536 lines
No EOL
208 KiB
HTML
536 lines
No EOL
208 KiB
HTML
<?xml version="1.0" encoding="utf-8"?>
|
||
<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
|
||
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">
|
||
|
||
<head><meta http-equiv="Content-Type" content="text/html; charset=utf-8" />
|
||
<!-- AppResources meta begin -->
|
||
<meta name="paf-app-resources" content="" />
|
||
<script type="text/javascript">var ncbi_startTime = new Date();</script>
|
||
|
||
<!-- AppResources meta end -->
|
||
|
||
<!-- TemplateResources meta begin -->
|
||
<meta name="paf_template" content="" />
|
||
|
||
<!-- TemplateResources meta end -->
|
||
|
||
<!-- Logger begin -->
|
||
<meta name="ncbi_db" content="books" /><meta name="ncbi_pdid" content="book-part" /><meta name="ncbi_acc" content="NBK98919" /><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="/NBK98919/" /><meta name="ncbi_pagename" content="Screen for RAS-Selective Lethal Compounds and VDAC Ligands - Probe 2 - Probe Reports from the NIH Molecular Libraries Program - NCBI Bookshelf" /><meta name="ncbi_bookparttype" content="chapter" /><meta name="ncbi_app" content="bookshelf" />
|
||
<!-- Logger end -->
|
||
|
||
<title>Screen for RAS-Selective Lethal Compounds and VDAC Ligands - Probe 2 - Probe Reports from the NIH Molecular Libraries Program - NCBI Bookshelf</title>
|
||
|
||
<!-- AppResources external_resources begin -->
|
||
<link rel="stylesheet" href="/core/jig/1.15.2/css/jig.min.css" /><script type="text/javascript" src="/core/jig/1.15.2/js/jig.min.js"></script>
|
||
|
||
<!-- AppResources external_resources end -->
|
||
|
||
<!-- Page meta begin -->
|
||
<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="Screen for RAS-Selective Lethal Compounds and VDAC Ligands - Probe 2" /><meta name="citation_publisher" content="National Center for Biotechnology Information (US)" /><meta name="citation_date" content="2011/12/12" /><meta name="citation_author" content="Joshua A Bittker" /><meta name="citation_author" content="Michel Weiwer" /><meta name="citation_author" content="Tim Lewis" /><meta name="citation_author" content="Kenichi Shimada" /><meta name="citation_author" content="Wan S Yang" /><meta name="citation_author" content="Lawrence MacPherson" /><meta name="citation_author" content="Sivaraman Dandapani" /><meta name="citation_author" content="Ben Munoz" /><meta name="citation_author" content="Michelle Palmer" /><meta name="citation_author" content="Brent R Stockwell" /><meta name="citation_author" content="Stuart L Schreiber" /><meta name="citation_pmid" content="22834036" /><meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK98919/" /><link rel="schema.DC" href="http://purl.org/DC/elements/1.0/" /><meta name="DC.Title" content="Screen for RAS-Selective Lethal Compounds and VDAC Ligands - Probe 2" /><meta name="DC.Type" content="Text" /><meta name="DC.Publisher" content="National Center for Biotechnology Information (US)" /><meta name="DC.Contributor" content="Joshua A Bittker" /><meta name="DC.Contributor" content="Michel Weiwer" /><meta name="DC.Contributor" content="Tim Lewis" /><meta name="DC.Contributor" content="Kenichi Shimada" /><meta name="DC.Contributor" content="Wan S Yang" /><meta name="DC.Contributor" content="Lawrence MacPherson" /><meta name="DC.Contributor" content="Sivaraman Dandapani" /><meta name="DC.Contributor" content="Ben Munoz" /><meta name="DC.Contributor" content="Michelle Palmer" /><meta name="DC.Contributor" content="Brent R Stockwell" /><meta name="DC.Contributor" content="Stuart L Schreiber" /><meta name="DC.Date" content="2011/12/12" /><meta name="DC.Identifier" content="https://www.ncbi.nlm.nih.gov/books/NBK98919/" /><meta name="description" content="Synthetic lethal screening is a chemical biology approach to identify small molecules that selectively kill oncogene-expressing engineered cell lines, with the goal of identifying pathways that provide specific targets against cancer cells. We performed a high-throughput screen of 303,282 compounds from the Molecular Libraries Small Molecule Repository (MLSMR) against immortalized BJ fibroblasts expressing HRASV12 followed by a counterscreen of lethal compounds in a series of isogenic cells lacking the oncogene. A new chemical class was identified that had improved potency compared with previously known RAS selective compounds. The most potent and selective of these, probe ML210, displayed nanomolar potency in the primary screening cell line while maintaining selectivity similar to previously identified probes. The probe is in a novel structural class in the field of RAS synthetically lethal compounds and will, therefore, be highly useful in identifying pathways that can potentially be used for selectively inhibiting cancer cells." /><meta name="og:title" content="Screen for RAS-Selective Lethal Compounds and VDAC Ligands - Probe 2" /><meta name="og:type" content="book" /><meta name="og:description" content="Synthetic lethal screening is a chemical biology approach to identify small molecules that selectively kill oncogene-expressing engineered cell lines, with the goal of identifying pathways that provide specific targets against cancer cells. We performed a high-throughput screen of 303,282 compounds from the Molecular Libraries Small Molecule Repository (MLSMR) against immortalized BJ fibroblasts expressing HRASV12 followed by a counterscreen of lethal compounds in a series of isogenic cells lacking the oncogene. A new chemical class was identified that had improved potency compared with previously known RAS selective compounds. The most potent and selective of these, probe ML210, displayed nanomolar potency in the primary screening cell line while maintaining selectivity similar to previously identified probes. The probe is in a novel structural class in the field of RAS synthetically lethal compounds and will, therefore, be highly useful in identifying pathways that can potentially be used for selectively inhibiting cancer cells." /><meta name="og:url" content="https://www.ncbi.nlm.nih.gov/books/NBK98919/" /><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/ml210/" /><link rel="canonical" href="https://www.ncbi.nlm.nih.gov/books/NBK98919/" /><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" />
|
||
|
||
<!-- Page meta end -->
|
||
<link rel="shortcut icon" href="//www.ncbi.nlm.nih.gov/favicon.ico" /><meta name="ncbi_phid" content="CE8C841E7D6653210000000000520046.m_13" />
|
||
<meta name='referrer' content='origin-when-cross-origin'/><link type="text/css" rel="stylesheet" href="//static.pubmed.gov/portal/portal3rc.fcgi/4216699/css/3852956/3985586/3808861/4121862/3974050/3917732/251717/4216701/14534/45193/4113719/3849091/3984811/3751656/4033350/3840896/3577051/3852958/4008682/4207974/4206132/4062871/12930/3964959/3854974/36029/4128070/9685/3549676/3609192/3609193/3609213/3395586.css" /><link type="text/css" rel="stylesheet" href="//static.pubmed.gov/portal/portal3rc.fcgi/4216699/css/3411343/3882866.css" media="print" /></head>
|
||
<body class="book-part">
|
||
<div class="grid">
|
||
<div class="col twelve_col nomargin shadow">
|
||
<!-- System messages like service outage or JS required; this is handled by the TemplateResources portlet -->
|
||
<div class="sysmessages">
|
||
<noscript>
|
||
<p class="nojs">
|
||
<strong>Warning:</strong>
|
||
The NCBI web site requires JavaScript to function.
|
||
<a href="/guide/browsers/#enablejs" title="Learn how to enable JavaScript" target="_blank">more...</a>
|
||
</p>
|
||
</noscript>
|
||
</div>
|
||
<!--/.sysmessage-->
|
||
<div class="wrap">
|
||
<div class="page">
|
||
<div class="top">
|
||
<div id="universal_header">
|
||
<section class="usa-banner">
|
||
<div class="usa-accordion">
|
||
<header class="usa-banner-header">
|
||
<div class="usa-grid usa-banner-inner">
|
||
<img src="https://www.ncbi.nlm.nih.gov/coreutils/uswds/img/favicons/favicon-57.png" alt="U.S. flag" />
|
||
<p>An official website of the United States government</p>
|
||
<button class="non-usa-accordion-button usa-banner-button" aria-expanded="false" aria-controls="gov-banner-top" type="button">
|
||
<span class="usa-banner-button-text">Here's how you know</span>
|
||
</button>
|
||
</div>
|
||
</header>
|
||
<div class="usa-banner-content usa-grid usa-accordion-content" id="gov-banner-top" aria-hidden="true">
|
||
<div class="usa-banner-guidance-gov usa-width-one-half">
|
||
<img class="usa-banner-icon usa-media_block-img" src="https://www.ncbi.nlm.nih.gov/coreutils/uswds/img/icon-dot-gov.svg" alt="Dot gov" />
|
||
<div class="usa-media_block-body">
|
||
<p>
|
||
<strong>The .gov means it's official.</strong>
|
||
<br />
|
||
Federal government websites often end in .gov or .mil. Before
|
||
sharing sensitive information, make sure you're on a federal
|
||
government site.
|
||
</p>
|
||
</div>
|
||
</div>
|
||
<div class="usa-banner-guidance-ssl usa-width-one-half">
|
||
<img class="usa-banner-icon usa-media_block-img" src="https://www.ncbi.nlm.nih.gov/coreutils/uswds/img/icon-https.svg" alt="Https" />
|
||
<div class="usa-media_block-body">
|
||
<p>
|
||
<strong>The site is secure.</strong>
|
||
<br />
|
||
The <strong>https://</strong> ensures that you are connecting to the
|
||
official website and that any information you provide is encrypted
|
||
and transmitted securely.
|
||
</p>
|
||
</div>
|
||
</div>
|
||
</div>
|
||
</div>
|
||
</section>
|
||
<div class="usa-overlay"></div>
|
||
<header class="ncbi-header" role="banner" data-section="Header">
|
||
|
||
<div class="usa-grid">
|
||
<div class="usa-width-one-whole">
|
||
|
||
<div class="ncbi-header__logo">
|
||
<a href="/" class="logo" aria-label="NCBI Logo" data-ga-action="click_image" data-ga-label="NIH NLM Logo">
|
||
<img src="https://www.ncbi.nlm.nih.gov/coreutils/nwds/img/logos/AgencyLogo.svg" alt="NIH NLM Logo" />
|
||
</a>
|
||
</div>
|
||
|
||
<div class="ncbi-header__account">
|
||
<a id="account_login" href="https://account.ncbi.nlm.nih.gov" class="usa-button header-button" style="display:none" data-ga-action="open_menu" data-ga-label="account_menu">Log in</a>
|
||
<button id="account_info" class="header-button" style="display:none" aria-controls="account_popup" type="button">
|
||
<span class="fa fa-user" aria-hidden="true">
|
||
<svg xmlns="http://www.w3.org/2000/svg" viewBox="0 0 24 24" width="20px" height="20px">
|
||
<g style="fill: #fff">
|
||
<ellipse cx="12" cy="8" rx="5" ry="6"></ellipse>
|
||
<path d="M21.8,19.1c-0.9-1.8-2.6-3.3-4.8-4.2c-0.6-0.2-1.3-0.2-1.8,0.1c-1,0.6-2,0.9-3.2,0.9s-2.2-0.3-3.2-0.9 C8.3,14.8,7.6,14.7,7,15c-2.2,0.9-3.9,2.4-4.8,4.2C1.5,20.5,2.6,22,4.1,22h15.8C21.4,22,22.5,20.5,21.8,19.1z"></path>
|
||
</g>
|
||
</svg>
|
||
</span>
|
||
<span class="username desktop-only" aria-hidden="true" id="uname_short"></span>
|
||
<span class="sr-only">Show account info</span>
|
||
</button>
|
||
</div>
|
||
|
||
<div class="ncbi-popup-anchor">
|
||
<div class="ncbi-popup account-popup" id="account_popup" aria-hidden="true">
|
||
<div class="ncbi-popup-head">
|
||
<button class="ncbi-close-button" data-ga-action="close_menu" data-ga-label="account_menu" type="button">
|
||
<span class="fa fa-times">
|
||
<svg xmlns="http://www.w3.org/2000/svg" viewBox="0 0 48 48" width="24px" height="24px">
|
||
<path d="M38 12.83l-2.83-2.83-11.17 11.17-11.17-11.17-2.83 2.83 11.17 11.17-11.17 11.17 2.83 2.83 11.17-11.17 11.17 11.17 2.83-2.83-11.17-11.17z"></path>
|
||
</svg>
|
||
</span>
|
||
<span class="usa-sr-only">Close</span></button>
|
||
<h4>Account</h4>
|
||
</div>
|
||
<div class="account-user-info">
|
||
Logged in as:<br />
|
||
<b><span class="username" id="uname_long">username</span></b>
|
||
</div>
|
||
<div class="account-links">
|
||
<ul class="usa-unstyled-list">
|
||
<li><a id="account_myncbi" href="/myncbi/" class="set-base-url" data-ga-action="click_menu_item" data-ga-label="account_myncbi">Dashboard</a></li>
|
||
<li><a id="account_pubs" href="/myncbi/collections/bibliography/" class="set-base-url" data-ga-action="click_menu_item" data-ga-label="account_pubs">Publications</a></li>
|
||
<li><a id="account_settings" href="/account/settings/" class="set-base-url" data-ga-action="click_menu_item" data-ga-label="account_settings">Account settings</a></li>
|
||
<li><a id="account_logout" href="/account/signout/" class="set-base-url" data-ga-action="click_menu_item" data-ga-label="account_logout">Log out</a></li>
|
||
</ul>
|
||
</div>
|
||
</div>
|
||
</div>
|
||
|
||
</div>
|
||
</div>
|
||
</header>
|
||
<div role="navigation" aria-label="access keys">
|
||
<a id="nws_header_accesskey_0" href="https://www.ncbi.nlm.nih.gov/guide/browsers/#ncbi_accesskeys" class="usa-sr-only" accesskey="0" tabindex="-1">Access keys</a>
|
||
<a id="nws_header_accesskey_1" href="https://www.ncbi.nlm.nih.gov" class="usa-sr-only" accesskey="1" tabindex="-1">NCBI Homepage</a>
|
||
<a id="nws_header_accesskey_2" href="/myncbi/" class="set-base-url usa-sr-only" accesskey="2" tabindex="-1">MyNCBI Homepage</a>
|
||
<a id="nws_header_accesskey_3" href="#maincontent" class="usa-sr-only" accesskey="3" tabindex="-1">Main Content</a>
|
||
<a id="nws_header_accesskey_4" href="#" class="usa-sr-only" accesskey="4" tabindex="-1">Main Navigation</a>
|
||
</div>
|
||
<section data-section="Alerts">
|
||
<div class="ncbi-alerts-placeholder"></div>
|
||
</section>
|
||
</div>
|
||
<div class="header">
|
||
<div class="res_logo"><h1 class="res_name"><a href="/books/" title="Bookshelf home">Bookshelf</a></h1><h2 class="res_tagline"></h2></div>
|
||
<div class="search"><form method="get" action="/books/"><div class="search_form"><label for="database" class="offscreen_noflow">Search database</label><select id="database"><optgroup label="Recent"><option value="books" selected="selected" data-ac_dict="bookshelf-search">Books</option><option value="pubmed">PubMed</option><option value="clinvar">ClinVar</option><option value="refseq" class="last">RefSeq</option></optgroup><optgroup label="All"><option value="gquery">All Databases</option><option value="assembly">Assembly</option><option value="biocollections">Biocollections</option><option value="bioproject">BioProject</option><option value="biosample">BioSample</option><option value="books" data-ac_dict="bookshelf-search">Books</option><option value="clinvar">ClinVar</option><option value="cdd">Conserved Domains</option><option value="gap">dbGaP</option><option value="dbvar">dbVar</option><option value="gene">Gene</option><option value="genome">Genome</option><option value="gds">GEO DataSets</option><option value="geoprofiles">GEO Profiles</option><option value="gtr">GTR</option><option value="ipg">Identical Protein Groups</option><option value="medgen">MedGen</option><option value="mesh">MeSH</option><option value="nlmcatalog">NLM Catalog</option><option value="nuccore">Nucleotide</option><option value="omim">OMIM</option><option value="pmc">PMC</option><option value="protein">Protein</option><option value="proteinclusters">Protein Clusters</option><option value="protfam">Protein Family Models</option><option value="pcassay">PubChem BioAssay</option><option value="pccompound">PubChem Compound</option><option value="pcsubstance">PubChem Substance</option><option value="pubmed">PubMed</option><option value="snp">SNP</option><option value="sra">SRA</option><option value="structure">Structure</option><option value="taxonomy">Taxonomy</option><option value="toolkit">ToolKit</option><option value="toolkitall">ToolKitAll</option><option value="toolkitbookgh">ToolKitBookgh</option></optgroup></select><div class="nowrap"><label for="term" class="offscreen_noflow" accesskey="/">Search term</label><div class="nowrap"><input type="text" name="term" id="term" title="Search Books. Use up and down arrows to choose an item from the autocomplete." value="" class="jig-ncbiclearbutton jig-ncbiautocomplete" data-jigconfig="dictionary:'bookshelf-search',disableUrl:'NcbiSearchBarAutoComplCtrl'" autocomplete="off" data-sbconfig="ds:'no',pjs:'no',afs:'no'" /></div><button id="search" type="submit" class="button_search nowrap" cmd="go">Search</button></div></div></form><ul class="searchlinks inline_list"><li>
|
||
<a href="/books/browse/">Browse Titles</a>
|
||
</li><li>
|
||
<a href="/books/advanced/">Advanced</a>
|
||
</li><li class="help">
|
||
<a href="/books/NBK3833/">Help</a>
|
||
</li><li class="disclaimer">
|
||
<a target="_blank" data-ga-category="literature_resources" data-ga-action="link_click" data-ga-label="disclaimer_link" href="https://www.ncbi.nlm.nih.gov/books/about/disclaimer/">Disclaimer</a>
|
||
</li></ul></div>
|
||
</div>
|
||
|
||
|
||
|
||
<!--<component id="Page" label="headcontent"/>-->
|
||
|
||
</div>
|
||
<div class="content">
|
||
<!-- site messages -->
|
||
<!-- Custom content 1 -->
|
||
<div class="col1">
|
||
|
||
</div>
|
||
|
||
<div class="container">
|
||
<div id="maincontent" class="content eight_col col">
|
||
<!-- Custom content in the left column above book nav -->
|
||
<div class="col2">
|
||
|
||
</div>
|
||
|
||
<!-- Book content -->
|
||
|
||
|
||
<!-- Custom content between navigation and content -->
|
||
<div class="col3">
|
||
|
||
</div>
|
||
|
||
<div class="document">
|
||
<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="#__NBK98919_dtls__">Show details</a><div style="display:none" class="ui-widget" id="__NBK98919_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/ml211/" title="Previous page in this title">< Prev</a><a class="active page_link next" href="/books/n/mlprobe/ml209/" title="Next page in this title">Next ></a></div></div></div></div></div>
|
||
