactam ring, rendering the antibiotic ineffective [<a class="bk_pop" href="#ml121.r8">8</a>, <a class="bk_pop" href="#ml121.r10">10</a>]. Increasingly, nosocomial beta-lactam antibiotic resistance arises in <i>P. aeruginosa, Enterobacteriaceae</i>, and other pathogenic bacteria via gene transfer of B1 MBLs [<a class="bk_pop" href="#ml121.r6">6</a>, <a class="bk_pop" href="#ml121.r11">11</a>], including IMP (active on IMiPenem) [<a class="bk_pop" href="#ml121.r12">12</a>] and VIM (Verona IMipenemase) [<a class="bk_pop" href="#ml121.r13">13</a>, <a class="bk_pop" href="#ml121.r14">14</a>]. For two of these enzymes, VIM-2 and IMP-1, no inhibitors exist for clinical use [<a class="bk_pop" href="#ml121.r8">8</a>, <a class="bk_pop" href="#ml121.r11">11</a>]. Thus, the identification of MBL inhibitors would provide useful tools for reducing nosocomial infections and elucidating their mechanism of action [<a class="bk_pop" href="#ml121.r2">2</a>, <a class="bk_pop" href="#ml121.r15">15</a>]. In the present report, we identify and characterize a compound belonging to a novel scaffold that inhibits VIM-2 selectively and with a submicromolar K<sub>i</sub>. This represents a significant advance in the field of selective beta-lactamase inhibitors.</p><div id="ml121.s3"><h3>2.1. Assays</h3><div id="ml121.tu2" class="table"><h3><span class="title">PubChem BioAssay Table</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK63598/table/ml121.tu2/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml121.tu2_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml121.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">AID</th><th id="hd_h_ml121.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Assay Name</th><th id="hd_h_ml121.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Assay Type</th><th id="hd_h_ml121.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Target</th><th id="hd_h_ml121.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Powder</th><th id="hd_h_ml121.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">[Compound]</th></tr></thead><tbody><tr><td headers="hd_h_ml121.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1527" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1527</a></td><td headers="hd_h_ml121.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Primary biochemical HTS assay to identify inhibitors of VIM-2.</td><td headers="hd_h_ml121.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Primary Assay (1X%ACT)</td><td headers="hd_h_ml121.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">VIM-2</td><td headers="hd_h_ml121.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">No</td><td headers="hd_h_ml121.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">5.6 μM</td></tr><tr><td headers="hd_h_ml121.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1556" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1556</a></td><td headers="hd_h_ml121.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Epi-absorbance primary biochemical HTS assay to identify inhibitors of IMP-1 metallo-beta-lactamase.</td><td headers="hd_h_ml121.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Primary Assay (1X%ACT)</td><td headers="hd_h_ml121.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">IMP-1</td><td headers="hd_h_ml121.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">No</td><td headers="hd_h_ml121.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">5.6 μM</td></tr><tr><td headers="hd_h_ml121.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1856" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1856</a></td><td headers="hd_h_ml121.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Epi-absorbance-based counterscreen for selective VIM-2 inhibitors: biochemical HTS assay to identify inhibitors of IMP-1 metallo-beta-lactamase.</td><td headers="hd_h_ml121.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Counterscreen Assay (3X%ACT)</td><td headers="hd_h_ml121.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">IMP-1</td><td headers="hd_h_ml121.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">No</td><td headers="hd_h_ml121.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">5.6 μM</td></tr><tr><td headers="hd_h_ml121.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1857" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1857</a></td><td headers="hd_h_ml121.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">FRET-based counterscreen assay for selective VIM-2 inhibitors: biochemical HTS assay to identify epi-absorbance assay artifacts.</td><td headers="hd_h_ml121.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Counterscreen Assay (3X%ACT)</td><td headers="hd_h_ml121.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">VIM-2 (CCF2)</td><td headers="hd_h_ml121.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">No</td><td headers="hd_h_ml121.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">5.6 μM</td></tr><tr><td headers="hd_h_ml121.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1860" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1860</a></td><td headers="hd_h_ml121.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Epi-absorbance-based confirmation biochemical HTS assay to identify selective inhibitors of VIM-2 metallo-beta-lactamase.</td><td headers="hd_h_ml121.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Confirmation Assay (3X %ACT)</td><td headers="hd_h_ml121.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">VIM-2</td><td headers="hd_h_ml121.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">No</td><td headers="hd_h_ml121.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">5.6 μM</td></tr><tr><td headers="hd_h_ml121.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1866" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1866</a></td><td headers="hd_h_ml121.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Epi-absorbance-based counterscreen assay for selective VIM-2 inhibitors: biochemical HTS assay to identify inhibitors of TEM-1 serine-beta-lactamase.</td><td headers="hd_h_ml121.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Counterscreen Assay (3X%ACT)</td><td headers="hd_h_ml121.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">TEM-1</td><td headers="hd_h_ml121.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">No</td><td headers="hd_h_ml121.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">5.6 μM</td></tr><tr><td headers="hd_h_ml121.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1919" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1919</a></td><td headers="hd_h_ml121.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Epi-absorbance-based dose response biochemical high throughput screening assay for selective inhibitors of VIM-2 metallo-beta-lactamase.</td><td headers="hd_h_ml121.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Dose Response (3X EC<sub>50</sub>)</td><td headers="hd_h_ml121.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">VIM-2</td><td headers="hd_h_ml121.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">No</td><td headers="hd_h_ml121.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">10-point, 1:3 dilution starting at 55.7 μM</td></tr><tr><td headers="hd_h_ml121.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1920" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1920</a></td><td headers="hd_h_ml121.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Epi-absorbance-based counterscreen for selective VIM-2 inhibitors: dose response biochemical HTS assay to identify inhibitors of IMP-1 metallo-beta-lactamase.</td><td headers="hd_h_ml121.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Dose Response Counterscreen (3X EC<sub>50</sub>)</td><td headers="hd_h_ml121.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">IMP-1</td><td headers="hd_h_ml121.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">No</td><td headers="hd_h_ml121.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">10-point, 1:3 dilution starting at 55.7 μM</td></tr><tr><td headers="hd_h_ml121.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1925" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1925</a></td><td headers="hd_h_ml121.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Epi-absorbance-based counterscreen for selective VIM-2 inhibitors: dose response biochemical HTS assay to identify inhibitors of TEM-1 serine-beta-lactamase.</td><td headers="hd_h_ml121.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Dose Response Counterscreen (3X EC<sub>50</sub>)</td><td headers="hd_h_ml121.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">TEM-1</td><td headers="hd_h_ml121.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">No</td><td headers="hd_h_ml121.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">10-point, 1:3 dilution starting at 55.7 μM</td></tr><tr><td headers="hd_h_ml121.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1926" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1926</a></td><td headers="hd_h_ml121.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">FRET-based counterscreen for selective VIM-2 inhibitors: dose response biochemical HTS assay to identify epi-absorbance assay artifacts.</td><td headers="hd_h_ml121.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Dose Response Counterscreen (3X EC<sub>50</sub>)</td><td headers="hd_h_ml121.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">VIM-2 (CCF2)</td><td headers="hd_h_ml121.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">No</td><td headers="hd_h_ml121.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">10-point, 1:3 dilution starting at 55.7 μM</td></tr><tr><td headers="hd_h_ml121.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1927" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1927</a></td><td headers="hd_h_ml121.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">FRET-based counterscreen for selective VIM-2 inhibitors: dose response biochemical HTS assay to identify inhibitors of IMP-1 metallo-beta-lactamase.</td><td headers="hd_h_ml121.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Dose Response Counterscreen (3X EC<sub>50</sub>)</td><td headers="hd_h_ml121.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">IMP-1 (CCF2)</td><td headers="hd_h_ml121.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">No</td><td headers="hd_h_ml121.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">10-point, 1:3 dilution starting at 55.7 μM</td></tr><tr><td headers="hd_h_ml121.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1854" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1854</a></td><td headers="hd_h_ml121.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Summary of probe development efforts to identify selective inhibitors of VIM-2 metallo-beta-lactamase.</td><td headers="hd_h_ml121.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Summary</td><td headers="hd_h_ml121.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">VIM-2</td><td headers="hd_h_ml121.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">No</td><td headers="hd_h_ml121.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">N/A</td></tr><tr><td headers="hd_h_ml121.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2128" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2128</a></td><td headers="hd_h_ml121.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Late stage results from the probe development efforts to identify inhibitors of VIM-2: probe results</td><td headers="hd_h_ml121.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Late Stage (probe)</td><td headers="hd_h_ml121.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">VIM-2</td><td headers="hd_h_ml121.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Yes</td><td headers="hd_h_ml121.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Various</td></tr><tr><td headers="hd_h_ml121.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2317" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2317</a></td><td headers="hd_h_ml121.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Late stage results from the probe development efforts to identify selective inhibitors of VIM-2 metallo-beta-lactamase: Prior art results.</td><td headers="hd_h_ml121.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Late Stage (prior art)</td><td headers="hd_h_ml121.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">VIM-2</td><td headers="hd_h_ml121.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Yes</td><td headers="hd_h_ml121.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Various</td></tr><tr><td headers="hd_h_ml121.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504620" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">504620</a></td><td headers="hd_h_ml121.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Late stage Assay provider assay to determine imipenem Synergy of probe</td><td headers="hd_h_ml121.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Late Stage (probe)</td><td headers="hd_h_ml121.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">VIM-2</td><td headers="hd_h_ml121.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Yes</td><td headers="hd_h_ml121.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Dose response starting at 100μM</td></tr></tbody></table></div></div><div id="ml121.tu3" class="table"><h3><span class="title">Table of Assay Rationale and Description</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK63598/table/ml121.tu3/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml121.tu3_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml121.tu3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">AID</th><th id="hd_h_ml121.tu3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Assay Rationale</th><th id="hd_h_ml121.tu3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Assay Description</th><th id="hd_h_ml121.tu3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Z′</th><th id="hd_h_ml121.tu3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">S:B</th></tr></thead><tbody><tr><td headers="hd_h_ml121.tu3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1527" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1527</a></td><td headers="hd_h_ml121.tu3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Primary screen to identify compounds that inhibit VIM-2.</td><td headers="hd_h_ml121.tu3_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">In this Primary assay, compounds are incubated with purified VIM-2 enzyme and <b>nitrocefin</b> in detergent-containing buffer at room temperature. The reaction is stopped by addition of EDTA, followed by measurement of well fluorescence. As designed, compounds that inhibit VIM-2 will inhibit nitrocefin hydrolysis, generation of red product, and quenching of plate fluorescence, resulting in increased well fluorescence. Compounds are tested in singlicate.