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<meta name="ncbi_db" content="books" /><meta name="ncbi_pdid" content="book-part" /><meta name="ncbi_acc" content="NBK56231" /><meta name="ncbi_domain" content="mlprobe" /><meta name="ncbi_report" content="record" /><meta name="ncbi_type" content="fulltext" /><meta name="ncbi_objectid" content="" /><meta name="ncbi_pcid" content="/NBK56231/" /><meta name="ncbi_pagename" content="Identification of Small-Molecule Inhibitors of Trypansoma cruzi Infection - Probe 1 - Probe Reports from the NIH Molecular Libraries Program - NCBI Bookshelf" /><meta name="ncbi_bookparttype" content="chapter" /><meta name="ncbi_app" content="bookshelf" />
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<title>Identification of Small-Molecule Inhibitors of Trypansoma cruzi Infection - 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-Molecule Inhibitors of Trypansoma cruzi Infection - 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="Leigh C Carmody" /><meta name="citation_author" content="Andrew Germain" /><meta name="citation_author" content="Douglas Barker" /><meta name="citation_author" content="Cristina Galan-Rodriguez" /><meta name="citation_author" content="Esther Bettiol" /><meta name="citation_author" content="Ana Rodriguez" /><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="21735600" /><meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK56231/" /><link rel="schema.DC" href="http://purl.org/DC/elements/1.0/" /><meta name="DC.Title" content="Identification of Small-Molecule Inhibitors of Trypansoma cruzi Infection - Probe 1" /><meta name="DC.Type" content="Text" /><meta name="DC.Publisher" content="National Center for Biotechnology Information (US)" /><meta name="DC.Contributor" content="Leigh C Carmody" /><meta name="DC.Contributor" content="Andrew Germain" /><meta name="DC.Contributor" content="Douglas Barker" /><meta name="DC.Contributor" content="Cristina Galan-Rodriguez" /><meta name="DC.Contributor" content="Esther Bettiol" /><meta name="DC.Contributor" content="Ana Rodriguez" /><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/NBK56231/" /><meta name="description" content="Chagas disease is a tropical disease caused by the parasite Trypanosoma cruzi (T. cruzi), which is endemic to Central and South America. Approximately 13 million people are infected with the parasite, and 25–30% of infected patients suffer from irreversible damage to the heart and digestive tract resulting in disability and death within 20 years of infection. Few treatments are available with limited effectiveness only against the early (acute) stages of disease, significant toxicity, and widespread drug resistance. We report the outcome of a high-throughput chemical library screen to identify novel, nontoxic, small-molecule inhibitors of T. cruzi, which will aid the development of more potent and selective therapies for both the acute and chronic stages of Chagas disease. Of the 303,224-screened compounds, 35 compounds were chosen based on their selectivity, potency, and chemical tractability. Of those, 27 dry powder-validated compounds were retested in the primary screen, secondary assays, and an orthogonal screen. Three scaffolds were prioritized to identify potential probes. One of these compounds (CID 6875690/ML164) displayed greater than 100-fold selective inhibition of T. cruzi confirmed in immunofluorescent imaging assays and is inactive against host cells at the highest tested dose. This new probe should be very useful in future cell-based investigations and in vivo studies of T. cruzi inhibition." /><meta name="og:title" content="Identification of Small-Molecule Inhibitors of Trypansoma cruzi Infection - Probe 1" /><meta name="og:type" content="book" /><meta name="og:description" content="Chagas disease is a tropical disease caused by the parasite Trypanosoma cruzi (T. cruzi), which is endemic to Central and South America. Approximately 13 million people are infected with the parasite, and 25–30% of infected patients suffer from irreversible damage to the heart and digestive tract resulting in disability and death within 20 years of infection. Few treatments are available with limited effectiveness only against the early (acute) stages of disease, significant toxicity, and widespread drug resistance. We report the outcome of a high-throughput chemical library screen to identify novel, nontoxic, small-molecule inhibitors of T. cruzi, which will aid the development of more potent and selective therapies for both the acute and chronic stages of Chagas disease. Of the 303,224-screened compounds, 35 compounds were chosen based on their selectivity, potency, and chemical tractability. Of those, 27 dry powder-validated compounds were retested in the primary screen, secondary assays, and an orthogonal screen. Three scaffolds were prioritized to identify potential probes. One of these compounds (CID 6875690/ML164) displayed greater than 100-fold selective inhibition of T. cruzi confirmed in immunofluorescent imaging assays and is inactive against host cells at the highest tested dose. This new probe should be very useful in future cell-based investigations and in vivo studies of T. cruzi inhibition." /><meta name="og:url" content="https://www.ncbi.nlm.nih.gov/books/NBK56231/" /><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/ml164/" /><link rel="canonical" href="https://www.ncbi.nlm.nih.gov/books/NBK56231/" /><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="#__NBK56231_dtls__">Show details</a><div style="display:none" class="ui-widget" id="__NBK56231_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/ml165/" title="Previous page in this title">< Prev</a><a class="active page_link next" href="/books/n/mlprobe/ml163/" 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="_NBK56231_"><span class="title" itemprop="name">Identification of Small-Molecule Inhibitors of <i>Trypansoma cruzi</i> Infection - Probe 1</span></h1><p class="contrib-group"><span itemprop="author">Leigh C Carmody</span>, <span itemprop="author">Andrew Germain</span>, <span itemprop="author">Douglas Barker</span>, <span itemprop="author">Cristina Galan-Rodriguez</span>, <span itemprop="author">Esther Bettiol</span>, <span itemprop="author">Ana Rodriguez</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="#__NBK56231_ai__" style="border:0;text-decoration:none">Author Information and Affiliations</a><div style="display:none" class="ui-widget" id="__NBK56231_ai__"><p class="contrib-group"><h4>Authors</h4><span itemprop="author">Leigh C Carmody</span>,<sup><img src="/corehtml/pmc/pmcgifs/corrauth.gif" alt="corresponding author" /></sup><sup>1</sup> <span itemprop="author">Andrew Germain</span>,<sup>1</sup> <span itemprop="author">Douglas Barker</span>,<sup>1</sup> <span itemprop="author">Cristina Galan-Rodriguez</span>,<sup>2</sup> <span itemprop="author">Esther Bettiol</span>,<sup>2</sup> <span itemprop="author">Ana Rodriguez</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>.</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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New York University, New York, NY</div><div class="affiliation"><sup>3</sup>
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Howard Hughes Medical Institute, Chemistry and 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@ydomracl" class="oemail">gro.etutitsnidaorb@ydomracl</a></div></div><p class="small">Received: <span itemprop="datePublished">March 26, 2010</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>Chagas disease is a tropical disease caused by the parasite <i>Trypanosoma cruzi (T. cruzi)</i>, which is endemic to Central and South America. Approximately 13 million people are infected with the parasite, and 25–30% of infected patients suffer from irreversible damage to the heart and digestive tract resulting in disability and death within 20 years of infection. Few treatments are available with limited effectiveness only against the early (acute) stages of disease, significant toxicity, and widespread drug resistance. We report the outcome of a high-throughput chemical library screen to identify novel, nontoxic, small-molecule inhibitors of <i>T. cruzi,</i> which will aid the development of more potent and selective therapies for both the acute and chronic stages of Chagas disease. Of the 303,224-screened compounds, 35 compounds were chosen based on their selectivity, potency, and chemical tractability. Of those, 27 dry powder-validated compounds were retested in the primary screen, secondary assays, and an orthogonal screen. Three scaffolds were prioritized to identify potential probes. One of these compounds (CID 6875690/<a href="/pcsubstance/?term=ML164[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML164</a>) displayed greater than 100-fold selective inhibition of <i>T. cruzi</i> confirmed in immunofluorescent imaging assays and is inactive against host cells at the highest tested dose. This new probe should be very useful in future cell-based investigations and <i>in vivo</i> studies of <i>T. cruzi</i> inhibition.</p></div><div class="h2"></div><p><b>Assigned Assay Grant No.:</b> 1R03MH085673-01</p><p><b>Screening Center Name and PI</b>: Broad Institute Probe Development Center, Stuart Schreiber</p><p><b>Chemistry Center Name and PI</b>: Broad Institute Probe Development Center, Stuart Schreiber</p><p><b>Assay Submitter and Institution:</b> Ana Rodriguez, New York University</p><p><b>PubChem Summary Bioassay Identifier (AID)</b>: <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1885" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1885</a></p><div id="ml164.s1"><h2 id="_ml164_s1_">Probe Structure and Characteristics</h2><div id="ml164.fu1" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56231/bin/ml164fu1.jpg" alt="ML164." /></div><h3><span class="title"><a href="/pcsubstance/?term=ML164[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML164</a></span></h3></div><div id="ml164.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/NBK56231/table/ml164.tu1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml164.tu1_lrgtbl__"><table class="no_top_margin"><tbody><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;">IUPAC Chemical Name</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"><i>N</i>-(3,4-dimethylphenyl)-<i>N</i>′-[(E)-(5-nitrofuran-2-yl)methylideneamino]butanediamide</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">PubChem CID</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">6875690</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Molecular Weight</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">358.34862 g/mol</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Molecular Formula</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">C<sub>17</sub>H<sub>18</sub>N<sub>4</sub>O<sub>5</sub></td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">XLogP3-AA</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">2.2</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">H-Bond Donor</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">2</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">H-Bond Acceptor</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">6</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Rotatable Bond Count</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">6</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Exact Mass</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">358.12772</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Topological Polar Surface Area</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">130</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Solubility (PBS, μM)</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">< 1</td></tr></tbody></table></div></div><div id="ml164.tu2" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK56231/table/ml164.tu2/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml164.tu2_lrgtbl__"><table class="no_bottom_margin"><thead><tr><th id="hd_h_ml164.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CID/ML No.</th><th id="hd_h_ml164.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Target Name</th><th id="hd_h_ml164.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">IC<sub>50</sub>/EC<sub>50</sub> (nM) [SID, AID]</th><th id="hd_h_ml164.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Anti-target</th><th id="hd_h_ml164.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">IC<sub>50</sub>/EC<sub>50</sub> (μM) [SID, AID]</th><th id="hd_h_ml164.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Fold Selective<sup><a class="bk_pop" href="#ml164.tfn1">*</a></sup></th><th id="hd_h_ml164.tu2_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Secondary Assay(s) IC<sub>50</sub>/EC<sub>50</sub> (nM) [SID, AID]</th></tr></thead><tbody><tr><td headers="hd_h_ml164.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">6875690/ <a href="/pcsubstance/?term=ML164[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML164</a></td><td headers="hd_h_ml164.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><i>Trypansoma cruzi</i> replication</td><td headers="hd_h_ml164.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">34.3 [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/87219036" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 87219036</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2630" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 2630</a>]</td><td headers="hd_h_ml164.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NIH/3T3 Cell Toxicity</td><td headers="hd_h_ml164.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Inactive [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/87219036" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 87219036</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2586" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 2586</a>]</td><td headers="hd_h_ml164.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">34.3 nM vs inactive</td><td headers="hd_h_ml164.tu2_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Immunofluorescence Static [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/87219036" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 87219036</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2630" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 2630</a>]</td></tr></tbody></table></div><div><div><dl class="temp-labeled-list small"><dt>*</dt><dd><div id="ml164.tfn1"><p class="no_margin">Selectivity = anti-target IC<sub>50</sub>/target IC<sub>50</sub>.</p></div></dd></dl></div></div></div></div><div id="ml164.s2"><h2 id="_ml164_s2_">Recommendations for scientific use of the probe</h2><p>The goal of this project is to identify a small-molecule inhibitor of the protist <i>Trypanosoma cruzi (T. cruzi)</i>, the causative parasite of Chagas disease. Although a few treatments for Chagas disease are available, these treatments are limited because they are only effective against the early (acute) stages of disease and have significant toxicity to the patient (<a class="bk_pop" href="#ml164.r1">1</a>, <a class="bk_pop" href="#ml164.r2">2</a>, <a class="bk_pop" href="#ml164.r3">3</a>). Novel antitrypanosomal agents will aid in developing a drug treatment for both the acute and chronic stages of Chagas disease. This probe (CID 6875690/<a href="/pcsubstance/?term=ML164[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML164</a>) is expected to inhibit the replication of <i>T. cruzi</i> without having effects on the viability of the mammalian host cell.</p><p>The probe (CID 6875690/<a href="/pcsubstance/?term=ML164[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML164</a>) described in this report inhibits the replication of <i>T. cruzi</i> with an IC<sub>50</sub> of 34.3 nM and is inactive toward mammalian cells (NIH/3T3) up to concentrations of 19,500 nM. In immunofluorescent imaging assays, the compound inhibited <i>T. cruzi</i> amastigote replication within host cells. Since amastigote morphology was maintained, this probe (CID 6875690/<a href="/pcsubstance/?term=ML164[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML164</a>) is categorized as a trypanostatic (static) compound.</p><p>This probe (CID 6875690/<a href="/pcsubstance/?term=ML164[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML164</a>) provides a valuable tool that will allow microbiologists investigating Chagas disease to identify new targets in <i>T. cruzi,</i> which may potentially lead to better drugs. Furthermore, the Assay Provider, Dr. Ana Rodriguez and her colleagues, will continue their own studies with this probe (CID 6875690/<a href="/pcsubstance/?term=ML164[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML164</a>) in a mouse model of Chagas disease.</p></div><div id="ml164.s3"><h2 id="_ml164_s3_">1. Introduction</h2><div id="ml164.s4"><h3>Scientific Rationale</h3><p>Chagas disease is a tropical parasitic disease caused by the parasite <i>Trypanosoma cruzi (T. cruzi)</i>. Approximately 13 million people are infected with the parasite, which is endemic to Central and South America and results in 14,000 deaths per year (<a class="bk_pop" href="#ml164.r2">2</a>, <a class="bk_pop" href="#ml164.r3">3</a>). Twenty-five to thirty percent of infected patients suffer from irreversible damage to the heart and digestive tract resulting in a high incidence of disability and death within 20 years of infection (<a class="bk_pop" href="#ml164.r1">1</a>, <a class="bk_pop" href="#ml164.r2">2</a>). Chagas disease is the leading cause of heart failure in the region.