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<title>Identification of small molecules that selectively inhibit streptokinase expression without suppression of viability in Group A streptococci - Probe 1 - Probe Reports from the NIH Molecular Libraries Program - NCBI Bookshelf</title>
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<meta name="robots" content="INDEX,FOLLOW,NOARCHIVE" /><meta name="citation_inbook_title" content="Probe Reports from the NIH Molecular Libraries Program [Internet]" /><meta name="citation_title" content="Identification of small molecules that selectively inhibit streptokinase expression without suppression of viability in Group A streptococci - Probe 1" /><meta name="citation_publisher" content="National Center for Biotechnology Information (US)" /><meta name="citation_date" content="2011/03/11" /><meta name="citation_author" content="W Frank An" /><meta name="citation_author" content="Shailesh R Metkar" /><meta name="citation_author" content="Partha P Nag" /><meta name="citation_author" content="Cathy L Hartland" /><meta name="citation_author" content="Douglas Barker" /><meta name="citation_author" content="Jose R Perez" /><meta name="citation_author" content="Hongmin Sun" /><meta name="citation_author" content="Lawrence MacPherson" /><meta name="citation_author" content="Michelle Palmer" /><meta name="citation_author" content="Stuart L Schreiber" /><meta name="citation_pmid" content="21735597" /><meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK56228/" /><link rel="schema.DC" href="http://purl.org/DC/elements/1.0/" /><meta name="DC.Title" content="Identification of small molecules that selectively inhibit streptokinase expression without suppression of viability in Group A streptococci - Probe 1" /><meta name="DC.Type" content="Text" /><meta name="DC.Publisher" content="National Center for Biotechnology Information (US)" /><meta name="DC.Contributor" content="W Frank An" /><meta name="DC.Contributor" content="Shailesh R Metkar" /><meta name="DC.Contributor" content="Partha P Nag" /><meta name="DC.Contributor" content="Cathy L Hartland" /><meta name="DC.Contributor" content="Douglas Barker" /><meta name="DC.Contributor" content="Jose R Perez" /><meta name="DC.Contributor" content="Hongmin Sun" /><meta name="DC.Contributor" content="Lawrence MacPherson" /><meta name="DC.Contributor" content="Michelle Palmer" /><meta name="DC.Contributor" content="Stuart L Schreiber" /><meta name="DC.Date" content="2011/03/11" /><meta name="DC.Identifier" content="https://www.ncbi.nlm.nih.gov/books/NBK56228/" /><meta name="description" content="Streptokinase is a major group A streptococcus (GAS) virulence factor and plays critical roles in GAS pathogenicity. We set out to identify and develop novel small molecule probes that inhibit streptokinase (SK) expression in GAS without suppressing cell growth to minimize potential drug resistance. We screened the Molecular Libraries Probe Centers Network (MLPCN) library of over 300,000 compounds and identified the first small molecular probes. The probe ML126 (CID-576989), carbazol-9-yl-(4-methylphenyl)methanone, selectively inhibits SK expression with an IC50 of 3.6 μM in a GAS strain and has no effect on bacterial growth. A series of analogs were synthesized to explore the structure activity relationships. These analogs showed over 10-fold range in potency of inhibition of SK expression, and revealed key structural requirements for optimal potency and selectivity. The probe ML126 (CID-576989) and its analogs are excellent molecular tools to investigate regulation of SK expression and mechanisms of GAS infection." /><meta name="og:title" content="Identification of small molecules that selectively inhibit streptokinase expression without suppression of viability in Group A streptococci - Probe 1" /><meta name="og:type" content="book" /><meta name="og:description" content="Streptokinase is a major group A streptococcus (GAS) virulence factor and plays critical roles in GAS pathogenicity. We set out to identify and develop novel small molecule probes that inhibit streptokinase (SK) expression in GAS without suppressing cell growth to minimize potential drug resistance. We screened the Molecular Libraries Probe Centers Network (MLPCN) library of over 300,000 compounds and identified the first small molecular probes. The probe ML126 (CID-576989), carbazol-9-yl-(4-methylphenyl)methanone, selectively inhibits SK expression with an IC50 of 3.6 μM in a GAS strain and has no effect on bacterial growth. A series of analogs were synthesized to explore the structure activity relationships. These analogs showed over 10-fold range in potency of inhibition of SK expression, and revealed key structural requirements for optimal potency and selectivity. The probe ML126 (CID-576989) and its analogs are excellent molecular tools to investigate regulation of SK expression and mechanisms of GAS infection." /><meta name="og:url" content="https://www.ncbi.nlm.nih.gov/books/NBK56228/" /><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/ml126/" /><link rel="canonical" href="https://www.ncbi.nlm.nih.gov/books/NBK56228/" /><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="#__NBK56228_dtls__">Show details</a><div style="display:none" class="ui-widget" id="__NBK56228_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/ml127/" title="Previous page in this title">< Prev</a><a class="active page_link next" href="/books/n/mlprobe/ml125/" 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="_NBK56228_"><span class="title" itemprop="name">Identification of small molecules that selectively inhibit streptokinase expression without suppression of viability in Group A streptococci - Probe 1</span></h1><p class="contrib-group"><span itemprop="author">W Frank An</span>, <span itemprop="author">Shailesh R Metkar</span>, <span itemprop="author">Partha P Nag</span>, <span itemprop="author">Cathy L Hartland</span>, <span itemprop="author">Douglas Barker</span>, <span itemprop="author">Jose R Perez</span>, <span itemprop="author">Hongmin Sun</span>, <span itemprop="author">Lawrence MacPherson</span>, <span itemprop="author">Michelle Palmer</span>, and <span itemprop="author">Stuart L Schreiber</span>.</p><a data-jig="ncbitoggler" href="#__NBK56228_ai__" style="border:0;text-decoration:none">Author Information and Affiliations</a><div style="display:none" class="ui-widget" id="__NBK56228_ai__"><p class="contrib-group"><h4>Authors</h4><span itemprop="author">W Frank An</span>,<sup>1</sup> <span itemprop="author">Shailesh R Metkar</span>,<sup>1</sup> <span itemprop="author">Partha P Nag</span>,<sup>1</sup> <span itemprop="author">Cathy L Hartland</span>,<sup>1</sup> <span itemprop="author">Douglas Barker</span>,<sup>1</sup> <span itemprop="author">Jose R Perez</span>,<sup>1</sup> <span itemprop="author">Hongmin Sun</span>,<sup>2</sup> <span itemprop="author">Lawrence MacPherson</span>,<sup>1</sup> <span itemprop="author">Michelle Palmer</span>,<sup>1</sup> and <span itemprop="author">Stuart L Schreiber</span><sup>1,3</sup>.<sup><img src="/corehtml/pmc/pmcgifs/corrauth.gif" alt="corresponding author" /></sup></p><h4>Affiliations</h4><div class="affiliation"><sup>1</sup>
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The Broad Institute Probe Development Center, Cambridge, MA</div><div class="affiliation"><sup>2</sup>
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University of Missouri, Columbia, MO</div><div class="affiliation"><sup>3</sup>
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Howard Hughes Medical Institute, Chemistry & Chemical Biology, Harvard University, Cambridge, MA</div><div class="affiliation"><sup><img src="/corehtml/pmc/pmcgifs/corrauth.gif" alt="corresponding author" /></sup>Corresponding author email:
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<a href="mailto:dev@null" data-email="gro.etutitsnidaorb@naf" class="oemail">gro.etutitsnidaorb@naf</a></div></div><p class="small">Received: <span itemprop="datePublished">November 16, 2009</span>; Last Update: <span itemprop="dateModified">March 11, 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>Streptokinase is a major group A streptococcus (GAS) virulence factor and plays critical roles in GAS pathogenicity. We set out to identify and develop novel small molecule probes that inhibit streptokinase (SK) expression in GAS without suppressing cell growth to minimize potential drug resistance. We screened the Molecular Libraries Probe Centers Network (MLPCN) library of over 300,000 compounds and identified the first small molecular probes. The probe <a href="/pcsubstance/?term=ML126[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML126</a> (CID-576989), carbazol-9-yl-(4-methylphenyl)methanone, selectively inhibits SK expression with an IC<sub>50</sub> of 3.6 μM in a GAS strain and has no effect on bacterial growth. A series of analogs were synthesized to explore the structure activity relationships. These analogs showed over 10-fold range in potency of inhibition of SK expression, and revealed key structural requirements for optimal potency and selectivity. The probe <a href="/pcsubstance/?term=ML126[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML126</a> (CID-576989) and its analogs are excellent molecular tools to investigate regulation of SK expression and mechanisms of GAS infection.</p></div><div class="h2"></div><p><b>Assigned Assay Grant No.:</b> 1R03DA026214-01</p><p><b>Screening Center Name and PI:</b> Broad Institute Probe Development Center, Stuart Schreiber, PhD</p><p><b>Chemistry Center Name and PI:</b> Broad Institute Probe Development Center, Stuart Schreiber, PhD</p><p><b>Assay Submitter & Institution:</b> Hongmin Sun, PhD. University of Missouri, Columbia</p><p><b>PubChem Summary Bioassay Identifier (AID):</b>
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<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1677" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1677</a></p><div id="ml126.s1"><h2 id="_ml126_s1_">Probe Structure & Characteristics</h2><div id="ml126.fu1" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56228/bin/ml126fu1.jpg" alt="ML126." /></div><h3><span class="title"><a href="/pcsubstance/?term=ML126[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML126</a></span></h3></div><div id="ml126.tu1" class="table"><h3><span class="title">Compound Summary in PubChem</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK56228/table/ml126.tu1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml126.tu1_lrgtbl__"><table class="no_top_margin"><tbody><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">IUPAC Chemical Name</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">carbazol-9-yl-(4-methylphenyl)methanone</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">PubChem CID</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">576989</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Molecular Weight</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">285.3392 g/mol</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Molecular Formula</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">C<sub>20</sub>H<sub>15</sub>NO</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">XLogP3-AA</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">5.2</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">H-Bond Donor</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">H-Bond Acceptor</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Rotatable Bond Count</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Exact Mass</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">285.115364</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Topological Polar Surface Area</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">22</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Heavy Atom Count</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">22</td></tr></tbody></table></div></div><div id="ml126.tu2" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK56228/table/ml126.tu2/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml126.tu2_lrgtbl__"><table><thead><tr><th id="hd_h_ml126.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CID/ML No.</th><th id="hd_h_ml126.