</td><td headers="hd_h_ml121.tu3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.94 ± 0.01</td><td headers="hd_h_ml121.tu3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">7.1 ± 0.72</td></tr><tr><td headers="hd_h_ml121.tu3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1556" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1556</a></td><td headers="hd_h_ml121.tu3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">To identify compounds which inhibit the related metallo-beta-lactamase enzyme, IMP-1. This assay serves as an <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1527" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1527</a> counterscreen, to determine whether compounds are selective VIM-2 inhibitors.</td><td headers="hd_h_ml121.tu3_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">In this Primary assay, compounds are incubated with purified IMP-1 enzyme and <b>nitrocefin</b> in detergent-containing buffer at room temperature. The reaction is stopped by addition of EDTA, followed by measurement of well fluorescence. As designed, compounds that inhibit IMP-1 will inhibit nitrocefin hydrolysis, generation of red product, and quenching of plate fluorescence, resulting in increased well fluorescence. Compounds are tested in singlicate.</td><td headers="hd_h_ml121.tu3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.90 ± 0.02</td><td headers="hd_h_ml121.tu3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">7.9 ± 1.7</td></tr><tr><td headers="hd_h_ml121.tu3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1856" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1856</a></td><td headers="hd_h_ml121.tu3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">To determine whether compounds were nonselective due to IMP-1 inhibition.</td><td headers="hd_h_ml121.tu3_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Same as <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1556" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1556</a> except that compounds are tested in triplicate.</td><td headers="hd_h_ml121.tu3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.93 ± 0.06</td><td headers="hd_h_ml121.tu3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">6.7 ± 0.1</td></tr><tr><td headers="hd_h_ml121.tu3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1857" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1857</a></td><td headers="hd_h_ml121.tu3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">To determine whether compounds were epi- absorbance assay artifacts.</td><td headers="hd_h_ml121.tu3_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">In this assay, compounds are incubated with purified VIM-2 enzyme and <b>CCF2</b> in detergent-containing buffer at room temperature. The reaction is stopped by addition of EDTA, followed by measurement of well FRET. As designed, compounds that inhibit VIM- 2 activity will prevent CCF2 hydrolysis, thereby preventing well FRET. Compounds were tested in triplicate.</td><td headers="hd_h_ml121.tu3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.93 ± 0.02</td><td headers="hd_h_ml121.tu3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3.9 ± 0.1</td></tr><tr><td headers="hd_h_ml121.tu3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1860" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1860</a></td><td headers="hd_h_ml121.tu3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">To confirm VIM-2 inhibition activity using nitrocefin substrate.</td><td headers="hd_h_ml121.tu3_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Same as <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1527" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1527</a> except that compounds tested in triplicate.</td><td headers="hd_h_ml121.tu3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.95 ± 0.01</td><td headers="hd_h_ml121.tu3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">6.3 ± 0.1</td></tr><tr><td headers="hd_h_ml121.tu3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1866" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1866</a></td><td headers="hd_h_ml121.tu3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">To determine whether compounds were nonselective due to inhibition of TEM-1 enzyme.</td><td headers="hd_h_ml121.tu3_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Same as <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1856" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1856</a> except that compounds are incubated with purified TEM-1 enzyme.</td><td headers="hd_h_ml121.tu3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.91 ± 0.01</td><td headers="hd_h_ml121.tu3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3.9 ± 0.1</td></tr><tr><td headers="hd_h_ml121.tu3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1919" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1919</a></td><td headers="hd_h_ml121.tu3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">To determine VIM-2 dose response curves using nitrocefin substrate.</td><td headers="hd_h_ml121.tu3_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Same as <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1527" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1527</a> except that compounds are tested in triplicate using a 10-point dilution series starting at 55.7 μM.</td><td headers="hd_h_ml121.tu3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.94 ± 0.02</td><td headers="hd_h_ml121.tu3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">7.2 ± 0.2</td></tr><tr><td headers="hd_h_ml121.tu3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1920" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1920</a></td><td headers="hd_h_ml121.tu3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">To determine IMP-1 dose response curves using nitrocefin substrate. This assay serves as a counterscreen for <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1919" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1919</a>.</td><td headers="hd_h_ml121.tu3_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Same as <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1556" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1556</a> except that compounds are tested in triplicate using a 10-point dilution series starting at 55.7 μM.</td><td headers="hd_h_ml121.tu3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.94 ± 0.01</td><td headers="hd_h_ml121.tu3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">7.5 ± 0.3</td></tr><tr><td headers="hd_h_ml121.tu3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1925" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1925</a></td><td headers="hd_h_ml121.tu3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">To determine TEM-1 dose response curves using nitrocefin substrate. This assay serves as a <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1919" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1919</a> counterscreen.</td><td headers="hd_h_ml121.tu3_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Same as <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1866" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1866</a> except that compounds are tested in triplicate using a 10-point dilution series starting at 55.7 μM.</td><td headers="hd_h_ml121.tu3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.91 ± 0.01</td><td headers="hd_h_ml121.tu3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3.9 ± 0.1</td></tr><tr><td headers="hd_h_ml121.tu3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1926" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1926</a></td><td headers="hd_h_ml121.tu3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">To determine VIM-2 dose response curves using CCF2 substrate. This assay serves as <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1919" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1919</a> counterscreen.</td><td headers="hd_h_ml121.tu3_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Same as <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1857" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1857</a> except that compounds are tested in triplicate using a 10-point dilution series starting at 55.7 μM.</td><td headers="hd_h_ml121.tu3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.92 ± 0.01</td><td headers="hd_h_ml121.tu3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">4.3 ± 0.1</td></tr><tr><td headers="hd_h_ml121.tu3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1927" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1927</a></td><td headers="hd_h_ml121.tu3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">To determine IMP-1 dose response curves using CCF2 substrate. This assay serves as a <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1919" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1919</a> counterscreen.</td><td headers="hd_h_ml121.tu3_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Same as <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1856" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1856</a> except that compounds are incubated with CCF2 substrate and in triplicate using a 10-point dilution series starting at 55.7 μM.</td><td headers="hd_h_ml121.tu3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.93 ± 0.02</td><td headers="hd_h_ml121.tu3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">25.3 ± 0.4</td></tr><tr><td headers="hd_h_ml121.tu3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1854" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1854</a></td><td headers="hd_h_ml121.tu3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Summarize probe development efforts.</td><td headers="hd_h_ml121.tu3_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">N/A</td><td headers="hd_h_ml121.tu3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">N/A</td><td headers="hd_h_ml121.tu3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">N/A</td></tr><tr><td headers="hd_h_ml121.tu3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2128" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2128</a></td><td headers="hd_h_ml121.tu3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Describes assays performed during probe development testing a probe candidate</td><td headers="hd_h_ml121.tu3_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">The probe candidate tested in VIM-2 assays, IMP-1 and TEM-1 counterscreens, VIM-2 Ki assays, and CCF2-format assays to identify nitrocefin assay artifacts.</td><td headers="hd_h_ml121.tu3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">N/A</td><td headers="hd_h_ml121.tu3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">N/A</td></tr><tr><td headers="hd_h_ml121.tu3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2317" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2317</a></td><td headers="hd_h_ml121.tu3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Describes the assays performed during the probe development effort which tested the prior art.</td><td headers="hd_h_ml121.tu3_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Prior art tested in VIM-2 assays, IMP-1 and TEM-1 counterscreen assays, VIM-2 Ki assays, and CCF2-format assays to identify nitrocefin assay artifacts.</td><td headers="hd_h_ml121.tu3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">N/A</td><td headers="hd_h_ml121.tu3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">N/A</td></tr><tr><td headers="hd_h_ml121.tu3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504620" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">504620</a></td><td headers="hd_h_ml121.tu3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Determines whether the probe can potentiate the activity of the known BLA inhibitor imipenem</td><td headers="hd_h_ml121.tu3_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Probe tested against VIM-2 transformed E. coli in the absence and presence of imipenem.</td><td headers="hd_h_ml121.tu3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">N/A</td><td headers="hd_h_ml121.tu3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">N/A</td></tr></tbody></table></div></div><p><i>(Click on the hyperlinks to obtain itemized protocols directly from PubChem; also see Summary</i>
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<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1854" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1854</a><i>)</i></p><div id="ml121.s4"><h4>VIM-2 Inhibition Assays (Epi-Absorbance-based; Nitrocefin) (PubChem AIDs <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1527" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1527</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1860" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1860</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1919" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1919</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2128" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2128 </a>and <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2317" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2317</a>)</h4><p>The purpose of these assays is to identify compounds that act as inhibitors of the VIM-2 beta-lactamase. This biochemical epi-absorbance-format assay employs the cephalosporin nitrocefin as the VIM-2 substrate, and takes advantage of the fluorescent properties of white microtiter plates [<a class="bk_pop" href="#ml121.r16">16</a>]. Nitrocefin is a yellow chromogenic substrate (Imax = 395 nm) that is hydrolyzed by beta-lactamases to yield a red product with increased absorbance properties (Imax = 495 nm) that quenches plate fluorescence by absorbing the plate's emission light [<a class="bk_pop" href="#ml121.r16">16</a>]. In this assay, test compounds are incubated with purified VIM-2 enzyme and nitrocefin in detergent-containing buffer at room temperature. The reaction is stopped by the addition of EDTA, followed by measurement of well fluorescence. As designed, compounds that inhibit VIM-2 will inhibit nitrocefin hydrolysis, inhibit generation of red product, and inhibit quenching of plate fluorescence, resulting in an increase in well fluorescence. Compounds were tested in singlicate (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1527" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID1527</a>) and triplicate (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1860" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1860</a>) at a final nominal concentration of 5.6 μM, in a 10-point 1:3 dilution series starting at a nominal concentration of 55.7 μM (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1919" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1919</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2128" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2128</a>, and <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2317" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2317</a>).