</p><p>In <a class="figpopup" href="/books/NBK56231/figure/ml164.f1/?report=objectonly" target="object" rid-figpopup="figml164f1" rid-ob="figobml164f1">Figure 1</a>, the drugs currently used to treat Chagas disease are: benznidazole <b>A</b> (Rochagan®, Radanil®, Hoffman-La Roche) and nifurtimox <b>B</b> (Lampit®, Bayer), whose anti-<i>T. cruzi</i> activities were discovered more than 30 years ago. Both of these drugs are effective only in the acute phase of infection, rather than the key chronic stage. They often do not completely eliminate the parasite despite long-term administration, while exhibiting unacceptable side effects (such as nausea, vomiting, weight loss, neurological effects, and signs of testicular and ovarian injury). Both treatments are actually nonspecific for <i>T cruzi;</i> in addition, resistance to these drugs is becoming more widespread. Thus, there is a clear need for more potent and selective therapies.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml164f1" co-legend-rid="figlgndml164f1"><a href="/books/NBK56231/figure/ml164.f1/?report=objectonly" target="object" title="Figure 1" class="img_link icnblk_img figpopup" rid-figpopup="figml164f1" rid-ob="figobml164f1"><img class="small-thumb" src="/books/NBK56231/bin/ml164f1.gif" src-large="/books/NBK56231/bin/ml164f1.jpg" alt="Figure 1. Current Treatments for Chagas Disease." /></a><div class="icnblk_cntnt" id="figlgndml164f1"><h4 id="ml164.f1"><a href="/books/NBK56231/figure/ml164.f1/?report=objectonly" target="object" rid-ob="figobml164f1">Figure 1</a></h4><p class="float-caption no_bottom_margin">Current Treatments for Chagas Disease. Benznidazole (Rochagen<sup>®</sup>, Radanil®, Hoffman-La Roche) (<i>A</i>); Nifurtimox (Lampit®, Bayer) (<i>B</i>) </p></div></div><p>Several recent reports have revealed promising new lead compounds that target <i>T. cruzi</i>, including inhibitors of the cysteine protease cruzain and inhibitors of sterol demethylase (<a class="bk_pop" href="#ml164.r4">4</a>). However, while the protease inhibitors (see <a class="figpopup" href="/books/NBK56231/figure/ml164.f2/?report=objectonly" target="object" rid-figpopup="figml164f2" rid-ob="figobml164f2">Figures 2A, 2B, and 2C</a>) exhibit excellent activity in biochemical assays (0.079 μM to 2 μM), they are inactive or demonstrate high micromolar activity in parasite inhibition in cell culture (1 μM to inactive) (<a class="bk_pop" href="#ml164.r4">4</a>,<a class="bk_pop" href="#ml164.r5">5</a>,<a class="bk_pop" href="#ml164.r6">6</a>). Thus, there is a continued unmet need to identify scaffolds that are potent in cell culture against <i>T. cruzi</i> and nontoxic to the mammalian host cell. Our approach overcomes this problem of lack of cellular activity by employing a phenotypic screen measuring parasite replication in a host cell. Counterscreening in the mammalian host filters out toxic compounds.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml164f2" co-legend-rid="figlgndml164f2"><a href="/books/NBK56231/figure/ml164.f2/?report=objectonly" target="object" title="Figure 2" class="img_link icnblk_img figpopup" rid-figpopup="figml164f2" rid-ob="figobml164f2"><img class="small-thumb" src="/books/NBK56231/bin/ml164f2.gif" src-large="/books/NBK56231/bin/ml164f2.jpg" alt="Figure 2. Recent Inhibitors of T. cruzi Cysteine Proteases." /></a><div class="icnblk_cntnt" id="figlgndml164f2"><h4 id="ml164.f2"><a href="/books/NBK56231/figure/ml164.f2/?report=objectonly" target="object" rid-ob="figobml164f2">Figure 2</a></h4><p class="float-caption no_bottom_margin">Recent Inhibitors of <i>T. cruzi</i> Cysteine Proteases. </p></div></div><p>There are currently four compounds in clinical trials for Chagas disease (see <a class="figpopup" href="/books/NBK56231/figure/ml164.f3/?report=objectonly" target="object" rid-figpopup="figml164f3" rid-ob="figobml164f3">Figure 3</a>). Benznidazole (see <a class="figpopup" href="/books/NBK56231/figure/ml164.f1/?report=objectonly" target="object" rid-figpopup="figml164f1" rid-ob="figobml164f1">Figure 1A</a>) initially developed for treatment of acute Chagas disease is in Phase III trials to expand its use to include the treatment of chronic Chagas cardiomyopathy. Three antifungal drugs from Merck (see <a class="figpopup" href="/books/NBK56231/figure/ml164.f3/?report=objectonly" target="object" rid-figpopup="figml164f3" rid-ob="figobml164f3">Figure 3A</a>), Takeda (see <a class="figpopup" href="/books/NBK56231/figure/ml164.f3/?report=objectonly" target="object" rid-figpopup="figml164f3" rid-ob="figobml164f3">Figure 3B</a>), and Eisai (structure not disclosed) are in Phase I or II trials for expanding their use to include treatment of Chagas disease. Two heart arrhythmia treatments (see <a class="figpopup" href="/books/NBK56231/figure/ml164.f3/?report=objectonly" target="object" rid-figpopup="figml164f3" rid-ob="figobml164f3">Figure 3C</a> and <a class="figpopup" href="/books/NBK56231/figure/ml164.f3/?report=objectonly" target="object" rid-figpopup="figml164f3" rid-ob="figobml164f3">Figure 3D</a>) are the most frequent treatments for the chronic stage heart issues associated with Chagas disease and have shown anti-parasitic activity in some patients. The only novel compound currently in the pipeline is a peptide mimic from the Sandler Center for Drug Discovery (see <a class="figpopup" href="/books/NBK56231/figure/ml164.f3/?report=objectonly" target="object" rid-figpopup="figml164f3" rid-ob="figobml164f3">Figure 3E</a>) which is in preparation for a Phase I safety trial. Since all of these compounds are nonspecific for <i>T. cruzi</i>, the side effects may be problematic as is the case for benzidazole in its current use. Therefore, a selective, nontoxic inhibitor of <i>T. cruzi</i> replication could be of significant use to the scientific community.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml164f3" co-legend-rid="figlgndml164f3"><a href="/books/NBK56231/figure/ml164.f3/?report=objectonly" target="object" title="Figure 3" class="img_link icnblk_img figpopup" rid-figpopup="figml164f3" rid-ob="figobml164f3"><img class="small-thumb" src="/books/NBK56231/bin/ml164f3.gif" src-large="/books/NBK56231/bin/ml164f3.jpg" alt="Figure 3. Compounds in Clinical Trials for Chagas Disease." /></a><div class="icnblk_cntnt" id="figlgndml164f3"><h4 id="ml164.f3"><a href="/books/NBK56231/figure/ml164.f3/?report=objectonly" target="object" rid-ob="figobml164f3">Figure 3</a></h4><p class="float-caption no_bottom_margin">Compounds in Clinical Trials for Chagas Disease. </p></div></div><p>This project aims to identify novel antitrypanosomal agents for the treatment of Chagas disease. One key design of the project is to target the parasites co-cultured with host cells and to demonstrate little to no toxicity to the host cell. This screening campaign has identified a probe (CID 6875690/<a href="/pcsubstance/?term=ML164[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML164</a>),N-(3,4-dimethylphenyl)-N′-[(E)-(5-nitrofuran-2-yl)methylideneamino]butanediamide, which has an IC<sub>50</sub> of 34.3 nM and has no toxicity towards NIH/3T3 cells at the highest concentration tested (i.e., 19.5 μM).</p></div></div><div id="ml164.s5"><h2 id="_ml164_s5_">2. Materials and Methods</h2><p>The methods in this section were either performed as described in Bettiol et al. (i.e., immunofluorescence) (<a class="bk_pop" href="#ml164.r7">7</a>) or modified for high throughput screening (co-culture and host cell toxicity).</p><div id="ml164.s6"><h3>Materials and Reagents</h3><p>T175 culture flasks with vented caps were obtained from BD Falcon, and hyperflasks were obtained from Corning (Corning, NY; Catalog no.10024). Disposable sterile filter units (500 ml or 1 L; pore size, 0.20 μm were obtained from Nalgene (Catalog no. 566-0020). Dulbecco’s modified Eagle’s medium (DMEM) with Phenol Red, high glucose, with L-glutamine and sodium pyruvate was obtained from Cellgro (Mediatech Inc, Manassas, VA; Catalog no. 10-013-CM).</p><p>Penicillin-streptomycin-L-glutamine (PSG, Catalog no. 10378-016), FBS-heat inactivated fetal bovine serum (FBS, Catalog no. 16140-089), and 0.25% Trypsin-EDTA 1X (Catalog no. 25200-072) were purchased from Gibco-Invitrogen. Sterile horse serum, from donor herd (if appearance of epimastigotes) was obtained from Sigma (Catalog no. H1270). Sterile, Ca++/ Mg++-free Phosphate Buffered Saline (PBS) 1X was prepared in house.</p><p>Nonidet P-40 (NP40, now called Igepal CA 360) was obtained from Fluka (Sigma-Aldrich, St. Louis, MO; Catalog no. 56741) and Gal-Screen<sup>®</sup> Buffer B was obtained from ABBiosciences (Allston, MA; Catalog no.T1031).</p></div><div id="ml164.s7"><h3>Cell Lines</h3><ul><li class="half_rhythm"><div>The following cell lines were used in this study: LLC-MK2 cells (rhesus monkey kidney epithelial cell line) and NIH/3T3 cells (mouse embryonic fibroblastic cell line) were initially obtained from the Assay Provider, then from ATCC. <i>T. cruzi</i> expressing β-galactosidase (<i>T. cruzi</i> -β-gal: Tulahuen strain, clone C4) was obtained from the Assay Provider with derivation as described in Buckner et al. (<a class="bk_pop" href="#ml164.r8">8</a>).</div></li><li class="half_rhythm"><div>Alexa Fluor 488 goat anti-rabbit IgG secondary antibody was from Molecular Probes<sup>®</sup>, Invitrogen (Carlsbad, CA).</div></li><li class="half_rhythm"><div>Polyclonal rabbit anti-<i>T. cruzi</i> was a gift from Dr. B. Burleigh, Harvard School of Public Health, Boston, MA).</div></li></ul></div><div id="ml164.s8"><h3>2.1. Assays</h3><p>A summary listing of completed assays and corresponding PubChem AID numbers is provided in <a href="#ml164.app1">Appendix A</a> (<a class="figpopup" href="/books/NBK56231/table/ml164.t5/?report=objectonly" target="object" rid-figpopup="figml164t5" rid-ob="figobml164t5">Table A1</a>). Refer to <a href="#ml164.app2">Appendix B</a> for the detailed assay protocols.</p><div id="ml164.s9"><h4>2.1.1. <i>T cruzi</i> Inhibition Assay (AID Nos.<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1885" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1885</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2044" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2044</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2294" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2294</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2630" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2630</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2396" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2396</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/493197" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">493197</a>)</h4><p><b>For cell propagation:</b> 90% DMEM, Phenol Red, 10% FBS, and 1% PSG were mixed and filtered through a 0.2-microns membrane. The cells were kept at 4°C, and then warmed up to 37°C in a water bath before use.</p><p><b>For</b>
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<b><i>T. cruzi</i></b>
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<b>culture and assays:</b> 98% DMEM, Phenol Red, 2% FBS, and 1% PSG were mixed and filtered through a 0.2-microns membrane. The cells were kept at 4°C, and then warmed up to 37°C in a water bath before use.</p><p>Solutions: Gal-Screen + 0.05% NP40. Using a Gal-Screen base kit, Buffer B (Catalog no. T2361) was mixed with 1:25 substrate (Catalog no. T2359) with a 1:400 dilution of 20% NP40.</p><p><b>NIH/3T3 Cell Culture.</b> NIH/3T3 cells were cultivated in DMEM supplemented with 10% FBS and 1% PSG in T175 in 50 ml total of medium.</p><p><b>LLC-MK2 Cell Culture.</b> LLC-MK2 cells were cultivated in DMEM supplemented with 10% FBS and 1% PSG in T175 flasks in 50 ml total of medium. Cells were usually passaged twice a week at 1:4 to 1:8 ratios.</p><p><b>Parasite Culture: <i>Tcruzi</i> β-gal (Tc).</b>
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<i>T. cruzi</i> -β-gal were cultivated in DMEM supplemented with 2% FBS and 1% PSG in T175 flasks with vented caps (important to avoid spills!) in 50 ml total of medium.</p></div><div id="ml164.s10"><h4>2.1.2. Growth Inhibition Assay for HTS (384-well plates)</h4><p>The medium was warmed up with 2% FBS/DMEM. The parasites were harvested in 50-ml tubes, and spun for 10 minutes at 2200 rpm. Approximately 15 ml of media was aspirated, and the samples were incubated for 3 to 5 hours. The NIH/3T3 cells were trypsinized (refer to cell culture protocol). When the NIH/3T3 cells were detached, the cells were harvested in DMEM, 2% FBS, and 1% PSG, then counted using the Nexcelom Cellometer. The cells were diluted to 166,667 cells/ml, and then added to a flask and plated 5,000 cells/30 μL per well using a standard cassette multiwell drop Combi. The cells were incubated for 3 hours, then <i>T. cruzi</i> cells were counted, diluted to 0.250 million cells/ml, and transferred to a 2-liter flask. Then, 50 nL compounds/DMSO were pinned to each well with NIH/3T3 cells. Next, 20 μL/well of parasites (5000 <i>T. cruzi</i>) were added with a standard cassette multiwell drop Combi on slow speed, and incubated for 4 days (or a minimum of 90 hours). Gal-Screen was prepared with 0.05% NP40, 30 μL per well were dispensed in a 384-well plate, incubated for 60 minutes, and the luminescence was read using Envision (Perkin-Elmer) at 0.1 sec/well.</p></div><div id="ml164.s11"><h4>2.1.3. Cell Toxicity Assay: NIH/3T3 Cells (AID Nos. <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2010" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2010</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2586" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2586</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/493247" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">493247</a>)</h4><p>For the cell toxicity assay with NIH/3T3 cells, the same materials as for <i>T. cruzi</i> co-culture assay were used. NIH/3T3 cells were cultivated in DMEM supplemented with 10% FBS and 1% PSG in T175 in 50 ml total of medium.</p></div><div id="ml164.s12"><h4>2.1.4. Intracellular <i>T. cruzi</i> Immunofluorescence Assay (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2632" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID No. 2632</a>)</h4><p>Fifty thousand NIH/3T3 cells were seeded on sterile glass coverslips in 12-well plates and allowed to adhere overnight. Five million <i>T. cruzi</i> parasites were added (mechanism of inhibition 100:1) and allowed to infect for 2 hours in DMEM plus 2% FBS and PSG. Parasites were rinsed out 3X with PBS, and compounds were added at 10X their IC<sub>50</sub> (as determined in <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2044" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 2044</a> and <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2294" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 2294</a>). Infected cells were further incubated for 4 days and fixed for 15 minutes with 4% paraformaldehyde.</p><p>Fixed cells on coverslips were rinsed with PBS and permeabilized for 15 minutes in PBS with 0.1% Triton X-100. After blocking for 20 minutes in PBS with 10% goat serum, 1% bovine serum albumin (BSA), 100 mM glycine, and 0.05% sodium azide, the cells were incubated for 1 hour at room temperature with a polyclonal rabbit anti-<i>T. cruzi</i> at 1:2000 dilution. After rinsing, an Alexa Fluor 488 goat anti-rabbit IgG secondary antibody was added for 1 hour at a 1:800 dilution. DNA was stained with DAPI, and coverslips were mounted with anti-fade mounting media. Images were taken using an inverted Olympus IX70 microscope with a 60X oil objective.</p></div></div><div id="ml164.s13"><h3>2.2. Probe Chemical Characterization</h3><p>The probe (CID 6875690/<a href="/pcsubstance/?term=ML164[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML164</a>) <b>1</b> was purchased from Enamine (ID T0512-1877); however it could be synthesized starting from 5-nitro-2-furancarboxaldehdye <b>2</b> in one step as shown in <a class="figpopup" href="/books/NBK56231/figure/ml164.f8/?report=objectonly" target="object" rid-figpopup="figml164f8" rid-ob="figobml164f8">Scheme 1</a>, based on literature precedent (<a class="bk_pop" href="#ml164.r9">9</a>). Treatment of <b>2</b> with <i>N</i>-(3,4-dimethylphenyl)-3-hydrazinylpropanamide <b>3</b> would provide the probe <b>1</b> (CID 6875690/<a href="/pcsubstance/?term=ML164[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML164</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/substance/87219052" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 87219052</a>), <i>N</i>-(3,4-dimethylphenyl)-<i>N</i>′-[(E)-(5-nitrofuran-2-yl)methylideneamino]butanediamide. The probe was determined to have a solubility of <1 μM in PBS at room temperature.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml164f8" co-legend-rid="figlgndml164f8"><a href="/books/NBK56231/figure/ml164.f8/?report=objectonly" target="object" title="Scheme 1" class="img_link icnblk_img figpopup" rid-figpopup="figml164f8" rid-ob="figobml164f8"><img class="small-thumb" src="/books/NBK56231/bin/ml164f8.gif" src-large="/books/NBK56231/bin/ml164f8.jpg" alt="Scheme 1. Synthesis of the Probe." /></a><div class="icnblk_cntnt" id="figlgndml164f8"><h4 id="ml164.f8"><a href="/books/NBK56231/figure/ml164.f8/?report=objectonly" target="object" rid-ob="figobml164f8">Scheme 1</a></h4><p class="float-caption no_bottom_margin">Synthesis of the Probe. </p></div></div><p>The probe (CID 6875690/<a href="/pcsubstance/?term=ML164[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML164</a>) and five analogs were submitted to the SMR collection (MLS002725693, MLS002725690, MLS002725681, MLS002725680, MLS002729069).</p><p>The <sup>1</sup>H NMR (300 MHz, DMSO-d6) spectra and LC-MS chromatograms of the probe (CID 6875690/<a href="/pcsubstance/?term=ML164[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML164</a>) and analogs (CID 6875684, CID 6884367, CID 6872741, CID 9689369, CID 9563094, CID 5344250, CID 6875617, CID 6322955, CID 9557463, CID 6875630, CID 5332109, CID 21210609, CID 9558304, CID 5334382, CID 5349954, CID 9640748) are provided in <a href="#ml164.app3">Appendix C</a>.</p></div><div id="ml164.s14"><h3>2.3. Probe Preparation</h3><p>Not applicable. The probe (CID 6875690/<a href="/pcsubstance/?term=ML164[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML164</a>) was purchased from Enamine (ID T5012-1877).