<div class="main-content lit-style" itemscope="itemscope" itemtype="http://schema.org/CreativeWork"><div class="meta-content fm-sec"><h1 id="_NBK98919_"><span class="title" itemprop="name">Screen for RAS-Selective Lethal Compounds and VDAC Ligands - Probe 2</span></h1><p class="contrib-group"><span itemprop="author">Joshua A Bittker</span>, <span itemprop="author">Michel Weiwer</span>, <span itemprop="author">Tim Lewis</span>, <span itemprop="author">Kenichi Shimada</span>, <span itemprop="author">Wan S Yang</span>, <span itemprop="author">Lawrence MacPherson</span>, <span itemprop="author">Sivaraman Dandapani</span>, <span itemprop="author">Ben Munoz</span>, <span itemprop="author">Michelle Palmer</span>, <span itemprop="author">Brent R Stockwell</span>, and <span itemprop="author">Stuart L Schreiber</span>.</p><a data-jig="ncbitoggler" href="#__NBK98919_ai__" style="border:0;text-decoration:none">Author Information and Affiliations</a><div style="display:none" class="ui-widget" id="__NBK98919_ai__"><p class="contrib-group"><h4>Authors</h4><span itemprop="author">Joshua A Bittker</span>,<sup>1</sup> <span itemprop="author">Michel Weiwer</span>,<sup>1</sup> <span itemprop="author">Tim Lewis</span>,<sup>1</sup> <span itemprop="author">Kenichi Shimada</span>,<sup>2</sup> <span itemprop="author">Wan S Yang</span>,<sup>2</sup> <span itemprop="author">Lawrence MacPherson</span>,<sup>1</sup> <span itemprop="author">Sivaraman Dandapani</span>,<sup>1</sup> <span itemprop="author">Ben Munoz</span>,<sup>1</sup> <span itemprop="author">Michelle Palmer</span>,<sup>1</sup> <span itemprop="author">Brent R Stockwell</span>,<sup>2</sup> and <span itemprop="author">Stuart L Schreiber</span><sup>1,3</sup>.</p><h4>Affiliations</h4><div class="affiliation"><sup>1</sup>
|
||
The Broad Institute Probe Development Center, Cambridge, MA</div><div class="affiliation"><sup>2</sup>
|
||
Howard Hughes Medical Institute, Department of Biological Sciences and
|
||
Department of Chemistry, Columbia University, New York, NY</div><div class="affiliation"><sup>3</sup>
|
||
Howard Hughes Medical Institute, Chemistry & Chemical Biology,
|
||
Harvard University, Cambridge, MA</div></div><p class="small">Received: <span itemprop="datePublished">February 10, 2011</span>; Last Update: <span itemprop="dateModified">December 12, 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>Synthetic lethal screening is a chemical biology approach to identify small molecules
|
||
that selectively kill oncogene-expressing engineered cell lines, with the goal of
|
||
identifying pathways that provide specific targets against cancer cells. We
|
||
performed a high-throughput screen of 303,282 compounds from the Molecular Libraries
|
||
Small Molecule Repository (MLSMR) against immortalized BJ fibroblasts expressing
|
||
HRAS<sup>V12</sup> followed by a counterscreen of lethal compounds in a series
|
||
of isogenic cells lacking the oncogene. A new chemical class was identified that had
|
||
improved potency compared with previously known RAS selective compounds. The most
|
||
potent and selective of these, probe <a href="/pcsubstance/?term=ML210[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML210</a>, displayed nanomolar
|
||
potency in the primary screening cell line while maintaining selectivity similar to
|
||
previously identified probes. The probe is in a novel structural class in the field
|
||
of RAS synthetically lethal compounds and will, therefore, be highly useful in
|
||
identifying pathways that can potentially be used for selectively inhibiting cancer
|
||
cells.</p></div><div class="h2"></div><p><b>Assigned Assay Grant No.:</b> 1R03MH084117-01</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 & Institution:</b> Brent R. Stockwell, Howard Hughes Medical Institute, Columbia University</p><p><b>PubChem Summary Bioassay Identifier (AID):</b>
|
||
<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1674" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1674</a></p><div id="ml210.s1"><h2 id="_ml210_s1_">Probe Structure & Characteristics</h2><div id="ml210.fu1" class="figure bk_fig"><div class="graphic"><img src="/books/NBK98919/bin/ml210fu1.jpg" alt="ML210." /></div><h3><span class="title"><a href="/pcsubstance/?term=ML210[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML210</a></span></h3></div><div id="ml210.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/NBK98919/table/ml210.tu1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml210.tu1_lrgtbl__"><table class="no_top_margin"><tbody><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">IUPAC Chemical Name</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">(4-(bis(4-chlorophenyl)methyl)piperazin-1-yl)(5-methyl-4-nitroisoxazol-3-yl)methanone</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;">CID 49766530</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;">475.32460 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>22</sub>H<sub>20</sub>Cl<sub>2</sub>N<sub>4</sub>O<sub>4</sub></td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">XLogP3</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">4.65</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">H-Bond Donor</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">H-Bond Acceptor</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">7</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Rotatable Bond Count</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">5</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Exact Mass</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">474.08616</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;">95.4</td></tr></tbody></table></div></div><div id="ml210.tu2" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK98919/table/ml210.tu2/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml210.tu2_lrgtbl__"><table><thead><tr><th id="hd_h_ml210.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CID/ML#</th><th id="hd_h_ml210.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Target Name</th><th id="hd_h_ml210.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_ml210.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Anti-target Name(s)</th><th id="hd_h_ml210.tu2_1_1_1_5" 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_ml210.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Fold Selective*</th><th id="hd_h_ml210.tu2_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Secondary Assay(s) Name: IC<sub>50</sub>/EC<sub>50</sub> (nM) [SID, AID]</th></tr></thead><tbody><tr><td headers="hd_h_ml210.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CID 49766530/<a href="/pcsubstance/?term=ML210[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML210</a></td><td headers="hd_h_ml210.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">BJeLR (HRAS<sup>V12</sup>)</td><td headers="hd_h_ml210.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">71 [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/103023280" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 103023280</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/493045" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 493045</a>]</td><td headers="hd_h_ml210.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">BJeH-LT (without HRAS<sup>V12</sup>)</td><td headers="hd_h_ml210.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">272 [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/103023280" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 103023280</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/493093" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 493093</a>]</td><td headers="hd_h_ml210.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">4</td><td headers="hd_h_ml210.tu2_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">DRD HRAS<sup>V12</sup>: 107 [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/103023280" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 103023280</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/493050" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 493050</a>];<br />BJeH (w/o HRAS<sup>V12</sup>): 628 [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/103023280" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 103023280</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/493051" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 493051</a>]</td></tr></tbody></table></div></div></div><div id="ml210.s2"><h2 id="_ml210_s2_">Recommendations for scientific use of the probe</h2><p>The goal of the project is to identify small molecules that are selectively lethal to tumor cells expressing RAS oncogenes. These probes will not necessarily interact with the RAS pathway, but may induce cell death by affecting cellular processes that, in conjunction with the activation of the RAS pathway, selectively kill the tumor cells.</p><p>One goal of identifying novel RAS selective probes is to determine additional pathways that may be targets for novel therapeutics. All three existing probes result in the same phenotype, with two of the three having confirmed activity against the same target, voltage dependent anion channels (VDACs.) Additional probes against other targets will assist in elucidating other targets or pathways of interest in RAS dependent tumors. In addition, novel probes that are similar to existing compounds that do target VDACs, but which have different selectivity or binding modes, will be valuable in determining the potential use of VDAC inhibition as a therapeutic method.</p><p>The probe <a href="/pcsubstance/?term=ML210[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML210</a> described herein has biological properties similar to a previously known compound (RSL-3), but has improved potency and is in a novel chemical series. Whereas RSL-3 and another probe identified in this project (probe <a href="/pcsubstance/?term=ML162[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML162</a>), have a common “warhead” that is potentially reactive, probe <a href="/pcsubstance/?term=ML210[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML210</a> has no such liability. This probe, therefore, represents an improvement over the state of the art (see <a href="#ml210.s29">Section 4.1</a>), and will be of benefit to the scientific community in determining the effects of RAS mutation on cellular pathways.</p><p>The probe <a href="/pcsubstance/?term=ML210[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML210</a> is stable in PBS for a minimum of 48h at room temperature and as a dry powder, in a desiccator, for a minimum of 1 year. The probe should be stored at room temperature in a dessicator and preferably protected from light (dark glass-container).</p></div><div id="ml210.s3"><h2 id="_ml210_s3_">1. Introduction</h2><p>The first rat sarcoma (RAS) oncogene was discovered as a genetic element from the Harvey and Kirsten rat sarcoma viruses with the ability to immortalize mammalian cells (<a class="bk_pop" href="#ml210.r1">1</a>,<a class="bk_pop" href="#ml210.r2">2</a>,<a class="bk_pop" href="#ml210.r3">3</a>). Mutated RAS oncogenes (i.e., HRAS, NRAS, and KRAS) are found in 10–20% of all human cancer. KRAS mutations are found in >90% of pancreatic cancers, 50% of colon cancers, and 25% of lung adenocarcinomas; NRAS mutations are found in 30% of liver cancers and 15% of melanomas; and HRAS mutations are found in 10% of kidney and bladder cancers (<a class="bk_pop" href="#ml210.r4">4</a>).</p><p>RAS proteins are guanine-nucleotide-binding proteins with GTPase activity and are associated with the plasma membrane. In the GTP-bound form, RAS proteins are mitogenic. Mutation of glycine-12 to other amino acids (including valine [RASG12V]) results in an oncogenic allele with constitutive mitogenic, transforming activity and reduced GTPase activity (<a class="bk_pop" href="#ml210.r5">5</a>). Four downstream pathways activated by RAS proteins are: 1) the RAF/MEK/ERK pathway, which regulates cell-cycle progression, 2) the PI3K/PDK/AKT pathway, which regulates cell survival, 3) the RalGDS pathway, which regulates membrane trafficking and vesicle formation, and 4) the PLCɛ/PKC pathway, which regulates Ca<sup>++</sup> signaling (<a class="bk_pop" href="#ml210.r5">5</a>,<a class="bk_pop" href="#ml210.r6">6</a>,<a class="bk_pop" href="#ml210.r7">7</a>).</p><p>Compounds that selectively kill cells expressing oncogenic RAS have the potential to eliminate tumor cells harboring specific oncogenic mutations while having minimal effects on normal cells lacking these mutations. This mode of action is known as synthetic lethality. Such synthetically lethal compounds can be used to elucidate the pathways that are involved in the oncogenesis of the mutant RAS gene whether directly in RAS-related pathways or in other pathways, such as metabolic function, that may be modulated by the activity of an oncogenic allele.</p><p>RAS synthetic lethal genes (such as TBK1, CCNA2, KIF2C, PLK1, APC/C, CDK4, and STK33) have recently been identified using RNAi screens (<a class="bk_pop" href="#ml210.r8">8</a>,<a class="bk_pop" href="#ml210.r9">9</a>,<a class="bk_pop" href="#ml210.r10">10</a>,<a class="bk_pop" href="#ml210.r11">11</a>,<a class="bk_pop" href="#ml210.r12">12</a>). Efforts were then undertaken to develop small molecule inhibitors of these targets, especially kinases. BI 6727, a PLK1 inhibitor developed by Boehringer Ingelheim (<a class="bk_pop" href="#ml210.r13">13</a>), BX 795, a TBK1 inhibitor developed by Berlex Biosciences (<a class="bk_pop" href="#ml210.r14">14</a>), and PD 0332991, a CDK4 inhibitor developed by Pfizer (<a class="bk_pop" href="#ml210.r15">15</a>), are presented in <a class="figpopup" href="/books/NBK98919/figure/ml210.f1/?report=objectonly" target="object" rid-figpopup="figml210f1" rid-ob="figobml210f1">Figure 1</a>.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml210f1" co-legend-rid="figlgndml210f1"><a href="/books/NBK98919/figure/ml210.f1/?report=objectonly" target="object" title="Figure 1" class="img_link icnblk_img figpopup" rid-figpopup="figml210f1" rid-ob="figobml210f1"><img class="small-thumb" src="/books/NBK98919/bin/ml210f1.gif" src-large="/books/NBK98919/bin/ml210f1.jpg" alt="Figure 1. RAS Synthetic Lethal Compounds Discovered Through RNAi Screens." /></a><div class="icnblk_cntnt" id="figlgndml210f1"><h4 id="ml210.f1"><a href="/books/NBK98919/figure/ml210.f1/?report=objectonly" target="object" rid-ob="figobml210f1">Figure 1</a></h4><p class="float-caption no_bottom_margin">RAS Synthetic Lethal Compounds Discovered Through RNAi Screens. BI 6727, a PLK1 inhibitor (Boehringer Ingelheim)(<i>a</i>); BX 795, a TBK1 inhibitor (Berlex Biosciences) (<i>b</i>); PD 0332991, a CDK4 inhibitor (Pfizer) (<i>c</i>). </p></div></div><p>In another approach, phenotypic screens allow the direct identification of small molecules that are selectively lethal to cell lines expressing a RAS oncogene. Further studies are then necessary to identify the target. Guo and colleagues (<a class="bk_pop" href="#ml210.r16">16</a>) have described a compound named oncrasin-1 (<a class="figpopup" href="/books/NBK98919/figure/ml210.f2/?report=objectonly" target="object" rid-figpopup="figml210f2" rid-ob="figobml210f2">Figure 2a</a>), which is synthetically lethal to cell lines harboring a KRAS oncogene but ineffective in HRAS mutant cell lines. Recently, Shaw et al. (<a class="bk_pop" href="#ml210.r17">17</a>) reported lanperisone (<a class="figpopup" href="/books/NBK98919/figure/ml210.f2/?report=objectonly" target="object" rid-figpopup="figml210f2" rid-ob="figobml210f2">Figure 2b</a>) as a small molecule inducer of oxidative stress that promote selective killing of KRAS mutant cancer cells. Stockwell and colleagues (<a class="bk_pop" href="#ml210.r18">18</a>,<a class="bk_pop" href="#ml210.r19">19</a>) have identified small molecules that are synthetically lethal to several HRAS and KRAS mutant cell lines (<a class="figpopup" href="/books/NBK98919/figure/ml210.f2/?report=objectonly" target="object" rid-figpopup="figml210f2" rid-ob="figobml210f2">Figure 2c</a>). They induce an oxidative, nonapoptotic cell death by targeting the RAS-Raf-MEK pathway. Erastin and RSL-5 have been shown to bind to voltage-dependent anion channels (VDAC) (<a class="bk_pop" href="#ml210.r18">18</a>) as opposed to RSL-3, which act in a VDAC-independent manner.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml210f2" co-legend-rid="figlgndml210f2"><a href="/books/NBK98919/figure/ml210.f2/?report=objectonly" target="object" title="Figure 2" class="img_link icnblk_img figpopup" rid-figpopup="figml210f2" rid-ob="figobml210f2"><img class="small-thumb" src="/books/NBK98919/bin/ml210f2.gif" src-large="/books/NBK98919/bin/ml210f2.jpg" alt="Figure 2. RAS Synthetic Lethal Compounds Discovered Through Phenotypic Screens." /></a><div class="icnblk_cntnt" id="figlgndml210f2"><h4 id="ml210.f2"><a href="/books/NBK98919/figure/ml210.f2/?report=objectonly" target="object" rid-ob="figobml210f2">Figure 2</a></h4><p class="float-caption no_bottom_margin">RAS Synthetic Lethal Compounds Discovered Through Phenotypic Screens. Compound identified by Guo and colleagues that is synthetically lethal to KRAS mutant cell lines (<i>a</i>); Compound identified by Shaw and colleagues that is synthetically lethal to KRAS <a href="/books/NBK98919/figure/ml210.f2/?report=objectonly" target="object" rid-ob="figobml210f2">(more...)</a></p></div></div><p>While erastin and RSL-3 compounds were used as on-target positive controls through this project (<a class="figpopup" href="/books/NBK98919/figure/ml210.f3/?report=objectonly" target="object" rid-figpopup="figml210f3" rid-ob="figobml210f3">Figure 3</a><b>,</b>
|
||