</p></div><div id="ml121.s5"><h4>IMP-1 Inhibition Counterscreens (Epi-absorbance-based; Nitrocefin) (AIDs <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1556" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1556</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1856" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1856</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1920" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1920</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2128" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2128 </a>and <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2317" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2317</a>)</h4><p>The purpose of this assay is to identify compounds that act as inhibitors of the IMP-1 beta-lactamase. This biochemical epi-absorbance-format assay employs the cephalosporin nitrocefin as the IMP-1 substrate, and takes advantage of the fluorescent properties of white microtiter plates [<a class="bk_pop" href="#ml121.r16">16</a>]. This assay also serves as a counterscreen to determine whether compounds identified as possible VIM-2 selective inhibitors are non-selective due to inhibition of IMP-1. Nitrocefin is a yellow chromogenic substrate (Imax = 395 nm) that is hydrolyzed by beta-lactamases to yield a red product with increased absorbance properties (Imax = 495 nm) that quenches plate fluorescence by absorbing the plate's emission light [<a class="bk_pop" href="#ml121.r16">16</a>]. In this assay, test compounds are incubated with purified IMP-1 enzyme and nitrocefin in detergent-containing buffer at room temperature. The reaction is stopped by the addition of EDTA, followed by measurement of well fluorescence. As designed, compounds that inhibit IMP-1 will inhibit nitrocefin hydrolysis, inhibit generation of red product, and inhibit quenching of plate fluorescence, resulting in an increase in well fluorescence. Compounds were tested in singlicate (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1556" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1556</a>) or in triplicate (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1856" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1856</a>) at a final nominal concentration of 5.6 μM, and in triplicate using a dilution series starting at a nominal test concentration of 60 micromolar (AIDs <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1920" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1920</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2128" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2128</a> and <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2317" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2317</a>).</p></div><div id="ml121.s6"><h4>VIM-2 Inhibition Counterscreens (FRET-based; CCF2) (AIDs <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1857" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1857</a> and <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1926" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1926</a>)</h4><p>The purpose of this assay is to confirm VIM-2 activity of compounds identified as active in a previous set of experiments entitled, “Primary biochemical high throughput screening assay to identify inhibitors of VIM-2 metallo-beta-lactamase” (PubChem <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1527" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1527</a>), and inactive in a set of experiments entitled, “Epi-absorbance primary biochemical high throughput screening assay to identify inhibitors of IMP-1 metallo-beta-lactamase” (PubChem <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1556" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1556</a>). This assay also serves as an orthogonal counterscreen to identify epi-absorbance assay artifacts. This biochemical FRET-based assay employs as the beta-lactamase substrate the cephalosporin scaffold CCF2 that has been tagged with a donor (7-hydroxycoumarin) and quencher/acceptor (fluorescein) FRET pair. VIM-2 mediated hydrolysis of the CCF2 beta-lactam bond releases the donor from the quencher, leading to increased donor fluorescence at 460nm and decreased acceptor fluorescence at 535nm. In this assay, test compounds are incubated with purified VIM-2 enzyme and CCF2 in detergent-containing buffer at room temperature. The reaction is stopped by the addition of EDTA, followed by measurement of well FRET. As designed, compounds that inhibit VIM-2 activity will prevent CCF2 hydrolysis, thereby preventing well FRET. Compounds were tested in triplicate at a final nominal concentration of 5.6 micromolar (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1857" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1857</a>), and in triplicate using a dilution series starting at a nominal test concentration of 60 micromolar (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1926" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1926</a>).</p></div><div id="ml121.s7"><h4>IMP-1 Inhibition Counterscreens (FRET-based; CCF2) (AIDs <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1927" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1927</a>)</h4><p>The purpose of this assay is to determine dose response curves for compounds identified as active in a previous set of experiments entitled, “Primary biochemical high throughput screening assay to identify inhibitors of VIM-2 metallo-beta-lactamase” (PubChem <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1527" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1527</a>), and inactive in a set of experiments entitled, “Epi-absorbance primary biochemical high throughput screening assay to identify inhibitors of IMP-1 metallo-beta-lactamase” (PubChem <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1556" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1556</a>). This assay also serves as an orthogonal counterscreen to identify epi-absorbance assay artifacts and to determine whether compounds were nonselective due to inhibition of IMP-1. This biochemical FRET-based assay employs the beta-lactamase substrate with cephalosporin scaffold CCF2 that has been tagged with a donor (7-hydroxycoumarin) and quencher/acceptor (fluorescein) FRET pair. IMP-1 mediated hydrolysis of the CCF2 beta-lactam increases donor fluorescence at 460nm and decreases acceptor fluorescence at 535nm. In this assay, test compounds are incubated with purified IMP-1 enzyme and CCF2 in detergent-containing buffer at room temperature. The reaction is stopped by the addition of EDTA, followed by measurement of well fluorescence. As designed, compounds that inhibit IMP-1 activity will prevent CCF2 hydrolysis, thereby preventing increase of donor fluorescence at 460nm and decrease of acceptor fluorescence at 535nm. Compounds were tested in triplicate using a dilution series starting at a nominal test concentration of 60 micromolar (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1927" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1927</a>).</p></div><div id="ml121.s8"><h4>TEM-1 Inhibition Counterscreens (FRET-based; CCF2) (AIDs <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1866" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1866</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1925" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1925</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2128" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2128</a>, and <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2317" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2317</a>)</h4><p>The purpose of this assay is to determine whether compounds identified as active in a previous set of experiments entitled, “Primary biochemical high throughput screening assay to identify inhibitors of VIM-2 metallo-beta-lactamase” (PubChem <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1527" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1527</a>), were nonselective or assay artifact due to inhibition of TEM-1, a serine beta-lactamase that hydrolyzes ampicillin, has negligible activity against extended-spectrum cephalosporins, and is inhibited by clavulanic acid [<a class="bk_pop" href="#ml121.r15">15</a>]. This biochemical epi-absorbance-format assay employs the cephalosporin nitrocefin as the TEM-1 substrate, and takes advantage of the fluorescent properties of white microtiter plates [<a class="bk_pop" href="#ml121.r16">16</a>]. Nitrocefin is a yellow chromogenic substrate (Imax = 395 nm) that is hydrolyzed by beta-lactamases to yield a red product with increased absorbance properties (Imax = 495 nm) that quenches plate fluorescence by absorbing the plate's emission light [<a class="bk_pop" href="#ml121.r16">16</a>]. In this assay, test compounds are incubated with purified TEM-1 enzyme and nitrocefin in detergent-containing buffer at room temperature. The reaction is stopped by the addition of potassium clavulanate, followed by measurement of well fluorescence. As designed, compounds that inhibit TEM-1 will inhibit nitrocefin hydrolysis, inhibit generation of red product, and inhibit quenching of plate fluorescence, resulting in an increase in well fluorescence. Compounds were tested in triplicate at a final nominal concentration of 5.6 micromolar (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1866" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1866</a>) and in triplicate using a dilution series starting at a nominal test concentration of 60 micromolar (AIDs <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1925" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1925</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2128" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2128</a> and <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2317" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2317</a>).</p></div><div id="ml121.s9"><h4>VIM-2 Ki Assay (AIDs <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2128" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2128</a> and <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2317" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2317</a>)</h4><p>The purpose of this assay is to determine the inhibition constant (Ki) and modality of probe candidate molecules. Kinetic assays were conducted by incubating a range of substrate concentrations (100-8 μM) with varying inhibitor concentrations and 0.1 nM enzyme at room temperature in buffer containing 50mM HEPES, 50μM ZnSO4, 0.05% Brij 35, pH 7.1. Absorbance was measured on a Tecan Safire2 monochromatic microplate reader at 495 nm. Initial velocities were obtained from plots of absorbance at 495 nm versus time, using data points from only the linear portion of the hydrolysis curve. Substrate hydrolysis was continuously monitored. Initial velocities were plotted vs. substrate concentration and kinetic parameters were calculated using Graphpad Prism version 5.01 suite of programs. All Ki values were determined by non-linear regression (hyperbolic equation) analysis using the mixed inhibition model which allows for simultaneous determination of mechanism of inhibition. Mechanism of inhibition was determined using the “alpha” parameter derived from a mixed-model inhibition by GraphPad Prism. The mechanism of inhibition was additionally evaluated by Hanes-Woolf and Lineweaver-Burke plots. Lineweaver-Burke best-fit lines are reported, along with Ki calculated from non-linear regression. The number of replicates for this assay is 3. Compounds tested in this assay are the prior art compound mitoxantrone (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2317" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 2317</a>) and the probe <a href="https://pubchem.ncbi.nlm.nih.gov/substance/85856282" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 85856282</a> (<a href="https://pubchem.ncbi.nlm.nih.gov/substance/2128" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2128</a>).</p></div></div><div id="ml121.s10"><h3>2.2. Probe Chemical Characterization</h3><p><i>Probe chemical structure including stereochemistry</i>. The synthesis of the probe and analytical characterization data are probe <a href="/pcsubstance/?term=ML121[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML121</a> are provided in <a href="#ml121.s11">Section 2.3</a> below. Data that establishes the stereochemistry of probe <a href="/pcsubstance/?term=ML121[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML121</a> (as a racemic compound) is also provided in <a href="#ml121.s11">Section 2.3</a>.</p><p><a href="/pcsubstance/?term=ML121[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML121</a> was obtained with >96% purity according to 1H NMR and LCMS analysis. However, <a href="/pcsubstance/?term=ML121[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML121</a> had relatively poor solubility characteristics, so we also generated a more soluble TFA salt, identified as <a href="/pcsubstance/?term=ML121[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML121</a>-TFA salt (<a href="https://pubchem.ncbi.nlm.nih.gov/substance/111978109" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID111978109</a>/CID50909789). The TFA salt was also obtained with >96% purity by 1H NMR and LCMS analysis.</p><p><i>Solubility</i>. The solubility of probe <a href="/pcsubstance/?term=ML121[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML121</a> (synthesized and registered by the SRIMSC as SR-03000001759-1/ <a href="https://pubchem.ncbi.nlm.nih.gov/substance/99343667" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID99343667</a>/ CID46891627) was measured in phosphate buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM sodium phosphate dibasic, 2 mM potassium phosphate monobasic and a pH of 7.4) at room temperature (23°C). The solubility of was found to be 16 μM. In contrast, the solubility of the TFA salt of <a href="/pcsubstance/?term=ML121[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML121</a> in the same assay system was found to be 152 μM.</p><p><i>Stability</i>. The stability of probe <a href="/pcsubstance/?term=ML121[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML121</a> and the TFA salt form of probe <a href="/pcsubstance/?term=ML121[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML121</a> (synthesized and registered by the SRISMC as SR-03000001759-1/ <a href="https://pubchem.ncbi.nlm.nih.gov/substance/99343667" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID99343667</a>/ CID46891627) were measured at room temperature (23°C) in PBS (no antioxidants or other protectants; DMSO concentration below 0.1%). The stability, represented by the half-life, was found to be > 48 hours for both compounds. Below are graphs showing loss of compound with time over a 48 hour period with a minimum of 6 time points. The table below indicates the percent of compound remaining at the end of the 48 hours.</p><p><i>The probe</i>