</p></div></div><div id="ml164.s15"><h2 id="_ml164_s15_">3. Results</h2><div id="ml164.s16"><h3>Probe Attributes</h3><ul><li class="half_rhythm"><div>Inhibits extracellular parasite invasion into host cell or inhibits intracellular parasitic replication.</div></li><li class="half_rhythm"><div>Non-toxic to host cells (100-fold selectivity towards parasite vs. host cells)</div></li></ul></div><div id="ml164.s17"><h3>3.1. Summary of Screening Results</h3><p><a class="figpopup" href="/books/NBK56231/figure/ml164.f4/?report=objectonly" target="object" rid-figpopup="figml164f4" rid-ob="figobml164f4">Figure 5</a> displays the critical path for probe development.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml164f4" co-legend-rid="figlgndml164f4"><a href="/books/NBK56231/figure/ml164.f4/?report=objectonly" target="object" title="Figure 5" class="img_link icnblk_img figpopup" rid-figpopup="figml164f4" rid-ob="figobml164f4"><img class="small-thumb" src="/books/NBK56231/bin/ml164f4.gif" src-large="/books/NBK56231/bin/ml164f4.jpg" alt="Figure 5. Critical Path for Probe Development." /></a><div class="icnblk_cntnt" id="figlgndml164f4"><h4 id="ml164.f4"><a href="/books/NBK56231/figure/ml164.f4/?report=objectonly" target="object" rid-ob="figobml164f4">Figure 5</a></h4><p class="float-caption no_bottom_margin">Critical Path for Probe Development. In the primary screen, 303,224 compounds were screened. Of these, 4,063 compounds were available for evaluation in the primary screen retest at dose and secondary assay. Of the 3,005 that passed this filter, 27 dry <a href="/books/NBK56231/figure/ml164.f4/?report=objectonly" target="object" rid-ob="figobml164f4">(more...)</a></p></div></div><p>A high-throughput screen of 303,224 compounds (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1885" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1885</a>) was performed in duplicate utilizing the recombinant Tulahuen strain of <i>T. cruzi</i> stably expressing beta-galactosidase reporter co-cultured with host cell, mouse fibroblast NIH/3T3 (<a class="bk_pop" href="#ml164.r8">8</a>). Signal was normalized to neutral (DMSO) controls, and a 55% inhibition cutoff at a screening concentration of 3.75 μM average was used to define a hit. Next, 4,394 hits were identified as inhibitors of <i>T. cruzi</i> replication when co-cultured with host cells and, of these, 4,063 were available as cherry picks. The cherry-picked compounds were retested in dose in the primary assay using co-cultured <i>T. cruzi</i> strain with host cells to confirm their inhibitory activity (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2044" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 2044</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2294" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2294</a>). From the 4,063 compounds retested at dose concentrations, 4,016 compounds (99%) re-confirmed in this assay. In parallel, these 4,063 compounds were included in toxicity assays against host cell NIH/3T3 (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2010" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 2010</a>) to determine if these compounds were cytotoxic to mammalian cells and thus, false positives. This secondary screen identified 1,011 compounds that reduced viability of NIH/3T3 cells; therefore, these were excluded as viable hits.</p><p>After completing retests and a secondary assay at dose from DMSO stocks, 3,005 compounds were clustered into groups with 70% similarity. The 25 clusters with at least five members were given to a team of chemists; 35 representative compounds, at least one from each cluster, were prioritized based on molecular weight, presumed solubility, and lack of toxic or reactive functionality. Of the 35 compounds selected, 27 authentic dry powders were obtained. From this set of dry powder compounds, a piperidine series was prioritized.</p><p>Additional analogs were ordered and tested in the primary and secondary toxicity assay. The results are reported in <a class="figpopup" href="/books/NBK56231/table/ml164.t1/?report=objectonly" target="object" rid-figpopup="figml164t1" rid-ob="figobml164t1">Table 1</a>, <a class="figpopup" href="/books/NBK56231/table/ml164.t2/?report=objectonly" target="object" rid-figpopup="figml164t2" rid-ob="figobml164t2">Table 2</a>, and <a class="figpopup" href="/books/NBK56231/table/ml164.t3/?report=objectonly" target="object" rid-figpopup="figml164t3" rid-ob="figobml164t3">Table 3</a>. Also, these compounds were tested in an additional immunofluorescence assay to determine the mode of action of the compounds (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2632" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 2632</a>).</p><p>Intracellular <i>T. cruzi</i> was treated with the probe compound (CID 6875690/<a href="/pcsubstance/?term=ML164[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML164</a>). The compound inhibited replication of <i>T. cruzi</i> within the mammalian host cell but did not lyse <i>T. cruzi</i>. Therefore, the compound was classified as a ‘static’ inhibitor. Related analogs were also tested (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2630" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AIDs 2630</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2586" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2586</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2632" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2632</a>).</p></div><div id="ml164.s18"><h3>3.2. Dose Response Curves for Probe</h3><p><a class="figpopup" href="/books/NBK56231/figure/ml164.f5/?report=objectonly" target="object" rid-figpopup="figml164f5" rid-ob="figobml164f5">Figure 6</a> displays dose response curves for the probe.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml164f5" co-legend-rid="figlgndml164f5"><a href="/books/NBK56231/figure/ml164.f5/?report=objectonly" target="object" title="Figure 6" class="img_link icnblk_img figpopup" rid-figpopup="figml164f5" rid-ob="figobml164f5"><img class="small-thumb" src="/books/NBK56231/bin/ml164f5.gif" src-large="/books/NBK56231/bin/ml164f5.jpg" alt="Figure 6. Dose-dependent Activities of the Probe (CID 6875690/ML164) in Target and Counterscreen Assays." /></a><div class="icnblk_cntnt" id="figlgndml164f5"><h4 id="ml164.f5"><a href="/books/NBK56231/figure/ml164.f5/?report=objectonly" target="object" rid-ob="figobml164f5">Figure 6</a></h4><p class="float-caption no_bottom_margin">Dose-dependent Activities of the Probe (CID 6875690/ML164) in Target and Counterscreen Assays. Primary screen from dry powders (IC<sub>50</sub> 34.3 nM) (AID 2630) (<i>A</i>); NIH/3T3 (inactive) Toxicity (AID 2586) (<i>B</i>). </p></div></div><p>The compound (CID 6875690/<a href="/pcsubstance/?term=ML164[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML164</a>) met the defined probe criteria by displaying greater than 100-fold selective inhibition of <i>T. cruzi</i> vs. NIH/3T3 (34.3 nM vs. inactive). Furthermore, the inhibition of <i>T. cruzi</i> replication was confirmed in immunofluorescent imaging assays (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2632" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 2632</a>). The compound inhibited replication as compared to DMSO control condition and the compound did not lyse the cells. The probe (CID 6875690/<a href="/pcsubstance/?term=ML164[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML164</a>) has acceptable solubility and stability in aqueous conditions.</p></div><div id="ml164.s19"><h3>3.3. Scaffold/Moiety Chemical Liabilities</h3><p>A search of PubChem for the probe compound (CID 6875690/<a href="/pcsubstance/?term=ML164[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML164</a>) revealed that the probe has been tested in 255 BioAssays and was confirmed as active only in our assays. Therefore, the compound is not promiscuous. The compound has a drug-like structure with no obvious chemical liabilities that would be a concern.</p><p>The PBS stability of the probe compound (CID 6875690/<a href="/pcsubstance/?term=ML164[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML164</a>) was monitored over 48 hours, and the data is presented in <a class="figpopup" href="/books/NBK56231/figure/ml164.f6/?report=objectonly" target="object" rid-figpopup="figml164f6" rid-ob="figobml164f6">Figure 7</a>. After 24 hours, 87.8% of the compound was remaining; however, the amount of compound decreased to 51.9% between 24 and 48 hours. Recovery with acetonitrile was done at each time point to account for any precipitation and showed that no precipitation occurred.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml164f6" co-legend-rid="figlgndml164f6"><a href="/books/NBK56231/figure/ml164.f6/?report=objectonly" target="object" title="Figure 7" class="img_link icnblk_img figpopup" rid-figpopup="figml164f6" rid-ob="figobml164f6"><img class="small-thumb" src="/books/NBK56231/bin/ml164f6.gif" src-large="/books/NBK56231/bin/ml164f6.jpg" alt="Figure 7. Stability of the Probe (CID 6875690/ML1164) in PBS." /></a><div class="icnblk_cntnt" id="figlgndml164f6"><h4 id="ml164.f6"><a href="/books/NBK56231/figure/ml164.f6/?report=objectonly" target="object" rid-ob="figobml164f6">Figure 7</a></h4><p class="float-caption no_bottom_margin">Stability of the Probe (CID 6875690/ML1164) in PBS. </p></div></div></div><div id="ml164.s20"><h3>3.4. SAR Tables</h3><p>The tables above reveal interesting SAR involving structural perturbations of the probe scaffold. For example, replacing the nitro group (entry 1, <a class="figpopup" href="/books/NBK56231/table/ml164.t1/?report=objectonly" target="object" rid-figpopup="figml164t1" rid-ob="figobml164t1">Table 1</a>) with hydrogen (entry 2) or methyl (entry 3) leads to complete loss of activity (<a class="figpopup" href="/books/NBK56231/table/ml164.t1/?report=objectonly" target="object" rid-figpopup="figml164t1" rid-ob="figobml164t1">Table 1</a>).</p><p>While the eastern portion of the scaffold is more tolerant to modification, anilines and amides derived from anilines seem to be favored over other substituents as can be seen by comparing <a class="figpopup" href="/books/NBK56231/table/ml164.t2/?report=objectonly" target="object" rid-figpopup="figml164t2" rid-ob="figobml164t2">Table 2</a> and <a class="figpopup" href="/books/NBK56231/table/ml164.t3/?report=objectonly" target="object" rid-figpopup="figml164t3" rid-ob="figobml164t3">Table 3</a>. In general, addition of substituents to the aromatic amine or amides results in greater potency (entries 1–4, <a class="figpopup" href="/books/NBK56231/table/ml164.t2/?report=objectonly" target="object" rid-figpopup="figml164t2" rid-ob="figobml164t2">Table 2</a>).</p><div id="ml164.t1" class="table"><h3><span class="label">Table 1</span><span class="title">SAR Showing the Nitrofuran Subunit is Essential for <i>T.Cruzi</i> Inhibition</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK56231/table/ml164.t1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml164.t1_lrgtbl__"><table class="no_margin"><thead><tr><th id="hd_h_ml164.t1_1_1_1_1" colspan="5" rowspan="1" style="text-align:center;vertical-align:middle;">SAR Analysis for <i>T. cruzi</i></th><th id="hd_h_ml164.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK56231/bin/ml164fu2.jpg" alt="Image ml164fu2.jpg" /></div></th><th id="hd_h_ml164.t1_1_1_1_3" colspan="4" rowspan="1" style="text-align:center;vertical-align:middle;">Potency (nM) mean ± S.E.M. (n=replicates)</th><th id="hd_h_ml164.t1_1_1_1_4" rowspan="2" colspan="1" headers="hd_h_ml164.t1_1_1_1_4" style="text-align:center;vertical-align:middle;">Target to Antitarget Fold Selectivity</th></tr><tr><th headers="hd_h_ml164.t1_1_1_1_1" id="hd_h_ml164.t1_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Entry</th><th headers="hd_h_ml164.t1_1_1_1_1" id="hd_h_ml164.t1_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CID</th><th headers="hd_h_ml164.t1_1_1_1_1" id="hd_h_ml164.t1_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SID</th><th headers="hd_h_ml164.t1_1_1_1_1" id="hd_h_ml164.t1_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Broad No.</th><th headers="hd_h_ml164.t1_1_1_1_1" id="hd_h_ml164.t1_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><sup><a class="bk_pop" href="#ml164.tfn3">*</a></sup></th><th headers="hd_h_ml164.t1_1_1_1_2" id="hd_h_ml164.t1_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">R</th><th headers="hd_h_ml164.t1_1_1_1_3" id="hd_h_ml164.t1_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">n</th><th headers="hd_h_ml164.t1_1_1_1_3" id="hd_h_ml164.t1_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><i>T. cruzi</i> inhibition</th><th headers="hd_h_ml164.t1_1_1_1_3" id="hd_h_ml164.t1_1_1_2_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">n</th><th headers="hd_h_ml164.t1_1_1_1_3" id="hd_h_ml164.t1_1_1_2_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Toxicity</th></tr></thead><tbody><tr><td headers="hd_h_ml164.t1_1_1_1_1 hd_h_ml164.t1_1_1_2_1" rowspan="2" colspan="1" style="text-align:center;vertical-align:middle;">1</td><td headers="hd_h_ml164.t1_1_1_1_1 hd_h_ml164.t1_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">6875690</td><td headers="hd_h_ml164.t1_1_1_1_1 hd_h_ml164.t1_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87219052" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87219052</a></td><td headers="hd_h_ml164.t1_1_1_1_1 hd_h_ml164.t1_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BRD-K64970062</td><td headers="hd_h_ml164.t1_1_1_1_1 hd_h_ml164.t1_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">P</td><td headers="hd_h_ml164.t1_1_1_1_2 hd_h_ml164.t1_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK56231/bin/ml164fu3.jpg" alt="Image ml164fu3.jpg" /></div></td><td headers="hd_h_ml164.t1_1_1_1_3 hd_h_ml164.t1_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml164.t1_1_1_1_3 hd_h_ml164.t1_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">34.3±3.1</td><td headers="hd_h_ml164.t1_1_1_1_3 hd_h_ml164.t1_1_1_2_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml164.t1_1_1_1_3 hd_h_ml164.t1_1_1_2_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">inactive</td><td headers="hd_h_ml164.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>500</td></tr><tr><td headers="hd_h_ml164.t1_1_1_1_1 hd_h_ml164.t1_1_1_2_2 hd_h_ml164.t1_1_1_2_3 hd_h_ml164.t1_1_1_2_4 hd_h_ml164.t1_1_1_2_5 hd_h_ml164.t1_1_1_1_2 hd_h_ml164.t1_1_1_2_6" colspan="5" rowspan="1" style="text-align:center;vertical-align:middle;">Solubility: < 1 μM</td><td headers="hd_h_ml164.t1_1_1_1_3 hd_h_ml164.t1_1_1_2_7 hd_h_ml164.t1_1_1_2_8 hd_h_ml164.t1_1_1_2_9 hd_h_ml164.t1_1_1_2_10 hd_h_ml164.t1_1_1_1_4" colspan="5" rowspan="1" style="text-align:left;vertical-align:middle;">Purity: >95%</td></tr><tr><td headers="hd_h_ml164.t1_1_1_1_1 hd_h_ml164.t1_1_1_2_1" rowspan="2" colspan="1" style="text-align:center;vertical-align:middle;">2</td><td headers="hd_h_ml164.t1_1_1_1_1 hd_h_ml164.t1_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">5349954</td><td headers="hd_h_ml164.t1_1_1_1_1 hd_h_ml164.t1_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87556790" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87556790</a></td><td headers="hd_h_ml164.t1_1_1_1_1 hd_h_ml164.t1_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BRD-K91047982</td><td headers="hd_h_ml164.t1_1_1_1_1 hd_h_ml164.t1_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">P</td><td headers="hd_h_ml164.t1_1_1_1_2 hd_h_ml164.t1_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK56231/bin/ml164fu4.jpg" alt="Image ml164fu4.jpg" /></div></td><td headers="hd_h_ml164.t1_1_1_1_3 hd_h_ml164.t1_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml164.t1_1_1_1_3 hd_h_ml164.t1_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">inactive</td><td headers="hd_h_ml164.t1_1_1_1_3 hd_h_ml164.t1_1_1_2_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">-</td><td headers="hd_h_ml164.t1_1_1_1_3 hd_h_ml164.t1_1_1_2_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">ND</td><td headers="hd_h_ml164.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">NA</td></tr><tr><td headers="hd_h_ml164.t1_1_1_1_1 hd_h_ml164.t1_1_1_2_2 hd_h_ml164.t1_1_1_2_3 hd_h_ml164.t1_1_1_2_4 hd_h_ml164.t1_1_1_2_5 hd_h_ml164.t1_1_1_1_2 hd_h_ml164.t1_1_1_2_6" colspan="5" rowspan="1" style="text-align:center;vertical-align:middle;">Solubility: 105.4 μM</td><td headers="hd_h_ml164.t1_1_1_1_3 hd_h_ml164.t1_1_1_2_7 hd_h_ml164.t1_1_1_2_8 hd_h_ml164.t1_1_1_2_9 hd_h_ml164.t1_1_1_2_10 hd_h_ml164.t1_1_1_1_4" colspan="5" rowspan="1" style="text-align:left;vertical-align:middle;">Purity: >95%</td></tr><tr><td headers="hd_h_ml164.t1_1_1_1_1 hd_h_ml164.t1_1_1_2_1" rowspan="2" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml164.t1_1_1_1_1 hd_h_ml164.t1_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">9640748</td><td headers="hd_h_ml164.t1_1_1_1_1 hd_h_ml164.t1_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87556791" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87556791</a></td><td headers="hd_h_ml164.t1_1_1_1_1 hd_h_ml164.t1_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BRD-K17628612</td><td headers="hd_h_ml164.t1_1_1_1_1 hd_h_ml164.t1_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">P</td><td headers="hd_h_ml164.t1_1_1_1_2 hd_h_ml164.t1_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK56231/bin/ml164fu5.jpg" alt="Image ml164fu5.jpg" /></div></td><td headers="hd_h_ml164.t1_1_1_1_3 hd_h_ml164.t1_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml164.t1_1_1_1_3 hd_h_ml164.t1_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">inactive</td><td headers="hd_h_ml164.t1_1_1_1_3 hd_h_ml164.t1_1_1_2_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">-</td><td headers="hd_h_ml164.t1_1_1_1_3 hd_h_ml164.t1_1_1_2_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">ND</td><td headers="hd_h_ml164.