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Target Name</th><th id="hd_h_ml126.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">IC<sub>50</sub>/EC<sub>50</sub> (nM) [SID, AID]</th><th id="hd_h_ml126.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Anti-target Name(s)</th><th id="hd_h_ml126.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">IC<sub>50</sub>/EC<sub>50</sub> (nM) [SID, AID]</th><th id="hd_h_ml126.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Selectivity</th><th id="hd_h_ml126.tu2_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Secondary Assay(s) Name: Viability IC<sub>50</sub>/EC<sub>50</sub> (nM) [SID, AID]</th></tr></thead><tbody><tr><td headers="hd_h_ml126.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">576989/<a href="/pcsubstance/?term=ML126[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML126</a></td><td headers="hd_h_ml126.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Streptokinase expression</td><td headers="hd_h_ml126.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3600 [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/85281160" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">85281160</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2137" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2137</a>]</td><td headers="hd_h_ml126.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Bacterial cell viability</td><td headers="hd_h_ml126.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">inactive [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/85281160" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">85281160</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2138" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2138</a>]</td><td headers="hd_h_ml126.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3600 nM versus. inactive</td><td headers="hd_h_ml126.tu2_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">inactive [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/85281160" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">85281160</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2138" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2138</a>]</td></tr></tbody></table></div></div></div><div id="ml126.s2"><h2 id="_ml126_s2_">Recommendations for scientific use of the probe</h2><p>The goal of the project is to identify and to develop novel small molecule probes that inhibit streptokinase (SK) expression in group A streptococcus (GAS). Streptokinase is a major GAS virulence factor that activates human plasminogen (<a class="bk_pop" href="#ml126.r1">1</a>,<a class="bk_pop" href="#ml126.r2">2</a>,<a class="bk_pop" href="#ml126.r3">3</a>). Novel antimicrobial agents that suppress pathogen virulence without selecting for drug-resistant mutants provide a promising alternative approach for treatment of infectious diseases (<a class="bk_pop" href="#ml126.r4">4</a>,<a class="bk_pop" href="#ml126.r5">5</a>,<a class="bk_pop" href="#ml126.r6">6</a>,<a class="bk_pop" href="#ml126.r7">7</a>). Based on the critical role of SK in GAS pathogenicity, we are attempting to identify chemical compounds that specifically reduce the expression of bacterial SK, without affecting bacterial viability.</p><p>The probe <a href="/pcsubstance/?term=ML126[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML126</a> (CID-576989) described in this report selectively inhibits the expression of bacterial SK in GAS without affecting bacterial cell viability. The probe <a href="/pcsubstance/?term=ML126[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML126</a> (CID-576989) inhibits SK expression with an IC<sub>50</sub> of 3.6 μM and was inactive in suppressing bacterial cell viability in the non-engineered UMAA2616 strain at concentrations at high as 50 μM.</p><p>The probe <a href="/pcsubstance/?term=ML126[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML126</a> (CID-576989) will be used to explore the contribution of SK to GAS infection. This probe may also serve as a starting point for trying to identify small molecules suitable for new mouse models. The Assay Provider, Dr. Hongmin Sun, has established that transgenic mice expressing human plasminogen (<i>Tg+</i>) are highly susceptible to GAS infection and are a useful model to test the impact of hit compounds in attenuating GAS virulence (<a class="bk_pop" href="#ml126.r8">8</a>,<a class="bk_pop" href="#ml126.r9">9</a>). <i>In vivo</i> efficacy of compounds is assessed in <i>Tg+</i> mice that are first infected with SK(+)GAS and then injected with doses of test compound intraperitoneally daily for 5 days, starting 1 day after infection.</p></div><div id="ml126.s3"><h2 id="_ml126_s3_">1. Introduction</h2><div id="ml126.s4"><h3>Scientific Rationale</h3><p>Group A streptococcus (GAS) is among the most common human pathogens affecting millions of people globally each year. Even though treatment with antibiotics has greatly reduced mortality in the developed world, complications of GAS infection are sometimes still lethal. Further, emergence of epidemic invasive GAS diseases poses another critical medical challenge, but little is known about the mechanism of invasiveness (<a class="bk_pop" href="#ml126.r3">3</a>). Knowledge of the underlying molecular mechanisms required for infection by this important pathogen will be crucial for the development of novel therapies for GAS infection.</p><p>Streptokinase (SK) is a key virulence factor for GAS infection through interacting with host plasminogen (<a class="bk_pop" href="#ml126.r2">2</a>,<a class="bk_pop" href="#ml126.r8">8</a>,<a class="bk_pop" href="#ml126.r9">9</a>). This project aims to take advantage of this observation and discover novel antimicrobial agents for the treatment of GAS infection. One key design of the project is to target specifically the bacterial virulence without impact on bacterial growth. Such agents provide a promising alternative approach for treatment of infectious diseases by reducing the risks of antibiotic resistance. Antibiotic resistance represents an increasing concern as a major global public health threat contributed by widespread use of antibiotics that cause death or growth arrest in the target bacteria. This screening campaign has identified a probe (CID-576989/<a href="/pcsubstance/?term=ML126[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML126</a>; carbazol-9-yl-(4-methylphenyl)methanone) with an IC<sub>50</sub> of 3.6 μM, which is inactive in the bacterial viability test within the range of testing concentrations up to 50 μM.</p></div></div><div id="ml126.s5"><h2 id="_ml126_s5_">2. Materials and Methods</h2><div id="ml126.s6"><h3>Materials and Reagents</h3><p>All reagents and solvents were purchased from commercial vendors and used as received. Phosphate-buffered saline (PBS) was acquired from the Broad Institute Supply and Quality Management (SQM; Cambridge, MA). SK assay solution was acquired from Innov Research (Novi, Michigan), and S2403 was acquired from Diapharma (West Chester, OH). BacTiter-Glo was purchased from Promega (catalog no. G8233; Madison, WI). White assay plates were purchased from Corning (catalog no. 3570; Corning, NY), and clear plates were purchased from Nunc (catalog no. 242757; Rochester, NY).</p><div id="ml126.s7"><h4>Growth Medium</h4><ul><li class="half_rhythm"><div>THY/S (30 g/L THB; Catalog no. 249240, Becton Dickinson, Sparks, MD/0.2% yeast extract; Catalog no. Y2010, US Biological, Swampscott, MA) with 100 μg/mL streptomycin (Catalog no. S9137; Sigma, St. Louis, MO)</div></li><li class="half_rhythm"><div>THY/S/S medium (30 g/L THB/0.2% yeast extract with 100 μg/mL streptomycin/100 μg/ml spectinomycin (Catalog no. S1317; Sigma, St. Louis, MO)</div></li><li class="half_rhythm"><div>THY/K medium (THY plus 40 μg/ml kanamycin) (Catalog no. S1637; Sigma, St. Louis, MO)</div></li></ul></div><div id="ml126.s8"><h4>Cell Lines</h4><p>SKKanGAS strain and UMAA 2616 were provided by the Assay Provider, Dr. Hongmin Sun (University of Missouri). Human plasma was obtained from Innovative Research (Novi, Michigan).</p></div></div><div id="ml126.s9"><h3>2.1. Assays</h3><p>A summary listing of completed assays and corresponding PubChem AID numbers is provided in <a href="#ml126.app1">Appendix A</a>. Refer to <a href="#ml126.app2">Appendix B</a> for the detailed assay protocols.</p><div id="ml126.s10"><h4>2.1.1. Primary Screen in SKKanGAS Strain (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1662" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1662</a>)</h4><p>On Day 1, the SKKanGAS strain was streaked out on THY/S/S medium plate (30 g/L THB/0.2% yeast extract with streptomycin 100 μg/mL, spectinomycin 100 μg/ml) agar (1.5%; Catalog no. 281230; Becton Dickinson, Sparks, MD), and incubated at 37°C overnight. On Day 2, a single colony was picked and grown overnight in THY/S/S medium at 37°C. On Day 3, the overnight culture was diluted 20-fold into fresh THY/S/S medium in a flask until it reached OD600 of 0.6–0.8. The culture was again diluted into cold THY/K (THY plus 40 μg/ml kanamycin) medium to result in OD600 value of 0.038, and the diluted culture was kept at 4°C overnight. On Day 4, the diluted culture was warmed to room temperature and 20 μl per well was dispensed with a Combi multidrop (Thermo Fisher Scientific Inc., Waltham, MA) into assay plates that contained 30 μl per well of THY/K. Next, 100 nL per well of compound was pinned with Cybi-Well (CyBio; Woburn, MA). Next, the plates were incubated at 37°C for 6 hours then cooled to room temperature for 30 minutes. Then, 30 μl per well of diluted BacTiter-Glo (25 μl per well of BacTiter-Glo and 5 μl per well of PBS) was added to the culture with a Combi multidrop. The plates were incubated at room temperature for 10 minutes, and luminescence was detected on an EnVision (Perkin Elmer, Waltham, MA) multimode reader.</p></div><div id="ml126.s11"><h4>2.1.2. Primary Retest in SKKanGAS Strain</h4><p>Repeat of primary screen at dose in SKKanGAS cells using BacTiter-Glo.</p></div><div id="ml126.s12"><h4>2.1.3. Secondary Screen for SK Expression in UMAA 2616 Strain (AID nos. <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1914" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1914</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2137" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2137</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504351" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">504351</a>)</h4><p>On Day 1, UMAA 2616 was streaked out on THY/S medium (30g/L THB/0.2% yeast extract with streptomycin 100 μg/mL), and incubated at 37°C overnight. On Day 2, a single colony was picked and grown overnight in THY/S medium at 37°C. On Day 3, the overnight culture was diluted 20-fold into fresh THY/S medium in a flask and grown until it reached OD600 of 0.6–0.8. The culture was again diluted into cold THY/S medium to result in OD600 value of 0.038, and the diluted culture was kept at 4°C overnight. On Day 4, the diluted culture was warmed to room temperature, and 20 μl per well of culture was dispensed with a Combi (Thermo Fisher Scientific Inc., Waltham, MA) into assay plates that contained 30 μl per well of THY/S medium. Next, 100 nL per well of compound was pinned with Cybi-Well (CyBio; Woburn, MA), and the plates were incubated at 37°C for 6 hours in a Liconic incubator. The cells were pelleted at 3000 rpm in a Sorvall centrifuge. Next, 10 μl per well of the supernatant was transferred twice but into separate clear plates with Cybi-Well Vario and stored at −20°C. The culture plates were warmed to room temperature for 30 minutes. Then, 50 μl per well of half strength BacTiter-Glo was added with a Combi multidrop. The plates were incubated at room temperature for 10 minutes, and luminescence was detected on an EnVision (Perkin Elmer; Waltham, MA) multimode reader for indications of bacterial cell viability.