<a class="figpopup" href="/books/NBK98919/figure/ml210.f4/?report=objectonly" target="object" rid-figpopup="figml210f4" rid-ob="figobml210f4">Figure 4</a>), additional compounds that produce the same phenotype would be useful in determining alternate modes of binding to VDACs. Other compounds that are synthetically lethal with HRAS<sup>V12</sup>, regardless of mechanism, would be beneficial for identifying additional targets for possible therapeutic intervention.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml210f3" co-legend-rid="figlgndml210f3"><a href="/books/NBK98919/figure/ml210.f3/?report=objectonly" target="object" title="Figure 3" class="img_link icnblk_img figpopup" rid-figpopup="figml210f3" rid-ob="figobml210f3"><img class="small-thumb" src="/books/NBK98919/bin/ml210f3.gif" src-large="/books/NBK98919/bin/ml210f3.jpg" alt="Figure 3. Growth Inhibition of HRASV12 Expressing and Non-expressing Cell Lines by the Positive Control Compound Erastin (CID 11214940) Identified in Pilot Screening." /></a><div class="icnblk_cntnt" id="figlgndml210f3"><h4 id="ml210.f3"><a href="/books/NBK98919/figure/ml210.f3/?report=objectonly" target="object" rid-ob="figobml210f3">Figure 3</a></h4><p class="float-caption no_bottom_margin">Growth Inhibition of HRAS<sup>V12</sup> Expressing and Non-expressing Cell Lines by the Positive Control Compound Erastin (CID 11214940) Identified in Pilot Screening. Growth inhibition concentration-response curves of erastin in engineered BJ fibroblasts expressing <a href="/books/NBK98919/figure/ml210.f3/?report=objectonly" target="object" rid-ob="figobml210f3">(more...)</a></p></div></div><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml210f4" co-legend-rid="figlgndml210f4"><a href="/books/NBK98919/figure/ml210.f4/?report=objectonly" target="object" title="Figure 4" class="img_link icnblk_img figpopup" rid-figpopup="figml210f4" rid-ob="figobml210f4"><img class="small-thumb" src="/books/NBK98919/bin/ml210f4.gif" src-large="/books/NBK98919/bin/ml210f4.jpg" alt="Figure 4. Growth Inhibition of HRASV12 Expressing and Non-expressing Cell Lines by the Positive Control Compound RSL-3 (CID 40911229) Identified in Pilot Screening." /></a><div class="icnblk_cntnt" id="figlgndml210f4"><h4 id="ml210.f4"><a href="/books/NBK98919/figure/ml210.f4/?report=objectonly" target="object" rid-ob="figobml210f4">Figure 4</a></h4><p class="float-caption no_bottom_margin">Growth Inhibition of HRAS<sup>V12</sup> Expressing and Non-expressing Cell Lines by the Positive Control Compound RSL-3 (CID 40911229) Identified in Pilot Screening. Growth inhibition concentration-response curves of RSL-3 in engineered BJ fibroblasts expressing <a href="/books/NBK98919/figure/ml210.f4/?report=objectonly" target="object" rid-ob="figobml210f4">(more...)</a></p></div></div></div><div id="ml210.s4"><h2 id="_ml210_s4_">2. Materials and Methods</h2><div id="ml210.s5"><h3>Materials and Reagents</h3><p>All reagents and solvents were purchased from commercial vendors and used as received.</p><p>Phosphate-buffered saline (PBS; catalog no. 08J07A0022) was acquired from the Broad Institute Supply and Quality Management (SQM; Cambridge, MA). CellTiter Glo (catalog no. G7573 lot 268563) was purchased from Promega (Fitchburg, WI), and Alamar Blue (catalog no. DAL1025) was acquired from Biosource/Invitrogen (Grand Island, NY). White, sterile, TC-treated, 384-well plates (catalog no. 3570) were acquired from Corning.</p><div id="ml210.s6"><h4>Growth Medium</h4><p>Dulbecco’s modified Eagle’s medium (DMEM; catalog no. 11995, Lot no. 476124) with 4 mM L-glutamine and fetal bovine serum (FBS; catalog no. 26140-079, Lot no. 302496) were purchased from Gibco (Grand Island, NY). M199 (catalog no. M7528, Lot no. 028K2403) was acquired from Sigma (St. Louis, MO). Trypsin (catalog no. 25-053-Cl; Lot no. 25053204) was purchased from MediaTech (Manassas, VA).</p></div><div id="ml210.s7"><h4>Cell Lines</h4><p>Throughout the project, four different cell types derived from BJ human fibroblasts were used to determine the effect of compounds on either HRAS<sup>V12</sup>-expressing or wild-type cell lines. The progenitor line was engineered into immortalized tumor lines by the method of Hahn (<a class="bk_pop" href="#ml210.r20">20</a>,<a class="bk_pop" href="#ml210.r21">21</a>,<a class="bk_pop" href="#ml210.r22">22</a>,<a class="bk_pop" href="#ml210.r23">23</a>,<a class="bk_pop" href="#ml210.r24">24</a>), which uses the expression of human telomerase (hTERT) and the Simian Virus 40 (SV40) large T (LT) and small T (ST) oncoproteins.</p><p>Three versions of these cells were used for screening. The primary screen was performed in fully transformed cells also expressing an oncogenic RAS allele, HRAS<sup>V12</sup>, a line referred to herein as BJeLR. For counterscreening, the isogenic cell line without HRAS<sup>V12</sup> was used, referred to as BJeH-LT. BJ fibroblasts with only hTERT expression were also used for non-HRAS<sup>V12</sup>-expressing counterscreening (BJeH). In addition, an alternative HRAS<sup>V12</sup>-expressing line was generated with different immortalizing factors to eliminate the possibility of compounds acting in a synthetically lethal manner with one of these other factors. These cells (referred to as DRD) are BJ fibroblasts expressing hTERT, SV40 small T oncoprotein, dominant negative p53, cyclin D1, and a mutant form of CDK4, along with the gene of interest, HRAS<sup>V12</sup>.</p></div></div><div id="ml210.s8"><h3>2.1. Assays</h3><p>A summary listing of completed assays and corresponding PubChem AID numbers is provided in <a href="#ml210.app1">Appendix A</a> (<a class="figpopup" href="/books/NBK98919/table/ml210.t10/?report=objectonly" target="object" rid-figpopup="figml210t10" rid-ob="figobml210t10">Table A1</a>). Refer to <a href="#ml210.app2">Appendix B</a> for the detailed assay protocols.</p><div id="ml210.s9"><h4>2.1.1. Primary Screen for BJeLR Cell Viability</h4><p>All cell lines were generated by the Stockwell lab as described previously (<a class="bk_pop" href="#ml210.r18">18</a>,<a class="bk_pop" href="#ml210.r19">19</a>). Cells were maintained in 35 ml of growth medium (per 1 Liter: 730 ml DMEM with 4 mM L-glutamine, 210 ml M199, 150 ml heat-inactivated fetal bovine serum (FBS) in a T175 cell culture flask (Corning) and incubated in a TC incubator (Thermo-Fisher) at 95% humidity, 5% CO<sub>2</sub>, 37°C. To passage, cells were harvested by first aspirating the media and rinsing the flask with 10 ml sterile PBS. Next, PBS was aspirated and 5 ml trypsin was added to the flask and incubated for 5 minutes at 22°C. Then, 8 ml of growth medium was added to the trypsin to quench the reaction. The cells were resuspended, counted, and 4.5 million cells in approximately 2 ml were transferred to 33 ml fresh growth medium in a new T175 flask. The lines were carried for no more than twenty passages.</p><p>For screening, the cells were harvested, and the concentration was adjusted to 33,000/ml. While gently stirring, cells were dispensed with a Combi multidrop (Thermo-Fisher) by adding 30 μL of suspension per well to white, sterile, TC-treated, 384-well plates (Corning) for a total of 1000 cells per well. The plates were incubated overnight in an automated TC incubator (Liconic) at 95% humidity, 5% CO<sub>2</sub>, 37°C.</p><p>For compound screening, 50 nL or 100 nL of compound were added, depending on the desired final compound concentration, using slotted steel pins (V&P Scientific) on a pin tool (HiRes Biosolutions). The plates were returned to the incubator for 48 hours. To read viability, the cells were removed from the incubator and cooled to room temperature for 30 minutes. Lids were removed, and 30 μL of diluted CellTiter Glo (1:3 dilution with PBS) was added to each with a Combi Multidrop. The plates were incubated for 10 minutes, and luminescence was detected on an Envision (Perkin-Elmer) multimode reader (0.1 seconds per well).</p></div><div id="ml210.s10"><h4>2.1.2. Primary Retest for BJeLR Cell Viability</h4><p>Repeat of primary screen at dose in BJeLR cells using Cell TiterGlo.</p></div><div id="ml210.s11"><h4>2.1.3. Secondary Counter screen for BJeH/LT/ST Cell Viability (Cell TiterGlo)</h4><p>As described in the primary screen in BJeLR cells but using the BJeH/LT/ST cell line.</p></div><div id="ml210.s12"><h4>2.1.3. Secondary Screen for DRD Cell Viability (Cell TiterGlo)</h4><p>As described in the primary screen in BJeLR cells but using the DRD cell line.</p></div><div id="ml210.s13"><h4>2.1.4. Secondary Counter screen for BJeH Cell Viability (Cell TiterGlo)</h4><p>As described in the primary screen in BJeLR cells but using the BJeH cell line.</p></div><div id="ml210.s14"><h4>2.1.5. Secondary Screen for BJeLR Cell Viability (Alamar Blue)</h4><p>The compounds were diluted into growth medium by adding 2 μL of DMSO compound solution to 148 μL of medium and mixing thoroughly. Dilutions were made in 384-well stock plates (Greiner, catalog no. 781270). Concentration-response curves were then made by further diluting this plate in series by adding 75 μL of solution to 75 μL of fresh growth medium, proceeding across the 384-well plate.</p><p>Next, 36 μL of cell suspension at 28,000 cells per well were added to the assay plates (1000 cells/well), and 4 μL of the medium containing the dilution series of compound were added to the cells. The cells were incubated for 48 hours in a TC incubator at 95% humidity, 5% CO<sub>2</sub>, 37°C. To measure viability, 10 μL Alamar Blue solution (50% in growth medium) was added to each well. The cells were incubated for 16 hours, and fluorescence intensity was read (544 nM excitation, 590 nM emission).</p></div><div id="ml210.s15"><h4>2.1.6. Secondary Screen for BJeH-LT/ST Cell Viability (Alamar Blue)</h4><p>As described in the secondary screen in BJeLR cells but using the BJeH-LT/ST cell line.</p></div><div id="ml210.s16"><h4>2.1.7. Secondary Screen for DRD Cell Viability (Alamar Blue)</h4><p>As described in the secondary screen in BJeLR cells but using the DRD cell line.</p></div><div id="ml210.s17"><h4>2.1.8. Secondary Screen for BJeH Cell Viability (Alamar Blue)</h4><p>As described in the primary screen in BJeLR cells but using the BJeH cell line.</p></div></div><div id="ml210.s18"><h3>2.2. Probe Chemical Characterization</h3><p>The probe molecule (<a href="/pcsubstance/?term=ML210[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML210</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/substance/103023280" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 103023280</a>, BRD-K01877528-001-01-6) was synthesized in six steps from bis(4-chlorophenyl)methanone, 5-methylisoxazole-3-carboxylic acid and piperazine (<a class="figpopup" href="/books/NBK98919/figure/ml210.f13/?report=objectonly" target="object" rid-figpopup="figml210f13" rid-ob="figobml210f13">Scheme 1</a>).</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml210f13" co-legend-rid="figlgndml210f13"><a href="/books/NBK98919/figure/ml210.f13/?report=objectonly" target="object" title="Scheme 1" class="img_link icnblk_img figpopup" rid-figpopup="figml210f13" rid-ob="figobml210f13"><img class="small-thumb" src="/books/NBK98919/bin/ml210f13.gif" src-large="/books/NBK98919/bin/ml210f13.jpg" alt="Scheme 1. Synthesis of the probe." /></a><div class="icnblk_cntnt" id="figlgndml210f13"><h4 id="ml210.f13"><a href="/books/NBK98919/figure/ml210.f13/?report=objectonly" target="object" rid-ob="figobml210f13">Scheme 1</a></h4><p class="float-caption no_bottom_margin">Synthesis of the probe. </p></div></div><p><b>General details.</b> Nitration of the 5-methylisoxazole-3-carboxylic acid building block was accomplished using concentrated sulfuric acid and potassium nitrate in 70% yield. The carboxylic acid <b>1</b> was then converted to the corresponding acid chloride, 5-methyl-4-nitroisoxazole-3-carbonyl chloride <b>2</b> in quantitative yield. Bis(4-chlorophenyl)methanone was reduced using sodium borohydride to the corresponding alcohol <b>3</b> in 94% yield. Chlorination using oxalyl chloride was followed by treatment with an excess of piperazine in refluxing acetonitrile and afforded 1-(bis(4-chlorophenyl)methyl)piperazine <b>5</b> in 68% yield. Coupling of secondary amine <b>5</b> with the acid chloride <b>2</b> in dichloromethane afforded the probe compound <b>6</b> in 74% yield. Full experimental details and characterization are provided below.</p><p>The solubility of the probe was measured in water and in PBS at room temperature and was found to be below 1 μM in both cases. The stability of the probe (<a href="/pcsubstance/?term=ML210[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML210</a>) in PBS (0.1% DMSO) was measured over 48 hours, and the data is shown in <a class="figpopup" href="/books/NBK98919/figure/ml210.f5/?report=objectonly" target="object" rid-figpopup="figml210f5" rid-ob="figobml210f5">Figure 5</a> (blue line). We suspected that poor solubility (and not instability) as the reason behind the dramatic drop in the amount of sample over time. To test this, we added acetonitrile to each well (final concentration 50%) and measured the amount of the probe. Amounts detected after addition of acetonitrile are shown in <a class="figpopup" href="/books/NBK98919/figure/ml210.f5/?report=objectonly" target="object" rid-figpopup="figml210f5" rid-ob="figobml210f5">Figure 5</a> (red line). From these results it can be concluded that the probe is stable in PBS and 100% of the probe is still present after 48 hours of incubation in PBS.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml210f5" co-legend-rid="figlgndml210f5"><a href="/books/NBK98919/figure/ml210.f5/?report=objectonly" target="object" title="Figure 5" class="img_link icnblk_img figpopup" rid-figpopup="figml210f5" rid-ob="figobml210f5"><img class="small-thumb" src="/books/NBK98919/bin/ml210f5.gif" src-large="/books/NBK98919/bin/ml210f5.jpg" alt="Figure 5. PBS Stability Data for the Probe (ML210)." /></a><div class="icnblk_cntnt" id="figlgndml210f5"><h4 id="ml210.f5"><a href="/books/NBK98919/figure/ml210.f5/?report=objectonly" target="object" rid-ob="figobml210f5">Figure 5</a></h4><p class="float-caption no_bottom_margin">PBS Stability Data for the Probe (ML210). Total ion count of the probe (ML210) over time (blue line). Total ion count of the probe upon addition of acetonitrile (red line). </p></div></div><p>The plasma protein binding was found to be 100% both in human and mouse. Plasma stability was 63.1% in human and 65.4% in mouse after a 5-hour incubation period. The probe and five analogs were submitted to the SMR collection [MLS003265661 (probe), MLS003265665, MLS003265666, MLS003265663, MLS003265664, MLS003265662].</p></div><div id="ml210.s19"><h3>2.3. Probe Preparation</h3><p><b>General details.</b> All reagents and solvents were purchased from commercial vendors and used as received. <sup>1</sup>H and <sup>13</sup>C NMR spectra were recorded on a Bruker 300 MHz or Varian UNITY INOVA 500 MHz spectrometer as indicated. Proton and carbon chemical shifts are reported in ppm (δ) relative to tetramethylsilane (δ = 0 for both <sup>1</sup>H and <sup>13</sup>C) or CDCl<sub>3</sub> solvent (<sup>1</sup>H δ 7.26, <sup>13</sup>C δ 77.0). NMR data are reported as follows: chemical shifts, multiplicity (obs. = obscured, br = broad, s = singlet, d = doublet, t = triplet, m = multiplet); coupling constant(s) in Hz; integration. Unless otherwise indicated NMR data were collected at 25 °C. Flash chromatography was performed using 40–60 μm Silica Gel (60 Å mesh) on a Teledyne Isco Combiflash R<i>f</i> system. Tandem Liquid Chromatography/Mass Spectrometry (LC/MS) was performed on a Waters 2795 separations module and 3100 mass detector. Analytical thin layer chromatography (TLC) was performed on EM Reagent 0.25 mm silica gel 60-F plates. Visualization was accomplished with ultraviolet (UV) light and aqueous potassium permanganate (KMnO<sub>4</sub>) stain followed by heating.</p><p><b>5-methyl-4-nitroisoxazole-3-carboxylic acid (<a class="bk_pop" href="#ml210.r1">1</a>):</b> 5-methylisoxazole-3-carboxylic acid (1.5 g, 12.04 mmol) was added to a mixture of potassium nitrate (1.83 g, 18.06 mmol) and sulfuric acid (5 ml) at room temperature. After complete dissolution, the mixture was warmed to 50°C and stirred for 4 hours. The mixture was then cooled to 0°C, ice was added, and the solution was neutralized with sodium bicarbonate. The mixture was extracted with ethyl acetate (3 × 30 ml), dried over sodium sulfate, filtered and concentrated to give 1.45 g of a white solid (8.43 mmol, 70%). The product could be further recrystallized from dichloromethane. <b><sup>1</sup>H NMR (300 MHz, DMSO-d6):</b> δ 6.72 (s, br, 1H), 2.28 (s, 3H); <b><sup>13</sup>C NMR (75 MHz, DMSO-d6):</b> δ 187.4, 124.2, 117.7, 107.3, 29.5.</p><p><b>5-methyl-4-nitroisoxazole-3-carbonyl chloride (2):</b> 5-methyl-4-nitroisoxazole-3-carboxylic acid (200 mg, 1.16 mmol) was dissolved in dichloromethane (4 ml) under argon atmosphere. Oxalyl chloride (202 μL, 2.32 mmol) was added dropwise at room temperature, followed by addition of one drop of DMF. After stirring overnight, the mixture was concentrated and co-evaporated with chloroform to afford 220 mg of a slightly yellow oil (1.15 mmol, 99%), which was carried on to the next step without further purification.</p><p><b>Bis(4-chlorophenyl)methanol (3):</b> Bis(4-chlorophenyl)methanone (3.0 g, 11.95 mmol) was dissolved in methanol/tetrahydrofuran 1:1 (100 ml) and cooled to 0°C. Sodium borohydride (452 mg, 11.95 mmol) was added in one portion, and the mixture was stirred for 30 minutes. After TLC showed complete conversion, the mixture was neutralized with acetic acid and concentrated. Dichloromethane was added, and the solution was washed with water (2 × 30 ml), dried on sodium sulfate and concentrated to afford 2.83 g of an off-white solid (11.18 mmol, 94%), which was carried to the next step without further purification. <b><sup>1</sup>H NMR (300 MHz, CDCl<sub>3</sub>):</b> δ 7.32 (d, <i>J</i> = 8.47 Hz, 4H), 7.28 (d, <i>J</i> = 7.28 Hz, 4H), 5.78 (s, 1H), 2.27 (s, 1H); <b>LRMS</b> (M + HCO<sub>2</sub>)<sup>−</sup>: 298.95.</p><p><b>4,4′-(Chloromethylene)bis(chlorobenzene) (<a class="bk_pop" href="#ml210.r4">4</a>):</b> Bis(4-chlorophenyl)methanol (2.83 g, 11.18 mmol) was dissolved in dichloromethane (30 ml) at room temperature. Oxalyl chloride (975 μL, 11.18 mmol) was added followed by addition of a drop of dimethylformamide. After stirring overnight, the mixture was concentrated and co-evaporated with chloroform to afford 3.0 g of an off-white solid (11.05 mmol, 99%), which was carried to the next step without further purification. <b><sup>1</sup>H NMR (300 MHz, CDCl<sub>3</sub>):</b> δ 7.32 (m, 8H), 6.06 (s, 1H).</p><p><b>1-(bis(4-chlorophenyl)methyl)piperazine (<a class="bk_pop" href="#ml210.r5">5</a>):</b> 4,4′-(Chloromethylene)bis(chlorobenzene) (3.0 g, 11.05 mmol) was dissolved in acetonitrile (100 ml) at room temperature. Piperazine (8.6 g, 100 mmol) was added, and the mixture was refluxed overnight. Acetonitrile was evaporated. The mixture was dissolved in ethyl acetate and washed three times with water to remove the excess piperazine. The organic phase was dried over sodium sulfate, filtered, and concentrated. The crude mixture was purified on silica gel using a gradient of (7N NH<sub>3</sub> in methanol) in dichloromethane to afford 2.4 g of an off-white solid (7.51 mmol, 68%). <b><sup>1</sup>H NMR (300 MHz, CDCl<sub>3</sub>):</b> δ 7.32 (d, <i>J</i> = 8.50 Hz, 4H), 7.25 (d, <i>J</i> = 8.56 Hz, 4H), 4.18 (s, 1H), 2.87 (t, <i>J</i> = 4.79 Hz, 4H), 2.32 (br s, 4H), 1.55 (br s, 1H); <b>LRMS</b> (M + H)<sup>+</sup>: 322.04.