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">NA</td></tr><tr><td headers="hd_h_ml164.t1_1_1_1_1 hd_h_ml164.t1_1_1_2_2 hd_h_ml164.t1_1_1_2_3 hd_h_ml164.t1_1_1_2_4 hd_h_ml164.t1_1_1_2_5 hd_h_ml164.t1_1_1_1_2 hd_h_ml164.t1_1_1_2_6" colspan="5" rowspan="1" style="text-align:center;vertical-align:middle;">Solubility: 17.3 μM</td><td headers="hd_h_ml164.t1_1_1_1_3 hd_h_ml164.t1_1_1_2_7 hd_h_ml164.t1_1_1_2_8 hd_h_ml164.t1_1_1_2_9 hd_h_ml164.t1_1_1_2_10 hd_h_ml164.t1_1_1_1_4" colspan="5" rowspan="1" style="text-align:left;vertical-align:middle;">Purity: >95%</td></tr></tbody></table></div><div><div><dl class="temp-labeled-list small"><dt></dt><dd><div id="ml164.tfn2"><p class="no_margin">NA=Not applicable; ND = Not determined</p></div></dd><dt>*</dt><dd><div id="ml164.tfn3"><p class="no_margin">P = Purchased</p></div></dd></dl></div></div></div><div id="ml164.t2" class="table"><h3><span class="label">Table 2</span><span class="title">SAR Showing the Influence of Aniline and Amide Substituents on <i>T.Cruzi</i> Inhibition</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK56231/table/ml164.t2/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml164.t2_lrgtbl__"><table class="no_margin"><thead><tr><th id="hd_h_ml164.t2_1_1_1_1" colspan="5" rowspan="1" style="text-align:center;vertical-align:middle;">SAR Analysis for <i>T. cruzi</i></th><th id="hd_h_ml164.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK56231/bin/ml164fu6.jpg" alt="Image ml164fu6.jpg" /></div></th><th id="hd_h_ml164.t2_1_1_1_3" colspan="4" rowspan="1" style="text-align:center;vertical-align:middle;">Potency (nM) mean ± S.E.M. (n=replicates)</th><th id="hd_h_ml164.t2_1_1_1_4" rowspan="2" colspan="1" headers="hd_h_ml164.t2_1_1_1_4" style="text-align:center;vertical-align:middle;">Target to Antitarget Fold Selectivity</th></tr><tr><th headers="hd_h_ml164.t2_1_1_1_1" id="hd_h_ml164.t2_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Entry</th><th headers="hd_h_ml164.t2_1_1_1_1" id="hd_h_ml164.t2_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CID</th><th headers="hd_h_ml164.t2_1_1_1_1" id="hd_h_ml164.t2_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SID</th><th headers="hd_h_ml164.t2_1_1_1_1" id="hd_h_ml164.t2_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Broad No.</th><th headers="hd_h_ml164.t2_1_1_1_1" id="hd_h_ml164.t2_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><sup><a class="bk_pop" href="#ml164.tfn5">*</a></sup></th><th headers="hd_h_ml164.t2_1_1_1_2" id="hd_h_ml164.t2_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">R</th><th headers="hd_h_ml164.t2_1_1_1_3" id="hd_h_ml164.t2_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">n</th><th headers="hd_h_ml164.t2_1_1_1_3" id="hd_h_ml164.t2_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><i>T. cruzi</i> inhibition</th><th headers="hd_h_ml164.t2_1_1_1_3" id="hd_h_ml164.t2_1_1_2_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">n</th><th headers="hd_h_ml164.t2_1_1_1_3" id="hd_h_ml164.t2_1_1_2_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Toxicity</th></tr></thead><tbody><tr><td headers="hd_h_ml164.t2_1_1_1_1 hd_h_ml164.t2_1_1_2_1" rowspan="2" colspan="1" style="text-align:center;vertical-align:middle;">1</td><td headers="hd_h_ml164.t2_1_1_1_1 hd_h_ml164.t2_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">6875690</td><td headers="hd_h_ml164.t2_1_1_1_1 hd_h_ml164.t2_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87219052" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87219052</a></td><td headers="hd_h_ml164.t2_1_1_1_1 hd_h_ml164.t2_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BRD-K64970062</td><td headers="hd_h_ml164.t2_1_1_1_1 hd_h_ml164.t2_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">P</td><td headers="hd_h_ml164.t2_1_1_1_2 hd_h_ml164.t2_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK56231/bin/ml164fu7.jpg" alt="Image ml164fu7.jpg" /></div></td><td headers="hd_h_ml164.t2_1_1_1_3 hd_h_ml164.t2_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml164.t2_1_1_1_3 hd_h_ml164.t2_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">34.3±3.1</td><td headers="hd_h_ml164.t2_1_1_1_3 hd_h_ml164.t2_1_1_2_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml164.t2_1_1_1_3 hd_h_ml164.t2_1_1_2_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">inactive</td><td headers="hd_h_ml164.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>500</td></tr><tr><td headers="hd_h_ml164.t2_1_1_1_1 hd_h_ml164.t2_1_1_2_2 hd_h_ml164.t2_1_1_2_3 hd_h_ml164.t2_1_1_2_4 hd_h_ml164.t2_1_1_2_5 hd_h_ml164.t2_1_1_1_2 hd_h_ml164.t2_1_1_2_6" colspan="5" rowspan="1" style="text-align:center;vertical-align:middle;">Solubility: < 1 μM</td><td headers="hd_h_ml164.t2_1_1_1_3 hd_h_ml164.t2_1_1_2_7 hd_h_ml164.t2_1_1_2_8 hd_h_ml164.t2_1_1_2_9 hd_h_ml164.t2_1_1_2_10 hd_h_ml164.t2_1_1_1_4" colspan="5" rowspan="1" style="text-align:left;vertical-align:middle;">Purity: >95%</td></tr><tr><td headers="hd_h_ml164.t2_1_1_1_1 hd_h_ml164.t2_1_1_2_1" rowspan="2" colspan="1" style="text-align:center;vertical-align:middle;">2</td><td headers="hd_h_ml164.t2_1_1_1_1 hd_h_ml164.t2_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">6875684</td><td headers="hd_h_ml164.t2_1_1_1_1 hd_h_ml164.t2_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99351071" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99351071</a></td><td headers="hd_h_ml164.t2_1_1_1_1 hd_h_ml164.t2_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BRD-K30246397</td><td headers="hd_h_ml164.t2_1_1_1_1 hd_h_ml164.t2_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">P</td><td headers="hd_h_ml164.t2_1_1_1_2 hd_h_ml164.t2_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK56231/bin/ml164fu8.jpg" alt="Image ml164fu8.jpg" /></div></td><td headers="hd_h_ml164.t2_1_1_1_3 hd_h_ml164.t2_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml164.t2_1_1_1_3 hd_h_ml164.t2_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3.00</td><td headers="hd_h_ml164.t2_1_1_1_3 hd_h_ml164.t2_1_1_2_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml164.t2_1_1_1_3 hd_h_ml164.t2_1_1_2_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">11400±0.847</td><td headers="hd_h_ml164.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>500</td></tr><tr><td headers="hd_h_ml164.t2_1_1_1_1 hd_h_ml164.t2_1_1_2_2 hd_h_ml164.t2_1_1_2_3 hd_h_ml164.t2_1_1_2_4 hd_h_ml164.t2_1_1_2_5 hd_h_ml164.t2_1_1_1_2 hd_h_ml164.t2_1_1_2_6" colspan="5" rowspan="1" style="text-align:center;vertical-align:middle;">Solubility: 5.9 μM</td><td headers="hd_h_ml164.t2_1_1_1_3 hd_h_ml164.t2_1_1_2_7 hd_h_ml164.t2_1_1_2_8 hd_h_ml164.t2_1_1_2_9 hd_h_ml164.t2_1_1_2_10 hd_h_ml164.t2_1_1_1_4" colspan="5" rowspan="1" style="text-align:left;vertical-align:middle;">Purity: 91%</td></tr><tr><td headers="hd_h_ml164.t2_1_1_1_1 hd_h_ml164.t2_1_1_2_1" rowspan="2" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml164.t2_1_1_1_1 hd_h_ml164.t2_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">6884367</td><td headers="hd_h_ml164.t2_1_1_1_1 hd_h_ml164.t2_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87219044" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87219044</a></td><td headers="hd_h_ml164.t2_1_1_1_1 hd_h_ml164.t2_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BRD-K85660637</td><td headers="hd_h_ml164.t2_1_1_1_1 hd_h_ml164.t2_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">P</td><td headers="hd_h_ml164.t2_1_1_1_2 hd_h_ml164.t2_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK56231/bin/ml164fu9.jpg" alt="Image ml164fu9.jpg" /></div></td><td headers="hd_h_ml164.t2_1_1_1_3 hd_h_ml164.t2_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml164.t2_1_1_1_3 hd_h_ml164.t2_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">30.8±9.7</td><td headers="hd_h_ml164.t2_1_1_1_3 hd_h_ml164.t2_1_1_2_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml164.t2_1_1_1_3 hd_h_ml164.t2_1_1_2_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">17550</td><td headers="hd_h_ml164.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>500</td></tr><tr><td headers="hd_h_ml164.t2_1_1_1_1 hd_h_ml164.t2_1_1_2_2 hd_h_ml164.t2_1_1_2_3 hd_h_ml164.t2_1_1_2_4 hd_h_ml164.t2_1_1_2_5 hd_h_ml164.t2_1_1_1_2 hd_h_ml164.t2_1_1_2_6" colspan="5" rowspan="1" style="text-align:center;vertical-align:middle;">Solubility: ND</td><td headers="hd_h_ml164.t2_1_1_1_3 hd_h_ml164.t2_1_1_2_7 hd_h_ml164.t2_1_1_2_8 hd_h_ml164.t2_1_1_2_9 hd_h_ml164.t2_1_1_2_10 hd_h_ml164.t2_1_1_1_4" colspan="5" rowspan="1" style="text-align:left;vertical-align:middle;">Purity: >95%</td></tr><tr><td headers="hd_h_ml164.t2_1_1_1_1 hd_h_ml164.t2_1_1_2_1" rowspan="2" colspan="1" style="text-align:center;vertical-align:middle;">4</td><td headers="hd_h_ml164.t2_1_1_1_1 hd_h_ml164.t2_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">6872741</td><td headers="hd_h_ml164.t2_1_1_1_1 hd_h_ml164.t2_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87556788" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87556788</a></td><td headers="hd_h_ml164.t2_1_1_1_1 hd_h_ml164.t2_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BRD-K73819439</td><td headers="hd_h_ml164.t2_1_1_1_1 hd_h_ml164.t2_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">P</td><td headers="hd_h_ml164.t2_1_1_1_2 hd_h_ml164.t2_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK56231/bin/ml164fu10.jpg" alt="Image ml164fu10.jpg" /></div></td><td headers="hd_h_ml164.t2_1_1_1_3 hd_h_ml164.t2_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml164.t2_1_1_1_3 hd_h_ml164.t2_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">61.3±14.8</td><td headers="hd_h_ml164.t2_1_1_1_3 hd_h_ml164.t2_1_1_2_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml164.t2_1_1_1_3 hd_h_ml164.t2_1_1_2_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">35600±14000</td><td headers="hd_h_ml164.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>500</td></tr><tr><td headers="hd_h_ml164.t2_1_1_1_1 hd_h_ml164.t2_1_1_2_2 hd_h_ml164.t2_1_1_2_3 hd_h_ml164.t2_1_1_2_4 hd_h_ml164.t2_1_1_2_5 hd_h_ml164.t2_1_1_1_2 hd_h_ml164.t2_1_1_2_6" colspan="5" rowspan="1" style="text-align:center;vertical-align:middle;">Solubility: ND</td><td headers="hd_h_ml164.t2_1_1_1_3 hd_h_ml164.t2_1_1_2_7 hd_h_ml164.t2_1_1_2_8 hd_h_ml164.t2_1_1_2_9 hd_h_ml164.t2_1_1_2_10 hd_h_ml164.t2_1_1_1_4" colspan="5" rowspan="1" style="text-align:left;vertical-align:middle;">Purity: 94%</td></tr><tr><td headers="hd_h_ml164.t2_1_1_1_1 hd_h_ml164.t2_1_1_2_1" rowspan="2" colspan="1" style="text-align:center;vertical-align:middle;">5</td><td headers="hd_h_ml164.t2_1_1_1_1 hd_h_ml164.t2_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">9689369</td><td headers="hd_h_ml164.t2_1_1_1_1 hd_h_ml164.t2_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99351075" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99351075</a></td><td headers="hd_h_ml164.t2_1_1_1_1 hd_h_ml164.t2_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BRD-K60134467</td><td headers="hd_h_ml164.t2_1_1_1_1 hd_h_ml164.t2_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">P</td><td headers="hd_h_ml164.t2_1_1_1_2 hd_h_ml164.t2_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK56231/bin/ml164fu11.jpg" alt="Image ml164fu11.jpg" /></div></td><td headers="hd_h_ml164.t2_1_1_1_3 hd_h_ml164.t2_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml164.t2_1_1_1_3 hd_h_ml164.t2_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">124±59.8</td><td headers="hd_h_ml164.t2_1_1_1_3 hd_h_ml164.t2_1_1_2_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml164.t2_1_1_1_3 hd_h_ml164.t2_1_1_2_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">5350±799</td><td headers="hd_h_ml164.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">43</td></tr><tr><td headers="hd_h_ml164.t2_1_1_1_1 hd_h_ml164.t2_1_1_2_2 hd_h_ml164.t2_1_1_2_3 hd_h_ml164.t2_1_1_2_4 hd_h_ml164.t2_1_1_2_5 hd_h_ml164.t2_1_1_1_2 hd_h_ml164.t2_1_1_2_6" colspan="5" rowspan="1" style="text-align:center;vertical-align:middle;">Solubility: ND</td><td headers="hd_h_ml164.t2_1_1_1_3 hd_h_ml164.t2_1_1_2_7 hd_h_ml164.t2_1_1_2_8 hd_h_ml164.t2_1_1_2_9 hd_h_ml164.t2_1_1_2_10 hd_h_ml164.t2_1_1_1_4" colspan="5" rowspan="1" style="text-align:left;vertical-align:middle;">Purity: 87%</td></tr><tr><td headers="hd_h_ml164.t2_1_1_1_1 hd_h_ml164.t2_1_1_2_1" rowspan="2" colspan="1" style="text-align:center;vertical-align:middle;">6</td><td headers="hd_h_ml164.t2_1_1_1_1 hd_h_ml164.t2_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">9563094</td><td headers="hd_h_ml164.t2_1_1_1_1 hd_h_ml164.t2_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99351068" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99351068</a></td><td headers="hd_h_ml164.t2_1_1_1_1 hd_h_ml164.t2_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BRD-K56690068</td><td headers="hd_h_ml164.t2_1_1_1_1 hd_h_ml164.t2_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">P</td><td headers="hd_h_ml164.t2_1_1_1_2 hd_h_ml164.t2_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK56231/bin/ml164fu12.jpg" alt="Image ml164fu12.jpg" /></div></td><td headers="hd_h_ml164.t2_1_1_1_3 hd_h_ml164.t2_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml164.t2_1_1_1_3 hd_h_ml164.t2_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">130±31.3</td><td headers="hd_h_ml164.t2_1_1_1_3 hd_h_ml164.t2_1_1_2_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml164.t2_1_1_1_3 hd_h_ml164.t2_1_1_2_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">23100±7230</td><td headers="hd_h_ml164.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">178</td></tr><tr><td headers="hd_h_ml164.t2_1_1_1_1 hd_h_ml164.t2_1_1_2_2 hd_h_ml164.t2_1_1_2_3 hd_h_ml164.t2_1_1_2_4 hd_h_ml164.t2_1_1_2_5 hd_h_ml164.t2_1_1_1_2 hd_h_ml164.t2_1_1_2_6" colspan="5" rowspan="1" style="text-align:center;vertical-align:middle;">Solubility: ND</td><td headers="hd_h_ml164.t2_1_1_1_3 hd_h_ml164.t2_1_1_2_7 hd_h_ml164.t2_1_1_2_8 hd_h_ml164.t2_1_1_2_9 hd_h_ml164.t2_1_1_2_10 hd_h_ml164.t2_1_1_1_4" colspan="5" rowspan="1" style="text-align:left;vertical-align:middle;">Purity: 79%</td></tr></tbody></table></div><div><div><dl class="temp-labeled-list small"><dt></dt><dd><div id="ml164.tfn4"><p class="no_margin">ND = Not determined</p></div></dd><dt>*</dt><dd><div id="ml164.tfn5"><p class="no_margin">P = Purchased</p></div></dd></dl></div></div></div><div id="ml164.t3" class="table"><h3><span class="label">Table 3</span><span class="title">SAR Showing the Influence of Other Substituents on <i>T.cruzi</i> Inhibition</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK56231/table/ml164.t3/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml164.t3_lrgtbl__"><table class="no_margin"><thead><tr><th id="hd_h_ml164.t3_1_1_1_1" colspan="5" rowspan="1" style="text-align:center;vertical-align:middle;">SAR Analysis for <i>T. cruzi</i></th><th id="hd_h_ml164.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK56231/bin/ml164fu13.jpg" alt="Image ml164fu13.jpg" /></div></th><th id="hd_h_ml164.t3_1_1_1_3" colspan="4" rowspan="1" style="text-align:center;vertical-align:middle;">Potency (nM) mean ± S.E.M. (n=replicates)</th><th id="hd_h_ml164.t3_1_1_1_4" rowspan="2" colspan="1" headers="hd_h_ml164.t3_1_1_1_4" style="text-align:center;vertical-align:middle;">Target to Antitarget Fold Selectivity</th></tr><tr><th headers="hd_h_ml164.t3_1_1_1_1" id="hd_h_ml164.t3_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Entry</th><th headers="hd_h_ml164.t3_1_1_1_1" id="hd_h_ml164.t3_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CID</th><th headers="hd_h_ml164.t3_1_1_1_1" id="hd_h_ml164.t3_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SID</th><th headers="hd_h_ml164.t3_1_1_1_1" id="hd_h_ml164.t3_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Broad No.</th><th headers="hd_h_ml164.t3_1_1_1_1" id="hd_h_ml164.t3_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><sup><a class="bk_pop" href="#ml164.tfn7">*</a></sup></th><th headers="hd_h_ml164.t3_1_1_1_2" id="hd_h_ml164.t3_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">R</th><th headers="hd_h_ml164.t3_1_1_1_3" id="hd_h_ml164.t3_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">n</th><th headers="hd_h_ml164.t3_1_1_1_3" id="hd_h_ml164.t3_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><i>T. cruzi</i> inhibition</th><th headers="hd_h_ml164.t3_1_1_1_3" id="hd_h_ml164.t3_1_1_2_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">n</th><th headers="hd_h_ml164.t3_1_1_1_3" id="hd_h_ml164.t3_1_1_2_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Toxicity</th></tr></thead><tbody><tr><td headers="hd_h_ml164.t3_1_1_1_1 hd_h_ml164.t3_1_1_2_1" rowspan="2" colspan="1" style="text-align:center;vertical-align:middle;">1</td><td headers="hd_h_ml164.t3_1_1_1_1 hd_h_ml164.t3_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">5344250</td><td headers="hd_h_ml164.t3_1_1_1_1 hd_h_ml164.t3_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87556789" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87556789</a></td><td headers="hd_h_ml164.t3_1_1_1_1 hd_h_ml164.t3_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BRD-K67470788</td><td headers="hd_h_ml164.t3_1_1_1_1 hd_h_ml164.t3_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">P</td><td headers="hd_h_ml164.t3_1_1_1_2 hd_h_ml164.t3_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK56231/bin/ml164fu14.jpg" alt="Image ml164fu14.jpg" /></div></td><td headers="hd_h_ml164.t3_1_1_1_3 hd_h_ml164.t3_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml164.t3_1_1_1_3 hd_h_ml164.t3_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">145±26</td><td headers="hd_h_ml164.t3_1_1_1_3 hd_h_ml164.t3_1_1_2_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml164.t3_1_1_1_3 hd_h_ml164.t3_1_1_2_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">inactive</td><td headers="hd_h_ml164.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>500</td></tr><tr><td headers="hd_h_ml164.t3_1_1_1_1 hd_h_ml164.t3_1_1_2_2 hd_h_ml164.t3_1_1_2_3 hd_h_ml164.t3_1_1_2_4 hd_h_ml164.t3_1_1_2_5 hd_h_ml164.t3_1_1_1_2 hd_h_ml164.t3_1_1_2_6" colspan="5" rowspan="1" style="text-align:center;vertical-align:middle;">Solubility: >500 μM</td><td headers="hd_h_ml164.t3_1_1_1_3 hd_h_ml164.t3_1_1_2_7 hd_h_ml164.t3_1_1_2_8 hd_h_ml164.t3_1_1_2_9 hd_h_ml164.t3_1_1_2_10 hd_h_ml164.t3_1_1_1_4" colspan="5" rowspan="1" style="text-align:left;vertical-align:middle;">Purity: >95%</td></tr><tr><td headers="hd_h_ml164.t3_1_1_1_1 hd_h_ml164.t3_1_1_2_1" rowspan="2" colspan="1" style="text-align:center;vertical-align:middle;">2</td><td headers="hd_h_ml164.t3_1_1_1_1 hd_h_ml164.t3_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">6875617</td><td headers="hd_h_ml164.t3_1_1_1_1 hd_h_ml164.t3_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87219027" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87219027</a></td><td headers="hd_h_ml164.t3_1_1_1_1 hd_h_ml164.t3_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BRD-K02558072</td><td headers="hd_h_ml164.t3_1_1_1_1 hd_h_ml164.t3_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">P</td><td headers="hd_h_ml164.t3_1_1_1_2 hd_h_ml164.t3_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK56231/bin/ml164fu15.jpg" alt="Image ml164fu15.jpg" /></div></td><td headers="hd_h_ml164.t3_1_1_1_3 hd_h_ml164.t3_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml164.t3_1_1_1_3 hd_h_ml164.t3_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">193±63</td><td headers="hd_h_ml164.t3_1_1_1_3 hd_h_ml164.t3_1_1_2_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml164.t3_1_1_1_3 hd_h_ml164.t3_1_1_2_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">19500</td><td headers="hd_h_ml164.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">101</td></tr><tr><td headers="hd_h_ml164.t3_1_1_1_1 hd_h_ml164.t3_1_1_2_2 hd_h_ml164.t3_1_1_2_3 hd_h_ml164.t3_1_1_2_4 hd_h_ml164.t3_1_1_2_5 hd_h_ml164.t3_1_1_1_2 hd_h_ml164.t3_1_1_2_6" colspan="5" rowspan="1" style="text-align:center;vertical-align:middle;">Solubility: >500 μM</td><td headers="hd_h_ml164.t3_1_1_1_3 hd_h_ml164.t3_1_1_2_7 hd_h_ml164.t3_1_1_2_8 hd_h_ml164.t3_1_1_2_9 hd_h_ml164.t3_1_1_2_10 hd_h_ml164.t3_1_1_1_4" colspan="5" rowspan="1" style="text-align:left;vertical-align:middle;">Purity: >95%</td></tr><tr><td headers="hd_h_ml164.t3_1_1_1_1 hd_h_ml164.t3_1_1_2_1" rowspan="2" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml164.t3_1_1_1_1 hd_h_ml164.t3_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">6322955</td><td headers="hd_h_ml164.t3_1_1_1_1 hd_h_ml164.t3_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99351074" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99351074</a></td><td headers="hd_h_ml164.t3_1_1_1_1 hd_h_ml164.t3_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BRD-K32049792</td><td headers="hd_h_ml164.t3_1_1_1_1 hd_h_ml164.t3_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">P</td><td headers="hd_h_ml164.t3_1_1_1_2 hd_h_ml164.t3_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