</p><p>SK expression was measured by adding 50 μl/well of SK assay solution (5 μl per well of human plasma, 1.2 μl to 2.4 μl per well of 4.2 mg/ml S2403, and 43.8 μl per well of PBS) to the 10 μl per well supernatant that had been thawed and warmed up to room temperature in clear SK assay plates. Compound CID-657963 was used as a positive control in the SK expression assay (see <a class="figpopup" href="/books/NBK56228/figure/ml126.f1/?report=objectonly" target="object" rid-figpopup="figml126f1" rid-ob="figobml126f1">Figure 1</a>). OD405 was taken at 0 h and after 1.5 h to 2.5 h of incubation at 37°C depending on the lot of the S2403.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml126f1" co-legend-rid="figlgndml126f1"><a href="/books/NBK56228/figure/ml126.f1/?report=objectonly" target="object" title="Figure 1" class="img_link icnblk_img figpopup" rid-figpopup="figml126f1" rid-ob="figobml126f1"><img class="small-thumb" src="/books/NBK56228/bin/ml126f1.gif" src-large="/books/NBK56228/bin/ml126f1.jpg" alt="Figure 1. Prior Art Compound CID-657963 for SK Expression." /></a><div class="icnblk_cntnt" id="figlgndml126f1"><h4 id="ml126.f1"><a href="/books/NBK56228/figure/ml126.f1/?report=objectonly" target="object" rid-ob="figobml126f1">Figure 1</a></h4><p class="float-caption no_bottom_margin">Prior Art Compound CID-657963 for SK Expression. Compound CID-657963 (positive control) in the SK expression assay. This compound did not significantly inhibit bacterial cell viability up to approximately 100 μM. </p></div></div></div><div id="ml126.s13"><h4>2.1.4. Secondary Screen for Bacterial Cell Viability in UMAA 2616 Strain (AID nos. <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1915" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1915</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2138" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2138</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504396" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">504396</a>)</h4><p>As described in the secondary screen for SK expression.</p></div></div><div id="ml126.s14"><h3>2.2. Probe Chemical Characterization</h3><p>After preparation as described in <a href="#ml126.s15">Section 2.3</a>, the probe (CID-576989/<a href="/pcsubstance/?term=ML126[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML126</a>) was analyzed by UPLC, <sup>1</sup>H NMR, <sup>13</sup>C NMR, and LC-MS. The data obtained from NMR spectroscopy were consistent with the structure of the probe (CID-576989/<a href="/pcsubstance/?term=ML126[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML126</a>), and UPLC indicated an isolated purity of 100%. The relevant data are provided in <a href="#ml126.app3">Appendix C</a>.</p><p>The observed solubility of the probe (CID-576989/<a href="/pcsubstance/?term=ML126[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML126</a>) was calculated to be less than 1 μM in PBS solution. The stability of the probe (CID-576989/<a href="/pcsubstance/?term=ML126[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML126</a>) in PBS (0.1% DMSO) was measured over 48 hours, and the data are shown in <a class="figpopup" href="/books/NBK56228/figure/ml126.f2/?report=objectonly" target="object" rid-figpopup="figml126f2" rid-ob="figobml126f2">Figure 2</a> (blue line). We suspected that poor solubility (and not instability) as the reason behind the dramatic drop in the amount of sample over time. To test this, we added acetonitrile to each well (final concentration 50%) and measured the amount of the probe. Amounts detected after addition of acetonitrile are shown in <a class="figpopup" href="/books/NBK56228/figure/ml126.f2/?report=objectonly" target="object" rid-figpopup="figml126f2" rid-ob="figobml126f2">Figure 2</a> (red line). Since addition of acetonitrile leads to increased detection of the probe, it is more likely that the compound is stable in PBS. The apparent instability as suggested by <a class="figpopup" href="/books/NBK56228/figure/ml126.f2/?report=objectonly" target="object" rid-figpopup="figml126f2" rid-ob="figobml126f2">Figure 2</a> (blue line) is an artifact resulting from poor solubility of the probe in PBS.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml126f2" co-legend-rid="figlgndml126f2"><a href="/books/NBK56228/figure/ml126.f2/?report=objectonly" target="object" title="Figure 2" class="img_link icnblk_img figpopup" rid-figpopup="figml126f2" rid-ob="figobml126f2"><img class="small-thumb" src="/books/NBK56228/bin/ml126f2.gif" src-large="/books/NBK56228/bin/ml126f2.jpg" alt="Figure 2. Stability of the Probe (CID-576989/ML126) in PBS." /></a><div class="icnblk_cntnt" id="figlgndml126f2"><h4 id="ml126.f2"><a href="/books/NBK56228/figure/ml126.f2/?report=objectonly" target="object" rid-ob="figobml126f2">Figure 2</a></h4><p class="float-caption no_bottom_margin">Stability of the Probe (CID-576989/ML126) in PBS. </p></div></div><p>Plasma protein binding (PPB) was determined to be 91.1% bound in human plasma. The probe (CID-576989/<a href="/pcsubstance/?term=ML126[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML126</a>) is moderately stable in human plasma with approximately 38.6% remaining after a 5-hour incubation period but unstable in murine plasma (less than 12.5% of the probe remains after 5 hours). The solubility, PPB, and plasma stability results are summarized in <a href="#ml126.s23">Section 3.4</a> (entry 1, <a class="figpopup" href="/books/NBK56228/table/ml126.t1/?report=objectonly" target="object" rid-figpopup="figml126t1" rid-ob="figobml126t1">Table 1</a>).</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml126t1"><a href="/books/NBK56228/table/ml126.t1/?report=objectonly" target="object" title="Table 1" class="img_link icnblk_img figpopup" rid-figpopup="figml126t1" rid-ob="figobml126t1"><img class="small-thumb" src="/books/NBK56228/table/ml126.t1/?report=thumb" src-large="/books/NBK56228/table/ml126.t1/?report=previmg" alt="Table 1. SAR Analysis and Properties of Various Phenyl Substituent Analogs." /></a><div class="icnblk_cntnt"><h4 id="ml126.t1"><a href="/books/NBK56228/table/ml126.t1/?report=objectonly" target="object" rid-ob="figobml126t1">Table 1</a></h4><p class="float-caption no_bottom_margin">SAR Analysis and Properties of Various Phenyl Substituent Analogs. </p></div></div><p>The probe (CID-576989/<a href="/pcsubstance/?term=ML126[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML126</a>) and four additional analogs were submitted to the SMR collection MLS002608919 (probe), MLS002608920 (CID-8852543), MLS002608921 (44247468), MLS002608922 (CID-85281167), and MLS002608923 (CID-85281172).</p></div><div id="ml126.s15"><h3>2.3. Probe Preparation</h3><div id="ml126.s16"><h4>Chemistry Experimental Methods</h4><p>The probe (CID-576989/<a href="/pcsubstance/?term=ML126[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML126</a>; <a href="https://pubchem.ncbi.nlm.nih.gov/substance/85281160" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-85281160</a>) was prepared in one step outlined in <a class="figpopup" href="/books/NBK56228/figure/ml126.f7/?report=objectonly" target="object" rid-figpopup="figml126f7" rid-ob="figobml126f7">Scheme 1</a>.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml126f7" co-legend-rid="figlgndml126f7"><a href="/books/NBK56228/figure/ml126.f7/?report=objectonly" target="object" title="Scheme 1" class="img_link icnblk_img figpopup" rid-figpopup="figml126f7" rid-ob="figobml126f7"><img class="small-thumb" src="/books/NBK56228/bin/ml126f7.gif" src-large="/books/NBK56228/bin/ml126f7.jpg" alt="Scheme 1. Synthesis of the Probe (CID-576989/ML126)." /></a><div class="icnblk_cntnt" id="figlgndml126f7"><h4 id="ml126.f7"><a href="/books/NBK56228/figure/ml126.f7/?report=objectonly" target="object" rid-ob="figobml126f7">Scheme 1</a></h4><p class="float-caption no_bottom_margin">Synthesis of the Probe (CID-576989/ML126). </p></div></div><p><b>General details.</b> All reagents and solvents were purchased from commercial vendors and used as received. Unless otherwise mentioned, all of the reactions were run in over-dried, round-bottom flasks or in regular vials under nitrogen. <sup>1</sup>H and <sup>13</sup>C NMR spectra were recorded on a Bruker 300 MHz spectrometer. Proton and carbon chemical shifts are reported in ppm (δ) relative to tetramethylsilane (<sup>1</sup>H δ 0.00) or residual chloroform in CDCl<sub>3</sub> solvent (<sup>1</sup>H δ 7.24, 13C δ 77.0). NMR data are reported as follows: chemical shifts, multiplicity (s = singlet, m = multiplet); coupling constant(s) in Hz; integration. Unless otherwise indicated NMR data were collected at 25°C. Flash chromatography was performed using 40 μm to 60 μm Silica Gel (60 Å mesh) on a Teledyne Isco Combiflash Rf system. Tandem Liquid Chromatography/Mass Spectrometry (LC/MS) was performed on a Waters 2795 separations module and 3100 mass detector. Analytical thin layer chromatography (TLC) was performed on EM Reagent 0.25 mm silica gel 60-F plates. Visualization was accomplished with UV light and aqueous potassium permanganate (KMnO<sub>4</sub>) stain followed by heating.</p><p><b>Synthesis of 9H-carbazol-9-yl)(p-tolyl)methanone:</b> A solution of carbazole (100 mg, 0.57 mmol) in anhydrous tetrahydrofuran (THF, 5 mL) was treated at 0°C with sodium hydride (NaH, 34.4 mg, 0.86 mmol, 1.5 equivalents, 60% dispersion in mineral oil). The reaction mixture was stirred at 0°C for 15 minutes and treated drop-wise with 4-methylbenzoyl chloride (107 mg, 0.91 mL, 0.69 mmol, 1.2 equivalents). The reaction mixture was slowly warmed to room temperature and was stirred for an additional 12 hours. The reaction was then quenched with saturated aqueous sodium bicarbonate (NaHCO<sub>3</sub>) solution, and extracted with ethyl acetate (EtOAc). The combined organic extracts were dried (sodium sulfate; Na<sub>2</sub>SO<sub>4</sub>), filtered, and concentrated. The crude material was purified by column chromatography over silica gel (hexanes/ethyl acetate: 100/0 to 90/10) to give the probe (CID-576989/<a href="/pcsubstance/?term=ML126[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML126</a>) as a white solid compound (123 mg, 75%).</p><p><b><sup>1</sup>H NMR</b> (300 MHz<b>, CDCl<sub>3</sub>):</b> δ 8.10–8.0 (m, 2H), 7.65–7.63 (m, 2H), 7.56–7.52 (m, 2H), 7.36–7.31 (m, 6H), 2.49 (s, 3H); <b><sup>13</sup>C NMR (</b>75 MHz, <b>CDCl<sub>3</sub>):</b> δ 169.8, 143.4, 139.4, 132.9, 129.6, 129.5, 126.8, 126.0, 123.3, 119.9, 115.4, 21.9; ESI-MS (M+H): m/z 286.</p><p>The <sup>1</sup>H NMR spectra, <sup>13</sup>C NMR spectra, and UPLC chromatograms of the probe (CID-576989/<a href="/pcsubstance/?term=ML126[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML126</a>) and analogs are provided in <a href="#ml126.app3">Appendix C</a>.</p></div></div><div id="ml126.s17"><h3>2.4. Additional Analytical Analysis</h3><p><b>Plasma Protein Binding.</b> Plasma protein binding was determined by equilibrium dialysis using the Rapid Equilibrium Dialysis (RED) device (Pierce Biotechnology, Rockford, IL) for both human and mouse plasma. Each compound was prepared in duplicate at 5 μM in plasma (0.95% acetonitrile, 0.05% DMSO) and added to one side of the membrane (200 μL) with PBS (350 μL; pH 7.4) added to the other side. The compounds were incubated at 37°C for 5 hours in an orbital shake at 250 rpm. Following the incubation, the samples were analyzed by UPLC-MS (Waters, Milford, MA) with compounds detected by SIR detection on a single quadrupole mass spectrometer.</p><p><b>Plasma Stability.</b> Plasma stability was determined at 37°C for 5 hours in both human and mouse plasma. Each compound was prepared in duplicate at 5 μM in plasma diluted 50/50 (v/v) with PBS pH 7.4 (0.95% acetonitrile, 0.05% DMSO). The compounds were incubated at 37°C for 5 hours with an orbital shake at 250 rpm with time points taken at 0 hours and 5 hours. The samples were analyzed by UPLC-MS (Waters, Milford, MA) with compounds detected by SIR detection on a single quadrupole mass spectrometer.</p></div></div><div id="ml126.s18"><h2 id="_ml126_s18_">3. Results</h2><div id="ml126.s19"><h3>Probe Attributes</h3><ul><li class="half_rhythm"><div>IC<sub>50</sub> ≤ 10 μM</div></li><li class="half_rhythm"><div>30- to 100-fold selectivity as defined by: IC<sub>50</sub> ratios of the inhibition of SK expression and the inhibition of cell growth where both IC<sub>50</sub> ratios are measurable.</div></li><li class="half_rhythm"><div>Compounds that are active in SK expression with IC<sub>50</sub> ≤ 10 μM but don’t result in cell growth inhibition at test concentrations no less than 50 μM.