</p><p><b>(4-(bis(4-chlorophenyl)methyl)piperazin-1-yl)(5-methyl-4-nitroisoxazol-3-yl)methanone (<a class="bk_pop" href="#ml210.r6">6</a>):</b> 5-methyl-4-nitroisoxazole-3-carbonyl chloride (33 mg, 0.17 mmol) was dissolved in dichloromethane (1 ml) at room temperature and 1-(bis(4-chlorophenyl)methyl)piperazine (55 mg, 0.17 mmol) was added, along with one drop of triethylamine. After stirring for 2 hours, TLC shows complete conversion. The mixture was dissolved in dichloromethane and washed with a saturated aqueous solution of sodium bicarbonate (2 × 10 ml). The organic phase was dried over sodium sulfate, filtered, and concentrated. The crude mixture was purified on silica gel using a gradient of ethyl acetate in hexanes to afford the final product (61 mg) as a white solid (0.128 mmol, 74%). <b><sup>1</sup>H NMR (500 MHz, CDCl<sub>3</sub>):</b> δ 7.32 (d, <i>J</i> = 8.58 Hz, 4H), 7.26 (d, <i>J</i> = 8.56 Hz, 4H), 4.25 (s, 1H), 3.84 (t, <i>J</i> = 4.96 Hz, 2H), 3.36 (t, <i>J</i> = 4.99 Hz, 2H), 2.85 (s, 3H), 2.52 (t, <i>J</i> = 5.08 Hz, 2H), 2.37 (t, <i>J</i> = 4.97 Hz, 2H); <b><sup>13</sup>C NMR (125 MHz, CDCl<sub>3</sub>):</b> δ 171.7, 156.4, 153.0, 139.9, 133.2, 129.0, 128.9, 94.7, 74.2, 51.5, 50.9, 46.9, 42.2, 13.4; <b>HRMS (ESI):</b> calculated mass for C<sub>22</sub>H<sub>20</sub>Cl<sub>2</sub>N<sub>4</sub>O<sub>4</sub> [M+H] 475.0934, found 475.0953.</p><p>The <sup>1</sup>H NMR and <sup>13</sup>C spectra and UPLC chromatograms of the probe (<a href="/pcsubstance/?term=ML210[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML210</a>) and analogs are provided in <a href="#ml210.app3">Appendix C</a>.</p></div></div><div id="ml210.s20"><h2 id="_ml210_s20_">3. Results</h2><div id="ml210.s21"><h3>Probe Attributes</h3><ul><li class="half_rhythm"><div>Confirmed activity in the RAS-dependent strains <2.5 μM.</div></li><li class="half_rhythm"><div>At least 4-fold weaker IC<sub>50</sub> activity in the non-RAS strains.</div></li></ul><p>The project included a primary high throughput screen of the entire MLSMR collection (greater than 300,000 substances). Approximately 0.4% of the compounds tested were selected for dose retest and selectivity. Of these, 14 compounds from the same chemical class were identified as having the desired selectivity. In addition to these commercially available compounds, multiple rounds of chemistry were performed to determine the SAR of several substituents on the scaffold, and the compound with the best combination of potency and selectivity was designated as the probe (<a href="/pcsubstance/?term=ML210[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML210</a>).</p></div><div id="ml210.s22"><h3>3.1. Summary of Screening Results</h3><p>A high throughput screen of 303,344 substances (303,282 unique compounds, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1832" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1832</a>) was performed in duplicate in a 7.5-μL reaction in 384-well plates seeded with 1000 cells per well in DMEM/FBS medium. Cells were grown overnight, then treated with compound at a final concentration of 7.5 μM for 48 hours, after which cell viability was measured through determination of ATP levels using Promega Cell-TiterGlo reagent. Compounds causing at least 50% reduction in ATP levels relative to DMSO-treated cells were considered active. This resulted in 516 active compounds, which were retested at dose along with several analogs of active families for a total of 1155 compounds (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1936" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1936</a>). These 1155 compounds were counterscreened against BJeH-LT (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1935" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1935</a>) and BJ-eH (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1933" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1933</a>) and also confirmed in a secondary assay for mechanism of action in DRD cells (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1934" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1934</a>.) The compounds that passed these four screens were analyzed by the Assay Provider using a related viability assay in which Alamar Blue was metabolized to a fluorescent product by living cells; however, the same selectivity profiles were not observed in these secondary assays, either due to differences in maintenance of cell lines or due to the different detection methods.</p><p>As a result of this discrepancy, the available most active compounds from the primary screen (435 compounds) were tested in BJeLR (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2610" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 2610</a>) and BJeH-LT (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2631" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 2631</a>) by the Assay Provider to determine selectivity. Of these, 73 of 435 compounds were then tested in the two additional cell lines: DRD (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2633" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 2633</a>) and BJeH (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2635" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 2635</a>). Of these compounds, 26 of 73 displayed the desired potency and selectivity in all four assays and were designated as probe candidates.</p><p>Two of these compounds were in the same chemical class and were commercially available, although
|
||
only one confirmed in the four cell lines (AIDs <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2607" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2607</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2608" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2608</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2609" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2609</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2611" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2611</a>; <a class="figpopup" href="/books/NBK98919/table/ml210.t1/?report=objectonly" target="object" rid-figpopup="figml210t1" rid-ob="figobml210t1">Table 1</a> and <a class="figpopup" href="/books/NBK98919/figure/ml210.f6/?report=objectonly" target="object" rid-figpopup="figml210f6" rid-ob="figobml210f6">Figure 6</a>) This core was used for further SAR studies to improve the potency and selectivity. A novel compound was identified with selectivity similar to the initial hit but with an approximate 10-fold improved potency. This compound was selected as the probe <a href="/pcsubstance/?term=ML210[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML210</a> ((4-(bis(4-chlorophenyl)methyl)piperazin-1-yl)(5-methyl-4-nitroisoxazol-3-yl)methanone).</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml210f6" co-legend-rid="figlgndml210f6"><a href="/books/NBK98919/figure/ml210.f6/?report=objectonly" target="object" title="Figure 6" class="img_link icnblk_img figpopup" rid-figpopup="figml210f6" rid-ob="figobml210f6"><img class="small-thumb" src="/books/NBK98919/bin/ml210f6.gif" src-large="/books/NBK98919/bin/ml210f6.jpg" alt="Figure 6. Critical Path for Probe Development." /></a><div class="icnblk_cntnt" id="figlgndml210f6"><h4 id="ml210.f6"><a href="/books/NBK98919/figure/ml210.f6/?report=objectonly" target="object" rid-ob="figobml210f6">Figure 6</a></h4><p class="float-caption no_bottom_margin">Critical Path for Probe Development. </p></div></div></div><div id="ml210.s23"><h3>3.2. Dose Response Curves for Probe</h3><p>The inhibition of cell viability curves for the probe (<a href="/pcsubstance/?term=ML210[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML210</a>) in four different cell lines are displayed in <a class="figpopup" href="/books/NBK98919/figure/ml210.f7/?report=objectonly" target="object" rid-figpopup="figml210f7" rid-ob="figobml210f7">Figure 7</a>.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml210f7" co-legend-rid="figlgndml210f7"><a href="/books/NBK98919/figure/ml210.f7/?report=objectonly" target="object" title="Figure 7" class="img_link icnblk_img figpopup" rid-figpopup="figml210f7" rid-ob="figobml210f7"><img class="small-thumb" src="/books/NBK98919/bin/ml210f7.gif" src-large="/books/NBK98919/bin/ml210f7.jpg" alt="Figure 7. Concentration-dependent Activities by the Probe (ML210) in HRASV12 Expressing and Non-expressing Cell Lines." /></a><div class="icnblk_cntnt" id="figlgndml210f7"><h4 id="ml210.f7"><a href="/books/NBK98919/figure/ml210.f7/?report=objectonly" target="object" rid-ob="figobml210f7">Figure 7</a></h4><p class="float-caption no_bottom_margin">Concentration-dependent Activities by the Probe (ML210) in HRAS<sup>V12</sup> Expressing and Non-expressing Cell Lines. Activity curves for probe (ML210): BJeLR (AID 493053) (<i>a</i>); DRD (AID 493050)(<i>b</i>); BJeH-LT (AID 493093)(<i>c</i>);BJeH (AID 493051)(<i>d</i>). </p></div></div></div><div id="ml210.s24"><h3>3.3. Scaffold/Moiety Chemical Liabilities</h3><p>A search of PubChem for the hit compound (CID 15945539, <a class="figpopup" href="/books/NBK98919/figure/ml210.f8/?report=objectonly" target="object" rid-figpopup="figml210f8" rid-ob="figobml210f8">Figure 8</a>) shows that it has been screened in 356 different assays and was
|
||
identified as active in only two other assays, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/485294" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 485294</a>: qHTS inhibitors of AmpC Beta-Lactamase (confirmatory) and <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/485297" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 485297</a>: qHTS Assay for Rab9 Promoter Activators (confirmatory). This shows that the probe molecule is very unlikely to be promiscuous, considering that the only difference between the hit compound and the probe molecule is the presence of a para-chloro substituent on both phenyl rings (<a class="figpopup" href="/books/NBK98919/figure/ml210.f8/?report=objectonly" target="object" rid-figpopup="figml210f8" rid-ob="figobml210f8">Figure 8</a>).</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml210f8" co-legend-rid="figlgndml210f8"><a href="/books/NBK98919/figure/ml210.f8/?report=objectonly" target="object" title="Figure 8" class="img_link icnblk_img figpopup" rid-figpopup="figml210f8" rid-ob="figobml210f8"><img class="small-thumb" src="/books/NBK98919/bin/ml210f8.gif" src-large="/books/NBK98919/bin/ml210f8.jpg" alt="Figure 8. Hit Compound and Probe Molecule Structures." /></a><div class="icnblk_cntnt" id="figlgndml210f8"><h4 id="ml210.f8"><a href="/books/NBK98919/figure/ml210.f8/?report=objectonly" target="object" rid-ob="figobml210f8">Figure 8</a></h4><p class="float-caption no_bottom_margin">Hit Compound and Probe Molecule Structures. </p></div></div><p>The benzhydrylpiperazine moiety is present in cetirizine (Zyrtec®, Pfizer, <a class="figpopup" href="/books/NBK98919/figure/ml210.f9/?report=objectonly" target="object" rid-figpopup="figml210f9" rid-ob="figobml210f9">Figure 9</a>), a second generation antihistamine used in the
|
||
treatment of allergies. Isoxazoles are also present in drugs, such as in valdecoxib (Bextra, G. D.
|
||
Searle & Company), a cyclooxygenase-2 selective inhibitor that was used as a nonsteroidal anti-inflammatory drug until 2005, and risperidone (Risperdal®, Johnson & Johnson, <a class="figpopup" href="/books/NBK98919/figure/ml210.f9/?report=objectonly" target="object" rid-figpopup="figml210f9" rid-ob="figobml210f9">Figure 9</a>), an atypical antipsychotic that is still one of the best-selling drugs for the treatment of schizoaffective disorders and bipolar disorders. Nitro groups are usually undesirable in drug discovery because they can be metabolized to toxic reactive intermediates such as nitroso or hydroxylamines. However, when present on an electron-rich, five-membered heterocyclic ring, they are often more tolerable. Several nitro-aromatic-containing drugs have been developed. For example, nifedipine (Procardia®, Bayer) is a calcium channel blocker used as an anti-anginal and antihypertensive agent, and clonazepam (Klonopin®, Roche, <a class="figpopup" href="/books/NBK98919/figure/ml210.f9/?report=objectonly" target="object" rid-figpopup="figml210f9" rid-ob="figobml210f9">Figure 9</a>) is used as an anticonvulsant, muscle relaxant, and anxiolytic.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml210f9" co-legend-rid="figlgndml210f9"><a href="/books/NBK98919/figure/ml210.f9/?report=objectonly" target="object" title="Figure 9" class="img_link icnblk_img figpopup" rid-figpopup="figml210f9" rid-ob="figobml210f9"><img class="small-thumb" src="/books/NBK98919/bin/ml210f9.gif" src-large="/books/NBK98919/bin/ml210f9.jpg" alt="Figure 9. Drugs Containing Benzhydrilpiperazine, Isoxazole and Nitro Groups." /></a><div class="icnblk_cntnt" id="figlgndml210f9"><h4 id="ml210.f9"><a href="/books/NBK98919/figure/ml210.f9/?report=objectonly" target="object" rid-ob="figobml210f9">Figure 9</a></h4><p class="float-caption no_bottom_margin">Drugs Containing Benzhydrilpiperazine, Isoxazole and Nitro Groups. </p></div></div></div><div id="ml210.s25"><h3>3.4. SAR Tables</h3><p>Even though the nitro group is present in several FDA-approved drugs, it can be a liability in
|
||
many instances. Thus, we first investigated the possibility of replacing the nitro group with other functional groups (<a class="figpopup" href="/books/NBK98919/table/ml210.t1/?report=objectonly" target="object" rid-figpopup="figml210t1" rid-ob="figobml210t1">Table 1</a>).</p><div id="ml210.t1" class="table"><h3><span class="label">Table 1</span><span class="title">Summary of SAR to Replace the Nitro Group</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK98919/table/ml210.t1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml210.t1_lrgtbl__"><table class="no_margin"><thead><tr><th id="hd_h_ml210.t1_1_1_1_1" colspan="4" rowspan="1" style="text-align:center;vertical-align:middle;">SAR Analysis to Replace the Nitro Group</th><th id="hd_h_ml210.t1_1_1_1_2" colspan="2" rowspan="1" style="text-align:center;vertical-align:middle;"></th><th id="hd_h_ml210.t1_1_1_1_3" colspan="4" rowspan="1" style="text-align:center;vertical-align:middle;">Potency (μM)<br />Mean ± S.E.M. (n=replicates)</th><th id="hd_h_ml210.t1_1_1_1_4" rowspan="3" colspan="1" headers="hd_h_ml210.t1_1_1_1_4" style="text-align:center;vertical-align:middle;">Target to AntiTarget Fold Selectivity</th></tr><tr><th headers="hd_h_ml210.t1_1_1_1_1 hd_h_ml210.t1_1_1_2_1" id="hd_h_ml210.t1_1_1_2_1" rowspan="2" colspan="1" style="text-align:center;vertical-align:middle;">No.</th><th headers="hd_h_ml210.t1_1_1_1_1 hd_h_ml210.t1_1_1_2_2" id="hd_h_ml210.t1_1_1_2_2" rowspan="2" colspan="1" style="text-align:center;vertical-align:middle;">CID</th><th headers="hd_h_ml210.t1_1_1_1_1 hd_h_ml210.t1_1_1_2_3" id="hd_h_ml210.t1_1_1_2_3" rowspan="2" colspan="1" style="text-align:center;vertical-align:middle;">SID</th><th headers="hd_h_ml210.t1_1_1_1_1 hd_h_ml210.t1_1_1_2_4" id="hd_h_ml210.t1_1_1_2_4" rowspan="2" colspan="1" style="text-align:center;vertical-align:middle;">Broad ID</th><th headers="hd_h_ml210.t1_1_1_1_2 hd_h_ml210.t1_1_1_2_5" id="hd_h_ml210.t1_1_1_2_5" rowspan="2" colspan="1" style="text-align:center;vertical-align:middle;"><sup><a class="bk_pop" href="#ml210.tfn1">*</a></sup></th><th headers="hd_h_ml210.t1_1_1_1_2 hd_h_ml210.t1_1_1_2_6" id="hd_h_ml210.t1_1_1_2_6" rowspan="2" colspan="1" style="text-align:center;vertical-align:middle;">R<sub>1</sub></th><th headers="hd_h_ml210.t1_1_1_1_3" id="hd_h_ml210.t1_1_1_2_7" colspan="2" rowspan="1" style="text-align:center;vertical-align:middle;">BJeLR</th><th headers="hd_h_ml210.t1_1_1_1_3" id="hd_h_ml210.t1_1_1_2_8" colspan="2" rowspan="1" style="text-align:center;vertical-align:middle;">BJeH-LT</th></tr><tr><th headers="hd_h_ml210.t1_1_1_1_3 hd_h_ml210.t1_1_1_2_7" id="hd_h_ml210.t1_1_1_3_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">n</th><th headers="hd_h_ml210.t1_1_1_1_3 hd_h_ml210.t1_1_1_2_7" id="hd_h_ml210.t1_1_1_3_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">IC<sub>50</sub></th><th headers="hd_h_ml210.t1_1_1_1_3 hd_h_ml210.t1_1_1_2_8" id="hd_h_ml210.t1_1_1_3_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">n</th><th headers="hd_h_ml210.t1_1_1_1_3 hd_h_ml210.t1_1_1_2_8" id="hd_h_ml210.t1_1_1_3_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">IC<sub>50</sub></th></tr></thead><tbody><tr><td headers="hd_h_ml210.t1_1_1_1_1 hd_h_ml210.t1_1_1_2_1" rowspan="2" colspan="1" style="text-align:center;vertical-align:middle;">1</td><td headers="hd_h_ml210.t1_1_1_1_1 hd_h_ml210.t1_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CID 15945539</td><td headers="hd_h_ml210.t1_1_1_1_1 hd_h_ml210.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/99351093" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 99351093</a></td><td headers="hd_h_ml210.t1_1_1_1_1 hd_h_ml210.t1_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BRD-K12393722</td><td headers="hd_h_ml210.t1_1_1_1_2 hd_h_ml210.t1_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">S</td><td headers="hd_h_ml210.t1_1_1_1_2 hd_h_ml210.t1_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">NO<sub>2</sub> (hit compound)</td><td headers="hd_h_ml210.t1_1_1_1_3 hd_h_ml210.t1_1_1_2_7 hd_h_ml210.t1_1_1_3_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2</td><td headers="hd_h_ml210.t1_1_1_1_3 hd_h_ml210.t1_1_1_2_7 hd_h_ml210.t1_1_1_3_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1.96 ± 0.93</td><td headers="hd_h_ml210.t1_1_1_1_3 hd_h_ml210.t1_1_1_2_8 hd_h_ml210.t1_1_1_3_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2</td><td headers="hd_h_ml210.t1_1_1_1_3 hd_h_ml210.t1_1_1_2_8 hd_h_ml210.t1_1_1_3_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">6.21 ± 1.24</td><td headers="hd_h_ml210.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3.71 ± 0.53</td></tr><tr><td headers="hd_h_ml210.t1_1_1_1_1 hd_h_ml210.t1_1_1_2_2 hd_h_ml210.t1_1_1_2_3 hd_h_ml210.t1_1_1_2_4 hd_h_ml210.t1_1_1_1_2 hd_h_ml210.t1_1_1_2_5 hd_h_ml210.t1_1_1_2_6 hd_h_ml210.t1_1_1_1_3 hd_h_ml210.t1_1_1_2_7 hd_h_ml210.t1_1_1_3_1 hd_h_ml210.t1_1_1_3_2 hd_h_ml210.t1_1_1_2_8 hd_h_ml210.t1_1_1_3_3 hd_h_ml210.t1_1_1_3_4 hd_h_ml210.t1_1_1_1_4" colspan="10" rowspan="1" style="text-align:center;vertical-align:middle;">Solubility (PBS): <0.5 μM; PPB (h): 98.2%, (m): 98.5%; PS (h): 63.2%, (m): 88.8%; Purity: 94%</td></tr><tr><td headers="hd_h_ml210.t1_1_1_1_1 hd_h_ml210.t1_1_1_2_1" rowspan="2" colspan="1" style="text-align:center;vertical-align:middle;">2</td><td headers="hd_h_ml210.t1_1_1_1_1 hd_h_ml210.t1_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CID 49766535</td><td headers="hd_h_ml210.t1_1_1_1_1 hd_h_ml210.