|
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<div class="graphic"><img src="/books/NBK56231/bin/ml164fu16.jpg" alt="Image ml164fu16.jpg" /></div></td><td headers="hd_h_ml164.t3_1_1_1_3 hd_h_ml164.t3_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml164.t3_1_1_1_3 hd_h_ml164.t3_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">317±103</td><td headers="hd_h_ml164.t3_1_1_1_3 hd_h_ml164.t3_1_1_2_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml164.t3_1_1_1_3 hd_h_ml164.t3_1_1_2_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">21200±611</td><td headers="hd_h_ml164.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">67</td></tr><tr><td headers="hd_h_ml164.t3_1_1_1_1 hd_h_ml164.t3_1_1_2_2 hd_h_ml164.t3_1_1_2_3 hd_h_ml164.t3_1_1_2_4 hd_h_ml164.t3_1_1_2_5 hd_h_ml164.t3_1_1_1_2 hd_h_ml164.t3_1_1_2_6" colspan="5" rowspan="1" style="text-align:center;vertical-align:middle;">Solubility: ND</td><td headers="hd_h_ml164.t3_1_1_1_3 hd_h_ml164.t3_1_1_2_7 hd_h_ml164.t3_1_1_2_8 hd_h_ml164.t3_1_1_2_9 hd_h_ml164.t3_1_1_2_10 hd_h_ml164.t3_1_1_1_4" colspan="5" rowspan="1" style="text-align:left;vertical-align:middle;">Purity: 96%</td></tr><tr><td headers="hd_h_ml164.t3_1_1_1_1 hd_h_ml164.t3_1_1_2_1" rowspan="2" colspan="1" style="text-align:center;vertical-align:middle;">4</td><td headers="hd_h_ml164.t3_1_1_1_1 hd_h_ml164.t3_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">9557463</td><td headers="hd_h_ml164.t3_1_1_1_1 hd_h_ml164.t3_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/104170274" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">104170274</a></td><td headers="hd_h_ml164.t3_1_1_1_1 hd_h_ml164.t3_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BRD-K38455523</td><td headers="hd_h_ml164.t3_1_1_1_1 hd_h_ml164.t3_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">P</td><td headers="hd_h_ml164.t3_1_1_1_2 hd_h_ml164.t3_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
|
||
<div class="graphic"><img src="/books/NBK56231/bin/ml164fu17.jpg" alt="Image ml164fu17.jpg" /></div></td><td headers="hd_h_ml164.t3_1_1_1_3 hd_h_ml164.t3_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml164.t3_1_1_1_3 hd_h_ml164.t3_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">335±121</td><td headers="hd_h_ml164.t3_1_1_1_3 hd_h_ml164.t3_1_1_2_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml164.t3_1_1_1_3 hd_h_ml164.t3_1_1_2_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">23600±5700</td><td headers="hd_h_ml164.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">70</td></tr><tr><td headers="hd_h_ml164.t3_1_1_1_1 hd_h_ml164.t3_1_1_2_2 hd_h_ml164.t3_1_1_2_3 hd_h_ml164.t3_1_1_2_4 hd_h_ml164.t3_1_1_2_5 hd_h_ml164.t3_1_1_1_2 hd_h_ml164.t3_1_1_2_6" colspan="5" rowspan="1" style="text-align:center;vertical-align:middle;">Solubility: 12.4 μM</td><td headers="hd_h_ml164.t3_1_1_1_3 hd_h_ml164.t3_1_1_2_7 hd_h_ml164.t3_1_1_2_8 hd_h_ml164.t3_1_1_2_9 hd_h_ml164.t3_1_1_2_10 hd_h_ml164.t3_1_1_1_4" colspan="5" rowspan="1" style="text-align:left;vertical-align:middle;">Purity: >95%</td></tr><tr><td headers="hd_h_ml164.t3_1_1_1_1 hd_h_ml164.t3_1_1_2_1" rowspan="2" colspan="1" style="text-align:center;vertical-align:middle;">5</td><td headers="hd_h_ml164.t3_1_1_1_1 hd_h_ml164.t3_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">6875630</td><td headers="hd_h_ml164.t3_1_1_1_1 hd_h_ml164.t3_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99351076" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99351076</a></td><td headers="hd_h_ml164.t3_1_1_1_1 hd_h_ml164.t3_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BRD-K52487866</td><td headers="hd_h_ml164.t3_1_1_1_1 hd_h_ml164.t3_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">P</td><td headers="hd_h_ml164.t3_1_1_1_2 hd_h_ml164.t3_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
|
||
<div class="graphic"><img src="/books/NBK56231/bin/ml164fu18.jpg" alt="Image ml164fu18.jpg" /></div></td><td headers="hd_h_ml164.t3_1_1_1_3 hd_h_ml164.t3_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml164.t3_1_1_1_3 hd_h_ml164.t3_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">501±234</td><td headers="hd_h_ml164.t3_1_1_1_3 hd_h_ml164.t3_1_1_2_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml164.t3_1_1_1_3 hd_h_ml164.t3_1_1_2_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">21500±1180</td><td headers="hd_h_ml164.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">43</td></tr><tr><td headers="hd_h_ml164.t3_1_1_1_1 hd_h_ml164.t3_1_1_2_2 hd_h_ml164.t3_1_1_2_3 hd_h_ml164.t3_1_1_2_4 hd_h_ml164.t3_1_1_2_5 hd_h_ml164.t3_1_1_1_2 hd_h_ml164.t3_1_1_2_6" colspan="5" rowspan="1" style="text-align:center;vertical-align:middle;">Solubility: 37.9 μM</td><td headers="hd_h_ml164.t3_1_1_1_3 hd_h_ml164.t3_1_1_2_7 hd_h_ml164.t3_1_1_2_8 hd_h_ml164.t3_1_1_2_9 hd_h_ml164.t3_1_1_2_10 hd_h_ml164.t3_1_1_1_4" colspan="5" rowspan="1" style="text-align:left;vertical-align:middle;">Purity: 87%</td></tr><tr><td headers="hd_h_ml164.t3_1_1_1_1 hd_h_ml164.t3_1_1_2_1" rowspan="2" colspan="1" style="text-align:center;vertical-align:middle;">6</td><td headers="hd_h_ml164.t3_1_1_1_1 hd_h_ml164.t3_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">5332109</td><td headers="hd_h_ml164.t3_1_1_1_1 hd_h_ml164.t3_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99351064" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99351064</a></td><td headers="hd_h_ml164.t3_1_1_1_1 hd_h_ml164.t3_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BRD-K75855222</td><td headers="hd_h_ml164.t3_1_1_1_1 hd_h_ml164.t3_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">P</td><td headers="hd_h_ml164.t3_1_1_1_2 hd_h_ml164.t3_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
|
||
<div class="graphic"><img src="/books/NBK56231/bin/ml164fu19.jpg" alt="Image ml164fu19.jpg" /></div></td><td headers="hd_h_ml164.t3_1_1_1_3 hd_h_ml164.t3_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml164.t3_1_1_1_3 hd_h_ml164.t3_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">680±491</td><td headers="hd_h_ml164.t3_1_1_1_3 hd_h_ml164.t3_1_1_2_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml164.t3_1_1_1_3 hd_h_ml164.t3_1_1_2_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">26700±9270</td><td headers="hd_h_ml164.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">39</td></tr><tr><td headers="hd_h_ml164.t3_1_1_1_1 hd_h_ml164.t3_1_1_2_2 hd_h_ml164.t3_1_1_2_3 hd_h_ml164.t3_1_1_2_4 hd_h_ml164.t3_1_1_2_5 hd_h_ml164.t3_1_1_1_2 hd_h_ml164.t3_1_1_2_6" colspan="5" rowspan="1" style="text-align:center;vertical-align:middle;">Solubility: ND</td><td headers="hd_h_ml164.t3_1_1_1_3 hd_h_ml164.t3_1_1_2_7 hd_h_ml164.t3_1_1_2_8 hd_h_ml164.t3_1_1_2_9 hd_h_ml164.t3_1_1_2_10 hd_h_ml164.t3_1_1_1_4" colspan="5" rowspan="1" style="text-align:left;vertical-align:middle;">Purity: 92%</td></tr><tr><td headers="hd_h_ml164.t3_1_1_1_1 hd_h_ml164.t3_1_1_2_1" rowspan="2" colspan="1" style="text-align:center;vertical-align:middle;">7</td><td headers="hd_h_ml164.t3_1_1_1_1 hd_h_ml164.t3_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">21210609</td><td headers="hd_h_ml164.t3_1_1_1_1 hd_h_ml164.t3_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/104170282" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">104170282</a></td><td headers="hd_h_ml164.t3_1_1_1_1 hd_h_ml164.t3_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BRD-K79949483</td><td headers="hd_h_ml164.t3_1_1_1_1 hd_h_ml164.t3_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">P</td><td headers="hd_h_ml164.t3_1_1_1_2 hd_h_ml164.t3_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
|
||
<div class="graphic"><img src="/books/NBK56231/bin/ml164fu20.jpg" alt="Image ml164fu20.jpg" /></div></td><td headers="hd_h_ml164.t3_1_1_1_3 hd_h_ml164.t3_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml164.t3_1_1_1_3 hd_h_ml164.t3_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">752±111</td><td headers="hd_h_ml164.t3_1_1_1_3 hd_h_ml164.t3_1_1_2_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml164.t3_1_1_1_3 hd_h_ml164.t3_1_1_2_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">inactive</td><td headers="hd_h_ml164.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>500</td></tr><tr><td headers="hd_h_ml164.t3_1_1_1_1 hd_h_ml164.t3_1_1_2_2 hd_h_ml164.t3_1_1_2_3 hd_h_ml164.t3_1_1_2_4 hd_h_ml164.t3_1_1_2_5 hd_h_ml164.t3_1_1_1_2 hd_h_ml164.t3_1_1_2_6" colspan="5" rowspan="1" style="text-align:center;vertical-align:middle;">Solubility: ND</td><td headers="hd_h_ml164.t3_1_1_1_3 hd_h_ml164.t3_1_1_2_7 hd_h_ml164.t3_1_1_2_8 hd_h_ml164.t3_1_1_2_9 hd_h_ml164.t3_1_1_2_10 hd_h_ml164.t3_1_1_1_4" colspan="5" rowspan="1" style="text-align:left;vertical-align:middle;">Purity: >95%</td></tr><tr><td headers="hd_h_ml164.t3_1_1_1_1 hd_h_ml164.t3_1_1_2_1" rowspan="2" colspan="1" style="text-align:center;vertical-align:middle;">8</td><td headers="hd_h_ml164.t3_1_1_1_1 hd_h_ml164.t3_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">9558304</td><td headers="hd_h_ml164.t3_1_1_1_1 hd_h_ml164.t3_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/104170276" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">104170276</a></td><td headers="hd_h_ml164.t3_1_1_1_1 hd_h_ml164.t3_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BRD-K41404496</td><td headers="hd_h_ml164.t3_1_1_1_1 hd_h_ml164.t3_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">P</td><td headers="hd_h_ml164.t3_1_1_1_2 hd_h_ml164.t3_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
|
||
<div class="graphic"><img src="/books/NBK56231/bin/ml164fu21.jpg" alt="Image ml164fu21.jpg" /></div></td><td headers="hd_h_ml164.t3_1_1_1_3 hd_h_ml164.t3_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml164.t3_1_1_1_3 hd_h_ml164.t3_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">838±513</td><td headers="hd_h_ml164.t3_1_1_1_3 hd_h_ml164.t3_1_1_2_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml164.t3_1_1_1_3 hd_h_ml164.t3_1_1_2_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">22300±5110</td><td headers="hd_h_ml164.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">27</td></tr><tr><td headers="hd_h_ml164.t3_1_1_1_1 hd_h_ml164.t3_1_1_2_2 hd_h_ml164.t3_1_1_2_3 hd_h_ml164.t3_1_1_2_4 hd_h_ml164.t3_1_1_2_5 hd_h_ml164.t3_1_1_1_2 hd_h_ml164.t3_1_1_2_6" colspan="5" rowspan="1" style="text-align:center;vertical-align:middle;">Solubility: ND</td><td headers="hd_h_ml164.t3_1_1_1_3 hd_h_ml164.t3_1_1_2_7 hd_h_ml164.t3_1_1_2_8 hd_h_ml164.t3_1_1_2_9 hd_h_ml164.t3_1_1_2_10 hd_h_ml164.t3_1_1_1_4" colspan="5" rowspan="1" style="text-align:left;vertical-align:middle;">Purity: 81%</td></tr><tr><td headers="hd_h_ml164.t3_1_1_1_1 hd_h_ml164.t3_1_1_2_1" rowspan="2" colspan="1" style="text-align:center;vertical-align:middle;">9</td><td headers="hd_h_ml164.t3_1_1_1_1 hd_h_ml164.t3_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">5334382</td><td headers="hd_h_ml164.t3_1_1_1_1 hd_h_ml164.t3_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/104170280" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">104170280</a></td><td headers="hd_h_ml164.t3_1_1_1_1 hd_h_ml164.t3_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BRD-K81091498</td><td headers="hd_h_ml164.t3_1_1_1_1 hd_h_ml164.t3_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">P</td><td headers="hd_h_ml164.t3_1_1_1_2 hd_h_ml164.t3_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
|
||
<div class="graphic"><img src="/books/NBK56231/bin/ml164fu22.jpg" alt="Image ml164fu22.jpg" /></div></td><td headers="hd_h_ml164.t3_1_1_1_3 hd_h_ml164.t3_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml164.t3_1_1_1_3 hd_h_ml164.t3_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1470±800</td><td headers="hd_h_ml164.t3_1_1_1_3 hd_h_ml164.t3_1_1_2_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml164.t3_1_1_1_3 hd_h_ml164.t3_1_1_2_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">28000±4080</td><td headers="hd_h_ml164.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">19</td></tr><tr><td headers="hd_h_ml164.t3_1_1_1_1 hd_h_ml164.t3_1_1_2_2 hd_h_ml164.t3_1_1_2_3 hd_h_ml164.t3_1_1_2_4 hd_h_ml164.t3_1_1_2_5 hd_h_ml164.t3_1_1_1_2 hd_h_ml164.t3_1_1_2_6" colspan="5" rowspan="1" style="text-align:center;vertical-align:middle;">Solubility: ND</td><td headers="hd_h_ml164.t3_1_1_1_3 hd_h_ml164.t3_1_1_2_7 hd_h_ml164.t3_1_1_2_8 hd_h_ml164.t3_1_1_2_9 hd_h_ml164.t3_1_1_2_10 hd_h_ml164.t3_1_1_1_4" colspan="5" rowspan="1" style="text-align:left;vertical-align:middle;">Purity: 94%</td></tr></tbody></table></div><div><div><dl class="temp-labeled-list small"><dt></dt><dd><div id="ml164.tfn6"><p class="no_margin">ND = Not determined</p></div></dd><dt>*</dt><dd><div id="ml164.tfn7"><p class="no_margin">P = Purchased</p></div></dd></dl></div></div></div><p>It is interesting that switching the xylene in (entry 1<b>, </b><a class="figpopup" href="/books/NBK56231/table/ml164.t2/?report=objectonly" target="object" rid-figpopup="figml164t2" rid-ob="figobml164t2">Table 2</a>) to naphthalene in (entry 3, <a class="figpopup" href="/books/NBK56231/table/ml164.t2/?report=objectonly" target="object" rid-figpopup="figml164t2" rid-ob="figobml164t2">Table 2</a>) or 2,4-dichlorophenyl in (entry 2, <a class="figpopup" href="/books/NBK56231/table/ml164.t2/?report=objectonly" target="object" rid-figpopup="figml164t2" rid-ob="figobml164t2">Table 2</a>) results in an increase in potency, but increases toxicity. Replacement of the aromatic amine with morpholine (entry 1, <a class="figpopup" href="/books/NBK56231/table/ml164.t3/?report=objectonly" target="object" rid-figpopup="figml164t3" rid-ob="figobml164t3">Table 3</a>) results in a 4-fold decrease in potency; however, it increases solubility to >500 μM.</p><p>We next investigated other types of substituents on the eastern side of the molecule. Replacement of the hydrazone amide with a carbamate (entry 2<b>, </b><a class="figpopup" href="/books/NBK56231/table/ml164.t3/?report=objectonly" target="object" rid-figpopup="figml164t3" rid-ob="figobml164t3">Table 3</a>) also resulted in an increase of solubility to >500 μM; however, it increases toxicity. Alkyl substituents result in a slight increase in solubility, but a greater than 10-fold decrease in potency (<a class="figpopup" href="/books/NBK56231/table/ml164.t3/?report=objectonly" target="object" rid-figpopup="figml164t3" rid-ob="figobml164t3">Table 3</a>). Compound CID 6875690 should serve as a useful probe for cell biology research because of its low molecular weight and good physical properties. Furthermore, the aromatic ring could be further substituted if an analog were sought with optimized drug metabolism pharmacokinetic (DMPK) properties.</p><p>The activity of the piperidine series of analogs in the <i>T. cruzi</i> inhibition assays are shown in <a class="figpopup" href="/books/NBK56231/figure/ml164.f7/?report=objectonly" target="object" rid-figpopup="figml164f7" rid-ob="figobml164f7">Figure 7</a>.