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<a href="/pcsubstance/?term=ML121[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML121</a>
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<i>and its TFA salt were measured for its ability to form glutathione adducts</i>. At concentrations of 100 μM reduced GSH, 10 μM of the probe does not appear to be a Michael acceptor [<a class="bk_pop" href="#ml121.r17">17</a>, <a class="bk_pop" href="#ml121.r18">18</a>].</p><div id="ml121.tu4" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK63598/table/ml121.tu4/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml121.tu4_lrgtbl__"><table><thead><tr><th id="hd_h_ml121.tu4_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Compound</th><th id="hd_h_ml121.tu4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MW</th><th id="hd_h_ml121.tu4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR Number</th><th id="hd_h_ml121.tu4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CID</th><th id="hd_h_ml121.tu4_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SID</th><th id="hd_h_ml121.tu4_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Solubility in PBS (μM)</th><th id="hd_h_ml121.tu4_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Michael Acceptor 100 μM GSH trap</th><th id="hd_h_ml121.tu4_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Stability in PBS t1/2 (hr)</th></tr></thead><tbody><tr><td headers="hd_h_ml121.tu4_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Probe <a href="/pcsubstance/?term=ML121[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML121</a> (Free base)</td><td headers="hd_h_ml121.tu4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">316</td><td headers="hd_h_ml121.tu4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-03000001759-1</td><td headers="hd_h_ml121.tu4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">46891627</td><td headers="hd_h_ml121.tu4_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99343667" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99343667</a> (synthesis)</td><td headers="hd_h_ml121.tu4_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">16</td><td headers="hd_h_ml121.tu4_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">No</td><td headers="hd_h_ml121.tu4_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">> 48 hr</td></tr><tr><td headers="hd_h_ml121.tu4_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Probe <a href="/pcsubstance/?term=ML121[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML121</a> (TFA salt)</td><td headers="hd_h_ml121.tu4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">430</td><td headers="hd_h_ml121.tu4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-03000001759-3</td><td headers="hd_h_ml121.tu4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">50909789</td><td headers="hd_h_ml121.tu4_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/111978109" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">111978109</a> (synthesis)</td><td headers="hd_h_ml121.tu4_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">152</td><td headers="hd_h_ml121.tu4_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">No</td><td headers="hd_h_ml121.tu4_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">> 48 hr</td></tr></tbody></table></div></div><div id="ml121.fu2" class="figure bk_fig"><div class="graphic"><img src="/books/NBK63598/bin/ml121fu2.jpg" alt="Stability of Selective VIM-2 Probe ML121 (SR3-1759)." /></div><h3><span class="title">Stability of Selective VIM-2 Probe <a href="/pcsubstance/?term=ML121[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML121</a> (SR3-1759)</span></h3></div><div id="ml121.tu5" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK63598/table/ml121.tu5/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml121.tu5_lrgtbl__"><table><thead><tr><th id="hd_h_ml121.tu5_1_1_1_1" colspan="3" rowspan="1" style="text-align:left;vertical-align:bottom;">Selective VIM-2 Inhibitor Probe <a href="/pcsubstance/?term=ML121[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML121</a></th></tr><tr><th headers="hd_h_ml121.tu5_1_1_1_1" id="hd_h_ml121.tu5_1_1_2_1" colspan="3" rowspan="1" style="text-align:left;vertical-align:bottom;">SR3-1759 Stability in PBS Buffer (pH 7.4)</th></tr><tr><th headers="hd_h_ml121.tu5_1_1_1_1 hd_h_ml121.tu5_1_1_2_1" id="hd_h_ml121.tu5_1_1_3_1" colspan="3" rowspan="1" style="text-align:left;vertical-align:bottom;">Sample concentration: 5 μM</th></tr><tr><th headers="hd_h_ml121.tu5_1_1_1_1 hd_h_ml121.tu5_1_1_2_1 hd_h_ml121.tu5_1_1_3_1" id="hd_h_ml121.tu5_1_1_4_1" colspan="3" rowspan="1" style="text-align:left;vertical-align:bottom;">Storage: Microcentrifuge Tube on Lab Bench</th></tr><tr><th headers="hd_h_ml121.tu5_1_1_1_1 hd_h_ml121.tu5_1_1_2_1 hd_h_ml121.tu5_1_1_3_1 hd_h_ml121.tu5_1_1_4_1" id="hd_h_ml121.tu5_1_1_5_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Time (hr)</th><th headers="hd_h_ml121.tu5_1_1_1_1 hd_h_ml121.tu5_1_1_2_1 hd_h_ml121.tu5_1_1_3_1 hd_h_ml121.tu5_1_1_4_1" id="hd_h_ml121.tu5_1_1_5_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">% remaining</th><th headers="hd_h_ml121.tu5_1_1_1_1 hd_h_ml121.tu5_1_1_2_1 hd_h_ml121.tu5_1_1_3_1 hd_h_ml121.tu5_1_1_4_1" id="hd_h_ml121.tu5_1_1_5_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">In(%remaining)</th></tr></thead><tbody><tr><td headers="hd_h_ml121.tu5_1_1_1_1 hd_h_ml121.tu5_1_1_2_1 hd_h_ml121.tu5_1_1_3_1 hd_h_ml121.tu5_1_1_4_1 hd_h_ml121.tu5_1_1_5_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0</td><td headers="hd_h_ml121.tu5_1_1_1_1 hd_h_ml121.tu5_1_1_2_1 hd_h_ml121.tu5_1_1_3_1 hd_h_ml121.tu5_1_1_4_1 hd_h_ml121.tu5_1_1_5_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">100</td><td headers="hd_h_ml121.tu5_1_1_1_1 hd_h_ml121.tu5_1_1_2_1 hd_h_ml121.tu5_1_1_3_1 hd_h_ml121.tu5_1_1_4_1 hd_h_ml121.tu5_1_1_5_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">4.61</td></tr><tr><td headers="hd_h_ml121.tu5_1_1_1_1 hd_h_ml121.tu5_1_1_2_1 hd_h_ml121.tu5_1_1_3_1 hd_h_ml121.tu5_1_1_4_1 hd_h_ml121.tu5_1_1_5_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">1</td><td headers="hd_h_ml121.tu5_1_1_1_1 hd_h_ml121.tu5_1_1_2_1 hd_h_ml121.tu5_1_1_3_1 hd_h_ml121.tu5_1_1_4_1 hd_h_ml121.tu5_1_1_5_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">97</td><td headers="hd_h_ml121.tu5_1_1_1_1 hd_h_ml121.tu5_1_1_2_1 hd_h_ml121.tu5_1_1_3_1 hd_h_ml121.tu5_1_1_4_1 hd_h_ml121.tu5_1_1_5_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">4.57</td></tr><tr><td headers="hd_h_ml121.tu5_1_1_1_1 hd_h_ml121.tu5_1_1_2_1 hd_h_ml121.tu5_1_1_3_1 hd_h_ml121.tu5_1_1_4_1 hd_h_ml121.tu5_1_1_5_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">2</td><td headers="hd_h_ml121.tu5_1_1_1_1 hd_h_ml121.tu5_1_1_2_1 hd_h_ml121.tu5_1_1_3_1 hd_h_ml121.tu5_1_1_4_1 hd_h_ml121.tu5_1_1_5_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">91</td><td headers="hd_h_ml121.tu5_1_1_1_1 hd_h_ml121.tu5_1_1_2_1 hd_h_ml121.tu5_1_1_3_1 hd_h_ml121.tu5_1_1_4_1 hd_h_ml121.tu5_1_1_5_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">4.51</td></tr><tr><td headers="hd_h_ml121.tu5_1_1_1_1 hd_h_ml121.tu5_1_1_2_1 hd_h_ml121.tu5_1_1_3_1 hd_h_ml121.tu5_1_1_4_1 hd_h_ml121.tu5_1_1_5_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">4</td><td headers="hd_h_ml121.tu5_1_1_1_1 hd_h_ml121.tu5_1_1_2_1 hd_h_ml121.tu5_1_1_3_1 hd_h_ml121.tu5_1_1_4_1 hd_h_ml121.tu5_1_1_5_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">88</td><td headers="hd_h_ml121.tu5_1_1_1_1 hd_h_ml121.tu5_1_1_2_1 hd_h_ml121.tu5_1_1_3_1 hd_h_ml121.tu5_1_1_4_1 hd_h_ml121.tu5_1_1_5_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">4.48</td></tr><tr><td headers="hd_h_ml121.tu5_1_1_1_1 hd_h_ml121.tu5_1_1_2_1 hd_h_ml121.tu5_1_1_3_1 hd_h_ml121.tu5_1_1_4_1 hd_h_ml121.tu5_1_1_5_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">8</td><td headers="hd_h_ml121.tu5_1_1_1_1 hd_h_ml121.tu5_1_1_2_1 hd_h_ml121.tu5_1_1_3_1 hd_h_ml121.tu5_1_1_4_1 hd_h_ml121.tu5_1_1_5_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">98</td><td headers="hd_h_ml121.tu5_1_1_1_1 hd_h_ml121.tu5_1_1_2_1 hd_h_ml121.tu5_1_1_3_1 hd_h_ml121.tu5_1_1_4_1 hd_h_ml121.tu5_1_1_5_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">4.58</td></tr><tr><td headers="hd_h_ml121.tu5_1_1_1_1 hd_h_ml121.tu5_1_1_2_1 hd_h_ml121.tu5_1_1_3_1 hd_h_ml121.tu5_1_1_4_1 hd_h_ml121.tu5_1_1_5_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">24</td><td headers="hd_h_ml121.tu5_1_1_1_1 hd_h_ml121.tu5_1_1_2_1 hd_h_ml121.tu5_1_1_3_1 hd_h_ml121.tu5_1_1_4_1 hd_h_ml121.tu5_1_1_5_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">89</td><td headers="hd_h_ml121.tu5_1_1_1_1 hd_h_ml121.tu5_1_1_2_1 hd_h_ml121.tu5_1_1_3_1 hd_h_ml121.tu5_1_1_4_1 hd_h_ml121.tu5_1_1_5_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">4.49</td></tr><tr><td headers="hd_h_ml121.tu5_1_1_1_1 hd_h_ml121.tu5_1_1_2_1 hd_h_ml121.tu5_1_1_3_1 hd_h_ml121.tu5_1_1_4_1 hd_h_ml121.tu5_1_1_5_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">48</td><td headers="hd_h_ml121.tu5_1_1_1_1 hd_h_ml121.tu5_1_1_2_1 hd_h_ml121.tu5_1_1_3_1 hd_h_ml121.tu5_1_1_4_1 hd_h_ml121.tu5_1_1_5_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">88</td><td headers="hd_h_ml121.tu5_1_1_1_1 hd_h_ml121.tu5_1_1_2_1 hd_h_ml121.tu5_1_1_3_1 hd_h_ml121.tu5_1_1_4_1 hd_h_ml121.tu5_1_1_5_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">4.48</td></tr></tbody></table></div></div></div><div id="ml121.s11"><h3>2.3. Probe Preparation</h3><p>The VIM-2 inhibitor probe <a href="/pcsubstance/?term=ML121[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML121</a> was synthesized by using a procedure for published for the synthesis of structurally related compounds [<a class="bk_pop" href="#ml121.r19">19</a>]. 4-Thioxo-thiazolidine-2-one (<b>A</b>) was synthesized following a literature procedure [<a class="bk_pop" href="#ml121.r20">20</a>]. Condensation of <b>A</b> with pyridine-3-carboxyaldehyde in acetic acid under standard conditions provided the thiazolidene-2-one <b>B</b>, which was obtained as red crystals. The (formal) hetero-Diels-Alder reaction of <b>B</b> with norbornene then provided the probe compound (<b>C</b>; <a href="/pcsubstance/?term=ML121[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML121</a>; <b>CID 46891627</b>). The stereochemistry of <a href="/pcsubstance/?term=ML121[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML121</a>, expected to be exo based on the 2006 paper [<a class="bk_pop" href="#ml121.r19">19</a>], was confirmed by the <sup>1</sup>H NOE data summarized below. Because the Probe <a href="/pcsubstance/?term=ML121[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML121</a> had poor solubility properties, it was converted to the trifluoroacetate salt <b>D</b> (<b>CID 50909789</b>).</p><div id="ml121.fu3" class="figure"><div class="graphic"><img src="/books/NBK63598/bin/ml121fu3.jpg" alt="Image ml121fu3" /></div></div><div id="ml121.fu4" class="figure bk_fig"><div class="graphic"><img src="/books/NBK63598/bin/ml121fu4.jpg" alt="1H NOE Data for C, ML121." /></div><h3><span class="title"><sup>1</sup>H NOE Data for <b>C</b>, <a href="/pcsubstance/?term=ML121[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML121</a></span></h3></div><p><b>Procedure for Synthesis of <a href="/pcsubstance/?term=ML121[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML121</a> (C;</b>
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<b><a href="https://pubchem.ncbi.nlm.nih.gov/substance/46891627" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 46891627</a>).</b> A mixture of <b>B</b> (0.5 mmol), norbornene (0.6 mmol), and a catalytic amount of hydroquinone in 1.0 mL of glacial acetic acid was refluxed for 1 h, then was cooled to ambient temperature and stirred for 12 h. The reaction mixture was concentrated under vacuum, and the residue was neutralized by treatment with 0.2 mL of Et<sub>3</sub>N. This mixture was then partitioned between dichloromethane and water. The organic layer was separated and dried over Na<sub>2</sub>SO<sub>4</sub> and the crude product obtained by evaporation of the filtrate was purified by column chromatography (MeOH/CH<sub>2</sub>Cl<sub>2</sub>). The product was obtained as a 4:1 mixture of isomers. This mixture was recrystallized using EtOAc/EtOH to afford single isomer <b>C</b> (>96% purity, confirmed by NMR and LC-MS), which is the exo-adduct of the hetero-Diels-Alder reaction.</p><p><b>Procedure for Synthesis of <a href="/pcsubstance/?term=ML121[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML121</a>-TFA Salt (D; CID 50909789)</b>. To a solution of <b>C</b> (20mg, 0.063 mmol) in 1 mL of water was added 5 μl of trifluoroacetic acid (TFA). This mixture was stirred at ambient temperature for 1 h, until the solid completely dissolved, then the solution was frozen in a dry ice-acetone bath, and lyophilized to give <b>D</b> as pale-yellow powder.</p><div id="ml121.fu5" class="figure bk_fig"><div class="graphic"><img src="/books/NBK63598/bin/ml121fu5.jpg" alt="B." /></div><h3><span class="title">B</span></h3></div><p><b>Data for intermediate B. <sup>1</sup>H NMR</b> (400 MHz, DMSO-d6 and Et<sub>3</sub>N) δ 8.76 (d, <i>J</i> = 2.3 Hz, 1 H), 8.50 (dd, <i>J</i> = 4.8, 1.6 Hz, 1 H), 7.95 (dddd, <i>J</i> = 8.0, 2.2, 1.6, 0.5 Hz, 1 H), 7.91 (s, 1 H), 7.48 (dd, <i>J</i> = 8.0, 4.8 Hz, 1 H); <b><sup>13</sup>C NMR</b> (100 MHz, DMSO-d6 and Et<sub>3</sub>N) δ 209.6, 172.6, 150.8, 148.5, 142.4, 135.0, 131.6, 124.0, 123.8, 21.8; <b>IR</b>: 3404, 2988, 1637, 1315, 924; <b>ESIMS</b>: C<sub>9</sub>H<sub>6</sub>N<sub>2</sub>OS<sub>2</sub> 222.0 (calculated), 222.2 (found).</p><div id="ml121.fu6" class="figure bk_fig"><div class="graphic"><img src="/books/NBK63598/bin/ml121fu6.jpg" alt="C, ML121 CID 46891627." /></div><h3><span class="title"><b>C</b>, <a href="/pcsubstance/?term=ML121[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML121</a><br /><b>CID 46891627</b></span></h3></div><p><b>Data for <a href="/pcsubstance/?term=ML121[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML121</a> as the Free Base (C):</b> >96% purity by 1H NMR and LCMS; <b>m.p.</b> 138–141°C; <b>Rf</b> = 0.44 (DCM:MeOH = 9:1); <b><sup>1</sup>H NMR</b> (400 MHz, DMSO- d<sub>6</sub>) δ 11.57 (s, 1 H), 8.63 (d, <i>J</i> = 1.9, 1 H), 8.55 (dd, <i>J</i> = 4.7, 1.5 Hz, 1 H), 7.85 (dt, <i>J</i> = 8.0, 2.0 Hz, 1 H), 7.44 (dd, <i>J</i> = 7.8, 4.8 Hz, 1 H), 3.55 (d, <i>J</i> = 10.3 Hz, 1 H), 3.45 (d, <i>J</i> = 6.9 Hz, 1 H), 2.30 (t, <i>J</i> = 9.0 Hz, 1 H), 2.23 (d, <i>J</i> = 4.0 Hz, 1 H), 2.12 (d, <i>J</i> = 10.1 Hz, 1 H), 1.89 (d, <i>J</i> = 3.5 Hz, 1 H), 1.55–1.65 (m, 1 H), 1.36–1.47 (m, 1 H), 1.26–1.36 (m, 1 H), 1.20 (d, <i>J</i> = 10.2 Hz, 1 H), 1.04–1.12 (m, 1 H); <b><sup>13</sup>C NMR</b> (100 MHz, DMSO-d6) δ 170.3, 150.0, 148.9, 136.8, 136.0, 123.8, 121.7, 113.1, 56.6, 51.3, 43.0, 41.2, 34.2, 28.9, 28.4; <b>IR</b>: 3430, 2959, 2876, 1666, 1427; <b>ESIMS</b>: C<sub>16</sub>H<sub>16</sub>N<sub>2</sub>OS<sub>2</sub> 316.07 (calculated), 316.02 (found).