</div></li></ul><p>The project included a primary high-throughput screen of the entire MLSMR collection (greater than 300,000 substances). Approximately 1% of the compounds tested were selected for dose retest and selectivity. Of these<b>,</b> approximately 60 compounds were identified as having the desired potency and selectivity. Resupply of these compounds was ordered from commercial sources for subsequent verification. In addition to these commercially available compounds, multiple rounds of chemistry were performed to generate new compounds for determination of the SAR of on the scaffolds. Compounds with the best combination of potency and selectivity and chemical properties were designated as the probes. This report concerns one structural series: Probe 1 (CID-576989/<a href="/pcsubstance/?term=ML126[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML126</a>).</p></div><div id="ml126.s20"><h3>3.1. Summary of Screening Results</h3><p><a class="figpopup" href="/books/NBK56228/figure/ml126.f3/?report=objectonly" target="object" rid-figpopup="figml126f3" rid-ob="figobml126f3">Figure 3</a> displays the critical path for probe development.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml126f3" co-legend-rid="figlgndml126f3"><a href="/books/NBK56228/figure/ml126.f3/?report=objectonly" target="object" title="Figure 3" class="img_link icnblk_img figpopup" rid-figpopup="figml126f3" rid-ob="figobml126f3"><img class="small-thumb" src="/books/NBK56228/bin/ml126f3.gif" src-large="/books/NBK56228/bin/ml126f3.jpg" alt="Figure 3. Critical Path for Probe Development." /></a><div class="icnblk_cntnt" id="figlgndml126f3"><h4 id="ml126.f3"><a href="/books/NBK56228/figure/ml126.f3/?report=objectonly" target="object" rid-ob="figobml126f3">Figure 3</a></h4><p class="float-caption no_bottom_margin">Critical Path for Probe Development. </p></div></div><p>A high-throughput screen of 303,546 compounds (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1662" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1662</a>) was performed in duplicate in the engineered strain SKKanGAS, in which the kanamycin resistance gene was placed under control of the SK promoter to identify compounds that can inhibit kanamycin resistance. Using a cutoff of 50% inhibition at a screening concentration of 7.5 μM average, 3962 hits were identified as SK expression inhibitors; among these, 3268 were available as cherry picks. These picked compounds were retested in dose in the SKKanGAs strain to confirm their inhibitory activity as well as in SK(-)GAS strain where the kanamycin gene is driven by an SK-independent promoter to investigate promoter dependence.</p><p>The next set of secondary tests provided a more physiologically relevant analysis of the compounds specificity and potency. Interference with SK expression was measured in the non-engineered UMAA 2616 strain by measuring SK activity in culture supernatant via activation of plasminogen enzymatic activity (AID nos. <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1914" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1914</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2137" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2137</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504351" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">504351</a>). Bacterial viability was also measured via ATP content from the assay sample (AID nos. <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1915" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1915</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2138" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2138</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504396" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">504396</a>). From this secondary panel, 95 compounds were prioritized, and 61 dry powders were obtained and tested multiple times in the secondary assays. From this set of dry powder compounds, a carbazole series was prioritized, and additional analogs were synthesized and tested in the secondary assays. This carbazole series became the probe 1 series with a group of 24 compounds (AID nos. <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2137" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2137</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2138" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2138</a>; <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504351" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">504351</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504396" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">504396</a>) (see <a class="figpopup" href="/books/NBK56228/table/ml126.t1/?report=objectonly" target="object" rid-figpopup="figml126t1" rid-ob="figobml126t1">Table 1</a>, <a href="#ml126.s23">Section 3.4</a>).</p></div><div id="ml126.s21"><h3>3.2. Dose Response Curves for Probe</h3><p>The probe (CID-576989/<a href="/pcsubstance/?term=ML126[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML126</a>) met the defined probe criteria and was not active in the bacterial cell viability assay. Approximately 100% inhibition of endogenous streptokinase in the non-engineered strain UMAA2616 was obtained. <a class="figpopup" href="/books/NBK56228/figure/ml126.f4/?report=objectonly" target="object" rid-figpopup="figml126f4" rid-ob="figobml126f4">Figure 4</a> displays the dose response curves for the probe (CID-576989/<a href="/pcsubstance/?term=ML126[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML126</a>).</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml126f4" co-legend-rid="figlgndml126f4"><a href="/books/NBK56228/figure/ml126.f4/?report=objectonly" target="object" title="Figure 4" class="img_link icnblk_img figpopup" rid-figpopup="figml126f4" rid-ob="figobml126f4"><img class="small-thumb" src="/books/NBK56228/bin/ml126f4.gif" src-large="/books/NBK56228/bin/ml126f4.jpg" alt="Figure 4. Concentration-dependent Activities (%) of Probe (CID-576989/ML126) in the Target Assay and Counterscreen Assays." /></a><div class="icnblk_cntnt" id="figlgndml126f4"><h4 id="ml126.f4"><a href="/books/NBK56228/figure/ml126.f4/?report=objectonly" target="object" rid-ob="figobml126f4">Figure 4</a></h4><p class="float-caption no_bottom_margin">Concentration-dependent Activities (%) of Probe (CID-576989/ML126) in the Target Assay and Counterscreen Assays. Probe CID-576989/ML126 concentration-dependent activities: SK expression (IC<sub>50</sub>=3.6 μM; AID-2137) (<i>A)</i>; Bacterial viability (inactive; <a href="/books/NBK56228/figure/ml126.f4/?report=objectonly" target="object" rid-ob="figobml126f4">(more...)</a></p></div></div></div><div id="ml126.s22"><h3>3.3. Scaffold/Moiety Chemical Liabilities</h3><p>The probe (CID-576989/<a href="/pcsubstance/?term=ML126[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML126</a>) does not have obvious chemical liabilities. It is stable over 48 hours in PBS buffer and shows moderate stability in human plasma. A PubChem activity survey performed on February 24, 2011 revealed only one confirmatory nonstreptokinase assay in which the probe (CID-576989/<a href="/pcsubstance/?term=ML126[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML126</a>) was active (out of 429 reported in PubChem). A structure-based search in SciFinder and Reaxys did not identify any publications or patents in which the probe compound appeared. Therefore, the probe is considered not to be a promiscuous binder.</p></div><div id="ml126.s23"><h3>3.4. SAR Tables</h3><p>The initial hit from the HTS campaign was (9<i>H</i>-carbazol-9-yl)(<i>p</i>-tolyl)methanone (entry 1, CID-576989, <a class="figpopup" href="/books/NBK56228/table/ml126.t1/?report=objectonly" target="object" rid-figpopup="figml126t1" rid-ob="figobml126t1">Table 1</a>). A collection of 23 structurally related analogs were synthesized and evaluated for their ability to inhibit SK expression in the primary assay and array of secondary assays. Three diversification points selected for chemical modification are depicted in <a class="figpopup" href="/books/NBK56228/figure/ml126.f5/?report=objectonly" target="object" rid-figpopup="figml126f5" rid-ob="figobml126f5">Figure 5</a>. The biological assay data and physical properties of the HTS hit and the analogs are presented below in <a class="figpopup" href="/books/NBK56228/table/ml126.t1/?report=objectonly" target="object" rid-figpopup="figml126t1" rid-ob="figobml126t1">Table 1</a>, <a class="figpopup" href="/books/NBK56228/table/ml126.t2/?report=objectonly" target="object" rid-figpopup="figml126t2" rid-ob="figobml126t2">Table 2</a>, and <a class="figpopup" href="/books/NBK56228/table/ml126.t3/?report=objectonly" target="object" rid-figpopup="figml126t3" rid-ob="figobml126t3">Table 3</a>.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml126f5" co-legend-rid="figlgndml126f5"><a href="/books/NBK56228/figure/ml126.f5/?report=objectonly" target="object" title="Figure 5" class="img_link icnblk_img figpopup" rid-figpopup="figml126f5" rid-ob="figobml126f5"><img class="small-thumb" src="/books/NBK56228/bin/ml126f5.gif" src-large="/books/NBK56228/bin/ml126f5.jpg" alt="Figure 5. Summary of SAR Performed on the Carbazole Scaffold." /></a><div class="icnblk_cntnt" id="figlgndml126f5"><h4 id="ml126.f5"><a href="/books/NBK56228/figure/ml126.f5/?report=objectonly" target="object" rid-ob="figobml126f5">Figure 5</a></h4><p class="float-caption no_bottom_margin">Summary of SAR Performed on the Carbazole Scaffold. Three diversity points (highlighted in red, blue, and green) were explored and the number of analogs screened for each site is specified. </p></div></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml126t2"><a href="/books/NBK56228/table/ml126.t2/?report=objectonly" target="object" title="Table 2" class="img_link icnblk_img figpopup" rid-figpopup="figml126t2" rid-ob="figobml126t2"><img class="small-thumb" src="/books/NBK56228/table/ml126.t2/?report=thumb" src-large="/books/NBK56228/table/ml126.t2/?report=previmg" alt="Table 2. SAR Analysis and Properties of Various Amide Side Chain Analogs." /></a><div class="icnblk_cntnt"><h4 id="ml126.t2"><a href="/books/NBK56228/table/ml126.t2/?report=objectonly" target="object" rid-ob="figobml126t2">Table 2</a></h4><p class="float-caption no_bottom_margin">SAR Analysis and Properties of Various Amide Side Chain Analogs. </p></div></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml126t3"><a href="/books/NBK56228/table/ml126.t3/?report=objectonly" target="object" title="Table 3" class="img_link icnblk_img figpopup" rid-figpopup="figml126t3" rid-ob="figobml126t3"><img class="small-thumb" src="/books/NBK56228/table/ml126.t3/?report=thumb" src-large="/books/NBK56228/table/ml126.t3/?report=previmg" alt="Table 3. SAR Analysis and Properties of Various Carbazole Replacement Analogs." /></a><div class="icnblk_cntnt"><h4 id="ml126.t3"><a href="/books/NBK56228/table/ml126.t3/?report=objectonly" target="object" rid-ob="figobml126t3">Table 3</a></h4><p class="float-caption no_bottom_margin">SAR Analysis and Properties of Various Carbazole Replacement Analogs. </p></div></div><p><a class="figpopup" href="/books/NBK56228/table/ml126.t1/?report=objectonly" target="object" rid-figpopup="figml126t1" rid-ob="figobml126t1">Table 1</a> presents the initial hit and several derivatives of the initial hit wherein the phenyl ring of the amide side chain of the carbazole scaffold was substituted with various electron-withdrawing and electron-donating groups on the <i>ortho-</i>, <i>meta-</i>, and <i>para-</i> positions to probe the SAR of this region. Presence of the electron-donating group at the <i>para-</i> position retains the potency (entries 1–3, <a class="figpopup" href="/books/NBK56228/table/ml126.t1/?report=objectonly" target="object" rid-figpopup="figml126t1" rid-ob="figobml126t1">Table 1</a>), whereas the potency is adversely affected when substituted at the <i>ortho</i>-position (entry 12, <a class="figpopup" href="/books/NBK56228/table/ml126.t1/?report=objectonly" target="object" rid-figpopup="figml126t1" rid-ob="figobml126t1">Table 1</a>). In contrast, electron-withdrawing groups at the <i>ortho</i>- position retain the activity (entries 4, 6, 9, <a class="figpopup" href="/books/NBK56228/table/ml126.t1/?report=objectonly" target="object" rid-figpopup="figml126t1" rid-ob="figobml126t1">Table 1</a>).