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml164f7" co-legend-rid="figlgndml164f7"><a href="/books/NBK56231/figure/ml164.f7/?report=objectonly" target="object" title="Figure 7" class="img_link icnblk_img figpopup" rid-figpopup="figml164f7" rid-ob="figobml164f7"><img class="small-thumb" src="/books/NBK56231/bin/ml164f7a.gif" src-large="/books/NBK56231/bin/ml164f7a.jpg" alt="Figure 7. Activity of Piperidine Series Analogs in T cruzi Inhibition Assays (AID 2630)." /></a><div class="icnblk_cntnt" id="figlgndml164f7"><h4 id="ml164.f7"><a href="/books/NBK56231/figure/ml164.f7/?report=objectonly" target="object" rid-ob="figobml164f7">Figure 7</a></h4><p class="float-caption no_bottom_margin">Activity of Piperidine Series Analogs in <i>T cruzi</i> Inhibition Assays (AID 2630). Representative curves of sixteen analogs tested in the primary screen at dose. CID 6875684 (<i>A</i>), CID 6884367 (<i>B</i>), CID 6872741 (<i>C</i>), CID 9689369 (<i>D</i>), CID 9563094 (<i>E</i>), CID 5344250 <a href="/books/NBK56231/figure/ml164.f7/?report=objectonly" target="object" rid-ob="figobml164f7">(more...)</a></p></div></div></div><div id="ml164.s21"><h3>3.5. Cellular Activity</h3><p>As the primary assay is a phenotypic, cell-based assay, the low nanomolar (nM) activity of the probe (CID 6875690/<a href="/pcsubstance/?term=ML164[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML164</a>) as described above by definition indicates excellent permeability and good physical properties.</p></div><div id="ml164.s22"><h3>3.6. Profiling Assays</h3><p>Not applicable.</p></div></div><div id="ml164.s23"><h2 id="_ml164_s23_">4. Discussion</h2><div id="ml164.s24"><h3>4.1. Comparison to Existing Art and Feature/Benefits of the New Probe</h3><p>The current state of the art for probe molecules in cell culture assays against <i>T. cruzi</i> is a potency of about 1 μM (<a class="bk_pop" href="#ml164.r6">6</a>), whereas our new probe (CID 6875690/<a href="/pcsubstance/?term=ML164[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML164</a>) is much more potent at 34.3 nM, a greater than 30-fold improvement. Also, the current treatments for Chagas disease are toxic to host cells. Probe (CID 6875690/<a href="/pcsubstance/?term=ML164[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML164</a>) is inactive against host cells at the highest tested dose (19.5 μM), a difference of greater than 500-fold compared with its’ activity against <i>T. cruzi</i>. These features should make this probe (CID 6875690/<a href="/pcsubstance/?term=ML164[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML164</a>) very useful as a probe molecule in cell assays and potentially facilitate future <i>in vivo</i> studies.</p><p>Investigation into relevant prior art entailed searching the following databases: SciFinder, Patent Lens, PubChem, and PubMed. The search terms applied and hit statistics are provided in <a class="figpopup" href="/books/NBK56231/table/ml164.t4/?report=objectonly" target="object" rid-figpopup="figml164t4" rid-ob="figobml164t4">Table 4</a>. Abstracts were obtained for all references returned and were analyzed for relevance to the current project. The searches were performed on and are current as of February 16, 2011.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml164t4"><a href="/books/NBK56231/table/ml164.t4/?report=objectonly" target="object" title="Table 4" class="img_link icnblk_img figpopup" rid-figpopup="figml164t4" rid-ob="figobml164t4"><img class="small-thumb" src="/books/NBK56231/table/ml164.t4/?report=thumb" src-large="/books/NBK56231/table/ml164.t4/?report=previmg" alt="Table 4. Search Strings and Databases Employed in the Prior Art Search." /></a><div class="icnblk_cntnt"><h4 id="ml164.t4"><a href="/books/NBK56231/table/ml164.t4/?report=objectonly" target="object" rid-ob="figobml164t4">Table 4</a></h4><p class="float-caption no_bottom_margin">Search Strings and Databases Employed in the Prior Art Search. </p></div></div></div><div id="ml164.s25"><h3>4.2. Mechanism of Action Studies</h3><p>In order to elucidate how this probe (CID 6875690/<a href="/pcsubstance/?term=ML164[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML164</a>) works, an immunofluorescent assay was employed to determine its mode of action and the extent of inhibition of <i>T. cruzi</i> replication in the host cell. To quantitate relative inhibition of parasite replication, 50 infected cells were selected for analysis for the probe (CID 6875690/<a href="/pcsubstance/?term=ML164[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML164</a>) and each analog. The number of infected cells with more than four amastigotes (where the parasite has invaded and is actively replicating) was divided by the total number of infected cells. As observed by immunofluorescence, all compounds in this series inhibited <i>T. cruzi</i> replication compared with control, but the parasite maintained defined amastigote bodies. Therefore, the mode of action was determined as static. No diffuse staining in the cytosol of host NIH/3T3 cells was observed in any case, suggesting no lysis of the amastigotes as described in Bettiol et al. (<a class="bk_pop" href="#ml164.r7">7</a>). Therefore, none of the compounds were categorized as lytic.</p><p>Additional experiments could be developed to identify the actual molecular target of this probe (CID 6875690/<a href="/pcsubstance/?term=ML164[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML164</a>). A phenotypic cell-based screening approach is extremely powerful for identifying novel compounds of interest when the exact mechanisms of parasite growth are not fully understood. At the Broad Institute, we have developed a robust, scalable method for confident identification of the protein targets of small molecules in their cellular context called <u>s</u>table <u>i</u>sotope <u>l</u>abeling by <u>a</u>mino acids in cell culture (SILAC). SILAC-based target identification technology overcomes prior difficulties with affinity-based target identification methods. This technology is routinely applied at the Broad Institute to identify targets of a variety of small molecules with drug-like properties, including kinase inhibitors, immunophilin modulators, and others.</p><p>Another method for target identification would be to develop a <i>T. cruzi</i> strain resistant to the probe (CID 6875690/<a href="/pcsubstance/?term=ML164[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML164</a>). In brief, <i>T. cruzi</i> could be co-cultured with the probe and compound-resistant parasites would be identified after culturing through many passages. A genomic approach would then be undertaken to identify the gene(s) that differed in the resistant strain versus the wild type. The identification of common mutated genes across multiple experiments could narrow the list of possible targets. Biochemical and biophysical approaches could then be applied for further characterize and optimize the probe (CID 6875690/<a href="/pcsubstance/?term=ML164[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML164</a>) for <i>in vivo</i> testing.</p></div><div id="ml164.s26"><h3>4.3. Planned Future Studies</h3><p>The probe (CID 6875690/<a href="/pcsubstance/?term=ML164[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML164</a>) will be tested for its inhibition of related parasites, <i>Leishmani</i> major and <i>Leishmania amazonensis</i>, with the aim of developing a treatment for <i>Leishmania</i> infections.</p><p>The probe (CID 6875690/<a href="/pcsubstance/?term=ML164[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML164</a>) and certain analogs are also being tested in a mouse model of Chagas disease using fluorescently tagged <i>T. cruzi</i>. Compounds are being dosed intravenously and the extent of <i>T. cruzi</i> replication is assessed by relative fluorescence in the infected area. These studies are ongoing at the University of Georgia by Drs. A. Rodriguez and R. Tarleton.</p></div></div><div id="ml164.s27"><h2 id="_ml164_s27_">5. References</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="ml164.r1">De Souza W. Basic cell biology of <em>Trypanosoma cruzi</em>. <span><span class="ref-journal">Curr Pharm Des. </span>2002;<span class="ref-vol">8</span>:269–285.</span> PubMed. [<a href="https://pubmed.ncbi.nlm.nih.gov/11860366" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 11860366</span></a>]</div></dd><dt>2.</dt><dd><div class="bk_ref" id="ml164.r2">Tarleton RL, Reithinger R, Urbina JA, Kitron U, Gürtler RE. The challenges of Chagas Disease: grim outlook or glimmer of hope. <span><span class="ref-journal">PLoS Med. </span>2007 Dec 27;<span class="ref-vol">4</span>(12):e332.</span> [<a href="/pmc/articles/PMC2222930/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2222930</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/18162039" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18162039</span></a>] [<a href="http://dx.crossref.org/10.1371/journal.pmed.0040332" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>3.</dt><dd><div class="bk_ref" id="ml164.r3">WHO Special Program for Research and Training in Tropical Diseases. <span class="ref-journal">Report of the Scientific Working Group on Chagas Disease.</span> World Health Organization; 2005. </div></dd><dt>4.</dt><dd><div class="bk_ref" id="ml164.r4">Konkle ME, Hargrove TY, Kleshchenko YY, von Kries JP, Ridenour W, Uddin MJ, Caprioli RM, Marnett LJ, Nes WD, Villalta F, et al. Indomethacin amides as a novel molecular scaffold for targeting <em>Trypanosoma cruzi</em> sterol 14α-demethylase. <span><span class="ref-journal">J Med Chem. </span>2009;<span class="ref-vol">52</span>(9):2846–2853.</span> PubMed. [<a href="/pmc/articles/PMC2744100/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2744100</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/19354253" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19354253</span></a>]</div></dd><dt>5.</dt><dd><div class="bk_ref" id="ml164.r5">Mott BT, Ferreira RS, Simeonov A, Jadhav A, Ang KK, Leister W, Shen M, Silveira JT, Doyle PS, Arkin MR, et al. Identification and optimization of inhibitors of trypanosomal cysteine proteases: cruzain, rhodesain, and TbCatB. <span><span class="ref-journal">J Med Chem. </span>2010;<span class="ref-vol">53</span>:52–60.</span> PubMed. [<a href="/pmc/articles/PMC2804034/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2804034</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/19908842" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19908842</span></a>]</div></dd><dt>6.</dt><dd><div class="bk_ref" id="ml164.r6">Brak K, Kerr ID, Barrett KT, Fuchi N, Debrath M, Ang K, et al. Nonpeptidic tetrafluorophenoxymehtyl ketone cruzain inhibitors as promising new leads for Chagas disease chemotherapy. <span><span class="ref-journal">J Med Chem. </span>2010;<span class="ref-vol">53</span>:1763–1773.</span> PubMed. [<a href="/pmc/articles/PMC2838180/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2838180</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/20088534" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 20088534</span></a>]</div></dd><dt>7.</dt><dd><div class="bk_ref" id="ml164.r7">Bettiol E, Samanovic M, Murkin AS, Raper J, Buckner F, Rodriguez A. Identification of three classes of heteroaromatic compounds with activity against intracellular Trypanosoma cruzi by chemical library screening. <span><span class="ref-journal">PLoS Negl Trop Dis. </span>2009;<span class="ref-vol">3</span>(2):e384.</span> Epub 2009 Feb 24. PubMed. [<a href="/pmc/articles/PMC2639639/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2639639</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/19238193" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19238193</span></a>] [<a href="http://dx.crossref.org/10.1371/journal.pntd.0000384" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>8.</dt><dd><div class="bk_ref" id="ml164.r8">Buckner FS, Verlinde CL, La Flamme AC, Van Voorhis WC. Efficient technique for screening drugs for activity against <em>Trypanosoma cruzi</em> using parasites expressing beta-galactosidase. <span><span class="ref-journal">Antimicrob Agents Chemother. </span>1996;<span class="ref-vol">40</span>(11):2592–2597.</span> PubMed. [<a href="/pmc/articles/PMC163582/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC163582</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/8913471" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 8913471</span></a>]</div></dd><dt>9.</dt><dd><div class="bk_ref" id="ml164.r9">Aguirre G, Cabrera E, Cerecetto H, Di Maio R, González M, Seoane G, Duffaut A, Denicola A, Gil MJ, Martínez-Merino V. Design, synthesis and biological evaluation of new potent 5-nitrofuryl derivatives as anti-<em>Trypanosoma cruzi</em> agents. Studies of trypanothione binding site of trypanothione reductase as target for rational design. <span><span class="ref-journal">Eur J Med Chem. </span>2004 May;<span class="ref-vol">39</span>(5):421–431.</span> PubMed. [<a href="https://pubmed.ncbi.nlm.nih.gov/15110968" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 15110968</span></a>]</div></dd></dl></div><div id="ml164.app1"><h2 id="_ml164_app1_">Appendix A. Assay Summary Table</h2><div id="ml164.t5" 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/NBK56231/table/ml164.t5/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml164.t5_lrgtbl__"><table class="no_margin"><thead><tr><th id="hd_h_ml164.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">PubChem AID</th><th id="hd_h_ml164.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Type</th><th id="hd_h_ml164.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Target</th><th id="hd_h_ml164.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Concentration Range (μM)</th><th id="hd_h_ml164.t5_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_ml164.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1885" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1885</a></td><td headers="hd_h_ml164.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Primary</td><td headers="hd_h_ml164.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><i>T. cruzi</i> co-culture</td><td headers="hd_h_ml164.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Average 3.75 μM</td><td headers="hd_h_ml164.t5_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">303,224</td></tr><tr><td headers="hd_h_ml164.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2044" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2044</a></td><td headers="hd_h_ml164.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Primary</td><td headers="hd_h_ml164.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><i>T. cruzi</i> co-culture</td><td headers="hd_h_ml164.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">100 nM-6.0 μM</td><td headers="hd_h_ml164.t5_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">4063</td></tr><tr><td headers="hd_h_ml164.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2294" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2294</a></td><td headers="hd_h_ml164.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Primary</td><td headers="hd_h_ml164.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><i>T. cruzi</i> co-culture</td><td headers="hd_h_ml164.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">4.0 nM-1.0 μM</td><td headers="hd_h_ml164.t5_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">319</td></tr><tr><td headers="hd_h_ml164.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2630" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2630</a></td><td headers="hd_h_ml164.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Primary</td><td headers="hd_h_ml164.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><i>T. cruzi</i> co-culture</td><td headers="hd_h_ml164.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.11 nM-19.5 μM</td><td headers="hd_h_ml164.t5_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">42</td></tr><tr><td headers="hd_h_ml164.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2396" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2396</a></td><td headers="hd_h_ml164.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Primary</td><td headers="hd_h_ml164.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><i>T. cruzi</i> co-culture</td><td headers="hd_h_ml164.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.11 nM-6.8 μM</td><td headers="hd_h_ml164.t5_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">27</td></tr><tr><td headers="hd_h_ml164.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/493197" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">493197</a></td><td headers="hd_h_ml164.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Primary</td><td headers="hd_h_ml164.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><i>T. cruzi</i> co-culture</td><td headers="hd_h_ml164.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">3 nM-19.5 μM</td><td headers="hd_h_ml164.t5_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">46</td></tr><tr><td headers="hd_h_ml164.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2010" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2010</a></td><td headers="hd_h_ml164.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Secondary</td><td headers="hd_h_ml164.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NIH3T3 Toxicity</td><td headers="hd_h_ml164.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">100 nM-6.0 μM</td><td headers="hd_h_ml164.t5_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">4063</td></tr><tr><td headers="hd_h_ml164.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2586" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2586</a></td><td headers="hd_h_ml164.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Secondary</td><td headers="hd_h_ml164.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NIH3T3 Toxicity</td><td headers="hd_h_ml164.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.11 nM-19.5 μM</td><td headers="hd_h_ml164.t5_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">27</td></tr><tr><td headers="hd_h_ml164.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/493247" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">493247</a></td><td headers="hd_h_ml164.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Secondary</td><td headers="hd_h_ml164.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NIH3T3 Toxicity</td><td headers="hd_h_ml164.