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/103023270" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 103023270</a></td><td headers="hd_h_ml210.t1_1_1_1_1 hd_h_ml210.t1_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BRD-K44168309</td><td headers="hd_h_ml210.t1_1_1_1_2 hd_h_ml210.t1_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">S</td><td headers="hd_h_ml210.t1_1_1_1_2 hd_h_ml210.t1_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">NH<sub>2</sub></td><td headers="hd_h_ml210.t1_1_1_1_3 hd_h_ml210.t1_1_1_2_7 hd_h_ml210.t1_1_1_3_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1</td><td headers="hd_h_ml210.t1_1_1_1_3 hd_h_ml210.t1_1_1_2_7 hd_h_ml210.t1_1_1_3_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">IA</td><td headers="hd_h_ml210.t1_1_1_1_3 hd_h_ml210.t1_1_1_2_8 hd_h_ml210.t1_1_1_3_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1</td><td headers="hd_h_ml210.t1_1_1_1_3 hd_h_ml210.t1_1_1_2_8 hd_h_ml210.t1_1_1_3_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">IA</td><td headers="hd_h_ml210.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">NA</td></tr><tr><td headers="hd_h_ml210.t1_1_1_1_1 hd_h_ml210.t1_1_1_2_2 hd_h_ml210.t1_1_1_2_3 hd_h_ml210.t1_1_1_2_4 hd_h_ml210.t1_1_1_1_2 hd_h_ml210.t1_1_1_2_5 hd_h_ml210.t1_1_1_2_6 hd_h_ml210.t1_1_1_1_3 hd_h_ml210.t1_1_1_2_7 hd_h_ml210.t1_1_1_3_1 hd_h_ml210.t1_1_1_3_2 hd_h_ml210.t1_1_1_2_8 hd_h_ml210.t1_1_1_3_3 hd_h_ml210.t1_1_1_3_4 hd_h_ml210.t1_1_1_1_4" colspan="10" rowspan="1" style="text-align:center;vertical-align:middle;">Solubility (PBS): <0.5 μM; PPB (h): 99.3%, (m): 99.1%; PS (h): 100%, (m): 100%; Purity: 90%</td></tr><tr><td headers="hd_h_ml210.t1_1_1_1_1 hd_h_ml210.t1_1_1_2_1" rowspan="2" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml210.t1_1_1_1_1 hd_h_ml210.t1_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CID 49766526</td><td headers="hd_h_ml210.t1_1_1_1_1 hd_h_ml210.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/103023277" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 103023277</a></td><td headers="hd_h_ml210.t1_1_1_1_1 hd_h_ml210.t1_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BRD-K72267627</td><td headers="hd_h_ml210.t1_1_1_1_2 hd_h_ml210.t1_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">S</td><td headers="hd_h_ml210.t1_1_1_1_2 hd_h_ml210.t1_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">NHAc</td><td headers="hd_h_ml210.t1_1_1_1_3 hd_h_ml210.t1_1_1_2_7 hd_h_ml210.t1_1_1_3_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1</td><td headers="hd_h_ml210.t1_1_1_1_3 hd_h_ml210.t1_1_1_2_7 hd_h_ml210.t1_1_1_3_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">IA</td><td headers="hd_h_ml210.t1_1_1_1_3 hd_h_ml210.t1_1_1_2_8 hd_h_ml210.t1_1_1_3_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1</td><td headers="hd_h_ml210.t1_1_1_1_3 hd_h_ml210.t1_1_1_2_8 hd_h_ml210.t1_1_1_3_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">IA</td><td headers="hd_h_ml210.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">NA</td></tr><tr><td headers="hd_h_ml210.t1_1_1_1_1 hd_h_ml210.t1_1_1_2_2 hd_h_ml210.t1_1_1_2_3 hd_h_ml210.t1_1_1_2_4 hd_h_ml210.t1_1_1_1_2 hd_h_ml210.t1_1_1_2_5 hd_h_ml210.t1_1_1_2_6 hd_h_ml210.t1_1_1_1_3 hd_h_ml210.t1_1_1_2_7 hd_h_ml210.t1_1_1_3_1 hd_h_ml210.t1_1_1_3_2 hd_h_ml210.t1_1_1_2_8 hd_h_ml210.t1_1_1_3_3 hd_h_ml210.t1_1_1_3_4 hd_h_ml210.t1_1_1_1_4" colspan="10" rowspan="1" style="text-align:center;vertical-align:middle;">Solubility (PBS): <0.5 μM; PPB (h): 95.5%, (m): 97.0%; PS (h): 100%, (m): 57.9%; Purity: 99%</td></tr><tr><td headers="hd_h_ml210.t1_1_1_1_1 hd_h_ml210.t1_1_1_2_1" rowspan="2" colspan="1" style="text-align:center;vertical-align:middle;">4</td><td headers="hd_h_ml210.t1_1_1_1_1 hd_h_ml210.t1_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CID 49766512</td><td headers="hd_h_ml210.t1_1_1_1_1 hd_h_ml210.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/103023275" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 103023275</a></td><td headers="hd_h_ml210.t1_1_1_1_1 hd_h_ml210.t1_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BRD-K26054379</td><td headers="hd_h_ml210.t1_1_1_1_2 hd_h_ml210.t1_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">S</td><td headers="hd_h_ml210.t1_1_1_1_2 hd_h_ml210.t1_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">NHSO<sub>2</sub>Me</td><td headers="hd_h_ml210.t1_1_1_1_3 hd_h_ml210.t1_1_1_2_7 hd_h_ml210.t1_1_1_3_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1</td><td headers="hd_h_ml210.t1_1_1_1_3 hd_h_ml210.t1_1_1_2_7 hd_h_ml210.t1_1_1_3_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">IA</td><td headers="hd_h_ml210.t1_1_1_1_3 hd_h_ml210.t1_1_1_2_8 hd_h_ml210.t1_1_1_3_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1</td><td headers="hd_h_ml210.t1_1_1_1_3 hd_h_ml210.t1_1_1_2_8 hd_h_ml210.t1_1_1_3_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">IA</td><td headers="hd_h_ml210.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">NA</td></tr><tr><td headers="hd_h_ml210.t1_1_1_1_1 hd_h_ml210.t1_1_1_2_2 hd_h_ml210.t1_1_1_2_3 hd_h_ml210.t1_1_1_2_4 hd_h_ml210.t1_1_1_1_2 hd_h_ml210.t1_1_1_2_5 hd_h_ml210.t1_1_1_2_6 hd_h_ml210.t1_1_1_1_3 hd_h_ml210.t1_1_1_2_7 hd_h_ml210.t1_1_1_3_1 hd_h_ml210.t1_1_1_3_2 hd_h_ml210.t1_1_1_2_8 hd_h_ml210.t1_1_1_3_3 hd_h_ml210.t1_1_1_3_4 hd_h_ml210.t1_1_1_1_4" colspan="10" rowspan="1" style="text-align:center;vertical-align:middle;">Solubility (PBS): ND; PPB (h): 97.5%, (m): 98.4%; PS (h): 100%, (m): 100%; Purity: 80%</td></tr><tr><td headers="hd_h_ml210.t1_1_1_1_1 hd_h_ml210.t1_1_1_2_1" rowspan="2" colspan="1" style="text-align:center;vertical-align:middle;">5</td><td headers="hd_h_ml210.t1_1_1_1_1 hd_h_ml210.t1_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CID 49766538</td><td headers="hd_h_ml210.t1_1_1_1_1 hd_h_ml210.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/103023296" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 103023296</a></td><td headers="hd_h_ml210.t1_1_1_1_1 hd_h_ml210.t1_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BRD-K50505048</td><td headers="hd_h_ml210.t1_1_1_1_2 hd_h_ml210.t1_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">S</td><td headers="hd_h_ml210.t1_1_1_1_2 hd_h_ml210.t1_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Br</td><td headers="hd_h_ml210.t1_1_1_1_3 hd_h_ml210.t1_1_1_2_7 hd_h_ml210.t1_1_1_3_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1</td><td headers="hd_h_ml210.t1_1_1_1_3 hd_h_ml210.t1_1_1_2_7 hd_h_ml210.t1_1_1_3_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">IA</td><td headers="hd_h_ml210.t1_1_1_1_3 hd_h_ml210.t1_1_1_2_8 hd_h_ml210.t1_1_1_3_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1</td><td headers="hd_h_ml210.t1_1_1_1_3 hd_h_ml210.t1_1_1_2_8 hd_h_ml210.t1_1_1_3_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">IA</td><td headers="hd_h_ml210.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">NA</td></tr><tr><td headers="hd_h_ml210.t1_1_1_1_1 hd_h_ml210.t1_1_1_2_2 hd_h_ml210.t1_1_1_2_3 hd_h_ml210.t1_1_1_2_4 hd_h_ml210.t1_1_1_1_2 hd_h_ml210.t1_1_1_2_5 hd_h_ml210.t1_1_1_2_6 hd_h_ml210.t1_1_1_1_3 hd_h_ml210.t1_1_1_2_7 hd_h_ml210.t1_1_1_3_1 hd_h_ml210.t1_1_1_3_2 hd_h_ml210.t1_1_1_2_8 hd_h_ml210.t1_1_1_3_3 hd_h_ml210.t1_1_1_3_4 hd_h_ml210.t1_1_1_1_4" colspan="10" rowspan="1" style="text-align:center;vertical-align:middle;">Solubility (PBS): <0.5 μM; PPB (h): 97.8%, (m): 99.3%; PS (h): 68.4%, (m): 44.9%; Purity: 99%</td></tr><tr><td headers="hd_h_ml210.t1_1_1_1_1 hd_h_ml210.t1_1_1_2_1" rowspan="2" colspan="1" style="text-align:center;vertical-align:middle;">6</td><td headers="hd_h_ml210.t1_1_1_1_1 hd_h_ml210.t1_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CID 49766527</td><td headers="hd_h_ml210.t1_1_1_1_1 hd_h_ml210.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/103023295" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 103023295</a></td><td headers="hd_h_ml210.t1_1_1_1_1 hd_h_ml210.t1_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BRD-K11438207</td><td headers="hd_h_ml210.t1_1_1_1_2 hd_h_ml210.t1_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">S</td><td headers="hd_h_ml210.t1_1_1_1_2 hd_h_ml210.t1_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Me</td><td headers="hd_h_ml210.t1_1_1_1_3 hd_h_ml210.t1_1_1_2_7 hd_h_ml210.t1_1_1_3_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1</td><td headers="hd_h_ml210.t1_1_1_1_3 hd_h_ml210.t1_1_1_2_7 hd_h_ml210.t1_1_1_3_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">IA</td><td headers="hd_h_ml210.t1_1_1_1_3 hd_h_ml210.t1_1_1_2_8 hd_h_ml210.t1_1_1_3_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1</td><td headers="hd_h_ml210.t1_1_1_1_3 hd_h_ml210.t1_1_1_2_8 hd_h_ml210.t1_1_1_3_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">IA</td><td headers="hd_h_ml210.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">NA</td></tr><tr><td headers="hd_h_ml210.t1_1_1_1_1 hd_h_ml210.t1_1_1_2_2 hd_h_ml210.t1_1_1_2_3 hd_h_ml210.t1_1_1_2_4 hd_h_ml210.t1_1_1_1_2 hd_h_ml210.t1_1_1_2_5 hd_h_ml210.t1_1_1_2_6 hd_h_ml210.t1_1_1_1_3 hd_h_ml210.t1_1_1_2_7 hd_h_ml210.t1_1_1_3_1 hd_h_ml210.t1_1_1_3_2 hd_h_ml210.t1_1_1_2_8 hd_h_ml210.t1_1_1_3_3 hd_h_ml210.t1_1_1_3_4 hd_h_ml210.t1_1_1_1_4" colspan="10" rowspan="1" style="text-align:center;vertical-align:middle;">Solubility (PBS): <0.5 μM; PPB (h): 98.7%, (m): 98.3%; PS (h): 86.2%, (m): 73.6%; Purity: 96%</td></tr><tr><td headers="hd_h_ml210.t1_1_1_1_1 hd_h_ml210.t1_1_1_2_1" rowspan="2" colspan="1" style="text-align:center;vertical-align:middle;">7</td><td headers="hd_h_ml210.t1_1_1_1_1 hd_h_ml210.t1_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CID 3244419</td><td headers="hd_h_ml210.t1_1_1_1_1 hd_h_ml210.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/85827279" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 85827279</a></td><td headers="hd_h_ml210.t1_1_1_1_1 hd_h_ml210.t1_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BRD-K16576106</td><td headers="hd_h_ml210.t1_1_1_1_2 hd_h_ml210.t1_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">P</td><td headers="hd_h_ml210.t1_1_1_1_2 hd_h_ml210.t1_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">H</td><td headers="hd_h_ml210.t1_1_1_1_3 hd_h_ml210.t1_1_1_2_7 hd_h_ml210.t1_1_1_3_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1</td><td headers="hd_h_ml210.t1_1_1_1_3 hd_h_ml210.t1_1_1_2_7 hd_h_ml210.t1_1_1_3_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">IA</td><td headers="hd_h_ml210.t1_1_1_1_3 hd_h_ml210.t1_1_1_2_8 hd_h_ml210.t1_1_1_3_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1</td><td headers="hd_h_ml210.t1_1_1_1_3 hd_h_ml210.t1_1_1_2_8 hd_h_ml210.t1_1_1_3_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">IA</td><td headers="hd_h_ml210.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">NA</td></tr><tr><td headers="hd_h_ml210.t1_1_1_1_1 hd_h_ml210.t1_1_1_2_2 hd_h_ml210.t1_1_1_2_3 hd_h_ml210.t1_1_1_2_4 hd_h_ml210.t1_1_1_1_2 hd_h_ml210.t1_1_1_2_5 hd_h_ml210.t1_1_1_2_6 hd_h_ml210.t1_1_1_1_3 hd_h_ml210.t1_1_1_2_7 hd_h_ml210.t1_1_1_3_1 hd_h_ml210.t1_1_1_3_2 hd_h_ml210.t1_1_1_2_8 hd_h_ml210.t1_1_1_3_3 hd_h_ml210.t1_1_1_3_4 hd_h_ml210.t1_1_1_1_4" colspan="10" rowspan="1" style="text-align:center;vertical-align:middle;">Solubility (PBS): <0.5 μM; PPB (h): ND, (m): ND; PS (h): ND, (m): ND; Purity: 100%</td></tr></tbody></table></div><div><div><dl class="temp-labeled-list small"><dt>*</dt><dd><div id="ml210.tfn1"><p class="no_margin">S = Synthesized, P = Purchased</p></div></dd><dt></dt><dd><div id="ml210.tfn2"><p class="no_margin">IA = Inactive; (m) = mouse; ND = Not determined; PPB (h) = Plasma protein binding in human; PS (h) = Plasma stability in human</p></div></dd></dl></div></div></div><p>Replacement of the nitro group by a primary amine, acetamide, sulfonamide, bromine, methyl, and
|
||
hydrogen (entries 2–7, respectively, <a class="figpopup" href="/books/NBK98919/table/ml210.t1/?report=objectonly" target="object" rid-figpopup="figml210t1" rid-ob="figobml210t1">Table 1</a>) led to completely inactive compounds, showing the importance of the nitro group for activity. We also tried to substitute the nitro group by a cyano group. Although cyanation of a variety of aromatic and heteroaromatic halides is known (<a class="bk_pop" href="#ml210.r25">25</a>), there are no reports of a direct conversion of 4-haloisoxazoles to the corresponding 4-cyanoisoxazoles. We investigated the possibility to synthesize the corresponding nitrile analog starting from a bromoisoxazole model compound using a wide variety of reported cyanation conditions (<a class="figpopup" href="/books/NBK98919/figure/ml210.f14/?report=objectonly" target="object" rid-figpopup="figml210f14" rid-ob="figobml210f14">Scheme 2</a>, <a class="figpopup" href="/books/NBK98919/table/ml210.t2/?report=objectonly" target="object" rid-figpopup="figml210t2" rid-ob="figobml210t2">Table 2</a>).</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml210f14" co-legend-rid="figlgndml210f14"><a href="/books/NBK98919/figure/ml210.f14/?report=objectonly" target="object" title="Scheme 2" class="img_link icnblk_img figpopup" rid-figpopup="figml210f14" rid-ob="figobml210f14"><img class="small-thumb" src="/books/NBK98919/bin/ml210f14.gif" src-large="/books/NBK98919/bin/ml210f14.jpg" alt="Scheme 2. Cyanation of a Bromoisoxazole Model Compound." /></a><div class="icnblk_cntnt" id="figlgndml210f14"><h4 id="ml210.f14"><a href="/books/NBK98919/figure/ml210.f14/?report=objectonly" target="object" rid-ob="figobml210f14">Scheme 2</a></h4><p class="float-caption no_bottom_margin">Cyanation of a Bromoisoxazole Model Compound. </p></div></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml210t2"><a href="/books/NBK98919/table/ml210.t2/?report=objectonly" target="object" title="Table 2" class="img_link icnblk_img figpopup" rid-figpopup="figml210t2" rid-ob="figobml210t2"><img class="small-thumb" src="/books/NBK98919/table/ml210.t2/?report=thumb" src-large="/books/NBK98919/table/ml210.t2/?report=previmg" alt="Table 2. Cyanation of a Bromoisoxazole Model Compound." /></a><div class="icnblk_cntnt"><h4 id="ml210.t2"><a href="/books/NBK98919/table/ml210.t2/?report=objectonly" target="object" rid-ob="figobml210t2">Table 2</a></h4><p class="float-caption no_bottom_margin">Cyanation of a Bromoisoxazole Model Compound. </p></div></div><p>Rosenmund-von Braun conditions (entry 1, <a class="figpopup" href="/books/NBK98919/table/ml210.t2/?report=objectonly" target="object" rid-figpopup="figml210t2" rid-ob="figobml210t2">Table 2</a>)
|
||
using an excess of copper (I) cyanide in refluxing N-methylpyrrolidone led to decomposition of the starting material. We then turned to palladium catalyzed cyanation reactions (entries 2–5, <a class="figpopup" href="/books/NBK98919/table/ml210.t2/?report=objectonly" target="object" rid-figpopup="figml210t2" rid-ob="figobml210t2">Table 2</a>), using copper (I) cyanide or zinc (II) cyanide and different phosphine ligands; but, in all cases, we either observed decomposition of the starting material or no reaction.</p><p>We also performed the cyanation reactions directly on the full probe that includes the
|
||
substituted piperazine moiety (<a class="figpopup" href="/books/NBK98919/figure/ml210.f15/?report=objectonly" target="object" rid-figpopup="figml210f15" rid-ob="figobml210f15">Scheme 3</a>), and these results are summarized in <a class="figpopup" href="/books/NBK98919/table/ml210.t3/?report=objectonly" target="object" rid-figpopup="figml210t3" rid-ob="figobml210t3">Table 3</a>.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml210f15" co-legend-rid="figlgndml210f15"><a href="/books/NBK98919/figure/ml210.f15/?report=objectonly" target="object" title="Scheme 3" class="img_link icnblk_img figpopup" rid-figpopup="figml210f15" rid-ob="figobml210f15"><img class="small-thumb" src="/books/NBK98919/bin/ml210f15.gif" src-large="/books/NBK98919/bin/ml210f15.jpg" alt="Scheme 3. Cyanation of the Bromoisoxazole Analog." /></a><div class="icnblk_cntnt" id="figlgndml210f15"><h4 id="ml210.f15"><a href="/books/NBK98919/figure/ml210.f15/?report=objectonly" target="object" rid-ob="figobml210f15">Scheme 3</a></h4><p class="float-caption no_bottom_margin">Cyanation of the Bromoisoxazole Analog. </p></div></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml210t3"><a href="/books/NBK98919/table/ml210.t3/?report=objectonly" target="object" title="Table 3" class="img_link icnblk_img figpopup" rid-figpopup="figml210t3" rid-ob="figobml210t3"><img class="small-thumb" src="/books/NBK98919/table/ml210.t3/?report=thumb" src-large="/books/NBK98919/table/ml210.t3/?report=previmg" alt="Table 3. Cyanation of the Bromoisoxazole Analog of the Probe (CID 3689413/ML210)." /></a><div class="icnblk_cntnt"><h4 id="ml210.t3"><a href="/books/NBK98919/table/ml210.t3/?report=objectonly" target="object" rid-ob="figobml210t3">Table 3</a></h4><p class="float-caption no_bottom_margin">Cyanation of the Bromoisoxazole Analog of the Probe (CID 3689413/ML210). </p></div></div><p>Unfortunately, no desired product could be detected in these experiments. The only product we could observe was the reductive opening of the isoxazole ring.</p><p>Next, we investigated the influence of the nitroisoxazole ring on the activity of the compound
|
||
(<a class="figpopup" href="/books/NBK98919/table/ml210.t4/?report=objectonly" target="object" rid-figpopup="figml210t4" rid-ob="figobml210t4">Table 4</a>). Replacing the methyl substituent on the
|
||
3-position of the isoxazole by an isopropyl group (entry 2, <a class="figpopup" href="/books/NBK98919/table/ml210.t4/?report=objectonly" target="object" rid-figpopup="figml210t4" rid-ob="figobml210t4">Table 4</a>) led to an inactive compound. Removing the nitro group at the
|
||
4-position led to a complete loss of activity, irrespective of the substituents on the 3-position of
|
||
the isoxazole ring (entries 3–6, <a class="figpopup" href="/books/NBK98919/table/ml210.t4/?report=objectonly" target="object" rid-figpopup="figml210t4" rid-ob="figobml210t4">Table 4</a>).