</p><p>Replacement of the phenyl ring in the amide side chain of the carbazole scaffold with heteroaryl groups resulted in significant loss of potency (entries1–2, <a class="figpopup" href="/books/NBK56228/table/ml126.t2/?report=objectonly" target="object" rid-figpopup="figml126t2" rid-ob="figobml126t2">Table 2</a>). In contrast, substitution of the p-toluoyl group with p-methylaniline moiety led to an ineffective analog suggesting the amide moiety is preferred over the urea functionality (entry 3, <a class="figpopup" href="/books/NBK56228/table/ml126.t2/?report=objectonly" target="object" rid-figpopup="figml126t2" rid-ob="figobml126t2">Table 2</a>).</p><p>When the carbazole moiety was replaced with other tricyclic heteroaromatic ring (entry 5, <a class="figpopup" href="/books/NBK56228/table/ml126.t3/?report=objectonly" target="object" rid-figpopup="figml126t3" rid-ob="figobml126t3">Table 3</a>) or bicyclic heteraromatic ring systems (entries 1 and 3, <a class="figpopup" href="/books/NBK56228/table/ml126.t3/?report=objectonly" target="object" rid-figpopup="figml126t3" rid-ob="figobml126t3">Table 3</a>), decrease in activity was observed. A similar result was observed when various positions of the carbazole ring were substituted with heteroatom (entries 2 and 4, <a class="figpopup" href="/books/NBK56228/table/ml126.t3/?report=objectonly" target="object" rid-figpopup="figml126t3" rid-ob="figobml126t3">Table 3</a>) and electron-donating functionality (entry 6, <a class="figpopup" href="/books/NBK56228/table/ml126.t3/?report=objectonly" target="object" rid-figpopup="figml126t3" rid-ob="figobml126t3">Table 3</a>).</p><p>In conclusion, several analogs of the original hit were synthesized and assayed to better understand the structure-activity relationships. It is important to note that several structurally related analogs have similar activities; however, the previously reported probe (entry 1, <a class="figpopup" href="/books/NBK56228/table/ml126.t1/?report=objectonly" target="object" rid-figpopup="figml126t1" rid-ob="figobml126t1">Table 1</a>; CID-576989/<a href="/pcsubstance/?term=ML126[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML126</a>), 9<i>H</i>-carbazol-9-yl)(<i>p</i>-tolyl)methanone, for the carbazole series remains as one of the most active analogs and will retain its’ status as the declared probe.</p></div><div id="ml126.s24"><h3>3.5. Cellular Activity</h3><p>The SK expression assay and the bacterial cell viability assay are bacterial cell-based phenotypic assays. Therefore, it is inferred that active compounds possess reasonable permeability across the bacterial membrane. No mammalian cell permeability was measured.</p></div><div id="ml126.s25"><h3>3.6. Profiling Assays</h3><p>A PubChem activity survey performed on February 24, 2011 using Broad’s internal algorithm revealed only one confirmatory nonstreptokinase assay (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1665" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1665</a>) in which the probe (CID-576989/<a href="/pcsubstance/?term=ML126[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML126</a>) was active (out of 429 reported in PubChem). Other profiling screens (e.g., CEREP) have not been performed with this compound to date.</p></div></div><div id="ml126.s26"><h2 id="_ml126_s26_">4. Discussion</h2><div id="ml126.s27"><h3>4.1. Comparison to Existing Art and How the New Probe is an Improvement</h3><p>The carbazole probe (CID-576989/<a href="/pcsubstance/?term=ML126[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML126</a>) has demonstrated selective inhibition of SK expression in a GAS strain without any effect on bacterial growth. The probe fulfilled all the probe criteria (<a class="figpopup" href="/books/NBK56228/table/ml126.t4/?report=objectonly" target="object" rid-figpopup="figml126t4" rid-ob="figobml126t4">Table 4</a>) and will serve quite well in further cell-based investigations concerning SK expression. It is interesting to see from <a class="figpopup" href="/books/NBK56228/table/ml126.t1/?report=objectonly" target="object" rid-figpopup="figml126t1" rid-ob="figobml126t1">Tables 1</a>–<a class="figpopup" href="/books/NBK56228/table/ml126.t3/?report=objectonly" target="object" rid-figpopup="figml126t3" rid-ob="figobml126t3">3</a> how potency is affected by the various benzoyl substituents and modification of the tricyclic ring. The range of potencies provides some confidence that the carbazole analogs are binding to one target specifically (i.e., a target that must control SK gene expression). The probe (CID-576989/<a href="/pcsubstance/?term=ML126[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML126</a>) and the structural analogs shown are fairly hydrophobic molecules, possibly reflecting the hydrophobic character of the site to which they bind.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml126t4"><a href="/books/NBK56228/table/ml126.t4/?report=objectonly" target="object" title="Table 4" class="img_link icnblk_img figpopup" rid-figpopup="figml126t4" rid-ob="figobml126t4"><img class="small-thumb" src="/books/NBK56228/table/ml126.t4/?report=thumb" src-large="/books/NBK56228/table/ml126.t4/?report=previmg" alt="Table 4. Probe Criteria." /></a><div class="icnblk_cntnt"><h4 id="ml126.t4"><a href="/books/NBK56228/table/ml126.t4/?report=objectonly" target="object" rid-ob="figobml126t4">Table 4</a></h4><p class="float-caption no_bottom_margin">Probe Criteria. </p></div></div><p>To support the novelty of the probe and its phenotypic effects, an investigation into the relevant prior art was performed by searching the following databases: SciFinder, Reaxys, PubChem, PubMed, US Patent and Trademark Office (USPTO), PatFT, AppFT, and World Intellectual Property Organization (WIPO). Specifically, we were looking for reports of chemical matters that demonstrated inhibition of streptokinase expression. The search terms applied and hit statistics are provided in <a class="figpopup" href="/books/NBK56228/table/ml126.t5/?report=objectonly" target="object" rid-figpopup="figml126t5" rid-ob="figobml126t5">Table 5</a>. Abstracts were obtained for all references returned including journal articles, patents, and other form of public disclosures and were analyzed for relevance to the current project. The searches were performed on, and are current as of, January 28, 2011.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml126t5"><a href="/books/NBK56228/table/ml126.t5/?report=objectonly" target="object" title="Table 5" class="img_link icnblk_img figpopup" rid-figpopup="figml126t5" rid-ob="figobml126t5"><img class="small-thumb" src="/books/NBK56228/table/ml126.t5/?report=thumb" src-large="/books/NBK56228/table/ml126.t5/?report=previmg" alt="Table 5. Search Strings and Databases Employed in the Prior Art Search." /></a><div class="icnblk_cntnt"><h4 id="ml126.t5"><a href="/books/NBK56228/table/ml126.t5/?report=objectonly" target="object" rid-ob="figobml126t5">Table 5</a></h4><p class="float-caption no_bottom_margin">Search Strings and Databases Employed in the Prior Art Search. </p></div></div><p>The literature and patent searches in <a class="figpopup" href="/books/NBK56228/table/ml126.t5/?report=objectonly" target="object" rid-figpopup="figml126t5" rid-ob="figobml126t5">Table 5</a> uncovered no known small molecule that is capable of inhibiting SK expression. The only compound (CID-657963) that is known to inhibit SK inhibition is shown in <a class="figpopup" href="/books/NBK56228/figure/ml126.f6/?report=objectonly" target="object" rid-figpopup="figml126f6" rid-ob="figobml126f6">Figure 6</a>. This compound was identified by the Assay Provider, Dr. Hongmin Sun, for this probe development project (Note: Initial results associated with the discovery of CID-657963 are not yet published). The moderate potency (≥ 10.0 μM) and the presence of a reactive alkylthioacetonitrile moiety in CID-657963 have motivated us to discover a superior scaffold with higher inhibitory potency from the MLPCN compound collection. The improved potency and no obvious chemical liabilities will make carbazole probe (CID-576989/<a href="/pcsubstance/?term=ML126[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML126</a>) a very useful tool molecule to better understand the biology of streptokinase expression in group A streptococci.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml126f6" co-legend-rid="figlgndml126f6"><a href="/books/NBK56228/figure/ml126.f6/?report=objectonly" target="object" title="Figure 6" class="img_link icnblk_img figpopup" rid-figpopup="figml126f6" rid-ob="figobml126f6"><img class="small-thumb" src="/books/NBK56228/bin/ml126f6.gif" src-large="/books/NBK56228/bin/ml126f6.jpg" alt="Figure 6. Compound (CID-657963) Inhibitor of SK Expression." /></a><div class="icnblk_cntnt" id="figlgndml126f6"><h4 id="ml126.f6"><a href="/books/NBK56228/figure/ml126.f6/?report=objectonly" target="object" rid-ob="figobml126f6">Figure 6</a></h4><p class="float-caption no_bottom_margin">Compound (CID-657963) Inhibitor of SK Expression. </p></div></div></div><div id="ml126.s28"><h3>4.2. Mechanism of Action Studies</h3><p>The experimental scope of this project did not include target identification due to the complexity of and considerably much more resource commitment to such tasks. Researchers can carry out mechanism of action (MOA) studies through the following approaches. First, determine if the decreased level of SK expression is due to transcriptional regulation. Second, if transcription is a control mechanism, quantitative gene expression measurement can be used to test the activities of known pathways and/or expression level of known pathway components that control SK expression. Third, based on the knowledge gained from the above experiments, one can generate specific hypotheses of possible protein targets and test activity of these targets biochemically. Alternatively, researchers can employ several affinity-based techniques using the probe (CID-576989/<a href="/pcsubstance/?term=ML126[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML126</a>), or its’ active but modified version, as “bait’ to fish out proteins that specifically interact with the probe (CID-576989/<a href="/pcsubstance/?term=ML126[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML126</a>). Inactive structurally similar analogs provided in this report are excellent control compounds for this purpose. The biological insight gained from the initial MOA studies should help narrow down the likelihood of the real targets if multiple specific binding proteins are identified.</p></div><div id="ml126.s29"><h3>4.3. Planned Future Studies</h3><p>In addition to the MOA studies proposed in <a href="#ml126.s28">Section 4.2</a>, researchers can further evaluate the probe (CID-576989/<a href="/pcsubstance/?term=ML126[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML126</a>) or activity of its’ active analogs in animal models if the pharmacokinetic parameters are favorable. An animal model successfully used in the Assay Provider’s laboratory to evaluate compound activity is provided in Sun et al. (<a class="bk_pop" href="#ml126.r2">2</a>).</p></div></div><div id="ml126.s30"><h2 id="_ml126_s30_">5. References</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="ml126.r1">Sun H, Ringdahl U, Homeister JW, Fay WP, Engleberg NC, Yang AY, Rozek LS, Wang X, Sjöbring U, Ginsburg D. Plasminogen is a critical host pathogenicity factor for group A streptococcal infection. <span><span class="ref-journal">Science. </span>2004 Aug 27;<span class="ref-vol">305</span>(5688:):1283–1286.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/15333838" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 15333838</span></a>]</div></dd><dt>2.</dt><dd><div class="bk_ref" id="ml126.r2">Christner R, Li Z, Raeder R, Podbielski A, Boyle MD. Identification of key gene products required for acquisition of plasmin-like enzymatic activity by group A streptococci. <span><span class="ref-journal">J Infect Dis. </span>1997 May;<span class="ref-vol">175</span>(5:):1115–1120.