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">3 nM-19.5 μM</td><td headers="hd_h_ml164.t5_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">46</td></tr><tr><td headers="hd_h_ml164.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2632" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2632</a></td><td headers="hd_h_ml164.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Orthogonal</td><td headers="hd_h_ml164.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Inhibition of <i>T. cruzi</i> replication</td><td headers="hd_h_ml164.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Single dose; 10x IC<sub>50</sub> as defined in <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2204" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 2204</a> and <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2294" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2294</a></td><td headers="hd_h_ml164.t5_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">27</td></tr><tr><td headers="hd_h_ml164.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1968" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1968</a></td><td headers="hd_h_ml164.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Summary</td><td headers="hd_h_ml164.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NA</td><td headers="hd_h_ml164.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NA</td><td headers="hd_h_ml164.t5_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NA</td></tr></tbody></table></div><div><div><dl class="temp-labeled-list small"><dt></dt><dd><div id="ml164.tfn8"><p class="no_margin">NA= not applicable</p></div></dd></dl></div></div></div></div><div id="ml164.app2"><h2 id="_ml164_app2_">Appendix B. Detailed Assay Protocols</h2><div id="ml164.s28"><h3><i>T cruzi</i> Inhibition Assay Protocol (AID Nos.<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1885" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1885</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2044" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2044</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2294" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2294</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2630" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2630</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2396" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2396</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/493197" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">493197</a>)</h3><div id="ml164.s29"><h4>Cell Propagation</h4><ol><li class="half_rhythm"><div>90% DMEM+Phenol red, 10% FBS, 1% PSG.</div></li><li class="half_rhythm"><div>Mix and filter through 0.2 microns membrane.</div></li><li class="half_rhythm"><div>Keep at 4°C. Warm up to 37°C in water bath before use.</div></li></ol></div><div id="ml164.s30"><h4>T. cruzi Culture and Assays</h4><ol><li class="half_rhythm"><div>98% DMEM+Phenol red, 2% FBS, 1% PSG.</div></li><li class="half_rhythm"><div>Mix and filter through 0.2 microns membrane.</div></li><li class="half_rhythm"><div>Keep at 4°C. Warm up to 37°C in water bath before use.</div></li></ol></div></div><div id="ml164.s31"><h3>Cell Culture Protocols</h3><div id="ml164.s32"><h4>NIH/3T3 Cell Culture</h4><ol><li class="half_rhythm"><div>Aspirate medium.</div></li><li class="half_rhythm"><div>Rinse cells with 10 ml PBS/plate.</div></li><li class="half_rhythm"><div>Aspirate PBS.</div></li><li class="half_rhythm"><div>Add 5 ml of pre-warmed trypsin-EDTA, swirl the dish to make sure the trypsin covers all the cells.</div></li><li class="half_rhythm"><div>Place the dishes back in the incubator for 5 minutes.</div></li><li class="half_rhythm"><div>Check that the cells are detaching.</div></li><li class="half_rhythm"><div>Add 20 ml of medium, pipette up and down to detach all the cells.</div></li><li class="half_rhythm"><div>Dilute into 2% FBS/DMEM for assay or 10% FBS/DMEM for propagation.</div></li></ol></div><div id="ml164.s33"><h4>LLC-MK2 Cell Culture</h4><ol><li class="half_rhythm"><div>Aspirate medium.</div></li><li class="half_rhythm"><div>Rinse cells with 10 ml PBS/plate.</div></li><li class="half_rhythm"><div>Aspirate PBS.</div></li><li class="half_rhythm"><div>Add 5 ml of pre-warmed trypsin-EDTA, swirl the dish to make sure the trypsin covers all the cells.</div></li><li class="half_rhythm"><div>Place the dishes back in the incubator for 5 minutes.</div></li><li class="half_rhythm"><div>Check that the cells are detaching.</div></li><li class="half_rhythm"><div>Add 20 ml of medium, pipette up and down to detach all the cells.</div></li><li class="half_rhythm"><div>Dilute into 2% FBS/DMEM for assay or 10% FBS/DMEM for propagation.</div></li></ol></div><div id="ml164.s34"><h4>T. cruzi β-gal (Tc) Parasite Culture</h4><ol><li class="half_rhythm"><div>The day before infection (or at least 2–3 hours before), plate 3 million LLC-MK2 cells/T175 in DMEM+10% FBS.</div></li><li class="half_rhythm"><div>Either thaw <i>T. cruzi</i> in from liquid nitrogen stock into 50 ml 2% FBS/DMEM or remove the media from a propagating LLC-MK2 co-culture; in other words, harvesting the medium containing the free <i>T. cruzi</i> in 50-ml Falcon tubes.</div></li><li class="half_rhythm"><div>Spin 10 minutes at 2200 rpm.</div></li><li class="half_rhythm"><div>Aspirate the supernatant until 15 ml is left.</div></li><li class="half_rhythm"><div>Place back the tubes in the incubator delicately (so as not to disturb the pellet).</div></li><li class="half_rhythm"><div>Incubate minimum 3 hours at 37°C.</div></li><li class="half_rhythm"><div>Take supernatant to fresh tube.</div></li><li class="half_rhythm"><div>To count: Mix 75 ml <i>T. cruzi</i> with 25 ml 16% PFA, mix, put 75 ml on Nexcelom cellometer cassette.</div></li><li class="half_rhythm"><div>Wait for 2–5 minutes to let the <i>T. cruzi</i> settle; use the <i>T. cruzi</i> method saved on the M10 machine, press display image, focus, and count.</div></li><li class="half_rhythm"><div>Aspirate LLC-MK2 media (used 10% for plating) and replace with 2% FBS/DMEM.</div></li><li class="half_rhythm"><div>Plate 17–35 million on <i>T. cruzi</i> on fresh LLC-MK2.</div></li><li class="half_rhythm"><div>Change medium after 2 days (Use 2% FBS/DMEM).</div></li><li class="half_rhythm"><div>Harvest <i>T. cruzi</i> trypomastigotes on Day 6 and Day 7.</div></li></ol></div></div><div id="ml164.s35"><h3>Growth Inhibition Assay Protocol for HTS (384-well plates)</h3><ol><li class="half_rhythm"><div>Warm up medium 2% FBS/DMEM.</div></li><li class="half_rhythm"><div>Harvest parasites in 50-ml tubes (1 flask per tube).</div></li><li class="half_rhythm"><div>Spin 10 minutes at 2200 rpm.</div></li><li class="half_rhythm"><div>Aspirate media approximately 15 ml and place them on a rack in the incubator to let trypomastigotes swim out of the pellet for 3–5 hours.</div></li><li class="half_rhythm"><div>In the meantime, trypsinize NIH/3T3 cells as described in cell culture protocol.</div></li><li class="half_rhythm"><div>When NIH/3T3 are detached, harvest them in DMEM- 2% FBS and 1% PSG and count using the NIH3T3 program using the Nexcelom Cellometer.</div></li><li class="half_rhythm"><div>Dilute cells to 166,667 cells /ml and add to flask.</div></li><li class="half_rhythm"><div>Plate 5,000 cells/30 μL per well using standard cassette multiwell drop Combi, adding at a fast speed.</div></li><li class="half_rhythm"><div>Put back in incubator for 3 hours to allow cells to attach.</div></li><li class="half_rhythm"><div>Count <i>T. cruzi</i> as described in the parasite culture protocol.</div></li><li class="half_rhythm"><div>Dilute to 0.250 million /ml and transfer to a 2-liter flask.</div></li><li class="half_rhythm"><div>Add to a 2- liter flask, stirring.</div></li><li class="half_rhythm"><div>Pin 50 nL compounds/DMSO to each well with NIH/3T3s.</div></li><li class="half_rhythm"><div>Add shortly after 20 μL per well of parasites (5000 <i>T. cruzi</i>) with standard cassette multiwell drop Combi on slow speed.</div></li><li class="half_rhythm"><div>Incubate for 4 days (or minimum 90 hours).</div></li><li class="half_rhythm"><div>Prepare Gal-Screen with 0.05% NP40.</div></li><li class="half_rhythm"><div>Add 30 μL per well of 384-well plate.</div></li><li class="half_rhythm"><div>Incubate for 60 minutes.</div></li><li class="half_rhythm"><div>Read using the ultrasensitive luminescence program on the Envision (Perkin-Elmer, 0.1 sec/well).</div></li></ol></div><div id="ml164.s36"><h3>Cell Toxicity Assay Protocol: NIH/3T3 Cells (AID Nos. <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2010" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2010</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2586" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2586</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/493247" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">493247</a>)</h3><ol><li class="half_rhythm"><div>Warm up medium 2% FBS/DMEM.</div></li><li class="half_rhythm"><div>Trypsinize NIH/3T3 cells as described in cell culture protocol.</div></li><li class="half_rhythm"><div>When NIH3T3s are detached, harvest them in DMEM- 2% FBS and 1% PSG and count using the NIH3T3 program using Cellometer Auto T4 (Nexcelom Biosciences).</div></li><li class="half_rhythm"><div>Dilute cells to 166,667 cells /ml and add to flask.</div></li><li class="half_rhythm"><div>Plate 5,000 cells/50 μL per well using a Thermo MultiDrop Combi liquid dispenser and a sterilized standard sized dispensing cassette adding at a fast speed in a tissue culture hood.</div></li><li class="half_rhythm"><div>Put back in incubator for 3 hours to allow cells to attach.</div></li><li class="half_rhythm"><div>Pin 50 nL compounds/DMSO to each well.</div></li><li class="half_rhythm"><div>Incubate for 4 days (or minimum 90 hours).</div></li><li class="half_rhythm"><div>On day of substrate addition, prepare CellTiter-Glo.</div></li></ol></div><div id="ml164.s37"><h3>CellTiter-Glo<sup>®</sup> Protocol</h3><ol><li class="half_rhythm"><div>Thaw the CellTiter-Glo Buffer, and equilibrate to room temperature prior to use. For convenience the CellTiter-Glo Buffer may be thawed and stored at room temperature for up to 48 hours prior to use.</div></li><li class="half_rhythm"><div>Transfer the appropriate volume of CellTiter-Glo Buffer into the amber bottle containing CellTiter-Glo Substrate to reconstitute the lyophilized enzyme/substrate mixture.</div></li><li class="half_rhythm"><div>Mix by gently vortexing, swirling, or by inverting the contents to obtain a homogeneous solution.</div></li><li class="half_rhythm"><div>Dilute solution 1:3 with PBS.</div></li><li class="half_rhythm"><div>Take out plates from incubator and incubate at room temperature for 30 minutes.</div></li><li class="half_rhythm"><div>Add 30 μL/well using a Thermo MultiDrop Combi liquid dispenser and a sterilized standard sized dispensing cassette adding at a fast speed.</div></li><li class="half_rhythm"><div>Allow the plate to incubate at room temperature for 10 minutes.</div></li><li class="half_rhythm"><div>Read using the ultrasensitive luminescence program on the Envision (Perkin Elmer, 0.1 sec/well).</div></li></ol></div><div id="ml164.s38"><h3>Intracellular <i>T cruzi</i> Immunofluorescence Assay Protocol (AID No. <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2632" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2632</a>)</h3><ol><li class="half_rhythm"><div>Seed NIH/3T3 cells on sterile glass coverslips in 12-well plates and allow cells to adhere overnight.</div></li><li class="half_rhythm"><div>Add <i>T. cruzi</i> parasites (MOI 100:1) and allow to infect for 2 hours in DMEM+2% FBS and PSG.</div></li><li class="half_rhythm"><div>Rinse parasites out 3 times with PBS, and add compounds at 10X their IC<sub>50</sub> (as determined in <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2044" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 2044</a> and <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2294" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 2294</a>).</div></li><li class="half_rhythm"><div>Incubate infected cells for 4 days and fix for 15 minutes with 4% paraformaldehyde.</div></li><li class="half_rhythm"><div>Rinse fixed cells on coverslips with PBS and permeabilize for 15 minutes in PBS with 0.1% Triton X-100.</div></li><li class="half_rhythm"><div>Block for 20 minutes in PBS with 10% goat serum, 1% bovine serum albumin (BSA), 100 mM glycine, and 0.05% sodium azide.</div></li><li class="half_rhythm"><div>Incubate cells for 1 hour at room temperature with a polyclonal rabbit anti-<i>T cruzi</i> at 1:2000 dilution.</div></li><li class="half_rhythm"><div>Rinse, then add an Alexa Fluor 488 goat anti-rabbit IgG secondary antibody (Molecular Probes, Invitrogen) for 1 hour at a 1:800 dilution.</div></li><li class="half_rhythm"><div>Stain DNA with DAPI, and mount coverslips with anti-fade mounting media.</div></li><li class="half_rhythm"><div>Take images using an inverted Olympus IX70 microscope with a 60X oil objective.</div></li></ol></div></div><div id="ml164.app3"><h2 id="_ml164_app3_">Appendix C. NMR and LC Data of Probe and Analogs</h2><div id="ml164.fu2" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56231/bin/ml164fu23.jpg" alt="1H NMR (300 MHz, DMSO-d6) Spectra of Probe (CID 6875690/ML164)." /></div><h3><span class="title"><sup>1</sup>H NMR (300 MHz, DMSO-d6) Spectra of Probe (CID 6875690/<a href="/pcsubstance/?term=ML164[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML164</a>)</span></h3></div><div id="ml164.fu3" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56231/bin/ml164fu24.jpg" alt="LC-MS Chromatogram of Probe (CID 6875690/ML164)." /></div><h3><span class="title">LC-MS Chromatogram of Probe (CID 6875690/<a href="/pcsubstance/?term=ML164[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML164</a>)</span></h3></div><div id="ml164.fu4" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56231/bin/ml164fu25.jpg" alt="1H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 6875684." /></div><h3><span class="title"><sup>1</sup>H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 6875684</span></h3></div><div id="ml164.fu5" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56231/bin/ml164fu26.jpg" alt="LC-MS Chromatogram of Analog CID 6875684." /></div><h3><span class="title">LC-MS Chromatogram of Analog CID 6875684</span></h3></div><div id="ml164.fu6" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56231/bin/ml164fu27.jpg" alt="1H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 6884367." /></div><h3><span class="title"><sup>1</sup>H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 6884367</span></h3></div><div id="ml164.fu7" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56231/bin/ml164fu28.jpg" alt="LC-MS Chromatogram of Analog CID 6884367." /></div><h3><span class="title">LC-MS Chromatogram of Analog CID 6884367</span></h3></div><div id="ml164.fu8" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=1H%20NMR%20(300%20MHz%2C%20DMSO-d6)%20Spectra%20of%20Analog%20CID%206872741.&p=BOOKS&id=56231_ml164fu29.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img src="/books/NBK56231/bin/ml164fu29.jpg" alt="1H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 6872741." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="title"><sup>1</sup>H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 6872741</span></h3></div><div id="ml164.fu9" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56231/bin/ml164fu30.jpg" alt="LC-MS Chromatogram of Analog CID 6872741." /></div><h3><span class="title">LC-MS Chromatogram of Analog CID 6872741</span></h3></div><div id="ml164.fu10" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=1H%20NMR%20(300%20MHz%2C%20DMSO-d6)%20Spectra%20of%20Analog%20CID%209689369.&p=BOOKS&id=56231_ml164fu31.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img src="/books/NBK56231/bin/ml164fu31.jpg" alt="1H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 9689369." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="title"><sup>1</sup>H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 9689369</span></h3></div><div id="ml164.fu11" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56231/bin/ml164fu32.jpg" alt="LC-MS Chromatogram of Analog CID 9689369." /></div><h3><span class="title">LC-MS Chromatogram of Analog CID 9689369</span></h3></div><div id="ml164.fu12" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=1H%20NMR%20(300%20MHz%2C%20DMSO-d6)%20Spectra%20of%20Analog%20CID%209563094.&p=BOOKS&id=56231_ml164fu33.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img src="/books/NBK56231/bin/ml164fu33.jpg" alt="1H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 9563094." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="title"><sup>1</sup>H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 9563094</span></h3></div><div id="ml164.fu13" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56231/bin/ml164fu34.jpg" alt="LC-MS Chromatogram of Analog CID 9563094." /></div><h3><span class="title">LC-MS Chromatogram of Analog CID 9563094</span></h3></div><div id="ml164.fu14" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56231/bin/ml164fu35.jpg" alt="1H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 5344250." /></div><h3><span class="title"><sup>1</sup>H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 5344250</span></h3></div><div id="ml164.fu15" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56231/bin/ml164fu36.jpg" alt="LC-MS Chromatogram of Analog CID 5344250." /></div><h3><span class="title">LC-MS Chromatogram of Analog CID 5344250</span></h3></div><div id="ml164.fu16" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=1H%20NMR%20(300%20MHz%2C%20DMSO-d6)%20Spectra%20of%20Analog%20CID%206875617.&p=BOOKS&id=56231_ml164fu37.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img src="/books/NBK56231/bin/ml164fu37.jpg" alt="1H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 6875617." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="title"><sup>1</sup>H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 6875617</span></h3></div><div id="ml164.fu17" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56231/bin/ml164fu38.jpg" alt="LC-MS Chromatogram of Analog CID 6875617." /></div><h3><span class="title">LC-MS Chromatogram of Analog CID 6875617</span></h3></div><div id="ml164.fu18" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=1H%20NMR%20(300%20MHz%2C%20DMSO-d6)%20Spectra%20of%20Analog%20CID%206322955.&p=BOOKS&id=56231_ml164fu39.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img src="/books/NBK56231/bin/ml164fu39.jpg" alt="1H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 6322955." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="title"><sup>1</sup>H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 6322955</span></h3></div><div id="ml164.fu19" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56231/bin/ml164fu40.jpg" alt="LC-MS Chromatogram of Analog CID 6322955." /></div><h3><span class="title">LC-MS Chromatogram of Analog CID 6322955</span></h3></div><div id="ml164.fu20" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=1H%20NMR%20(300%20MHz%2C%20DMSO-d6)%20Spectra%20of%20Analog%20CID%209557463.&p=BOOKS&id=56231_ml164fu41.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img src="/books/NBK56231/bin/ml164fu41.jpg" alt="1H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 9557463." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="title"><sup>1</sup>H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 9557463</span></h3></div><div id="ml164.fu21" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56231/bin/ml164fu42.jpg" alt="LC-MS Chromatogram of Analog CID 9557463." /></div><h3><span class="title">LC-MS Chromatogram of Analog CID 9557463</span></h3></div><div id="ml164.fu22" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=1H%20NMR%20(300%20MHz%2C%20DMSO-d6)%20Spectra%20of%20Analog%20CID%206875630.&p=BOOKS&id=56231_ml164fu43.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img src="/books/NBK56231/bin/ml164fu43.jpg" alt="1H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 6875630." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="title"><sup>1</sup>H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 6875630</span></h3></div><div id="ml164.fu23" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56231/bin/ml164fu44.jpg" alt="LC-MS Chromatogram of Analog CID 6875630." /></div><h3><span class="title">LC-MS Chromatogram of Analog CID 6875630</span></h3></div><div id="ml164.fu24" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=1H%20NMR%20(300%20MHz%2C%20DMSO-d6)%20Spectra%20of%20Analog%20CID%205332109.&p=BOOKS&id=56231_ml164fu45.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img src="/books/NBK56231/bin/ml164fu45.jpg" alt="1H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 5332109." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="title"><sup>1</sup>H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 5332109</span></h3></div><div id="ml164.fu25" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56231/bin/ml164fu46.jpg" alt="LC-MS Chromatogram of Analog CID 5332109." /></div><h3><span class="title">LC-MS Chromatogram of Analog CID 5332109</span></h3></div><div id="ml164.fu26" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=1H%20NMR%20(300%20MHz%2C%20DMSO-d6)%20Spectra%20of%20Analog%20CID%2021210609.&p=BOOKS&id=56231_ml164fu47.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img src="/books/NBK56231/bin/ml164fu47.jpg" alt="1H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 21210609." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="title"><sup>1</sup>H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 21210609</span></h3></div><div id="ml164.fu27" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=LC-MS%20Chromatogram%20of%20Analog%20CID%2021210609.&p=BOOKS&id=56231_ml164fu48.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img src="/books/NBK56231/bin/ml164fu48.jpg" alt="LC-MS Chromatogram of Analog CID 21210609." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="title">LC-MS Chromatogram of Analog CID 21210609</span></h3></div><div id="ml164.fu28" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56231/bin/ml164fu49.jpg" alt="1H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 9558304." /></div><h3><span class="title"><sup>1</sup>H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 9558304</span></h3></div><div id="ml164.fu29" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56231/bin/ml164fu50.jpg" alt="LC-MS Chromatogram of Analog CID 9558304." /></div><h3><span class="title">LC-MS Chromatogram of Analog CID 9558304</span></h3></div><div id="ml164.fu30" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=1H%20NMR%20(300%20MHz%2C%20DMSO-d6)%20Spectra%20of%20Analog%20CID%205334382.&p=BOOKS&id=56231_ml164fu51.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img src="/books/NBK56231/bin/ml164fu51.jpg" alt="1H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 5334382." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="title"><sup>1</sup>H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 5334382</span></h3></div><div id="ml164.fu31" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56231/bin/ml164fu52.jpg" alt="LC-MS Chromatogram of Analog CID 5334382." /></div><h3><span class="title">LC-MS Chromatogram of Analog CID 5334382</span></h3></div><div id="ml164.fu32" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=1H%20NMR%20(300%20MHz%2C%20DMSO-d6)%20Spectra%20of%20Analog%20CID%205349954.&p=BOOKS&id=56231_ml164fu53.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img src="/books/NBK56231/bin/ml164fu53.jpg" alt="1H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 5349954." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="title"><sup>1</sup>H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 5349954</span></h3></div><div id="ml164.fu33" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56231/bin/ml164fu54.jpg" alt="LC-MS Chromatogram of Analog CID 5349954." /></div><h3><span class="title">LC-MS Chromatogram of Analog CID 5349954</span></h3></div><div id="ml164.fu34" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=1H%20NMR%20(300%20MHz%2C%20DMSO-d6)%20Spectra%20of%20Analog%20CID%209640748.&p=BOOKS&id=56231_ml164fu55.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img src="/books/NBK56231/bin/ml164fu55.jpg" alt="1H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 9640748." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="title"><sup>1</sup>H NMR (300 MHz, DMSO-d6) Spectra of Analog CID 9640748</span></h3></div><div id="ml164.fu35" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56231/bin/ml164fu56.jpg" alt="LC-MS Chromatogram of Analog CID 9640748." /></div><h3><span class="title">LC-MS Chromatogram of Analog CID 9640748</span></h3></div></div><div id="ml164.app4"><h2 id="_ml164_app4_">Appendix D. Compounds Submitted to BioFocus</h2><div id="ml164.t6" 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/NBK56231/table/ml164.t6/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml164.t6_lrgtbl__"><table class="no_margin"><thead><tr><th id="hd_h_ml164.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BRD</th><th id="hd_h_ml164.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SID</th><th id="hd_h_ml164.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CID</th><th id="hd_h_ml164.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">P/A</th><th id="hd_h_ml164.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLSID</th><th id="hd_h_ml164.t6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">ML</th></tr></thead><tbody><tr><td headers="hd_h_ml164.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BRD-K64970062-001-01-5</td><td headers="hd_h_ml164.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87219052" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87219052</a></td><td headers="hd_h_ml164.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">6875690</td><td headers="hd_h_ml164.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">P</td><td headers="hd_h_ml164.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS002725687</td><td headers="hd_h_ml164.t6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="/pcsubstance/?term=ML164[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML164</a></td></tr><tr><td headers="hd_h_ml164.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BRD-K85660637-001-01-3</td><td headers="hd_h_ml164.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87219044" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87219044</a></td><td headers="hd_h_ml164.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">884367</td><td headers="hd_h_ml164.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">A</td><td headers="hd_h_ml164.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS002725693</td><td headers="hd_h_ml164.t6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">NA</td></tr><tr><td headers="hd_h_ml164.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BRD-K67470788-001-01-6</td><td headers="hd_h_ml164.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87556789" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87556789</a></td><td headers="hd_h_ml164.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">5344250</td><td headers="hd_h_ml164.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">A</td><td headers="hd_h_ml164.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS002725680</td><td headers="hd_h_ml164.t6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">NA</td></tr><tr><td headers="hd_h_ml164.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BRD-K02558072-001-01-1</td><td headers="hd_h_ml164.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/7219027" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">7219027</a></td><td headers="hd_h_ml164.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">6875617</td><td headers="hd_h_ml164.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">A</td><td headers="hd_h_ml164.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS002725690</td><td headers="hd_h_ml164.t6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">NA</td></tr><tr><td headers="hd_h_ml164.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BRD-K91047982-001-01-6</td><td headers="hd_h_ml164.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/7556790" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">7556790</a></td><td headers="hd_h_ml164.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">5349954</td><td headers="hd_h_ml164.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">A</td><td headers="hd_h_ml164.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS002725681</td><td headers="hd_h_ml164.t6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">NA</td></tr><tr><td headers="hd_h_ml164.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BRD-K17628612-001-01-2</td><td headers="hd_h_ml164.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87556791" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87556791</a></td><td headers="hd_h_ml164.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">9640748</td><td headers="hd_h_ml164.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">A</td><td headers="hd_h_ml164.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS002729069</td><td headers="hd_h_ml164.t6_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="ml164.tfn9"><p class="no_margin">NA= Not applicable; A = analog; P = probe</p></div></dd></dl></div></div></div></div><div id="bk_toc_contnr"></div></div></div>
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<div class="post-content"><div><div class="half_rhythm"><a href="/books/about/copyright/">Copyright Notice</a></div><div class="small"><span class="label">Bookshelf ID: NBK56231</span><span class="label">PMID: <a href="https://pubmed.ncbi.nlm.nih.gov/21735600" title="PubMed record of this page" ref="pagearea=meta&targetsite=entrez&targetcat=link&targettype=pubmed">21735600</a></span></div><div style="margin-top:2em" class="bk_noprnt"><a class="bk_cntns" href="/books/n/mlprobe/">Contents</a><div class="pagination bk_noprnt"><a class="active page_link prev" href="/books/n/mlprobe/ml165/" title="Previous page in this title">< Prev</a><a class="active page_link next" href="/books/n/mlprobe/ml163/" title="Next page in this title">Next ></a></div></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/NBK56231/?report=reader">PubReader</a></li><li><a href="/books/NBK56231/?report=printable">Print View</a></li><li><a data-jig="ncbidialog" href="#_ncbi_dlg_citbx_NBK56231" data-jigconfig="width:400,modal:true">Cite this Page</a><div id="_ncbi_dlg_citbx_NBK56231" style="display:none" title="Cite this Page"><div class="bk_tt">Carmody LC, Germain A, Barker D, et al. Identification of Small-Molecule Inhibitors of Trypansoma cruzi Infection - Probe 1. 2010 Mar 26 [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/NBK56231/pdf/Bookshelf_NBK56231.pdf">PDF version of this page</a> (3.0M)</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="#ml164.s1" ref="log$=inpage&link_id=inpage">Probe Structure and Characteristics</a></li><li><a href="#ml164.s2" ref="log$=inpage&link_id=inpage">Recommendations for scientific use of the probe</a></li><li><a href="#ml164.s3" ref="log$=inpage&link_id=inpage">Introduction</a></li><li><a href="#ml164.s5" ref="log$=inpage&link_id=inpage">Materials and Methods</a></li><li><a href="#ml164.s15" ref="log$=inpage&link_id=inpage">Results</a></li><li><a href="#ml164.s23" ref="log$=inpage&link_id=inpage">Discussion</a></li><li><a href="#ml164.s27" ref="log$=inpage&link_id=inpage">References</a></li><li><a href="#ml164.app1" ref="log$=inpage&link_id=inpage">Assay Summary Table</a></li><li><a href="#ml164.app2" ref="log$=inpage&link_id=inpage">Detailed Assay Protocols</a></li><li><a href="#ml164.app3" ref="log$=inpage&link_id=inpage">NMR and LC Data of Probe and Analogs</a></li><li><a href="#ml164.app4" ref="log$=inpage&link_id=inpage">Compounds Submitted to BioFocus</a></li></ul></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Related information</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="discovery_db_links" id="Shutter"></a></div><div class="portlet_content"><ul><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pmc&DbFrom=books&Cmd=Link&LinkName=books_pmc_refs&IdsFromResult=2509354" ref="log$=recordlinks">PMC</a><div class="brieflinkpop offscreen_noflow">PubMed Central citations</div></li><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pcsubstance&DbFrom=books&Cmd=Link&LinkName=books_pcsubstance&IdsFromResult=2509354" ref="log$=recordlinks">PubChem Substance</a><div class="brieflinkpop offscreen_noflow">Related PubChem Substances</div></li><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pubmed&DbFrom=books&Cmd=Link&LinkName=books_pubmed_refs&IdsFromResult=2509354" ref="log$=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/21735611" ref="ordinalpos=1&linkpos=1&log$=relatedreviews&logdbfrom=pubmed"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> Identification of Small-Molecule Inhibitors of Trypansoma cruzi Infection - Probe 3.</a><span class="source">[Probe Reports from the NIH Mol...]</span><div class="brieflinkpop offscreen_noflow"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> Identification of Small-Molecule Inhibitors of Trypansoma cruzi Infection - Probe 3.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Carmody LC, Germain A, Barker D, Galan-Rodriguez C, Bettiol E, Rodriguez A, MacPherson L, Palmer M, Schreiber SL. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Probe Reports from the NIH Molecular Libraries Program. 2010</em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/21634083" ref="ordinalpos=1&linkpos=2&log$=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-Molecule Inhibitors of Trypansoma cruzi Infection.</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> Identification of Small-Molecule Inhibitors of Trypansoma cruzi Infection.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Carmody LC, Germain A, Barker D, Galan-Rodriguez C, Bettiol E, Rodriguez A, MacPherson L, Palmer M, Schreiber SL. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Probe Reports from the NIH Molecular Libraries Program. 2010</em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/24479197" ref="ordinalpos=1&linkpos=3&log$=relatedreviews&logdbfrom=pubmed"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> Identification of Diversity-Oriented Synthesis Derived Small Molecule, ML341, with Cidal Activity Against Trypanosoma cruzi.</a><span class="source">[Probe Reports from the NIH Mol...]</span><div class="brieflinkpop offscreen_noflow"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> Identification of Diversity-Oriented Synthesis Derived Small Molecule, ML341, with Cidal Activity Against Trypanosoma cruzi.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Carmody LC, Germain AR, Engel JC, Gut J, Kaiser M, Jewett I, LeQuemen S, Marie JC, Dandapani S, Rodriguez A, et al. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Probe Reports from the NIH Molecular Libraries Program. 2010</em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/26964673" ref="ordinalpos=1&linkpos=4&log$=relatedarticles&logdbfrom=pubmed">Compound profiling and 3D-QSAR studies of hydrazone derivatives with activity against intracellular Trypanosoma cruzi.</a><span class="source">[Bioorg Med Chem. 2016]</span><div class="brieflinkpop offscreen_noflow">Compound profiling and 3D-QSAR studies of hydrazone derivatives with activity against intracellular Trypanosoma cruzi.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Costa LB, Cardoso MV, de Oliveira Filho GB, de Moraes Gomes PA, Espíndola JW, de Jesus Silva TG, Torres PH, Silva Junior FP, Martin J, de Figueiredo RC, et al. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Bioorg Med Chem. 2016 Apr 15; 24(8):1608-18. 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