</p><div id="ml121.fu7" class="figure bk_fig"><div class="graphic"><img src="/books/NBK63598/bin/ml121fu7.jpg" alt="D, SR-03000001759-3 CID 50909789." /></div><h3><span class="title"><b>D</b>, SR-03000001759-3<br /><b>CID 50909789</b></span></h3></div><p><b>Data for <a href="/pcsubstance/?term=ML121[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML121</a> as the TFA Salt (D; CID 50909789):</b> >96% purity by <sup>1</sup>H NMR and LCMS; m.p. 135–137°C; <b><sup>1</sup>H NMR</b> (400 MHz, D<sub>2</sub>O/DMSO-d<sub>6</sub> = 5:1 ) δ 8.94 (s, 1 H), 8.85 (d, <i>J</i> = 5.7 Hz, 1 H), 8.77 (d, <i>J</i> = 8.2 Hz, 1 H), 8.19 (dd, <i>J</i> = 8.0, 6.0 Hz, 1 H), 7.48 (dd, <i>J</i> = 8.0, 4.8 Hz, 1 H), 3.98 (d, <i>J</i> = 9.4 Hz, 1 H), 3.54 (d, <i>J</i> = 7.8 Hz, 1 H), 2.45 (t, <i>J</i> = 8.6 Hz, 1 H), 2.38 (d, <i>J</i> = 3.9 Hz, 1 H), 2.04–2.19 (m, 2 H), 1.66–1.76 (m, 1 H), 1.48–1.58 (m, 1 H), 1.31–1.40 (m, 2 H), 1.15–1.24 (m, 1 H); <b><sup>13</sup>C NMR</b> (100 MHz, D<sub>2</sub>O/DMSO-d6=5:1) δ 174.1, 147.2, 141.4, 141.1, 140.7, 127.9, 123.9, 117.9, 115.0, 112.6, 56.7, 51.6, 48.9, 44.1, 43.1, 41.7, 34.2, 29.0, 28.3.</p></div></div><div id="ml121.s12"><h2 id="_ml121_s12_">3. Results</h2><div id="ml121.s13"><h3>3.1. Summary of Screening Results</h3><p>Following nitrocefin-based primary HTS in singlicate to identify selective VIM-2 inhibitors (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1527" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1527</a>), nitrocefin-based counterscreening in singlicate against the IMP-1 metallo-beta-lactamase (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1556" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1556</a>), nitrocefin-based confirmation of hit activity in triplicate (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1860" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1860</a>), nitrocefin-based counterscreening in triplicate against IMP-1 (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1856" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1856</a>) and TEM-1 (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1866" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1866</a>), VIM-2 CCF2-based counterscreening in triplicate to identify nitrocefin-based assay artifacts (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1857" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID1857</a>), nitrocefin-based dose response assays to determine VIM-2 potency (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1919" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1919</a>) and selectivity against IMP-1 (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1920" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1920</a> and <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1927" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1927</a>), TEM-1 (1925), and CCF2-based dose response counterscreening in triplicate to identify nitrocefin-based assay artifacts (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1926" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1926</a>), certain compounds were identified as possible candidates for probe development. A TFA salt form of probe <a href="/pcsubstance/?term=ML121[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML121</a> was tested in synergy assays to determine if it could potentiate the activity of the antibiotic imipenem, and was found active. Probes were identified (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2128" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 2128</a>) and are an improvement over the prior art, mitoxantrone (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2317" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 2317</a>). All HTS and assay provider results have been provided to the PubChem database.</p><div id="ml121.fu8" class="figure bk_fig"><div class="graphic"><img src="/books/NBK63598/bin/ml121fu8.jpg" alt="VIM-2 Selective Inhibitor." /></div><h3><span class="title">VIM-2 Selective Inhibitor</span></h3></div></div><div id="ml121.s14"><h3>3.2. Dose Response Curves for Probes</h3><p>We resynthesized a sample of the MBL VIM-2 selective probe. A loss of 4-fold in potency was observed as was insolubility at 10mM concentration in DMSO. Another sample was then prepared as a TFA salt and submitted for testing in the VIM-2 Nitrocefin assay using the identical method to those found in <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1919" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1919</a>. These results, shown below, reproduce the original sample results and demonstrate that we have resynthesized the original MBL probe.</p><div id="ml121.tu6" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK63598/table/ml121.tu6/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml121.tu6_lrgtbl__"><table><thead><tr><th id="hd_h_ml121.tu6_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Final Ic50 [M]</th><th id="hd_h_ml121.tu6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Averaged Max % Response</th><th id="hd_h_ml121.tu6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">StdDev Max % Response</th><th id="hd_h_ml121.tu6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Hill Slope</th><th id="hd_h_ml121.tu6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Curve</th></tr></thead><tbody><tr><td headers="hd_h_ml121.tu6_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">49.7E-9</td><td headers="hd_h_ml121.tu6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">113.68</td><td headers="hd_h_ml121.tu6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">0.57</td><td headers="hd_h_ml121.tu6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">1.90</td><td headers="hd_h_ml121.tu6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">
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<div class="graphic"><img src="/books/NBK63598/bin/ml121fu9.jpg" alt="Image ml121fu9.jpg" /></div></td></tr></tbody></table></div></div></div><div id="ml121.s15"><h3>3.3. Scaffold/Moiety Chemical Liabilities</h3><p>There are no known instability or chemical liabilities associated with the chemical scaffold of probe <a href="/pcsubstance/?term=ML121[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML121</a>.</p></div><div id="ml121.s16"><h3>3.4. SAR Tables</h3><p><b>Describe SAR & chemistry strategy (including structure and data) that led to the probe.</b> Following the above HTS target and counterscreening assays, a compound belonging to the <b>6, 7-dihydro-5H-thiopyrano[2,3-d]thiazole</b> scaffold was identified: <b>CID4870494/<a href="https://pubchem.ncbi.nlm.nih.gov/substance/24790728" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 24790728</a></b>. This compound does not share structural similarities with the prior art compound mitoxantrone (see SAR Table and [<a class="bk_pop" href="#ml121.r2">2</a>]). Analogs were purchased in powder form or re-ordered from the MLSMR in liquid form and tested in dose response assays against both VIM-2 and IMP-1 enzymes using nitrocefin and CCF2 substrate assays. Additional studies to determine the Ki and mode of action of the probe were also performed. Results for these assays are summarized in the SAR Table below and in <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2128" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 2128</a> and <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2317" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 2317</a>.</p><p>The synthesis of <a href="/pcsubstance/?term=ML121[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML121</a>, presented in <a href="#ml121.s11">Section 2.3</a> of this Probe Report, is amenable to synthesis of additional analogs by substituting different aldehydes for the pyridine-3-carboxaldehyde used in the synthesis of <a href="/pcsubstance/?term=ML121[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML121</a>, and, in principle, by replacing norbornene with other reactive olefins in the hetero-Diels-Alder reaction used in the synthesis of <a href="/pcsubstance/?term=ML121[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML121</a>.[<a class="bk_pop" href="#ml121.r19">19</a>]</p><div id="ml121.tu7" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK63598/table/ml121.tu7/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml121.tu7_lrgtbl__"><table class="no_bottom_margin"><thead><tr><th id="hd_h_ml121.tu7_1_1_1_1" colspan="10" rowspan="1" style="text-align:center;vertical-align:middle;">Compound Information</th><th id="hd_h_ml121.tu7_1_1_1_2" colspan="9" rowspan="1" style="text-align:center;vertical-align:middle;">Screening and Probe Development Assays</th></tr><tr><th headers="hd_h_ml121.tu7_1_1_1_1" id="hd_h_ml121.tu7_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Compound</th><th headers="hd_h_ml121.tu7_1_1_1_1" id="hd_h_ml121.tu7_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Structure</th><th headers="hd_h_ml121.tu7_1_1_1_1" id="hd_h_ml121.tu7_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CID</th><th headers="hd_h_ml121.tu7_1_1_1_1" id="hd_h_ml121.tu7_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SID</th><th headers="hd_h_ml121.tu7_1_1_1_1" id="hd_h_ml121.tu7_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS</th><th headers="hd_h_ml121.tu7_1_1_1_1" id="hd_h_ml121.tu7_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Source: MLS</th><th headers="hd_h_ml121.tu7_1_1_1_1" id="hd_h_ml121.tu7_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Sourced Outside ML</th><th headers="hd_h_ml121.tu7_1_1_1_1" id="hd_h_ml121.tu7_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Chem Synth</th><th headers="hd_h_ml121.tu7_1_1_1_1" id="hd_h_ml121.tu7_1_1_2_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Vendor</th><th headers="hd_h_ml121.tu7_1_1_1_1" id="hd_h_ml121.tu7_1_1_2_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Vendor Cat ID</th><th headers="hd_h_ml121.tu7_1_1_1_2" id="hd_h_ml121.tu7_1_1_2_11" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">VIM-2 Primary HTS [<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1527" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1527</a>] (%Inhibition)</th><th headers="hd_h_ml121.tu7_1_1_1_2" id="hd_h_ml121.tu7_1_1_2_12" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">IMP-1 Primary HTS [<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1556" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1556</a>] (%Inhibition)</th><th headers="hd_h_ml121.tu7_1_1_1_2" id="hd_h_ml121.tu7_1_1_2_13" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">VIM-2 Dose Response [<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1919" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1919</a>] (IC50, μM)</th><th headers="hd_h_ml121.tu7_1_1_1_2" id="hd_h_ml121.tu7_1_1_2_14" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">VIM-2 CCF2 Dose Response [<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1926" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1926</a>] (IC50, μM)</th><th headers="hd_h_ml121.tu7_1_1_1_2" id="hd_h_ml121.tu7_1_1_2_15" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">IMP-1 CCF2 Dose Response [<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1927" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1927</a>] (IC50, μM)</th><th headers="hd_h_ml121.tu7_1_1_1_2" id="hd_h_ml121.tu7_1_1_2_16" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">VIM-2 Ki Assay [<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2128" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 2128</a>] (Ki)</th><th headers="hd_h_ml121.tu7_1_1_1_2" id="hd_h_ml121.tu7_1_1_2_17" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">VIM-2 Powder Assay [<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2128" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 2128</a>] (IC50, μM)</th><th headers="hd_h_ml121.tu7_1_1_1_2" id="hd_h_ml121.tu7_1_1_2_18" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">IMP-1 Dose Response [<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2128" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 2128</a>] (IC50, μM)</th><th headers="hd_h_ml121.tu7_1_1_1_2" id="hd_h_ml121.tu7_1_1_2_19" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">TEM-1 Dose Response [<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2128" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 2128</a>] (IC50, μM)</th></tr></thead><tbody><tr><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b>VIM-2 Selective PROBE</b></td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK63598/bin/ml121fu10.jpg" alt="Image ml121fu10.jpg" /></div></td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">4870494</td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/24790728" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">24790728</a></td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS 000680027</td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Yes</td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Yes</td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">No</td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Life Chem</td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">F1734-0049</td><td headers="hd_h_ml121.tu7_1_1_1_2 hd_h_ml121.tu7_1_1_2_11" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Active (84.22)</td><td headers="hd_h_ml121.tu7_1_1_1_2 hd_h_ml121.tu7_1_1_2_12" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Inactive (4.15)</td><td headers="hd_h_ml121.tu7_1_1_1_2 hd_h_ml121.tu7_1_1_2_13" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Active (0.054)</td><td headers="hd_h_ml121.tu7_1_1_1_2 hd_h_ml121.tu7_1_1_2_14" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Active (0.034)</td><td headers="hd_h_ml121.tu7_1_1_1_2 hd_h_ml121.tu7_1_1_2_15" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Inactive (>60.0)</td><td headers="hd_h_ml121.tu7_1_1_1_2 hd_h_ml121.tu7_1_1_2_16" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Active (148 ± 14 nM) (non-competitive)</td><td headers="hd_h_ml121.tu7_1_1_1_2 hd_h_ml121.tu7_1_1_2_17" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Active <sup><a class="bk_pop" href="#ml121.tfn2">#</a></sup> (0.223 ± 0.003)</td><td headers="hd_h_ml121.tu7_1_1_1_2 hd_h_ml121.tu7_1_1_2_18" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Inactive (>60)</td><td headers="hd_h_ml121.tu7_1_1_1_2 hd_h_ml121.tu7_1_1_2_19" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Inactive (>60)</td></tr><tr><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b>Analog 1</b></td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK63598/bin/ml121fu11.jpg" alt="Image ml121fu11.jpg" /></div></td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3243007</td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/4249000" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">4249000</a></td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS 000085438</td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Yes</td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">No</td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">No</td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Chem