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/9129074" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 9129074</span></a>]</div></dd><dt>3.</dt><dd><div class="bk_ref" id="ml126.r3">Sun H, Wang X, Degen JL, Ginsburg D. Reduced thrombin generation increases host susceptibility to group A streptococcal infection. <span><span class="ref-journal">Blood. </span>2009 Feb 5;<span class="ref-vol">113</span>(6:):1358–1364.</span> Epub 2008 Dec 3. [<a href="/pmc/articles/PMC2637198/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2637198</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/19056689" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19056689</span></a>]</div></dd><dt>4.</dt><dd><div class="bk_ref" id="ml126.r4">Escaich S. Antivirulence as a new antibacterial approach for chemotherapy. <span><span class="ref-journal">Curr Opin Chem Biol. </span>2008 Aug;<span class="ref-vol">12</span>(4:):400–408.</span> Epub 2008 Jul 17. Review. [<a href="https://pubmed.ncbi.nlm.nih.gov/18639647" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18639647</span></a>]</div></dd><dt>5.</dt><dd><div class="bk_ref" id="ml126.r5">Keyser P, Elofsson M, Rosell S, Wolf-Watz H. Virulence blockers as alternatives to antibiotics: type III secretion inhibitors against Gram-negative bacteria. <span><span class="ref-journal">J Intern Med. </span>2008 Jul;<span class="ref-vol">264</span>(1:):17–29.</span> Epub 2008 Apr 3. [<a href="https://pubmed.ncbi.nlm.nih.gov/18393958" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18393958</span></a>]</div></dd><dt>6.</dt><dd><div class="bk_ref" id="ml126.r6">Oxoby M, Moreau F, Durant L, Denis A, Genevard JM, Vongsouthi V, Escaich S, Gerusz V. Towards gram-positive antivirulence drugs: new inhibitors of Streptococcus agalactiae Stk1. <span><span class="ref-journal">Bioorg Med Chem Lett. </span>2010 Jun 15;<span class="ref-vol">20</span>(12:):3486–3490.</span> [<a href="/pmc/articles/PMC2923816/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2923816</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/20529681" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 20529681</span></a>]</div></dd><dt>7.</dt><dd><div class="bk_ref" id="ml126.r7">Aberg V, Almqvist F. Pilicides-small molecules targeting bacterial virulence. <span><span class="ref-journal">Org Biomol Chem. </span>2007 Jun 21;<span class="ref-vol">5</span>(12:):1827–1834.</span> Epub 2007 Apr 20. [<a href="https://pubmed.ncbi.nlm.nih.gov/17551629" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17551629</span></a>]</div></dd><dt>8.</dt><dd><div class="bk_ref" id="ml126.r8">Engleberg NC, Heath A, Miller A, Rivera C, DiRita VJ. Spontaneous mutations in the CsrRS two-component regulatory system of Streptococcus pyogenes result in enhanced virulence in a murine model of skin and soft tissue infection. <span><span class="ref-journal">J Infect Dis. </span>2001 Apr 1;<span class="ref-vol">183</span>(7:):1043–1054.</span> Epub 2001 Mar 1. [<a href="https://pubmed.ncbi.nlm.nih.gov/11237829" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 11237829</span></a>]</div></dd><dt>9.</dt><dd><div class="bk_ref" id="ml126.r9">Bisno AL, Stevens DL. Streptococcal infections of skin and soft tissues. <span><span class="ref-journal">N Engl J Med. </span>1996 Jan 25;<span class="ref-vol">334</span>(4:):240–245.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/8532002" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 8532002</span></a>]</div></dd></dl></div><div id="ml126.app1"><h2 id="_ml126_app1_">Appendix A. Assay Summary Table</h2><div id="ml126.t6" class="table"><h3><span class="label">Table A1</span><span class="title">Summary of Completed Assays and AIDs</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK56228/table/ml126.t6/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml126.t6_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml126.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">PubChem AID</th><th id="hd_h_ml126.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Type</th><th id="hd_h_ml126.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Target</th><th id="hd_h_ml126.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Concentration Range (μM)</th><th id="hd_h_ml126.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Samples Tested</th></tr></thead><tbody><tr><td headers="hd_h_ml126.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1662" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1662</a></td><td headers="hd_h_ml126.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Primary</td><td headers="hd_h_ml126.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Growth inhibition, Ska promoter</td><td headers="hd_h_ml126.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Average 7.5</td><td headers="hd_h_ml126.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">303,546</td></tr><tr><td headers="hd_h_ml126.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1900" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1900</a></td><td headers="hd_h_ml126.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Counterscreen</td><td headers="hd_h_ml126.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Non-Ska promoter</td><td headers="hd_h_ml126.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">15-0.06</td><td headers="hd_h_ml126.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">3,268</td></tr><tr><td headers="hd_h_ml126.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1902" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1902</a></td><td headers="hd_h_ml126.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">DR in Primary</td><td headers="hd_h_ml126.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Ska promoter</td><td headers="hd_h_ml126.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">15-0.06</td><td headers="hd_h_ml126.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">3,268</td></tr><tr><td headers="hd_h_ml126.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1914" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1914</a></td><td headers="hd_h_ml126.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Secondary</td><td headers="hd_h_ml126.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">SK expression</td><td headers="hd_h_ml126.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">15-0.06</td><td headers="hd_h_ml126.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">3,268</td></tr><tr><td headers="hd_h_ml126.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1915" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1915</a></td><td headers="hd_h_ml126.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Secondary</td><td headers="hd_h_ml126.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Bacterial Viability</td><td headers="hd_h_ml126.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">15-0.06</td><td headers="hd_h_ml126.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">3,268</td></tr><tr><td headers="hd_h_ml126.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2137" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2137</a></td><td headers="hd_h_ml126.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Secondary</td><td headers="hd_h_ml126.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">SK expression</td><td headers="hd_h_ml126.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">50-0.02</td><td headers="hd_h_ml126.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">61</td></tr><tr><td headers="hd_h_ml126.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2138" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2138</a></td><td headers="hd_h_ml126.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Secondary</td><td headers="hd_h_ml126.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Bacterial Viability</td><td headers="hd_h_ml126.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">50-0.02</td><td headers="hd_h_ml126.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">61</td></tr><tr><td headers="hd_h_ml126.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504351" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">504351</a></td><td headers="hd_h_ml126.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Secondary</td><td headers="hd_h_ml126.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">SK expression</td><td headers="hd_h_ml126.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">50-0.01</td><td headers="hd_h_ml126.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">57</td></tr><tr><td headers="hd_h_ml126.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504396" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">504396</a></td><td headers="hd_h_ml126.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Secondary</td><td headers="hd_h_ml126.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Bacterial Viability</td><td headers="hd_h_ml126.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">50-0.01</td><td headers="hd_h_ml126.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">57</td></tr><tr><td headers="hd_h_ml126.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1677" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1677</a></td><td headers="hd_h_ml126.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Summary</td><td headers="hd_h_ml126.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NA</td><td headers="hd_h_ml126.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NA</td><td headers="hd_h_ml126.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NA</td></tr></tbody></table></div></div></div><div id="ml126.app2"><h2 id="_ml126_app2_">Appendix B. Detailed Assay Protocols</h2><div id="ml126.s31"><h3>Primary Screen Protocol in SKKanGAS Strain (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1662" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1662</a>)</h3><ol><li class="half_rhythm"><div>Day 1: Streak out SKKanGAS for colonies on THY/S/S medium (30 g/L THB/0.2% yeast extract with streptomycin 100 μg/ml, spectinomycin 100 μg/ml) plate. Incubate at 37°C overnight.</div></li><li class="half_rhythm"><div>Day 2: Grow an overnight culture in THY/S/S medium from a single colony. Incubate at 37°C overnight.</div></li><li class="half_rhythm"><div>Day 3: Measure OD600 of the overnight culture, and then dilute the culture into fresh THY/S/S medium (1:20) in a flask. Monitor OD600 until it reaches 0.6–0.8 (between 3–5 hours).</div></li><li class="half_rhythm"><div>Dispense assay plates at 30 μl/well of THY/kanamycin (THY plus 40 μg/ml kanamycin) with Combi. Load plates into incubators.</div></li><li class="half_rhythm"><div>Dilute the 0.8 OD culture down to OD 0.038 in cold THY/kanamycin; keep the diluted culture at 4°C overnight.</div></li><li class="half_rhythm"><div>Day 4: Warm the diluted culture to room temperature. Dispense 20 μl/well of diluted culture with a Combi onto the pinned assay plates containing 30 μl/well of THY/S medium.</div></li><li class="half_rhythm"><div>Pin approximately 100 nL/well of compounds with Cybi-Well, and incubate the plates at 37°C for 6 hours.</div></li><li class="half_rhythm"><div>Cool plates to room temperature for 30 minutes.</div></li><li class="half_rhythm"><div>Add 30 μl/well solution (25 μl/well BacTiter-Glo and 5 μl/well PBS) to the culture, and incubate at room temperature for 10 minutes.</div></li><li class="half_rhythm"><div>Read on an EnVision plate reader.</div></li></ol></div><div id="ml126.s32"><h3>Secondary Assay Protocols: SK Expression (AID nos. <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1914" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1914</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2137" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2137</a>) and Bacterial Cell Viability (AID nos. <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1915" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1915</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2138" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2138</a>)</h3><ol><li class="half_rhythm"><div>Day 1: Streak out UMAA 2616 for colonies on THY/S medium (30 g/L THB/0.2% yeast extract with streptomycin 100 μg/mL) plate. Incubate at 37°C overnight.