|
||
Benzoisoxazole (entry 7, <a class="figpopup" href="/books/NBK98919/table/ml210.t4/?report=objectonly" target="object" rid-figpopup="figml210t4" rid-ob="figobml210t4">Table 4</a>) and
|
||
2,4,5-trimethylfuran (entry 8, <a class="figpopup" href="/books/NBK98919/table/ml210.t4/?report=objectonly" target="object" rid-figpopup="figml210t4" rid-ob="figobml210t4">Table 4</a>) analogs were
|
||
also found to be inactive, further confirming the necessity of the nitro substituent for activity. Replacing the nitroisoxazole ring with other nitroaromatics such as 4-methyl-2-nitrophenyl (entry 9, <a class="figpopup" href="/books/NBK98919/table/ml210.t4/?report=objectonly" target="object" rid-figpopup="figml210t4" rid-ob="figobml210t4">Table 4</a><b>)</b> or 4-nitropyrazole (entry 10, <a class="figpopup" href="/books/NBK98919/table/ml210.t4/?report=objectonly" target="object" rid-figpopup="figml210t4" rid-ob="figobml210t4">Table 4</a>) led to completely inactive compounds. Hence, the presence of a nitro group is necessary for activity but not sufficient. Thus, the nitroisoxazole moiety of the molecule is important for activity and was conserved intact for further SAR studies. Interestingly, replacing the nitroisoxazole ring with a 2-nitrofuran ring (entry 11, <a class="figpopup" href="/books/NBK98919/table/ml210.t4/?report=objectonly" target="object" rid-figpopup="figml210t4" rid-ob="figobml210t4">Table 4</a>) led to a reverse selectivity, and the compound was selectively toxic to the HRAS wild-type cell line BJeH-LT versus the HRAS<sup>V12</sup> cell line BJeLR.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml210t4"><a href="/books/NBK98919/table/ml210.t4/?report=objectonly" target="object" title="Table 4" class="img_link icnblk_img figpopup" rid-figpopup="figml210t4" rid-ob="figobml210t4"><img class="small-thumb" src="/books/NBK98919/table/ml210.t4/?report=thumb" src-large="/books/NBK98919/table/ml210.t4/?report=previmg" alt="Table 4. Summary of SAR on the Nitroisoxazole Ring." /></a><div class="icnblk_cntnt"><h4 id="ml210.t4"><a href="/books/NBK98919/table/ml210.t4/?report=objectonly" target="object" rid-ob="figobml210t4">Table 4</a></h4><p class="float-caption no_bottom_margin">Summary of SAR on the Nitroisoxazole Ring. </p></div></div><p>To improve the solubility of the probe, we then investigated the influence of the
|
||
benzhydryl-piperazine portion of the molecule (<a class="figpopup" href="/books/NBK98919/table/ml210.t5/?report=objectonly" target="object" rid-figpopup="figml210t5" rid-ob="figobml210t5">Table
|
||
5</a>). Introduction of a solubilizing agent such as morpholine (entry 1, <a class="figpopup" href="/books/NBK98919/table/ml210.t5/?report=objectonly" target="object" rid-figpopup="figml210t5" rid-ob="figobml210t5">Table 5</a>) improved solubility up to >500 μM, but led to an inactive compound. A shorter amide like the <i>p</i>-fluorobenzyl analog (entry 2, <a class="figpopup" href="/books/NBK98919/table/ml210.t5/?report=objectonly" target="object" rid-figpopup="figml210t5" rid-ob="figobml210t5">Table 5</a>) was also inactive.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml210t5"><a href="/books/NBK98919/table/ml210.t5/?report=objectonly" target="object" title="Table 5" class="img_link icnblk_img figpopup" rid-figpopup="figml210t5" rid-ob="figobml210t5"><img class="small-thumb" src="/books/NBK98919/table/ml210.t5/?report=thumb" src-large="/books/NBK98919/table/ml210.t5/?report=previmg" alt="Table 5. Summary of SAR on the Benzhydryl-Piperazine Portion." /></a><div class="icnblk_cntnt"><h4 id="ml210.t5"><a href="/books/NBK98919/table/ml210.t5/?report=objectonly" target="object" rid-ob="figobml210t5">Table 5</a></h4><p class="float-caption no_bottom_margin">Summary of SAR on the Benzhydryl-Piperazine Portion. </p></div></div><p>Keeping the piperazine ring in place and only replacing the benzhydryl portion with an
|
||
ethylcarbamate (entry 3, <a class="figpopup" href="/books/NBK98919/table/ml210.t5/?report=objectonly" target="object" rid-figpopup="figml210t5" rid-ob="figobml210t5">Table 5</a>) or a
|
||
<i>o</i>-methoxyphenyl ring (entry 4, <a class="figpopup" href="/books/NBK98919/table/ml210.t5/?report=objectonly" target="object" rid-figpopup="figml210t5" rid-ob="figobml210t5">Table
|
||
5</a>) also led to inactive compounds. Quite surprisingly, even removing one of the two phenyl rings (entry 5, <a class="figpopup" href="/books/NBK98919/table/ml210.t5/?report=objectonly" target="object" rid-figpopup="figml210t5" rid-ob="figobml210t5">Table 5</a>) led to a completely inactive compound, showing that the benzhydryl portion is necessary for activity.</p><p>Realizing that both phenyl groups are required for activity, we next investigated the influence
|
||
of the piperazine linker (<a class="figpopup" href="/books/NBK98919/table/ml210.t6/?report=objectonly" target="object" rid-figpopup="figml210t6" rid-ob="figobml210t6">Table 6</a>). Replacing the
|
||
piperazine linker with homopiperazine (entry 2, <a class="figpopup" href="/books/NBK98919/table/ml210.t6/?report=objectonly" target="object" rid-figpopup="figml210t6" rid-ob="figobml210t6">Table
|
||
6</a>) led to a small decrease in activity, showing that homopiperazine is tolerated at that position. The ethylene diamine analog (entry 3, <a class="figpopup" href="/books/NBK98919/table/ml210.t6/?report=objectonly" target="object" rid-figpopup="figml210t6" rid-ob="figobml210t6">Table 6</a>) was almost completely inactive, showing that some rigidity as well as the absence of hydrogen bond donors is preferred for activity.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml210t6"><a href="/books/NBK98919/table/ml210.t6/?report=objectonly" target="object" title="Table 6" class="img_link icnblk_img figpopup" rid-figpopup="figml210t6" rid-ob="figobml210t6"><img class="small-thumb" src="/books/NBK98919/table/ml210.t6/?report=thumb" src-large="/books/NBK98919/table/ml210.t6/?report=previmg" alt="Table 6. Summary of SAR on the Piperazine Ring." /></a><div class="icnblk_cntnt"><h4 id="ml210.t6"><a href="/books/NBK98919/table/ml210.t6/?report=objectonly" target="object" rid-ob="figobml210t6">Table 6</a></h4><p class="float-caption no_bottom_margin">Summary of SAR on the Piperazine Ring. </p></div></div><p>To improve activity and solubility of the probe, the substitution on both phenyl rings of the
|
||
benzhydryl group was investigated (<a class="figpopup" href="/books/NBK98919/table/ml210.t7/?report=objectonly" target="object" rid-figpopup="figml210t7" rid-ob="figobml210t7">Table 7</a>).
|
||
Replacement of both phenyl group by 2-pyridyl (entry 2, <a class="figpopup" href="/books/NBK98919/table/ml210.t7/?report=objectonly" target="object" rid-figpopup="figml210t7" rid-ob="figobml210t7">Table
|
||
7</a>) or 4-pyridyl groups (entry 3, <a class="figpopup" href="/books/NBK98919/table/ml210.t7/?report=objectonly" target="object" rid-figpopup="figml210t7" rid-ob="figobml210t7">Table 7</a>) dramatically increased the PBS solubility up to 500 μM and reduced plasma protein binding down to 40–60%. Unfortunately, both of these compounds were found to be inactive.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml210t7"><a href="/books/NBK98919/table/ml210.t7/?report=objectonly" target="object" title="Table 7" class="img_link icnblk_img figpopup" rid-figpopup="figml210t7" rid-ob="figobml210t7"><img class="small-thumb" src="/books/NBK98919/table/ml210.t7/?report=thumb" src-large="/books/NBK98919/table/ml210.t7/?report=previmg" alt="Table 7. Summary of SAR on the Benzhydryl Portion of the Molecule." /></a><div class="icnblk_cntnt"><h4 id="ml210.t7"><a href="/books/NBK98919/table/ml210.t7/?report=objectonly" target="object" rid-ob="figobml210t7">Table 7</a></h4><p class="float-caption no_bottom_margin">Summary of SAR on the Benzhydryl Portion of the Molecule. </p></div></div><p>Introducing <i>p</i>-fluoro substituents on both phenyl rings (entry 4, <a class="figpopup" href="/books/NBK98919/table/ml210.t7/?report=objectonly" target="object" rid-figpopup="figml210t7" rid-ob="figobml210t7">Table 7</a>) led to a 4-fold increase in activity. It had no
|
||
effect on solubility and plasma protein binding but slightly improved the plasma stability.
|
||
Introduction of <i>p</i>-methoxy substituents on both phenyl groups (entry 5, <a class="figpopup" href="/books/NBK98919/table/ml210.t7/?report=objectonly" target="object" rid-figpopup="figml210t7" rid-ob="figobml210t7">Table 7</a>) led to a decrease in activity although it improved plasma stability. The introduction of <i>p</i>-chloro substituents on both phenyl rings (entry 6, <a class="figpopup" href="/books/NBK98919/table/ml210.t7/?report=objectonly" target="object" rid-figpopup="figml210t7" rid-ob="figobml210t7">Table 7</a>) led to a 10-fold increase in activity at the cost of a slight decrease in selectivity. However, this analog was chosen as the probe. Taking clues from the structure of the drug Cetirizine (see <a class="figpopup" href="/books/NBK98919/figure/ml210.f9/?report=objectonly" target="object" rid-figpopup="figml210f9" rid-ob="figobml210f9">Figure 9</a>), we made an analog that contains an unsymmetrical benzhydryl unit where one phenyl ring is unsubstituted while the other phenyl ring has a 4-chloro substituent (entry 7, <a class="figpopup" href="/books/NBK98919/table/ml210.t7/?report=objectonly" target="object" rid-figpopup="figml210t7" rid-ob="figobml210t7">Table 7</a>). Unfortunately, this modification led to a decrease in activity.</p><p>Overall, studying the effect of structural changes of the benzhydryl portion of the molecule on
|
||
activity turned out to be productive, and we identified a synthetic compound (entry 6, <a class="figpopup" href="/books/NBK98919/table/ml210.t7/?report=objectonly" target="object" rid-figpopup="figml210t7" rid-ob="figobml210t7">Table 7</a>) with the 4-chlorophenyl substituent as the probe. The probe compound was also found to have an IC<sub>50</sub> of 179 ± 72 nM in the DRD cell line, which represents a 15-fold increase in activity compared to the hit compound. It also shows an IC<sub>50</sub> of 21.4 ± 20.8 μM in the BJeH cell line.</p><p>During the course of our SAR investigation, we found that a 2-nitrofuran analog had a selective
|
||
toxicity for the HRAS wild-type cell line BJeH-LT. Thus, we synthesized and screened several
|
||
2-nitrofuran analogs having different linker nature and different substitution pattern on the
|
||
benzhydryl portions (<a class="figpopup" href="/books/NBK98919/table/ml210.t8/?report=objectonly" target="object" rid-figpopup="figml210t8" rid-ob="figobml210t8">Table 8</a>). The selective toxicity
|
||
toward the BJeH-LT cell line versus the BJeLR cell line was conserved when both phenyl group of the
|
||
benzhydryl portion were substituted with a <i>p</i>-fluoro (entry 1, <a class="figpopup" href="/books/NBK98919/table/ml210.t8/?report=objectonly" target="object" rid-figpopup="figml210t8" rid-ob="figobml210t8">Table 8</a>) or a <i>p</i>-methoxy group (entry 2, <a class="figpopup" href="/books/NBK98919/table/ml210.t8/?report=objectonly" target="object" rid-figpopup="figml210t8" rid-ob="figobml210t8">Table 8</a>). The same tendency was observed when the piperazine linker was replaced by a homopiperazine linker (entry 3, <a class="figpopup" href="/books/NBK98919/table/ml210.t8/?report=objectonly" target="object" rid-figpopup="figml210t8" rid-ob="figobml210t8">Table 8</a>) or an ethylene diamine linker (entry 4, <a class="figpopup" href="/books/NBK98919/table/ml210.t8/?report=objectonly" target="object" rid-figpopup="figml210t8" rid-ob="figobml210t8">Table 8</a>). When the amide portion was reduced to an amine (entry 5, <a class="figpopup" href="/books/NBK98919/table/ml210.t8/?report=objectonly" target="object" rid-figpopup="figml210t8" rid-ob="figobml210t8">Table 8</a>), the compound was found to be inactive in both cell lines. This switch in the selectivity profile for nitrofuran analogs versus nitroisoxazole analogs is somewhat interesting and probably implies a different mode of action.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml210t8"><a href="/books/NBK98919/table/ml210.t8/?report=objectonly" target="object" title="Table 8" class="img_link icnblk_img figpopup" rid-figpopup="figml210t8" rid-ob="figobml210t8"><img class="small-thumb" src="/books/NBK98919/table/ml210.t8/?report=thumb" src-large="/books/NBK98919/table/ml210.t8/?report=previmg" alt="Table 8. Summary of SAR on Several Nitrofuran Analogs." /></a><div class="icnblk_cntnt"><h4 id="ml210.t8"><a href="/books/NBK98919/table/ml210.t8/?report=objectonly" target="object" rid-ob="figobml210t8">Table 8</a></h4><p class="float-caption no_bottom_margin">Summary of SAR on Several Nitrofuran Analogs. </p></div></div><p><a class="figpopup" href="/books/NBK98919/figure/ml210.f10/?report=objectonly" target="object" rid-figpopup="figml210f10" rid-ob="figobml210f10">Figure 10</a> presents a visual, high-level summary of the SAR performed on the nitroisoxazole scaffold. All positions of the hit compounds were investigated: the nitroisoxazole portion, which proved to be required for activity; the linker nature, where piperazine was found to be preferred and homopiperazine tolerated; and the benzhydryl portion, where both phenyl groups are necessary for activity and <i>p</i>-chloro substitution is preferred.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml210f10" co-legend-rid="figlgndml210f10"><a href="/books/NBK98919/figure/ml210.f10/?report=objectonly" target="object" title="Figure 10" class="img_link icnblk_img figpopup" rid-figpopup="figml210f10" rid-ob="figobml210f10"><img class="small-thumb" src="/books/NBK98919/bin/ml210f10.gif" src-large="/books/NBK98919/bin/ml210f10.jpg" alt="Figure 10. Summary of SAR performed on the Hit Compound (24S/11P)." /></a><div class="icnblk_cntnt" id="figlgndml210f10"><h4 id="ml210.f10"><a href="/books/NBK98919/figure/ml210.f10/?report=objectonly" target="object" rid-ob="figobml210f10">Figure 10</a></h4><p class="float-caption no_bottom_margin">Summary of SAR performed on the Hit Compound (24S/11P). </p></div></div></div><div id="ml210.s26"><h3>3.5. Cellular Activity</h3><p>All assays used in this project were cellular cytotoxicity assays, with the probe selected on the basis of selective cellular growth inhibition. Therefore, no additional testing of cell toxicity or cell permeability was necessary.</p></div><div id="ml210.s27"><h3>3.6. Profiling Assays</h3><p>The probe compound (<a href="/pcsubstance/?term=ML210[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML210</a>) is a novel compound and has not been reported previously in PubChem. As described above, a search of PubChem for the initial hit compound (CID 15945539), which has similar selectivity to the final probe, shows that it has been screened in 356 different assays and was identified as active in only two other assays (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/485294" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 485294</a> and <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/485297" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 485297</a>). Given the similarity of the probe to the initial hit, it is, therefore, unlikely that the probe is a promiscuous compound. We plan to perform further off-target testing through a standard commercial panel (CEREP/Ricerca).</p><p>The compound will be submitted to further cancer cell-line profiling. A previously reported HRAS selective probe developed as part of this project, <a href="/pcsubstance/?term=ML162[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML162</a>, showed significant selectivity when tested in the NCI60 cell-line panel and in cancer cell-lines as part of another initiative underway at the Broad Institute (<a class="bk_pop" href="#ml210.r26">26</a>). This new probe, <a href="/pcsubstance/?term=ML210[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML210</a>, has similarly been added to the compound set for profiling across hundreds of cancer cell-lines.</p><p>To date, <a href="/pcsubstance/?term=ML210[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML210</a> has been tested in a small subset (approximately 20) of these cell lines to determine its’ potency and selectivity. It displays differential toxicity comparable to probe <a href="/pcsubstance/?term=ML162[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML162</a>, with an IC<sub>50</sub> difference of over two log units between certain cancer lineages and tumor types. Further analysis will determine how similar the pattern of selectivity of <a href="/pcsubstance/?term=ML210[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML210</a>is to <a href="/pcsubstance/?term=ML162[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML162</a> and to other known compounds and will provide insight into the potential uniqueness of the molecular mechanism of this probe.</p></div></div><div id="ml210.s28"><h2 id="_ml210_s28_">4. Discussion</h2><p>A novel, selective, potent probe (<a href="/pcsubstance/?term=ML210[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML210</a>) was identified by high throughput screening of the NIH molecular libraries screening collection. The activity profile of probe <a href="/pcsubstance/?term=ML210[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML210</a> is similar to those of previous probes in this field of research that have proven to be very useful, while the new probe displays improved potency and is in a novel chemical class.</p><div id="ml210.s29"><h3>4.1. Comparison to Existing Art and How the New Probe is an Improvement</h3><p>Probe <a href="/pcsubstance/?term=ML210[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML210</a> is an improvement over the existing art. The two compounds previously identified using the same screening methods are: erastin and RSL-3. Erastin effectively inhibits the engineered HRAS<sup>V12</sup>-expressing cell lines with a low micromolar IC<sub>50</sub> and 4-fold selectivity over isogenic non-HRAS<sup>V12</sup>-expressing cells. A molecular target, voltage-dependent anion channel (VDAC) has also been determined for erastin. RSL-3 is reported to be more potent (IC<sub>50</sub> ~100 nM) and somewhat more selective, although the molecular target is not known. In our hands, erastin was found to have an IC<sub>50</sub> of 1.1 μM in BJeLR and 3.2 μM in BJeHLT, which gives a 2.9-fold selectivity for BJeLR. RSL-3 was found to have an IC<sub>50</sub> of 0.75 μM in BJeLR and 1.5 μM in BJeHLT, which gives a 2-fold selectivity for BJeLR. Probe <a href="/pcsubstance/?term=ML210[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML210</a> is five times more potent than erastin and 3.7 times more potent than RSL-3 in BJeLR and its selectivity (2.6-fold) is superior to RSL-3 and comparable to erastin. Probe <a href="/pcsubstance/?term=ML210[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML210</a> displays an improved potency over one existing probe, erastin, while maintaining similar selectivity. It is also a novel chemotype compared to existing probe RSL-3 and our other probe <a href="/pcsubstance/?term=ML162[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML162</a>, lacking the potential chemical liability of an electrophilic alpha-chloro amide. Although day to day variability in the response of engineered cell lines was observed due to some instability of the constructs, <a href="/pcsubstance/?term=ML210[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML210</a> consistently displayed equal or better potency and selectivity than the prior art probes erastin and RSL3 when all molecules were run in parallel experiments.</p><p>Furthermore, <a href="/pcsubstance/?term=ML210[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML210</a> appears to cause a phenotype similar to the other probe
|
||