Div</td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">6194-0087</td><td headers="hd_h_ml121.tu7_1_1_1_2 hd_h_ml121.tu7_1_1_2_11" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Active (8.85)</td><td headers="hd_h_ml121.tu7_1_1_1_2 hd_h_ml121.tu7_1_1_2_12" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Inactive (2.84)</td><td headers="hd_h_ml121.tu7_1_1_1_2 hd_h_ml121.tu7_1_1_2_13 hd_h_ml121.tu7_1_1_2_14 hd_h_ml121.tu7_1_1_2_15 hd_h_ml121.tu7_1_1_2_16 hd_h_ml121.tu7_1_1_2_17 hd_h_ml121.tu7_1_1_2_18 hd_h_ml121.tu7_1_1_2_19" colspan="7" rowspan="1" style="text-align:center;vertical-align:middle;">See below<sup><a class="bk_pop" href="#ml121.tfn3">^</a></sup></td></tr><tr><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b>Analog 2</b></td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK63598/bin/ml121fu12.jpg" alt="Image ml121fu12.jpg" /></div></td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3708465</td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/14727633" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">14727633</a></td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS 000549994</td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Yes</td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">No</td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">No</td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Specs</td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">AF-399/ 42057249</td><td headers="hd_h_ml121.tu7_1_1_1_2 hd_h_ml121.tu7_1_1_2_11" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Inactive (6.76)</td><td headers="hd_h_ml121.tu7_1_1_1_2 hd_h_ml121.tu7_1_1_2_12" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Inactive (−3.32)</td><td headers="hd_h_ml121.tu7_1_1_1_2 hd_h_ml121.tu7_1_1_2_13 hd_h_ml121.tu7_1_1_2_14 hd_h_ml121.tu7_1_1_2_15 hd_h_ml121.tu7_1_1_2_16 hd_h_ml121.tu7_1_1_2_17 hd_h_ml121.tu7_1_1_2_18 hd_h_ml121.tu7_1_1_2_19" colspan="7" rowspan="4" style="text-align:center;vertical-align:middle;">See below<sup><a class="bk_pop" href="#ml121.tfn4">†</a></sup></td></tr><tr><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b>Analog 3</b></td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK63598/bin/ml121fu13.jpg" alt="Image ml121fu13.jpg" /></div></td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3243512</td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/4249579" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">4249579</a></td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS 000085437</td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Yes</td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">No</td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">No</td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Chem Div</td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">6194-0084</td><td headers="hd_h_ml121.tu7_1_1_1_2 hd_h_ml121.tu7_1_1_2_11" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Inactive (5.97)</td><td headers="hd_h_ml121.tu7_1_1_1_2 hd_h_ml121.tu7_1_1_2_12" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Inactive (−2.38)</td></tr><tr><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b>Analog 4</b></td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
|
||
<div class="graphic"><img src="/books/NBK63598/bin/ml121fu14.jpg" alt="Image ml121fu14.jpg" /></div></td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">4414227</td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/14733407" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">14733407</a></td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS 000529675</td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Yes</td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">No</td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">No</td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Life Chem</td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">F1422-0004</td><td headers="hd_h_ml121.tu7_1_1_1_2 hd_h_ml121.tu7_1_1_2_11" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Inactive (4.01)</td><td headers="hd_h_ml121.tu7_1_1_1_2 hd_h_ml121.tu7_1_1_2_12" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Inactive (−0.56)</td></tr><tr><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b>Analog 5</b></td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
|
||
<div class="graphic"><img src="/books/NBK63598/bin/ml121fu15.jpg" alt="Image ml121fu15.jpg" /></div></td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3235825</td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/4240758" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">4240758</a></td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS 000094210</td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Yes</td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">No</td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">No</td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Chem Div</td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">D008-0255</td><td headers="hd_h_ml121.tu7_1_1_1_2 hd_h_ml121.tu7_1_1_2_11" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Inactive (1.92)</td><td headers="hd_h_ml121.tu7_1_1_1_2 hd_h_ml121.tu7_1_1_2_12" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Inactive (−1.86)</td></tr><tr><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b><i>Prior Art:</i></b>
|
||
<i>Mitoxantrone</i></td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
|
||
<div class="graphic"><img src="/books/NBK63598/bin/ml121fu16.jpg" alt="Image ml121fu16.jpg" /></div></td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><i>5458171 (4212)</i></td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/56424031" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">56424031 </a>
|
||
<i>(</i><a href="https://pubchem.ncbi.nlm.nih.gov/substance/85856281" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">85856281</a><i>)</i></td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><i>MLS 001333711</i></td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><i>Yes</i></td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><i>Yes</i></td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><i>No</i></td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><i>Sigma Aldrich</i></td><td headers="hd_h_ml121.tu7_1_1_1_1 hd_h_ml121.tu7_1_1_2_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><i>M6545</i></td><td headers="hd_h_ml121.tu7_1_1_1_2 hd_h_ml121.tu7_1_1_2_11 hd_h_ml121.tu7_1_1_2_12 hd_h_ml121.tu7_1_1_2_13 hd_h_ml121.tu7_1_1_2_14 hd_h_ml121.tu7_1_1_2_15" colspan="5" rowspan="1" style="text-align:center;vertical-align:middle;">This compound was not part of the MLSMR collection</td><td headers="hd_h_ml121.tu7_1_1_1_2 hd_h_ml121.tu7_1_1_2_16" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><i>Active</i><sup><a class="bk_pop" href="#ml121.tfn1">*</a></sup>
|
||
<i>(1.5</i> ± 0.2 μM) <i>(non- competitive)</i></td><td headers="hd_h_ml121.tu7_1_1_1_2 hd_h_ml121.tu7_1_1_2_17" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><i>Active</i><sup><a class="bk_pop" href="#ml121.tfn2">#</a></sup>
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||
<sup><a class="bk_pop" href="#ml121.tfn1">*</a></sup>
|
||
<i>(0.63 ± 0.04)</i></td><td headers="hd_h_ml121.tu7_1_1_1_2 hd_h_ml121.tu7_1_1_2_18" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><i>Inactive (>56)</i></td><td headers="hd_h_ml121.tu7_1_1_1_2 hd_h_ml121.tu7_1_1_2_19" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><i>Inactive (>25)</i></td></tr></tbody></table></div><div><div><dl class="temp-labeled-list small"><dt>*</dt><dd><div id="ml121.tfn1"><p class="no_margin">These values are taken from Reference Minond et al 2009 and can be found in PubChem <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2317" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 2317</a>.</p></div></dd><dt>#</dt><dd><div id="ml121.tfn2"><p class="no_margin">Please refer to the Ki values (rather than these IC50 values) for a more complete representation of the mechanism of action and potency for these inhibitors.</p></div></dd><dt>^</dt><dd><div id="ml121.tfn3"><p class="no_margin"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/4249000" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 4249000</a> met the 7.06% inhibition cutoff to be Active in <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1527" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID1527</a>. However, it was not tested in subsequent assays due to its low % inhibition, compared to the probe (84.22%).</p></div></dd><dt>†</dt><dd><div id="ml121.tfn4"><p class="no_margin">Compounds were not tested due to inactivity in the VIM2 Primary assay [<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1527" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1527</a>].</p></div></dd></dl></div></div></div></div><div id="ml121.s17"><h3>3.5. Cellular Activity</h3><p>The probe was tested in a variety of cell-based assays performed by the SRIMSC and assay provider (see above assays section). These assays were performed to determine the probe’s selectivity and mechanism of action. The results of these studies demonstrated that probe <a href="/pcsubstance/?term=ML121[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML121</a> (<b>CID4870494)</b> is a non-competitive inhibitor, with a submicromolar K<sub>i</sub> (K<sub>i</sub> = 148 ± 14 nM). This represents a 10-fold improvement in potency over the prior art probe, Mitoxantrone.</p><div id="ml121.s18"><h4>VIM-2 Ki Assay (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2128" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 2128</a>)</h4><p>The purpose of these assays is to determine inhibition constant (Ki) and modality of probe candidate molecules. Kinetic assays were conducted by incubating the range of substrate concentrations (100-8 μM) with varying inhibitor concentrations and 0.1 nM enzyme at room temperature in buffer containing 50mM HEPES, 50μM ZnSO4, 0.05% Brij 35, pH 7.1. Absorbance was measured on a Tecan Safire<sup>2</sup> monochromatic microplate reader at 495 nm. Initial velocities were obtained from plots of absorbance at 495 nm versus time, using data points from only the linear portion of the hydrolysis curve. Substrate hydrolysis was continuously monitored.</p><p>Initial velocities were plotted vs. substrate concentration and kinetic parameters were calculated using Graphpad Prism version 5.01 suite of programs. Mode of inhibition was determined using fit comparison capability of Graphpad Prism version 5.01 and additionally evaluated by Lineweaver-Burke plot. K<sub>i</sub> values were determined by non-linear regression analysis. The number of replicates for this assay is 3.</p><div id="ml121.tu8" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK63598/table/ml121.tu8/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml121.tu8_lrgtbl__"><table><thead><tr><th id="hd_h_ml121.tu8_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Corp ID</th><th id="hd_h_ml121.tu8_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SID</th><th id="hd_h_ml121.tu8_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CID</th><th id="hd_h_ml121.tu8_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Modality</th><th id="hd_h_ml121.tu8_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Ki, nM</th><th id="hd_h_ml121.tu8_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Dose</th><th id="hd_h_ml121.tu8_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0</th><th id="hd_h_ml121.tu8_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">40 nM</th><th id="hd_h_ml121.tu8_1_1_1_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">80 nM</th><th id="hd_h_ml121.tu8_1_1_1_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">100 nM</th><th id="hd_h_ml121.tu8_1_1_1_11" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">150 nM</th></tr></thead><tbody><tr><td headers="hd_h_ml121.tu8_1_1_1_1" rowspan="5" colspan="1" style="text-align:center;vertical-align:middle;">SR-01000775688-3</td><td headers="hd_h_ml121.tu8_1_1_1_2" rowspan="5" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/85856282" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">85856282</a></td><td headers="hd_h_ml121.tu8_1_1_1_3" rowspan="5" colspan="1" style="text-align:center;vertical-align:middle;">4870494</td><td headers="hd_h_ml121.tu8_1_1_1_4" rowspan="5" colspan="1" style="text-align:center;vertical-align:middle;">Non-competitive inhibition</td><td headers="hd_h_ml121.tu8_1_1_1_5" rowspan="5" colspan="1" style="text-align:center;vertical-align:middle;">148</td><td headers="hd_h_ml121.tu8_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b>Slope</b></td><td headers="hd_h_ml121.tu8_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">109300</td><td headers="hd_h_ml121.tu8_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">100700</td><td headers="hd_h_ml121.tu8_1_1_1_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">147700</td><td headers="hd_h_ml121.tu8_1_1_1_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">155600</td><td headers="hd_h_ml121.tu8_1_1_1_11" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">180000</td></tr><tr><td headers="hd_h_ml121.tu8_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b>Slope SE</b></td><td headers="hd_h_ml121.tu8_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">9767</td><td headers="hd_h_ml121.tu8_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">8928</td><td headers="hd_h_ml121.tu8_1_1_1_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">18280</td><td headers="hd_h_ml121.tu8_1_1_1_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">11390</td><td headers="hd_h_ml121.tu8_1_1_1_11" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">16040</td></tr><tr><td headers="hd_h_ml121.tu8_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b>Y intercept</b></td><td headers="hd_h_ml121.tu8_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">6284</td><td headers="hd_h_ml121.tu8_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">6506</td><td headers="hd_h_ml121.tu8_1_1_1_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">8809</td><td headers="hd_h_ml121.tu8_1_1_1_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">9733</td><td headers="hd_h_ml121.tu8_1_1_1_11" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">10960</td></tr><tr><td headers="hd_h_ml121.tu8_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b>Y intercept SE</b></td><td headers="hd_h_ml121.tu8_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">575.9</td><td headers="hd_h_ml121.tu8_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">526.4</td><td headers="hd_h_ml121.tu8_1_1_1_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1053</td><td headers="hd_h_ml121.tu8_1_1_1_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">671.3</td><td headers="hd_h_ml121.tu8_1_1_1_11" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">890.6</td></tr><tr><td headers="hd_h_ml121.tu8_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b>r2</b></td><td headers="hd_h_ml121.tu8_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.8172</td><td headers="hd_h_ml121.tu8_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.8196</td><td headers="hd_h_ml121.tu8_1_1_1_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.8031</td><td headers="hd_h_ml121.tu8_1_1_1_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.8696</td><td headers="hd_h_ml121.tu8_1_1_1_11" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.8235</td></tr></tbody></table></div></div></div><div id="ml121.s19"><h4>MIC Assays</h4><p>Mitoxantrone, <i>p</i>CMB, as well as <b>probe <a href="/pcsubstance/?term=ML121[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML121</a></b> were further tested for their inherent antimicrobial activity. Minimal inhibitory concentration (MIC) values were measured in both resistant (BL21/VIM-2) and non-resistant (BL21) <i>E. coli</i>, with imipenem as a positive control. Mitoxantrone exhibited antibacterial activity (MIC = 8.4 μg/mL) against both resistant (BL21/VIM-2) and non-resistant (BL21) <i>E. coli</i>. Similarly, <i>p</i>CMB demonstrated antibacterial activity (MIC = 17.9 μg/mL) against resistant (BL21/VIM-2) and non-resistant (BL21) <i>E. coli</i>. For comparison, the MIC of imipenem against resistant (BL21/VIM-2) and non-resistant (BL21) <i>E. coli</i> was 1.9 μg/mL and 0.2 μg/mL, respectively.