</div></li><li class="half_rhythm"><div>Day 2: Grow an overnight culture in THY/S medium from a single colony. Incubate at 37°C overnight.</div></li><li class="half_rhythm"><div>Day 3: Dilute the overnight culture into fresh THY/S medium (1:20) in a flask. Monitor OD600 until it reaches 0.6–0.8 (between 3–5 hours).</div></li><li class="half_rhythm"><div>Dilute the 0.8 OD culture down to OD 0.038 in cold THY/S medium; keep the diluted culture at 4 °C overnight.</div></li><li class="half_rhythm"><div>Day 4: Warm the diluted culture to room temperature. Dispense 20 μl/well of diluted culture with a Combi onto the pinned assay plates containing 30 μl/well of THY/S medium.</div></li><li class="half_rhythm"><div>Pin 100 nL/well of compounds with Cybi-Well, and incubate the plates at 37°C for 6 hours in a Liconic incubator.</div></li><li class="half_rhythm"><div>Pellet the cells at 3000 rpm in a Sorvall centrifuge. Transfer 10 μl/well of supernatant twice into separate, clear plates for assay with Cybi-Well Vario. Store at −20°C.</div></li><li class="half_rhythm"><div>Warm the culture plates to room temperature for 30 minutes, then add 50 μl/well of BacTiter-Glo (1/2x).</div></li><li class="half_rhythm"><div>Incubate at room temperature for 10 minutes. Read on an EnVision plate reader.</div></li><li class="half_rhythm"><div>Add 50 μl/well of SK assay solution (5 μl/well of human plasma, 1.2 μl/well of 4.2 mg/ml S2403, 43.8 μl/well of PBS) to the 10 μl/well supernatant (thawed and warmed to room temperature) in clear SK assay plates with a Combi.</div></li><li class="half_rhythm"><div>Read OD405 at 0 hours and after incubation at 37°C for 1.5–2.5 hours depending on the specific lot of substrate.</div></li></ol></div></div><div id="ml126.app3"><h2 id="_ml126_app3_">Appendix C. NMR and LC Data of Probe and Analogs</h2><div id="ml126.fu2" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56228/bin/ml126fu14.jpg" alt="1H NMR (300 MHz, CDCl3) Spectrum of Probe CID-576989/ML126." /></div><h3><span class="title"><sup>1</sup>H NMR (300 MHz, CDCl3) Spectrum of Probe CID-576989/<a href="/pcsubstance/?term=ML126[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML126</a></span></h3></div><div id="ml126.fu3" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56228/bin/ml126fu15.jpg" alt="13 C NMR (75 MHz, CDCl3) Spectrum of Probe CID-576989/ML126." /></div><h3><span class="title"><sup>13</sup> C NMR (75 MHz, CDCl3) Spectrum of Probe CID-576989/<a href="/pcsubstance/?term=ML126[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML126</a></span></h3></div><div id="ml126.fu4" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56228/bin/ml126fu16.jpg" alt="UPLC Chromatogram of Probe CID-576989/ML126." /></div><h3><span class="title">UPLC Chromatogram of Probe CID-576989/<a href="/pcsubstance/?term=ML126[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML126</a></span></h3></div><div id="ml126.s36"><h3>Spectroscopic Data for SAR Analogs</h3><div id="ml126.fu5" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56228/bin/ml126fu17.jpg" alt="1H NMR (300 MHz, CDCl3) Spectrum of Analog CID-13900925." /></div><h3><span class="title"><sup>1</sup>H NMR (300 MHz, CDCl3) Spectrum of Analog CID-13900925</span></h3></div><div id="ml126.fu6" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56228/bin/ml126fu18.jpg" alt="UPLC Chromatogram of Analog CID-13900925." /></div><h3><span class="title">UPLC Chromatogram of Analog CID-13900925</span></h3></div><div id="ml126.fu7" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56228/bin/ml126fu19.jpg" alt="1H NMR (300 MHz, CDCl3) Spectrum of Analog CID-44247470." /></div><h3><span class="title"><sup>1</sup>H NMR (300 MHz, CDCl3) Spectrum of Analog CID-44247470</span></h3></div><div id="ml126.fu8" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56228/bin/ml126fu20.jpg" alt="UPLC Chromatogram of Analog CID-44247470." /></div><h3><span class="title">UPLC Chromatogram of Analog CID-44247470</span></h3></div><div id="ml126.fu9" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56228/bin/ml126fu21.jpg" alt="1H NMR (300 MHz, CDCl3) Spectrum of Analog CID-49843039." /></div><h3><span class="title"><sup>1</sup>H NMR (300 MHz, CDCl3) Spectrum of Analog CID-49843039</span></h3></div><div id="ml126.fu10" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56228/bin/ml126fu22.jpg" alt="UPLC Chromatogram of Analog CID-49843039." /></div><h3><span class="title">UPLC Chromatogram of Analog CID-49843039</span></h3></div><div id="ml126.fu11" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56228/bin/ml126fu23.jpg" alt="1H NMR (300 MHz, CDCl3) Spectrum of Analog CID-23935237." /></div><h3><span class="title"><sup>1</sup>H NMR (300 MHz, CDCl3) Spectrum of Analog CID-23935237</span></h3></div><div id="ml126.fu12" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56228/bin/ml126fu24.jpg" alt="UPLC Chromatogram of Analog CID-23935237." /></div><h3><span class="title">UPLC Chromatogram of Analog CID-23935237</span></h3></div><div id="ml126.fu13" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56228/bin/ml126fu25.jpg" alt="1H NMR (300 MHz, CDCl3) Spectrum of Analog CID-13900924." /></div><h3><span class="title"><sup>1</sup>H NMR (300 MHz, CDCl3) Spectrum of Analog CID-13900924</span></h3></div><div id="ml126.fu14" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56228/bin/ml126fu26.jpg" alt="UPLC Chromatogram of Analog CID-13900924." /></div><h3><span class="title">UPLC Chromatogram of Analog CID-13900924</span></h3></div><div id="ml126.fu15" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56228/bin/ml126fu27.jpg" alt="1H NMR (300 MHz, CDCl3) Spectrum of Analog CID-8852543." /></div><h3><span class="title"><sup>1</sup>H NMR (300 MHz, CDCl3) Spectrum of Analog CID-8852543</span></h3></div><div id="ml126.fu16" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56228/bin/ml126fu28.jpg" alt="UPLC Chromatogram of Analog CID-8852543." /></div><h3><span class="title">UPLC Chromatogram of Analog CID-8852543</span></h3></div><div id="ml126.fu17" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56228/bin/ml126fu29.jpg" alt="1H NMR (300 MHz, CDCl3) Spectrum of Analog CID-44247468." /></div><h3><span class="title"><sup>1</sup>H NMR (300 MHz, CDCl3) Spectrum of Analog CID-44247468</span></h3></div><div id="ml126.fu18" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56228/bin/ml126fu30.jpg" alt="UPLC Chromatogram of Analog CID-44247468." /></div><h3><span class="title">UPLC Chromatogram of Analog CID-44247468</span></h3></div><div id="ml126.fu19" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56228/bin/ml126fu31.jpg" alt="1H NMR (300 MHz, CDCl3) Spectrum of Analog CID-44247472." /></div><h3><span class="title"><sup>1</sup>H NMR (300 MHz, CDCl3) Spectrum of Analog CID-44247472</span></h3></div><div id="ml126.fu20" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56228/bin/ml126fu32.jpg" alt="UPLC Chromatogram of Analog CID-44247472." /></div><h3><span class="title">UPLC Chromatogram of Analog CID-44247472</span></h3></div><div id="ml126.fu21" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56228/bin/ml126fu33.jpg" alt="1H NMR (300 MHz, CDCl3) Spectrum of Analog CID-95052." /></div><h3><span class="title"><sup>1</sup>H NMR (300 MHz, CDCl3) Spectrum of Analog CID-95052</span></h3></div><div id="ml126.fu22" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56228/bin/ml126fu34.jpg" alt="UPLC Chromatogram of Analog CID-95052." /></div><h3><span class="title">UPLC Chromatogram of Analog CID-95052</span></h3></div><div id="ml126.fu23" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56228/bin/ml126fu35.jpg" alt="1H NMR (300 MHz, CDCl3) Spectrum of Analog CID-49843154." /></div><h3><span class="title"><sup>1</sup>H NMR (300 MHz, CDCl<sub>3</sub>) Spectrum of Analog CID-49843154</span></h3></div><div id="ml126.fu24" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56228/bin/ml126fu36.jpg" alt="UPLC Chromatogram of Analog CID-49843154." /></div><h3><span class="title">UPLC Chromatogram of Analog CID-49843154</span></h3></div><div id="ml126.fu25" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56228/bin/ml126fu37.jpg" alt="1H NMR (300 MHz, CDCl3) Spectrum of Analog CID-13227242." /></div><h3><span class="title"><sup>1</sup>H NMR (300 MHz, CDCl<sub>3</sub>) Spectrum of Analog CID-13227242</span></h3></div><div id="ml126.fu26" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56228/bin/ml126fu38.jpg" alt="UPLC Chromatogram of Analog CID-13227242." /></div><h3><span class="title">UPLC Chromatogram of Analog CID-13227242</span></h3></div><div id="ml126.fu27" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56228/bin/ml126fu39.jpg" alt="1H NMR (300 MHz, CDCl3) Spectrum of Analog CID-24102740." /></div><h3><span class="title"><sup>1</sup>H NMR (300 MHz, CDCl<sub>3</sub>) Spectrum of Analog CID-24102740</span></h3></div><div id="ml126.fu28" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56228/bin/ml126fu40.jpg" alt="UPLC Chromatogram of Analog CID-24102740." /></div><h3><span class="title">UPLC Chromatogram of Analog CID-24102740</span></h3></div><div id="ml126.fu29" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56228/bin/ml126fu41.jpg" alt="1H NMR (300 MHz, CDCl3) Spectrum of Analog CID-1848658." /></div><h3><span class="title"><sup>1</sup>H NMR (300 MHz, CDCl<sub>3</sub>) Spectrum of Analog CID-1848658</span></h3></div><div id="ml126.fu30" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56228/bin/ml126fu42.jpg" alt="UPLC Chromatogram of Analog CID-1848658." /></div><h3><span class="title">UPLC Chromatogram of Analog CID-1848658</span></h3></div><div id="ml126.fu31" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56228/bin/ml126fu43.jpg" alt="1H NMR (300 MHz, CDCl3) Spectrum of Analog CID-44247467." /></div><h3><span class="title"><sup>1</sup>H NMR (300 MHz, CDCl<sub>3</sub>) Spectrum of Analog CID-44247467</span></h3></div><div id="ml126.fu32" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56228/bin/ml126fu44.jpg" alt="UPLC Chromatogram of Analog CID-44247467." /></div><h3><span class="title">UPLC Chromatogram of Analog CID-44247467</span></h3></div><div id="ml126.fu33" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56228/bin/ml126fu45.jpg" alt="1H NMR (300 MHz, CDCl3) Spectrum of Analog CID-49843195." /></div><h3><span class="title"><sup>1</sup>H NMR (300 MHz, CDCl<sub>3</sub>) Spectrum of Analog CID-49843195</span></h3></div><div id="ml126.fu34" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56228/bin/ml126fu46.jpg" alt="UPLC Chromatogram of Analog CID-49843195." /></div><h3><span class="title">UPLC Chromatogram of Analog CID-49843195</span></h3></div><div id="ml126.fu35" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56228/bin/ml126fu47.jpg" alt="1H NMR (300 MHz, CDCl3) Spectrum of Analog CID-49843019." /></div><h3><span class="title"><sup>1</sup>H NMR (300 MHz, CDCl<sub>3</sub>) Spectrum of Analog CID-49843019</span></h3></div><div id="ml126.fu36" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56228/bin/ml126fu48.jpg" alt="UPLC Chromatogram of Analog CID-49843019." /></div><h3><span class="title">UPLC Chromatogram of Analog CID-49843019</span></h3></div><div id="ml126.fu37" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56228/bin/ml126fu49.jpg" alt="1H NMR (300 MHz, CDCl3) Spectrum of Analog CID-49843256." /></div><h3><span class="title"><sup>1</sup>H NMR (300 MHz, CDCl<sub>3</sub>) Spectrum of Analog CID-49843256</span></h3></div><div id="ml126.fu38" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56228/bin/ml126fu50.jpg" alt="UPLC Chromatogram of Analog CID-49843256." /></div><h3><span class="title">UPLC Chromatogram of Analog CID-49843256</span></h3></div><div id="ml126.fu39" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56228/bin/ml126fu51.jpg" alt="1H NMR (300 MHz, CDCl3) Spectrum of Analog CID-49843032." /></div><h3><span class="title"><sup>1</sup>H NMR (300 MHz, CDCl<sub>3</sub>) Spectrum of Analog CID-49843032</span></h3></div><div id="ml126.fu40" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56228/bin/ml126fu52.jpg" alt="UPLC Chromatogram of Analog CID-49843032." /></div><h3><span class="title">UPLC Chromatogram of Analog CID-49843032</span></h3></div><div