arising from this screening campaign, <a href="/pcsubstance/?term=ML162[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML162</a>, while belonging to an entirely different structural class. Preliminary profiling experiments in the NCI60 cell line panel show that while <a href="/pcsubstance/?term=ML210[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML210</a> is less potent than <a href="/pcsubstance/?term=ML162[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML162</a>, as expected from the observed screening results, it shows a similar pattern of activity in the NCI60 cell lines, with the inter-replicate variability of <a href="/pcsubstance/?term=ML162[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML162</a> (<a class="figpopup" href="/books/NBK98919/figure/ml210.f11/?report=objectonly" target="object" rid-figpopup="figml210f11" rid-ob="figobml210f11">Figure 11A</a>) being similar to the inter-compound variability of <a href="/pcsubstance/?term=ML162[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML162</a> and <a href="/pcsubstance/?term=ML210[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML210</a> (<a class="figpopup" href="/books/NBK98919/figure/ml210.f11/?report=objectonly" target="object" rid-figpopup="figml210f11" rid-ob="figobml210f11">Figure 11B and 11C</a>). The existence of two probes of completely different structure that have similar patterns of biological activity is very useful for identifying potential targets for further study and will advance the field of cancer and specifically Ras biology.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml210f11" co-legend-rid="figlgndml210f11"><a href="/books/NBK98919/figure/ml210.f11/?report=objectonly" target="object" title="Figure 11" class="img_link icnblk_img figpopup" rid-figpopup="figml210f11" rid-ob="figobml210f11"><img class="small-thumb" src="/books/NBK98919/bin/ml210f11.gif" src-large="/books/NBK98919/bin/ml210f11.jpg" alt="Figure 11. ML210 vs ML162 activity in the NCI60 cell lines." /></a><div class="icnblk_cntnt" id="figlgndml210f11"><h4 id="ml210.f11"><a href="/books/NBK98919/figure/ml210.f11/?report=objectonly" target="object" rid-ob="figobml210f11">Figure 11</a></h4><p class="float-caption no_bottom_margin">ML210
|
||
<i>vs</i>
|
||
ML162 activity in the NCI60 cell lines. Log GI<sub>50</sub> values of ML210 against the NCI60 panel of cell lines. Replicate comparison of ML162 (panel A). ML210 compared to two replicate tests of ML162 (panel B and C). GI<sub>50</sub>s weaker than 100 μM <a href="/books/NBK98919/figure/ml210.f11/?report=objectonly" target="object" rid-ob="figobml210f11">(more...)</a></p></div></div><p>Chemically, probe <a href="/pcsubstance/?term=ML210[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML210</a> is in a novel structural class not previously reported in the field of RAS selective compounds. Further profiling activity in nonengineered cancer cells will demonstrate the importance of multiple probes with different activity patterns and may lead to identification of novel pathways important for small-molecule susceptibility of cancers.</p><p>Investigation into relevant prior art entailed searching the following databases: SciFinder,
|
||
Reaxys, PubChem, PubMed, US Patent and Trademark Office (USPTO), PatFT, AppFT, and World Intellectual Property Organization (WIPO). The search terms applied and hit statistics are provided in <a class="figpopup" href="/books/NBK98919/table/ml210.t9/?report=objectonly" target="object" rid-figpopup="figml210t9" rid-ob="figobml210t9">Table 9</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 January 26, 2011.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml210t9"><a href="/books/NBK98919/table/ml210.t9/?report=objectonly" target="object" title="Table 9" class="img_link icnblk_img figpopup" rid-figpopup="figml210t9" rid-ob="figobml210t9"><img class="small-thumb" src="/books/NBK98919/table/ml210.t9/?report=thumb" src-large="/books/NBK98919/table/ml210.t9/?report=previmg" alt="Table 9. Search Strings and Databases Employed in the Prior Art Search." /></a><div class="icnblk_cntnt"><h4 id="ml210.t9"><a href="/books/NBK98919/table/ml210.t9/?report=objectonly" target="object" rid-ob="figobml210t9">Table 9</a></h4><p class="float-caption no_bottom_margin">Search Strings and Databases Employed in the Prior Art Search. </p></div></div><p>The literature and patent searches summarized in <a class="figpopup" href="/books/NBK98919/table/ml210.t9/?report=objectonly" target="object" rid-figpopup="figml210t9" rid-ob="figobml210t9">Table 9</a> uncovered three small molecules that are known to be selectively lethal to cell lines harboring an HRAS mutation (HRAS<sup>V12</sup>), namely erastin, RSL-3, and RSL-5 (see <a class="figpopup" href="/books/NBK98919/figure/ml210.f2/?report=objectonly" target="object" rid-figpopup="figml210f2" rid-ob="figobml210f2">Figure 2</a>).</p></div><div id="ml210.s30"><h3>4.2. Mechanism of Action Studies</h3><p>The putative mechanism of action, based on the design of the primary screen and counterscreens, involves a target related to expression of oncogenic HRAS. As is demonstrated by the prior art, this does not mean the target is HRAS or on the RAS pathway; rather, the direct molecular target may be involved in processes that are merely more sensitive to inhibition when oncogenic HRAS is expressed. In the case of erastin, such a target has been shown to be VDACs.</p><p>In regard to the generality of the probe’s activity, we have shown that MEK inhibitor
|
||
U0126 suppresses the activity of <a href="/pcsubstance/?term=ML210[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML210</a> over 30 fold in two cell lines, BJeLR (<a class="figpopup" href="/books/NBK98919/figure/ml210.f12/?report=objectonly" target="object" rid-figpopup="figml210f12" rid-ob="figobml210f12">figure 12A</a>) and human lung carcinoma Calu-1 (<a class="figpopup" href="/books/NBK98919/figure/ml210.f12/?report=objectonly" target="object" rid-figpopup="figml210f12" rid-ob="figobml210f12">Figure 12B</a>), which carry mutant HRAS and KRAS, respectively. This result indicates that <a href="/pcsubstance/?term=ML210[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML210</a> likely targets common biological characteristics induced by mutations in different isoforms of Ras. Although we have not yet tested whether previously reported Ras synthetic lethal genes are involved in the mechanism of action of <a href="/pcsubstance/?term=ML210[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML210</a>, many of these genes were identified in cancer type and Ras isoform specific contexts (<a class="bk_pop" href="#ml210.r9" data-bk-pop-others="ml210.r10 ml210.r11 ml210.r12">9–12</a>), and may not be active generically. Therefore, further genetic suppressor screens using this new probe in multiple cell lines with Ras mutations may discover potential drug targets in common among different Ras isoform mutations.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml210f12" co-legend-rid="figlgndml210f12"><a href="/books/NBK98919/figure/ml210.f12/?report=objectonly" target="object" title="Figure 12" class="img_link icnblk_img figpopup" rid-figpopup="figml210f12" rid-ob="figobml210f12"><img class="small-thumb" src="/books/NBK98919/bin/ml210f12.gif" src-large="/books/NBK98919/bin/ml210f12.jpg" alt="Figure 12. Activity of ML210 in BJeLR (HRAS mutant) and Calu-1 (KRAS mutant) in the presence and absence of a MEK inhibitor (U0126)." /></a><div class="icnblk_cntnt" id="figlgndml210f12"><h4 id="ml210.f12"><a href="/books/NBK98919/figure/ml210.f12/?report=objectonly" target="object" rid-ob="figobml210f12">Figure 12</a></h4><p class="float-caption no_bottom_margin">Activity of ML210 in BJeLR (HRAS mutant) and Calu-1 (KRAS mutant) in the presence and absence of a MEK inhibitor (U0126). </p></div></div><p>A genetic method to identify a target is the use of RNAi in conjunction with the probe compound. An analysis of the target of probe <a href="/pcsubstance/?term=ML210[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML210</a> may result in a similar activity profile in various cancer cell lines and will synergize with lower doses of the probe. The Broad Institute has created an RNAi platform dedicated to genome-scale experiments for the systematic application of RNA knockdown methods. Both historical data mining and future experiments can be used to identify candidate genes and pathways that are responsible for the selective phenotype of this molecule.</p><p>We are also using profile experiments to determine additional correlations with sensitivity to the probe. The compound was recently submitted to the NCI for testing in the NCI60 cell panel. Upon completion of NCI60 panel profiling, correlations will be attempted to the known genetic characteristics of these cell lines. We have also added this probe to a small set of approximately 300 compounds that are being profiled against a larger (1000) panel of cell lines as part of another initiative underway at the Broad Institute (<a class="bk_pop" href="#ml210.r26">26</a>). This profiling analysis will correlate effects of the probe on cell viability and other cellular markers with gene expression data. Additional hypotheses can then be generated as to the molecular pathway or target of probe <a href="/pcsubstance/?term=ML210[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML210</a>.</p></div><div id="ml210.s31"><h3>4.3. Planned Future Studies</h3><p>Probe <a href="/pcsubstance/?term=ML210[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML210</a> was identified through a series of phenotypic screens. Therefore, one of the key uses of this probe will be the identification of a molecular target or pathway, as described above, which confers selective toxicity in cancer cells with certain genetic expression profiles. This can be done through biochemical and proteomic studies in an attempt to capture and identify the direct binding partner.</p><p>Another approach involves additional profiling in a sufficient number of well characterized cell lines to correlate sensitivity to the probe with genetic features. This is the goal of the 1000-cell line profiling already underway using probe <a href="/pcsubstance/?term=ML210[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML210</a>.</p><p>As a designated probe compound, <a href="/pcsubstance/?term=ML210[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML210</a> will by default be included in several planned studies at the Broad Institute that will characterize the MLSMR probe set. The Broad Institute Center Driven Research Project (CDRP) will profile probes, analogs, and a subset of less characterized compounds from the MLSMR using both multi-parametric, image-based analysis and 1000-plex Luminex gene expression arrays in a well-defined cancer cell line, U2OS. In addition, probe compounds will be further characterized in 1000-plex gene expression arrays in up to 20 additional cell lines. These and other profiling approaches will continue to enhance the understanding of the mechanism of action and applicability of probes such as <a href="/pcsubstance/?term=ML210[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML210</a>.</p><p>We envision that as this probe continues to be developed, it will become a common tool for testing susceptibility of cancers to specific inhibition and differentiating between genetic features. Additional characterization of probe <a href="/pcsubstance/?term=ML210[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML210</a> through more widespread use will further define its phenotypic properties, continuing to enhance its utility as a research tool for cancer biology.</p></div></div><div id="ml210.s32"><h2 id="_ml210_s32_">5. References</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="ml210.r1">Harvey JJ. An unidentified virus which causes the rapid production of tumours in mice. <span><span class="ref-journal">Nature. </span>1964 Dec 12;<span class="ref-vol">204</span>:1104–1105.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/14243400" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 14243400</span></a>]</div></dd><dt>2.</dt><dd><div class="bk_ref" id="ml210.r2">Kirsten WH, Mayer LA. Morphological responses to a murine erythroblastosis virus. <span><span class="ref-journal">J Natl Cancer Inst. </span>1967 Aug;<span class="ref-vol">39</span>(2):311–335.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/18623947" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18623947</span></a>]</div></dd><dt>3.</dt><dd><div class="bk_ref" id="ml210.r3">Barbacid M. Ras genes. <span><span class="ref-journal">Annu Rev Biochem. </span>1987;<span class="ref-vol">56</span>:779–827.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/3304147" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 3304147</span></a>]</div></dd><dt>4.</dt><dd><div class="bk_ref" id="ml210.r4">Bos JL. Ras oncogenes in human cancer: a review. <span><span class="ref-journal">Cancer Res. </span>1989 Sep 1;<span class="ref-vol">49</span>(17):4682–4689.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/2547513" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 2547513</span></a>]</div></dd><dt>5.</dt><dd><div class="bk_ref" id="ml210.r5">Malumbre M, Barbacid M. Ras oncogenes: the first 30 years. <span><span class="ref-journal">Nat Rev Cancer. </span>2003 Jun;<span class="ref-vol">3</span>(6):459–465.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/12778136" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 12778136</span></a>]</div></dd><dt>6.</dt><dd><div class="bk_ref" id="ml210.r6">Downward J. Targeting RAS signalling pathways in cancer therapy. <span><span class="ref-journal">Nat Rev Cancer. </span>2003 Jan;<span class="ref-vol">3</span>(1):11–22.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/12509763" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 12509763</span></a>]</div></dd><dt>7.</dt><dd><div class="bk_ref" id="ml210.r7">Coleman ML, Marshall CJ, Olson MF. RAS and RHO GTPases in G1-phase cell-cycle regulation. <span><span class="ref-journal">Nat Rev Mol Cell Biol. </span>2004 May;<span class="ref-vol">5</span>(5):355–366.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/15122349" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 15122349</span></a>]</div></dd><dt>8.</dt><dd><div class="bk_ref" id="ml210.r8">Mizuarai S, Kotani H. Synthetic lethal interactions for the development of cancer therapeutics: biological and methodological advancements. <span><span class="ref-journal">Hum Genet. </span>2010 Dec;<span class="ref-vol">128</span>(6):567–575.</span> Epub 2010 Oct 26. [<a href="https://pubmed.ncbi.nlm.nih.gov/20976469" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 20976469</span></a>]</div></dd><dt>9.</dt><dd><div class="bk_ref" id="ml210.r9">Luo J, Emanuele MJ, Li D, Creighton CJ, Schlabach MR, Westbrook TF, Wong KK, Elledge SJ. A genome-wide RNAi screen identifies multiple synthetic lethal interactions with the Ras oncogene. <span><span class="ref-journal">Cell. </span>2009 May 29;<span class="ref-vol">137</span>(5):835–848.</span> [<a href="/pmc/articles/PMC2768667/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2768667</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/19490893" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19490893</span></a>]</div></dd><dt>10.</dt><dd><div class="bk_ref" id="ml210.r10">Puyol M, Martýn A, Dubus P, Mulero F, Pizcueta P, Khan G, Guerra C, Santamarýa D, Barbacid M. A synthetic lethal interaction between K-Ras oncogenes and Cdk4 unveils a therapeutic strategy for non-small cell lung carcinoma. <span><span class="ref-journal">Cancer Cell. </span>2010 Jul 13;<span class="ref-vol">18</span>:63–73.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/20609353" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 20609353</span></a>]</div></dd><dt>11.</dt><dd><div class="bk_ref" id="ml210.r11">Scholl C, Frohling S, Dunn IF, Schinzel AC, Barbie DA, Kim SY, Silver SJ, Tamayo P, Wadlow RC, Ramaswamy S, et al. Synthetic lethal interaction between oncogenic KRAS dependency and STK33 suppression in human cancer cells. <span><span class="ref-journal">Cell. </span>2009 May 29;<span class="ref-vol">137</span>(5):821–834.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/19490892" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19490892</span></a>]</div></dd><dt>12.</dt><dd><div class="bk_ref" id="ml210.r12">Barbie DA, Tamayo P, Boehm JS, Kim SY, Moody SE, Dunn IF, Schinzel AC, Sandy P, Meylan E, Scholl C, et al. Systematic RNA interference reveals that oncogenic KRAS-driven cancers require TBK1. <span><span class="ref-journal">Nature. </span>2009 Nov 5;<span class="ref-vol">462</span>:108–114.</span> Epub 2009 Oct 21. [<a href="/pmc/articles/PMC2783335/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2783335</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/19847166" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19847166</span></a>]</div></dd><dt>13.</dt><dd><div class="bk_ref" id="ml210.r13">Rudolph D, Steegmaier M, Hoffmann M, Grauert M, Baum A, Quant J, Haslinger C, Garin-Chesa P, Adolf GR. BI 6727, a Polo-like kinase inhibitor with improved pharmacokinetic profile and broad antitumor activity. <span><span class="ref-journal">Clin Cancer Res. </span>2009 May 1;<span class="ref-vol">15</span>(9):3094–3102.</span> Epub 2009 Apr 21. [<a href="https://pubmed.ncbi.nlm.nih.gov/19383823" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19383823</span></a>]</div></dd><dt>14.</dt><dd><div class="bk_ref" id="ml210.r14">Clark K, Plater L, Peggie M, Cohen P. Use of the pharmacological inhibitor BX795 to study the regulation and physiological roles of TBK1 and IkappaB kinase epsilon: a distinct upstream kinase mediates Ser-172 phosphorylation and activation. <span><span class="ref-journal">J Biol Chem. </span>2009 May 22;<span class="ref-vol">284</span>(21):14136–14146.</span> Epub 2009 Mar 22. [<a href="/pmc/articles/PMC2682862/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2682862</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/19307177" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19307177</span></a>]</div></dd><dt>15.</dt><dd><div class="bk_ref" id="ml210.r15">Menu E, Garcia J, Huang X, Di Liberto M, Toogood PL, Chen I, Vanderkerken K, Chen-Kiang S. A novel therapeutic combination using PD 0332991 and bortezomib: study in the 5T33MM myeloma model. <span><span class="ref-journal">Cancer Res. </span>2008 Jul 15;<span class="ref-vol">68</span>(14):5519–5523.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/18632601" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18632601</span></a>]</div></dd><dt>16.</dt><dd><div class="bk_ref" id="ml210.r16">Guo W, Wu S, Liu J, Fang B. Identification of a small molecule with synthetic lethality for K-Ras and protein kinase C Iota. <span><span class="ref-journal">Cancer Res. </span>2008 Sep 15;<span class="ref-vol">68</span>(18):7403–7408.</span> [<a href="/pmc/articles/PMC2678915/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2678915</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/18794128" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18794128</span></a>]</div></dd><dt>17.</dt><dd><div class="bk_ref" id="ml210.r17">Shaw AT, Winslow MM, Magendantz M, Ouyang C, Dowdle J, Subramanian A, Lewis TA, Maglathin RL, Tolliday N, Jacks T. Selective killing of K-ras mutant cancer cells by small molecule inducers of oxidative stress. <span><span class="ref-journal">Proc. Natl. Acad. Sci. U.S.A. </span>2011;<span class="ref-vol">108</span>(21):8773–8778.</span> [<a href="/pmc/articles/PMC3102385/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3102385</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/21555567" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 21555567</span></a>]</div></dd><dt>18.</dt><dd><div class="bk_ref" id="ml210.r18">Dolma S, Lessnick SL, Hanh WC, Stockwell BR. Identification of genotype-selective antitumor agents using synthetic lethal chemical screening in engineered human tumor cells. <span><span class="ref-journal">Cancer Cell. </span>2003 Mar;<span class="ref-vol">3</span>(3):285–296.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/12676586" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 12676586</span></a>]</div></dd><dt>19.</dt><dd><div class="bk_ref" id="ml210.r19">Yang WS, Stockwell BR. Synthetic lethal screening identifies compounds activating iron-dependent, nonapoptotic cell death in oncogenic-RAS-harboring cancer cells. <span><span class="ref-journal">Chem Biol. </span>2008 Mar;<span class="ref-vol">15</span>(3):234–245.</span> [<a href="/pmc/articles/PMC2683762/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2683762</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/18355723" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18355723</span></a>]</div></dd><dt>20.</dt><dd><div class="bk_ref" id="ml210.r20">Hanh WC. Immortalization and transformation of human cells. <span><span class="ref-journal">Mol Cells. </span>2002 Jun 30;<span class="ref-vol">13</span>(3):351–361.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/12132573" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 12132573</span></a>]</div></dd><dt>21.</dt><dd><div class="bk_ref" id="ml210.r21">Hanh WC, Counter CM, Lundberg AS, Beijersbergen RL, Brooks MW, Weinberg RA. Creation of human tumour cells with defined genetic elements. <span><span class="ref-journal">Nature. </span>1999 Jul 29;<span class="ref-vol">400</span>(6743):464–468.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/10440377" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 10440377</span></a>]</div></dd><dt>22.