</p><p>MIC determination for <a href="/pcsubstance/?term=ML121[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML121</a> done on transformed BL21-VIM-2 E. coli and separately on untransformed BL21 E. coli parental cell yielded no toxicity in either strain up to the highest concentration tested (MIC>31.6μg/ml or 100μM).</p></div><div id="ml121.s20"><h4>Antibiotic Synergy Assays</h4><p>Using a checkerboard microdilution method, the probe was also tested for its ability to potentiate the efficacy of imipenem on VIM-2-expressing <i>E. coli</i> (BL21/VIM-2). Results for prior art compounds <i>p</i>CMB and mitoxantrone are also shown (see Minond 2009 for details). As determined by the methods described previously [<a class="bk_pop" href="#ml121.r21">21</a>] both <i>p</i>CMB and mitoxantrone exhibited synergy with imipenem when present at concentrations as low as 2.2 μg/mL and 2.1 μg/mL, respectively (see also [<a class="bk_pop" href="#ml121.r2">2</a>]).</p><p>For the probe, imipenem MIC values were calculated in the presence of a 10-point, 2-fold serial dilution (0.195–100 μM) of probe <b><a href="/pcsubstance/?term=ML121[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML121</a> (TFA salt)</b>. Testing the combined effect of <a href="/pcsubstance/?term=ML121[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML121</a> with imipenem was accomplished using the checkerboard microdilution method [<a class="bk_pop" href="#ml121.r21">21</a>–<a class="bk_pop" href="#ml121.r23">23</a>]. <a href="/pcsubstance/?term=ML121[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML121</a> partially restored susceptibility of BL21/VIM2 bacteria to imipenem. The synergistic effect of <a href="/pcsubstance/?term=ML121[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML121</a> is described as a factor or fold effect. The amount <a href="/pcsubstance/?term=ML121[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML121</a> lowers the MIC of imipenem compared to that of imipenem alone yields this result. These results have been submitted to PubChem.</p><div id="ml121.tu9" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK63598/table/ml121.tu9/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml121.tu9_lrgtbl__"><table><thead><tr><th id="hd_h_ml121.tu9_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">PID</th><th id="hd_h_ml121.tu9_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Structure</th><th id="hd_h_ml121.tu9_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">VIM-2 IC<sub>50,</sub><br />(μM)</th><th id="hd_h_ml121.tu9_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">VIM-2 K<sub>i,</sub><br />(μM)</th><th id="hd_h_ml121.tu9_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Mechanism<sup>c</sup></th><th id="hd_h_ml121.tu9_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BL21/VIM-2 Imipenem MIC,<br />μg/mL</th><th id="hd_h_ml121.tu9_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BL21/VIM2 SR MIC,<br />(μM)</th><th id="hd_h_ml121.tu9_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BL21/VIM-2 Imipenem MIC with[Max BLI],<br />μg/mL</th><th id="hd_h_ml121.tu9_1_1_1_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Imipenem MIC Potentiation,<br />Fold</th><th id="hd_h_ml121.tu9_1_1_1_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2X/4X/8X Synergy in BL21VIM2<br />(μM)</th></tr></thead><tbody><tr><td headers="hd_h_ml121.tu9_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;"><a href="/pcsubstance/?term=ML121[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML121</a></td><td headers="hd_h_ml121.tu9_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK63598/bin/ml121fu17.jpg" alt="Image ml121fu17.jpg" /></div></td><td headers="hd_h_ml121.tu9_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.223 ± 0.006</td><td headers="hd_h_ml121.tu9_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.148 ± 0.014</td><td headers="hd_h_ml121.tu9_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">NC</td><td headers="hd_h_ml121.tu9_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2.67 ± 1.15</td><td headers="hd_h_ml121.tu9_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>100</td><td headers="hd_h_ml121.tu9_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.67 ± 0.29</td><td headers="hd_h_ml121.tu9_1_1_1_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">4</td><td headers="hd_h_ml121.tu9_1_1_1_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">12.5/25/>100</td></tr></tbody></table></div></div></div></div><div id="ml121.s21"><h3>3.6. Profiling Assays</h3><p>The identified VIM-2 selective inhibitor probe <a href="/pcsubstance/?term=ML121[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML121</a> was tested in biochemical and bacterial cell-based assays to determine inhibition of VIM-2, IMP-1, TEM-1, as well as assays to assess its ability to potentiate imipenem. Based on the results of these assays, along with the extensive findings of the 309 Bioassays in PubChem, it was agreed by the SRIMSC and assay provider that additional profiling was not required at this time.</p></div></div><div id="ml121.s22"><h2 id="_ml121_s22_">4. Discussion</h2><div id="ml121.s23"><h3>4.1. Comparison to existing art and how the new probe is an improvement</h3><p>The new probe <a href="/pcsubstance/?term=ML121[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML121</a> exhibits an improved potency and selectivity over the existing art, mitoxantrone. Mitoxantrone has been found active in over 28% of assays tested, where as the new VIM-2 selective probe was found active in less than 2% of PubChem Bioassays. This activity profile demonstrates the greatly improved selectivity of the <a href="/pcsubstance/?term=ML121[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML121</a> probe. A search of the patent literature identified no additional VIM-2 inhibitors.</p><div id="ml121.tu10" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK63598/table/ml121.tu10/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml121.tu10_lrgtbl__"><table><thead><tr><th id="hd_h_ml121.tu10_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Name</th><th id="hd_h_ml121.tu10_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Structure</th><th id="hd_h_ml121.tu10_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR Number</th><th id="hd_h_ml121.tu10_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CID</th><th id="hd_h_ml121.tu10_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SID</th><th id="hd_h_ml121.tu10_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS ID</th><th id="hd_h_ml121.tu10_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">VIM-2 IC50</th><th id="hd_h_ml121.tu10_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">IMP-1 IC50</th><th id="hd_h_ml121.tu10_1_1_1_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">VIM-2 Ki</th><th id="hd_h_ml121.tu10_1_1_1_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">TEM-1 IC50</th><th id="hd_h_ml121.tu10_1_1_1_11" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Synergy</th><th id="hd_h_ml121.tu10_1_1_1_12" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">PubChem Activity</th></tr></thead><tbody><tr><td headers="hd_h_ml121.tu10_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Probe <a href="/pcsubstance/?term=ML121[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML121</a></td><td headers="hd_h_ml121.tu10_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK63598/bin/ml121fu18.jpg" alt="Image ml121fu18.jpg" /></div></td><td headers="hd_h_ml121.tu10_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR- 01000775688-1</td><td headers="hd_h_ml121.tu10_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">4870494</td><td headers="hd_h_ml121.tu10_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/24790728" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">24790728 </a> (MLSMR); <a href="https://pubchem.ncbi.nlm.nih.gov/substance/85856282" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">85856282 </a> (purch); <a href="https://pubchem.ncbi.nlm.nih.gov/substance/103911139" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">103911139 </a> (synth)</td><td headers="hd_h_ml121.tu10_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS- 000680027</td><td headers="hd_h_ml121.tu10_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">223 nM</td><td headers="hd_h_ml121.tu10_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>60 μM</td><td headers="hd_h_ml121.tu10_1_1_1_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">148 nM</td><td headers="hd_h_ml121.tu10_1_1_1_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>60 μM</td><td headers="hd_h_ml121.tu10_1_1_1_11" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Active (12.5 μM) (TFA salt)</td><td headers="hd_h_ml121.tu10_1_1_1_12" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">6/ 309 (1.9%)</td></tr><tr><td headers="hd_h_ml121.tu10_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Prior art Mitoxantrone</td><td headers="hd_h_ml121.tu10_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK63598/bin/ml121fu19.jpg" alt="Image ml121fu19.jpg" /></div></td><td headers="hd_h_ml121.tu10_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR- 01000076001-7</td><td headers="hd_h_ml121.tu10_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">4212 (5458171)</td><td headers="hd_h_ml121.tu10_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/56424031" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">56424031 </a> (<a href="https://pubchem.ncbi.nlm.nih.gov/substance/85856281" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">85856281</a>)</td><td headers="hd_h_ml121.tu10_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS 001333711</td><td headers="hd_h_ml121.tu10_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">powder 0.63 μM</td><td headers="hd_h_ml121.tu10_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">> 56 μM</td><td headers="hd_h_ml121.tu10_1_1_1_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1.5 μM (non- competitive):</td><td headers="hd_h_ml121.tu10_1_1_1_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">> 56 μM</td><td headers="hd_h_ml121.tu10_1_1_1_11" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Not applicable</td><td headers="hd_h_ml121.tu10_1_1_1_12" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">40/139 (28.8%)</td></tr></tbody></table></div></div></div><div id="ml121.s24"><h3>4.2. Mechanism of Action Studies</h3><p>The assay provider has performed assays to elucidate the mode of inhibition and the probe’s inhibitory constant (Ki) against VIM-2. The results of these probe development efforts determined that probe <a href="/pcsubstance/?term=ML121[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML121</a> is a selective, non-competitive inhibitor probe of the metallo-beta-lactamase VIM-2. This probe exhibits a submicromolar Ki value for VIM-2 with minimal activity against IMP-1 and TEM-1 enzymes, as determined using epi-absorbance and fluorescence-based biochemical assays. These assays suggest that the probe is a non-competitive inhibitor of VIM-2.</p></div><div id="ml121.s25"><h3>4.3. Planned Future Studies</h3><p>The assay provider and SRIMSC are currently developing more potent novel compounds as well as compounds that act as broad (non-selective) beta-lactamase inhibitors. These efforts are in progress and will be reported in an upcoming probe report.</p></div></div><div id="ml121.s26"><h2 id="_ml121_s26_">5. References</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="ml121.r1">Mazerski J, Martelli S, Borowski E. The geometry of intercalation complex of antitumor mitoxantrone and ametantrone with DNA: molecular dynamics simulations. <span><span class="ref-journal">Acta Biochim Pol. </span>1998;<span class="ref-vol">45</span>(1):1–11.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/9701490" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 9701490</span></a>]</div></dd><dt>2.</dt><dd><div class="bk_ref" id="ml121.r2">Minond D, Saldanha SA, Subramaniam P, Spaargaren M, Spicer T, Fotsing JR, Weide T, Fokin VV, Sharpless KB, Galleni M, Bebrone C, Lassaux P, Hodder P. Inhibitors of VIM-2 by screening pharmacologically active and click-chemistry compound libraries. <span><span class="ref-journal">Bioorg Med Chem. </span>2009;<span class="ref-vol">17</span>(14):5027–37.</span> [<a href="/pmc/articles/PMC2759276/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2759276</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/19553129" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19553129</span></a>]</div></dd><dt>3.</dt><dd><div class="bk_ref" id="ml121.r3">Siegel RE. Emerging gram-negative antibiotic resistance: daunting challenges, declining sensitivities, and dire consequences. <span><span class="ref-journal">Respir Care. </span>2008;<span class="ref-vol">53</span>(4):471–9.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/18364060" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18364060</span></a>]</div></dd><dt>4.</dt><dd><div class="bk_ref" id="ml121.r4">Gupta V. An update on newer beta-lactamases. <span><span class="ref-journal">Indian J Med Res. </span>2007;<span class="ref-vol">126</span>(5):417–27.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/18160745" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18160745</span></a>]</div></dd><dt>5.