id="ml126.fu41" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56228/bin/ml126fu53.jpg" alt="1H NMR (300 MHz, CDCl3) Spectrum of Analog CID-46926576." /></div><h3><span class="title"><sup>1</sup>H NMR (300 MHz, CDCl<sub>3</sub>) Spectrum of Analog CID-46926576</span></h3></div><div id="ml126.fu42" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56228/bin/ml126fu54.jpg" alt="UPLC Chromatogram of Analog CID-46926576." /></div><h3><span class="title">UPLC Chromatogram of Analog CID-46926576</span></h3></div><div id="ml126.fu43" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56228/bin/ml126fu55.jpg" alt="1H NMR (300 MHz, CDCl3) Spectrum of Analog CID-49843096." /></div><h3><span class="title"><sup>1</sup>H NMR (300 MHz, CDCl<sub>3</sub>) Spectrum of Analog CID-49843096</span></h3></div><div id="ml126.fu44" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56228/bin/ml126fu56.jpg" alt="UPLC Chromatogram of Analog CID-49843096." /></div><h3><span class="title">UPLC Chromatogram of Analog CID-49843096</span></h3></div><div id="ml126.fu45" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56228/bin/ml126fu57.jpg" alt="1H NMR (300 MHz, CDCl3) Spectrum of Analog CID-46926570." /></div><h3><span class="title"><sup>1</sup>H NMR (300 MHz, CDCl<sub>3</sub>) Spectrum of Analog CID-46926570</span></h3></div><div id="ml126.fu46" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56228/bin/ml126fu58.jpg" alt="UPLC Chromatogram of Analog CID-46926570." /></div><h3><span class="title">UPLC Chromatogram of Analog CID-46926570</span></h3></div><div id="ml126.fu47" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56228/bin/ml126fu59.jpg" alt="1H NMR (300 MHz, CDCl3) Spectrum of Analog CID-3473393." /></div><h3><span class="title"><sup>1</sup>H NMR (300 MHz, CDCl<sub>3</sub>) Spectrum of Analog CID-3473393</span></h3></div><div id="ml126.fu48" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56228/bin/ml126fu60.jpg" alt="UPLC Chromatogram of Analog CID-3473393." /></div><h3><span class="title">UPLC Chromatogram of Analog CID-3473393</span></h3></div><div id="ml126.fu49" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56228/bin/ml126fu61.jpg" alt="1H NMR (300 MHz, CDCl3) Spectrum of Analog CID-46926571." /></div><h3><span class="title"><sup>1</sup>H NMR (300 MHz, CDCl<sub>3</sub>) Spectrum of Analog CID-46926571</span></h3></div><div id="ml126.fu50" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56228/bin/ml126fu62.jpg" alt="UPLC Chromatogram of Analog CID-46926571." /></div><h3><span class="title">UPLC Chromatogram of Analog CID-46926571</span></h3></div></div></div><div id="ml126.app4"><h2 id="_ml126_app4_">Appendix D. Compounds Submitted to BioFocus</h2><div id="ml126.t7" class="table"><h3><span class="label">Table A2</span><span class="title">Probe and Analog Information</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK56228/table/ml126.t7/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml126.t7_lrgtbl__"><table class="no_margin"><thead><tr><th id="hd_h_ml126.t7_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BROAD ID</th><th id="hd_h_ml126.t7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">PUBCHEM SID</th><th id="hd_h_ml126.t7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">PUBCHEM CID</th><th id="hd_h_ml126.t7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">P/A</th><th id="hd_h_ml126.t7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS No.</th><th id="hd_h_ml126.t7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">ML No.</th></tr></thead><tbody><tr><td headers="hd_h_ml126.t7_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BRD-K71265179-001</td><td headers="hd_h_ml126.t7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/85281160" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">85281160</a></td><td headers="hd_h_ml126.t7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">576989</td><td headers="hd_h_ml126.t7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">P</td><td headers="hd_h_ml126.t7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS002608919</td><td headers="hd_h_ml126.t7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="/pcsubstance/?term=ML126[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML126</a></td></tr><tr><td headers="hd_h_ml126.t7_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BRD-K19704225-001</td><td headers="hd_h_ml126.t7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/85281135" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">85281135</a></td><td headers="hd_h_ml126.t7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">8852543</td><td headers="hd_h_ml126.t7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">A</td><td headers="hd_h_ml126.t7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS002608920</td><td headers="hd_h_ml126.t7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">NA</td></tr><tr><td headers="hd_h_ml126.t7_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BRD-K83315615-001</td><td headers="hd_h_ml126.t7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/85281165" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">85281165</a></td><td headers="hd_h_ml126.t7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">44247468</td><td headers="hd_h_ml126.t7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">A</td><td headers="hd_h_ml126.t7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS002608921</td><td headers="hd_h_ml126.t7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">NA</td></tr><tr><td headers="hd_h_ml126.t7_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BRD-K85868893-001</td><td headers="hd_h_ml126.t7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/85281167" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">85281167</a></td><td headers="hd_h_ml126.t7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">44247472</td><td headers="hd_h_ml126.t7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">A</td><td headers="hd_h_ml126.t7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS002608922</td><td headers="hd_h_ml126.t7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">NA</td></tr><tr><td headers="hd_h_ml126.t7_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BRD-K94919853-001</td><td headers="hd_h_ml126.t7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/85281172" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">85281172</a></td><td headers="hd_h_ml126.t7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3473393</td><td headers="hd_h_ml126.t7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">A</td><td headers="hd_h_ml126.t7_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS002608923</td><td headers="hd_h_ml126.t7_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">NA</td></tr></tbody></table></div><div><div><dl class="temp-labeled-list small"><dt></dt><dd><div id="ml126.tfn9"><p class="no_margin">A=analog;NA=not applicable;P=probe</p></div></dd></dl></div></div></div></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/NBK56228/?report=reader">PubReader</a></li><li><a href="/books/NBK56228/?report=printable">Print View</a></li><li><a data-jig="ncbidialog" href="#_ncbi_dlg_citbx_NBK56228" data-jigconfig="width:400,modal:true">Cite this Page</a><div id="_ncbi_dlg_citbx_NBK56228" style="display:none" title="Cite this Page"><div class="bk_tt">An WF, Metkar SR, Nag PP, et al. Identification of small molecules that selectively inhibit streptokinase expression without suppression of viability in Group A streptococci - Probe 1. 2009 Nov 16 [Updated 2011 Mar 11]. 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/NBK56228/pdf/Bookshelf_NBK56228.pdf">PDF version of this page</a> (1.2M)</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="#ml126.s1" ref="log$=inpage&link_id=inpage">Probe Structure & Characteristics</a></li><li><a href="#ml126.s2" ref="log$=inpage&link_id=inpage">Recommendations for scientific use of the probe</a></li><li><a href="#ml126.s3" ref="log$=inpage&link_id=inpage">Introduction</a></li><li><a href="#ml126.s5" ref="log$=inpage&link_id=inpage">Materials and Methods</a></li><li><a href="#ml126.s18" ref="log$=inpage&link_id=inpage">Results</a></li><li><a href="#ml126.s26" ref="log$=inpage&link_id=inpage">Discussion</a></li><li><a href="#ml126.s30" ref="log$=inpage&link_id=inpage">References</a></li><li><a href="#ml126.app1" ref="log$=inpage&link_id=inpage">Assay Summary Table</a></li><li><a href="#ml126.app2" ref="log$=inpage&link_id=inpage">Detailed Assay Protocols</a></li><li><a href="#ml126.app3" ref="log$=inpage&link_id=inpage">NMR and LC Data of Probe and Analogs</a></li><li><a href="#ml126.app4" ref="log$=inpage&link_id=inpage">Compounds Submitted to BioFocus</a></li></ul></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Related information</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="discovery_db_links" id="Shutter"></a></div><div class="portlet_content"><ul><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pmc&DbFrom=books&Cmd=Link&LinkName=books_pmc_refs&IdsFromResult=2509080" ref="log$=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=2509080" ref="log$=recordlinks">PubChem BioAssay for Chemical Probe</a><div class="brieflinkpop offscreen_noflow">PubChem BioAssay records reporting screening data for the development of the chemical probe(s) described in this book chapter</div></li><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pcsubstance&DbFrom=books&Cmd=Link&LinkName=books_pcsubstance&IdsFromResult=2509080" ref="log$=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=2509080" ref="log$=recordlinks">PubMed</a><div class="brieflinkpop offscreen_noflow">Links to PubMed</div></li></ul></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Similar articles in PubMed</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="PBooksDiscovery_RA" id="Shutter"></a></div><div class="portlet_content"><ul><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/21735596" ref="ordinalpos=1&linkpos=1&log$=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> Identification of small molecules that selectively inhibit streptokinase expression without suppression of viability in Group A streptococci - 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Epub 2003 Aug 5.</em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/21735608" ref="ordinalpos=1&linkpos=5&log$=relatedreviews&logdbfrom=pubmed"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> Campaign to identify novel modulators of the Retinoic acid receptor-related Orphan Receptors (ROR).</a><span class="source">[Probe Reports from the NIH Mol...]</span><div class="brieflinkpop offscreen_noflow"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> Campaign to identify novel modulators of the Retinoic acid receptor-related Orphan Receptors (ROR).<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Kumar N, Solt LA, Conkright J, Wang Y, Istrate MA, Busby SA, Garcia-Ordonez RD, Nuhant P, Burris T, Mercer BA, et al. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Probe Reports from the NIH Molecular Libraries Program. 2010</em></div></div></li></ul><a class="seemore" href="/sites/entrez?db=pubmed&cmd=link&linkname=pubmed_pubmed_reviews&uid=21735597" ref="ordinalpos=1&log$=relatedreviews_seeall&logdbfrom=pubmed">See reviews...</a><a class="seemore" href="/sites/entrez?db=pubmed&cmd=link&linkname=pubmed_pubmed&uid=21735597" 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=67d66c1084f3725e592dc46c">Identification of small molecules that selectively inhibit streptokinase express...</a><div class="ralinkpop offscreen_noflow">Identification of small molecules that selectively inhibit streptokinase expression without suppression of viability in Group A streptococci - 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<li>
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<a class="text-white" href="https://www.nih.gov/">NIH</a>
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<a class="text-white" href="https://www.hhs.gov/">HHS</a>
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<li>
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<a class="text-white" href="https://www.usa.gov/">USA.gov</a>
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