</dt><dd><div class="bk_ref" id="ml121.r5">Bradford PA. Extended-spectrum beta-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. <span><span class="ref-journal">Clin Microbiol Rev. </span>2001;<span class="ref-vol">14</span>(4):933–51.</span> table of contents. [<a href="/pmc/articles/PMC89009/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC89009</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/11585791" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 11585791</span></a>]</div></dd><dt>6.</dt><dd><div class="bk_ref" id="ml121.r6">Sacha P, Wieczorek P, Hauschild T, Zorawski M, Olszanska D, Tryniszewska E. Metallo-beta-lactamases of Pseudomonas aeruginosa--a novel mechanism resistance to beta-lactam antibiotics. <span><span class="ref-journal">Folia Histochem Cytobiol. </span>2008;<span class="ref-vol">46</span>(2):137–42.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/18519228" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18519228</span></a>]</div></dd><dt>7.</dt><dd><div class="bk_ref" id="ml121.r7">Koch AL. Bacterial wall as target for attack: past, present, and future research. <span><span class="ref-journal">Clin Microbiol Rev. </span>2003;<span class="ref-vol">16</span>(4):673–87.</span> [<a href="/pmc/articles/PMC207114/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC207114</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/14557293" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 14557293</span></a>]</div></dd><dt>8.</dt><dd><div class="bk_ref" id="ml121.r8">Jin W, Arakawa Y, Yasuzawa H, Taki T, Hashiguchi R, Mitsutani K, Shoga A, Yamaguchi Y, Kurosaki H, Shibata N, Ohta M, Goto M. Comparative study of the inhibition of metallo-beta-lactamases (IMP-1 and VIM-2) by thiol compounds that contain a hydrophobic group. <span><span class="ref-journal">Biol Pharm Bull. </span>2004;<span class="ref-vol">27</span>(6):851–6.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/15187432" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 15187432</span></a>]</div></dd><dt>9.</dt><dd><div class="bk_ref" id="ml121.r9">Abeylath SC, Turos E. Drug delivery approaches to overcome bacterial resistance to beta-lactam antibiotics. <span><span class="ref-journal">Expert Opin Drug Deliv. </span>2008;<span class="ref-vol">5</span>(9):931–49.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/18754746" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18754746</span></a>]</div></dd><dt>10.</dt><dd><div class="bk_ref" id="ml121.r10">Wang Z, Fast W, Valentine AM, Benkovic SJ. Metallo-beta-lactamase: structure and mechanism. <span><span class="ref-journal">Curr Opin Chem Biol. </span>1999;<span class="ref-vol">3</span>(5):614–22.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/10508665" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 10508665</span></a>]</div></dd><dt>11.</dt><dd><div class="bk_ref" id="ml121.r11">Walsh TR, Toleman MA, Poirel L, Nordmann P. Metallo-beta-lactamases: the quiet before the storm? <span><span class="ref-journal">Clin Microbiol Rev. </span>2005;<span class="ref-vol">18</span>(2):306–25.</span> [<a href="/pmc/articles/PMC1082798/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC1082798</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/15831827" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 15831827</span></a>]</div></dd><dt>12.</dt><dd><div class="bk_ref" id="ml121.r12">Hirakata Y, Izumikawa K, Yamaguchi T, Takemura H, Tanaka H, Yoshida R, Matsuda J, Nakano M, Tomono K, Maesaki S, Kaku M, Yamada Y, Kamihira S, Kohno S. Rapid detection and evaluation of clinical characteristics of emerging multiple-drug-resistant gram-negative rods carrying the metallo-beta-lactamase gene blaIMP. <span><span class="ref-journal">Antimicrob Agents Chemother. </span>1998;<span class="ref-vol">42</span>(8):2006–11.</span> [<a href="/pmc/articles/PMC105724/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC105724</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/9687398" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 9687398</span></a>]</div></dd><dt>13.</dt><dd><div class="bk_ref" id="ml121.r13">Lauretti L, Riccio ML, Mazzariol A, Cornaglia G, Amicosante G, Fontana R, Rossolini GM. Cloning and characterization of blaVIM, a new integron-borne metallo-beta-lactamase gene from a Pseudomonas aeruginosa clinical isolate. <span><span class="ref-journal">Antimicrob Agents Chemother. </span>1999;<span class="ref-vol">43</span>(7):1584–90.</span> [<a href="/pmc/articles/PMC89328/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC89328</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/10390207" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 10390207</span></a>]</div></dd><dt>14.</dt><dd><div class="bk_ref" id="ml121.r14">Wang CX, Mi ZH. Imipenem-resistant Pseudomonas aeruginosa producing IMP-1 metallo-beta-lactamases and lacking the outer-membrane protein OprD. <span><span class="ref-journal">J Med Microbiol. </span>2006;<span class="ref-vol">55</span>(Pt 3):353–4.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/16476803" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 16476803</span></a>]</div></dd><dt>15.</dt><dd><div class="bk_ref" id="ml121.r15">Paterson DL, Bonomo RA. Extended-spectrum beta-lactamases: a clinical update. <span><span class="ref-journal">Clin Microbiol Rev. </span>2005;<span class="ref-vol">18</span>(4):657–86.</span> [<a href="/pmc/articles/PMC1265908/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC1265908</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/16223952" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 16223952</span></a>]</div></dd><dt>16.</dt><dd><div class="bk_ref" id="ml121.r16">Zuck P, O’Donnell GT, Cassaday J, Chase P, Hodder P, Strulovici B, Ferrer M. Miniaturization of absorbance assays using the fluorescent properties of white microplates. <span><span class="ref-journal">Anal Biochem. </span>2005;<span class="ref-vol">342</span>(2):254–9.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/15949786" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 15949786</span></a>]</div></dd><dt>17.</dt><dd><div class="bk_ref" id="ml121.r17">Li X, He Y, Ruiz CH, Koenig M, Cameron MD, Vojkovsky T. Characterization of dasatinib and its structural analogs as CYP3A4 mechanism-based inactivators and the proposed bioactivation pathways. <span><span class="ref-journal">Drug Metab Dispos. </span>2009;<span class="ref-vol">37</span>(6):1242–50.</span> [<a href="/pmc/articles/PMC3202349/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3202349</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/19282395" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19282395</span></a>]</div></dd><dt>18.</dt><dd><div class="bk_ref" id="ml121.r18">Li X, Kamenecka TM, Cameron MD. Bioactivation of the epidermal growth factor receptor inhibitor gefitinib: implications for pulmonary and hepatic toxicities. <span><span class="ref-journal">Chem Res Toxicol. </span>2009;<span class="ref-vol">22</span>(10):1736–42.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/19803472" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19803472</span></a>]</div></dd><dt>19.</dt><dd><div class="bk_ref" id="ml121.r19">Lesyk R, Zimenkovsky B, Atamanyuk D, Jensen F, Kiec-Kononowicz K, Gzella A. Anticancer thiopyrano[2,3-d][1,3]thiazol-2-ones with norbornane moiety. Synthesis, cytotoxicity, physico-chemical properties, and computational studies. <span><span class="ref-journal">Bioorg Med Chem. </span>2006;<span class="ref-vol">14</span>(15):5230–40.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/16632367" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 16632367</span></a>]</div></dd><dt>20.</dt><dd><div class="bk_ref" id="ml121.r20">Gouveia FL, de Oliveira RM, de Oliveira TB, da Silva IM, do Nascimento SC, de Sena KX, de Albuquerque JF. Synthesis, antimicrobial and cytotoxic activities of some 5-arylidene-4-thioxo-thiazolidine-2-ones. <span><span class="ref-journal">Eur J Med Chem. </span>2009;<span class="ref-vol">44</span>(5):2038–43.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/19027993" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19027993</span></a>]</div></dd><dt>21.</dt><dd><div class="bk_ref" id="ml121.r21">Bonapace CR, Bosso JA, Friedrich LV, White RL. Comparison of methods of interpretation of checkerboard synergy testing. <span><span class="ref-journal">Diagn Microbiol Infect Dis. </span>2002;<span class="ref-vol">44</span>(4):363–6.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/12543542" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 12543542</span></a>]</div></dd><dt>22.</dt><dd><div class="bk_ref" id="ml121.r22">Bajaksouzian S, Visalli MA, Jacobs MR, Appelbaum PC. Activities of levofloxacin, ofloxacin, and ciprofloxacin, alone and in combination with amikacin, against acinetobacters as determined by checkerboard and time-kill studies. <span><span class="ref-journal">Antimicrob Agents Chemother. </span>1997;<span class="ref-vol">41</span>(5):1073–6.</span> [<a href="/pmc/articles/PMC163853/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC163853</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/9145872" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 9145872</span></a>]</div></dd><dt>23.</dt><dd><div class="bk_ref" id="ml121.r23">Barry AL, Craig WA, Nadler H, Reller LB, Sanders CC, Swenson JM, et al. <span class="ref-journal">Methods for Determining Bactericidal Activity of Antimicrobial Agents; Approved Guideline.</span> National Committee on Clinical Laboratory Standards; 1999. M26-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/NBK63598/?report=reader">PubReader</a></li><li><a href="/books/NBK63598/?report=printable">Print View</a></li><li><a data-jig="ncbidialog" href="#_ncbi_dlg_citbx_NBK63598" data-jigconfig="width:400,modal:true">Cite this Page</a><div id="_ncbi_dlg_citbx_NBK63598" style="display:none" title="Cite this Page"><div class="bk_tt">Minond D, Saldanha SA, Spicer T, et al. HTS Assay for Discovery of Novel Metallo-Beta-lactamase (MBL) Inhibitors. 2009 Nov 13 [Updated 2011 May 5]. 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/NBK63598/pdf/Bookshelf_NBK63598.pdf">PDF version of this page</a> (710K)</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="#ml121.s1" ref="log$=inpage&link_id=inpage">Recommendations for scientific use of the probe (ML121)</a></li><li><a href="#ml121.s2" ref="log$=inpage&link_id=inpage">Introduction</a></li><li><a href="#ml121.s12" ref="log$=inpage&link_id=inpage">Results</a></li><li><a href="#ml121.s22" ref="log$=inpage&link_id=inpage">Discussion</a></li><li><a href="#ml121.s26" ref="log$=inpage&link_id=inpage">References</a></li></ul></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Related information</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="discovery_db_links" id="Shutter"></a></div><div class="portlet_content"><ul><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pmc&DbFrom=books&Cmd=Link&LinkName=books_pmc_refs&IdsFromResult=2580688" ref="log$=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=2580688" ref="log$=recordlinks">PubChem BioAssay for Chemical Probe</a><div class="brieflinkpop offscreen_noflow">PubChem BioAssay records reporting screening data for the development of the chemical probe(s) described in this book chapter</div></li><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pcsubstance&DbFrom=books&Cmd=Link&LinkName=books_pcsubstance&IdsFromResult=2580688" ref="log$=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=2580688" ref="log$=recordlinks">PubMed</a><div class="brieflinkpop offscreen_noflow">Links to PubMed</div></li></ul></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Similar articles in PubMed</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="PBooksDiscovery_RA" id="Shutter"></a></div><div class="portlet_content"><ul><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/23762954" ref="ordinalpos=1&linkpos=1&log$=relatedreviews&logdbfrom=pubmed"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> ML302, a Novel Beta-lactamase (BLA) Inhibitor.</a><span class="source">[Probe Reports from the NIH Mol...]</span><div class="brieflinkpop offscreen_noflow"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> ML302, a Novel Beta-lactamase (BLA) Inhibitor.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Spicer T, Minond D, Enogieru I, Saldanha SA, Allais C, Liu Q, Mercer BA, Roush WR, Hodder P. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Probe Reports from the NIH Molecular Libraries Program. 2010</em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/19553129" ref="ordinalpos=1&linkpos=2&log$=relatedarticles&logdbfrom=pubmed">Inhibitors of VIM-2 by screening pharmacologically active and click-chemistry compound libraries.</a><span class="source">[Bioorg Med Chem. 2009]</span><div class="brieflinkpop offscreen_noflow">Inhibitors of VIM-2 by screening pharmacologically active and click-chemistry compound libraries.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Minond D, Saldanha SA, Subramaniam P, Spaargaren M, Spicer T, Fotsing JR, Weide T, Fokin VV, Sharpless KB, Galleni M, et al. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Bioorg Med Chem. 2009 Jul 15; 17(14):5027-37. 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Epub 2015 Dec 7.</em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/11304992" ref="ordinalpos=1&linkpos=5&log$=relatedreviews&logdbfrom=pubmed"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> [Metallo-beta-lactamase producing bacteria].</a><span class="source">[Nihon Rinsho. 2001]</span><div class="brieflinkpop offscreen_noflow"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> [Metallo-beta-lactamase producing bacteria].<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Iyobe S. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Nihon Rinsho. 2001 Apr; 59(4):701-6. </em></div></div></li></ul><a class="seemore" href="/sites/entrez?db=pubmed&cmd=link&linkname=pubmed_pubmed_reviews&uid=21961119" ref="ordinalpos=1&log$=relatedreviews_seeall&logdbfrom=pubmed">See reviews...</a><a class="seemore" href="/sites/entrez?db=pubmed&cmd=link&linkname=pubmed_pubmed&uid=21961119" ref="ordinalpos=1&log$=relatedarticles_seeall&logdbfrom=pubmed">See all...</a></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Recent Activity</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="recent_activity" id="Shutter"></a></div><div class="portlet_content"><div xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" id="HTDisplay" class=""><div class="action"><a href="javascript:historyDisplayState('ClearHT')">Clear</a><a href="javascript:historyDisplayState('HTOff')" class="HTOn">Turn Off</a><a href="javascript:historyDisplayState('HTOn')" class="HTOff">Turn On</a></div><ul id="activity"><li class="ra_rcd ralinkpopper two_line"><a class="htb ralinkpopperctrl" ref="log$=activity&linkpos=1" href="/portal/utils/pageresolver.fcgi?recordid=67d66c14cde49f3df7dcd0ad">HTS Assay for Discovery of Novel Metallo-Beta-lactamase (MBL) Inhibitors - 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