</dt><dd><div class="bk_ref" id="ml210.r22">Hanh WC, Dessain SK, Brooks MW, King JE, Elenbaas B, Sabatini DM, DeCaprio JA, Weinberg RA. Enumeration of the simian virus 40 early region elements necessary for human cell transformation. <span><span class="ref-journal">Mol Cell Biol. </span>2002 Apr;<span class="ref-vol">22</span>(7):2111–2123.</span> [<a href="/pmc/articles/PMC133688/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC133688</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/11884599" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 11884599</span></a>]</div></dd><dt>23.</dt><dd><div class="bk_ref" id="ml210.r23">Hanh WC, Weinberg RA. Modelling the molecular circuitry of cancer. <span><span class="ref-journal">Nat Rev Cancer. </span>2002 May;<span class="ref-vol">2</span>(5):331–341.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/12044009" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 12044009</span></a>]</div></dd><dt>24.</dt><dd><div class="bk_ref" id="ml210.r24">Hanh WC, Weinberg RA. Rules for making human tumor cells. <span><span class="ref-journal">N Engl J Med. </span>2002 Nov 14;<span class="ref-vol">347</span>(20):1593–1603.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/12432047" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 12432047</span></a>]</div></dd><dt>25.</dt><dd><div class="bk_ref" id="ml210.r25">Jones LH, Summerhill NW, Swain NA, Mills JE. Aromatic chloride to nitrile transformation: medicinal and synthetic chemistry. <span><span class="ref-journal">Med. Chem. Commun. </span>2010 Dec;<span class="ref-vol">1</span>(5):309–318.</span></div></dd><dt>26.</dt><dd><div class="bk_ref" id="ml210.r26">Schreiber SL, Shamji AF, Clemons PA, Hon C, Koehler AN, Munoz B, Palmer M, Stern AM, Wagner BK, Powers S. for the Cancer Target Discovery and Development Network. Towards patient-based cancer therapeutics. <span><span class="ref-journal">Nat Biotechnol. </span>2010 Sep;<span class="ref-vol">28</span>(9):904–906.</span> [Available on 2011/3/1] [<a href="/pmc/articles/PMC2939009/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2939009</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/20829823" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 20829823</span></a>]</div></dd></dl></div><div id="ml210.app1"><h2 id="_ml210_app1_">Appendix A. Summary of Completed Assays and AIDs</h2><div id="ml210.t10" 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/NBK98919/table/ml210.t10/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml210.t10_lrgtbl__"><table class="no_margin"><thead><tr><th id="hd_h_ml210.t10_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">PubChem AID No.</th><th id="hd_h_ml210.t10_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Type</th><th id="hd_h_ml210.t10_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Target</th><th id="hd_h_ml210.t10_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Concentration Range</th><th id="hd_h_ml210.t10_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_ml210.t10_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1674" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1674</a></td><td headers="hd_h_ml210.t10_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Summary</td><td headers="hd_h_ml210.t10_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">NA</td><td headers="hd_h_ml210.t10_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">NA</td><td headers="hd_h_ml210.t10_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">NA</td></tr><tr><td headers="hd_h_ml210.t10_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1554" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1554</a></td><td headers="hd_h_ml210.t10_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Primary</td><td headers="hd_h_ml210.t10_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BJeLR</td><td headers="hd_h_ml210.t10_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">7.5 μM</td><td headers="hd_h_ml210.t10_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">303344</td></tr><tr><td headers="hd_h_ml210.t10_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1936" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1936</a></td><td headers="hd_h_ml210.t10_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">DR in primary</td><td headers="hd_h_ml210.t10_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BJeLR</td><td headers="hd_h_ml210.t10_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">15 μM – 0.12 μM</td><td headers="hd_h_ml210.t10_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1155</td></tr><tr><td headers="hd_h_ml210.t10_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1935" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1935</a></td><td headers="hd_h_ml210.t10_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Counterscreen</td><td headers="hd_h_ml210.t10_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BJeH-LT</td><td headers="hd_h_ml210.t10_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">15μM – 0.12 μM</td><td headers="hd_h_ml210.t10_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1155</td></tr><tr><td headers="hd_h_ml210.t10_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1934" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1934</a></td><td headers="hd_h_ml210.t10_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Secondary</td><td headers="hd_h_ml210.t10_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">DRD</td><td headers="hd_h_ml210.t10_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">15 μM – 0.12 μM</td><td headers="hd_h_ml210.t10_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1155</td></tr><tr><td headers="hd_h_ml210.t10_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1933" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1933</a></td><td headers="hd_h_ml210.t10_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Counterscreen</td><td headers="hd_h_ml210.t10_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BJeH</td><td headers="hd_h_ml210.t10_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">15 μM – 0.12 μM</td><td headers="hd_h_ml210.t10_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1155</td></tr><tr><td headers="hd_h_ml210.t10_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2610" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2610</a></td><td headers="hd_h_ml210.t10_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Secondary</td><td headers="hd_h_ml210.t10_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BJeLR</td><td headers="hd_h_ml210.t10_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">13.3 μM – 0.10 μM<sup><a class="bk_pop" href="#ml210.tfn15">*</a></sup></td><td headers="hd_h_ml210.t10_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">435</td></tr><tr><td headers="hd_h_ml210.t10_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2631" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2631</a></td><td headers="hd_h_ml210.t10_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Secondary</td><td headers="hd_h_ml210.t10_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BJeH-LT</td><td headers="hd_h_ml210.t10_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">13.3 μM – 0.10 μM<sup><a class="bk_pop" href="#ml210.tfn15">*</a></sup></td><td headers="hd_h_ml210.t10_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">435</td></tr><tr><td headers="hd_h_ml210.t10_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2633" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2633</a></td><td headers="hd_h_ml210.t10_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Secondary</td><td headers="hd_h_ml210.t10_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">DRD</td><td headers="hd_h_ml210.t10_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">6.7 μM – 0.013 μM<sup><a class="bk_pop" href="#ml210.tfn15">*</a></sup></td><td headers="hd_h_ml210.t10_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">73</td></tr><tr><td headers="hd_h_ml210.t10_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2635" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2635</a></td><td headers="hd_h_ml210.t10_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Secondary</td><td headers="hd_h_ml210.t10_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BJeH</td><td headers="hd_h_ml210.t10_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">6.7 μM – 0.013 μM<sup><a class="bk_pop" href="#ml210.tfn15">*</a></sup></td><td headers="hd_h_ml210.t10_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">73</td></tr><tr><td headers="hd_h_ml210.t10_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2608" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2608</a></td><td headers="hd_h_ml210.t10_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Secondary-powder</td><td headers="hd_h_ml210.t10_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BJeLR</td><td headers="hd_h_ml210.t10_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">20 μM – 20 nM</td><td headers="hd_h_ml210.t10_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">14</td></tr><tr><td headers="hd_h_ml210.t10_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2609" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2609</a></td><td headers="hd_h_ml210.t10_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Secondary-powder</td><td headers="hd_h_ml210.t10_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BJeH-LT</td><td headers="hd_h_ml210.t10_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">20 μM – 20 nM</td><td headers="hd_h_ml210.t10_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">14</td></tr><tr><td headers="hd_h_ml210.t10_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2607" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2607</a></td><td headers="hd_h_ml210.t10_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Secondary-powder</td><td headers="hd_h_ml210.t10_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">DRD</td><td headers="hd_h_ml210.t10_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">20 μM – 20 nM</td><td headers="hd_h_ml210.t10_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">14</td></tr><tr><td headers="hd_h_ml210.t10_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2611" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2611</a></td><td headers="hd_h_ml210.t10_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Secondary-powder</td><td headers="hd_h_ml210.t10_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BJeH</td><td headers="hd_h_ml210.t10_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">20 μM – 20 nM</td><td headers="hd_h_ml210.t10_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">14</td></tr><tr><td headers="hd_h_ml210.t10_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/493053" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">493053</a></td><td headers="hd_h_ml210.t10_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Secondary-powder</td><td headers="hd_h_ml210.t10_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BJeLR</td><td headers="hd_h_ml210.t10_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">45 μM – 45 nM</td><td headers="hd_h_ml210.t10_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">31</td></tr><tr><td headers="hd_h_ml210.t10_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/493093" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">493093</a></td><td headers="hd_h_ml210.t10_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Secondary-powder</td><td headers="hd_h_ml210.t10_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BJeH-LT</td><td headers="hd_h_ml210.t10_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">45 μM – 45 nM</td><td headers="hd_h_ml210.t10_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">31</td></tr><tr><td headers="hd_h_ml210.t10_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/493050" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">493050</a></td><td headers="hd_h_ml210.t10_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Secondary-powder</td><td headers="hd_h_ml210.t10_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">DRD</td><td headers="hd_h_ml210.t10_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">45 μM – 45 nM</td><td headers="hd_h_ml210.t10_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">31</td></tr><tr><td headers="hd_h_ml210.t10_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/493051" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">493051</a></td><td headers="hd_h_ml210.t10_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Secondary-powder</td><td headers="hd_h_ml210.t10_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BJeH</td><td headers="hd_h_ml210.t10_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">45 μM – 45 nM</td><td headers="hd_h_ml210.t10_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">31</td></tr></tbody></table></div><div><div><dl class="temp-labeled-list small"><dt>*</dt><dd><div id="ml210.tfn15"><p class="no_margin">Select superactive compounds were retested at a range of 210 nM – 0.41 nM (32X lower)</p></div></dd></dl></div></div></div></div><div id="ml210.app2"><h2 id="_ml210_app2_">Appendix B. Detailed Assay Protocols</h2><div id="ml210.s33"><h3>Primary Screen for BJeLR Cell Viability</h3><ol><li class="half_rhythm"><div>Maintain cells in 35 ml of growth medium (per 1 Liter: 730 ml DMEM with 4 mM L-glutamine, 210 ml M199, 150 ml heat-inactivated fetal bovine serum (FBS) in a T175 cell culture flask.</div></li><li class="half_rhythm"><div>Incubate in a TC incubator at 95% humidity, 5% CO<sub>2</sub>, 37°C.</div></li><li class="half_rhythm"><div>To passage, harvest cells by first aspirating the media and rinsing the flask with 10 ml sterile PBS.</div></li><li class="half_rhythm"><div>Aspirate PBS and add 5 ml trypsin to the flask. Incubate for 5 minutes at 22°C.</div></li><li class="half_rhythm"><div>Add 8 ml of growth medium to the trypsin to quench the reaction.</div></li><li class="half_rhythm"><div>Resuspend the cells, count, and transfer 4.5 million cells in approximately 2 ml to 33 ml fresh growth medium in a new T175 flask. Carry the lines for no more than 20 passages.</div></li><li class="half_rhythm"><div>For screening, harvest the cells, and adjust the concentration to 33,000/ml. While gently stirring, disperse the cells with a Combi multidrop by adding 30 μL of suspension per well to white, sterile, TC-treated, 384-well plates for a total of 1000 cells per well. Incubate the plates overnight in an automated TC incubator at 95% humidity, 5% CO<sub>2</sub>, 37°C.</div></li><li class="half_rhythm"><div>For compound screening, add 50 nL or 100 nL of compound, depending on the desired final compound concentration, using slotted steel pins on a pin tool.</div></li><li class="half_rhythm"><div>Return the plates to the incubator for 48 hours. To read viability, remove the cells from the incubator and cool to room temperature for 30 minutes. Remove the lids, and add 30 μL of diluted CellTiter Glo (1:3 dilution with PBS) to each with a Combi Multidrop. Incubate the plates for 10 minutes, and detect luminescence on an Envision (Perkin-Elmer) multimode reader (0.1 seconds per well).</div></li></ol></div><div id="ml210.s34"><h3>Secondary Screen for BJeLR Cell Viability (Alamar Blue)</h3><ol><li class="half_rhythm"><div>Dilute the compounds into growth medium by adding 2 μL of DMSO compound solution to 148 μL of medium and mix thoroughly. Make dilutions in 384-well stock plates (Greiner, catalog no. 781270).</div></li><li class="half_rhythm"><div>Further dilute these plates in series by adding 75 μL of solution to 75 μL of fresh growth medium, proceeding across the 384-well plate, and generate concentration-response curves.</div></li><li class="half_rhythm"><div>Next, add 36 μL of cell suspension at 28,000 cells per well to the assay plates (1000 cells/well), and add 4 μL of the medium containing the dilution series of compound to the cells.</div></li><li class="half_rhythm"><div>Incubate the cells for 48 hours in a TC incubator at 95% humidity, 5% CO<sub>2</sub>, 37°C.</div></li><li class="half_rhythm"><div>To measure viability, add 10 μL Alamar Blue solution (50% in growth medium) to each well.</div></li><li class="half_rhythm"><div>Incubate the cells for 16 hours, and read fluorescence intensity (544 nM excitation, 590 nM emission.)</div></li></ol></div></div><div id="ml210.app3"><h2 id="_ml210_app3_">Appendix C. NMR and LC Data of Probe and Analogs</h2><p id="ml210.fu2"><a href="/books/NBK98919/figure/ml210.fu2/?report=objectonly" target="object"><sup>1</sup>H NMR Spectrum (500 MHz, CDCl<sub>3</sub>) of the probe (CID 49766530/ML210)</a></p><p id="ml210.fu3"><a href="/books/NBK98919/figure/ml210.fu3/?report=objectonly" target="object"><sup>13</sup>C NMR Spectrum (125 MHz, CDCl<sub>3</sub>) of the probe</a></p><p id="ml210.fu4"><a href="/books/NBK98919/figure/ml210.fu4/?report=objectonly" target="object">UPLC chromatogram of the probe showing >93% purity</a></p><div id="ml210.s35"><h3>Spectroscopic Data for SAR Analogs</h3><p id="ml210.fu5"><a href="/books/NBK98919/figure/ml210.fu5/?report=objectonly" target="object"><sup>1</sup>HNMR Spectrum (300 MHz, CDCl<sub>3</sub>) of Analog CID 15945539</a></p><p id="ml210.fu6"><a href="/books/NBK98919/figure/ml210.fu6/?report=objectonly" target="object">UPLC Chromatogram of Analog CID 15945539</a></p><p id="ml210.fu7"><a href="/books/NBK98919/figure/ml210.fu7/?report=objectonly" target="object"><sup>1</sup>HNMR Spectrum (300 MHz, CDCl<sub>3</sub>) of Analog CID 49766535</a></p><p id="ml210.fu8"><a href="/books/NBK98919/figure/ml210.fu8/?report=objectonly" target="object">LCMS Chromatogram of Analog CID 49766535</a></p><p id="ml210.fu9"><a href="/books/NBK98919/figure/ml210.fu9/?report=objectonly" target="object"><sup>1</sup>HNMR Spectrum (300 MHz, CDCl<sub>3</sub>) of Analog CID 49766526</a></p><p id="ml210.fu10"><a href="/books/NBK98919/figure/ml210.fu10/?report=objectonly" target="object">UPLC Chromatogram of Analog CID 49766526</a></p><p id="ml210.fu11"><a href="/books/NBK98919/figure/ml210.fu11/?report=objectonly" target="object"><sup>1</sup>HNMR Spectrum (300 MHz, CDCl<sub>3</sub>) of Analog CID 49766512</a></p><p id="ml210.fu12"><a href="/books/NBK98919/figure/ml210.fu12/?report=objectonly" target="object">UPLC Chromatogram of Analog CID 49766512</a></p><p id="ml210.fu13"><a href="/books/NBK98919/figure/ml210.fu13/?report=objectonly" target="object"><sup>1</sup>HNMR Spectrum (300 MHz, CDCl<sub>3</sub>) of Analog CID 49766538</a></p><p id="ml210.fu14"><a href="/books/NBK98919/figure/ml210.fu14/?report=objectonly" target="object">UPLC Chromatogram of Analog CID 49766538</a></p><p id="ml210.fu15"><a href="/books/NBK98919/figure/ml210.fu15/?report=objectonly" target="object"><sup>1</sup>HNMR Spectrum (300 MHz, CDCl<sub>3</sub>) of Analog CID 49766527</a></p><p id="ml210.fu16"><a href="/books/NBK98919/figure/ml210.fu16/?report=objectonly" target="object">UPLC Chromatogram of Analog CID 49766527</a></p><p id="ml210.fu17"><a href="/books/NBK98919/figure/ml210.fu17/?report=objectonly" target="object"><sup>1</sup>HNMR Spectrum (300 MHz, CDCl<sub>3</sub>) of Analog CID 3244419</a></p><p id="ml210.fu18"><a href="/books/NBK98919/figure/ml210.fu18/?report=objectonly" target="object">UPLC Chromatogram of Analog CID 3244419</a></p><p id="ml210.fu19"><a href="/books/NBK98919/figure/ml210.fu19/?report=objectonly" target="object"><sup>1</sup>HNMR Spectrum (300 MHz, CDCl<sub>3</sub>) of Analog CID 15945537</a></p><p id="ml210.fu20"><a href="/books/NBK98919/figure/ml210.fu20/?report=objectonly" target="object">UPLC Chromatogram of Analog CID 15945537</a></p><p id="ml210.fu21"><a href="/books/NBK98919/figure/ml210.fu21/?report=objectonly" target="object"><sup>1</sup>HNMR Spectrum (300 MHz, CDCl<sub>3</sub>) of Analog CID 3242450</a></p><p id="ml210.fu22"><a href="/books/NBK98919/figure/ml210.fu22/?report=objectonly" target="object">UPLC Chromatogram of Analog CID 3242450</a></p><p id="ml210.fu23"><a href="/books/NBK98919/figure/ml210.fu23/?report=objectonly" target="object"><sup>1</sup>HNMR Spectrum (300 MHz, CDCl<sub>3</sub>) of Analog CID 5307213</a></p><p id="ml210.fu24"><a href="/books/NBK98919/figure/ml210.fu24/?report=objectonly" target="object">UPLC Chromatogram of Analog CID 5307213</a></p><p id="ml210.fu25"><a href="/books/NBK98919/figure/ml210.fu25/?report=objectonly" target="object"><sup>1</sup>HNMR Spectrum (300 MHz, CDCl<sub>3</sub>) of Analog CID 9550629</a></p><p id="ml210.fu26"><a href="/books/NBK98919/figure/ml210.fu26/?report=objectonly" target="object">UPLC Chromatogram of Analog CID 9550629</a></p><p id="ml210.fu27"><a href="/books/NBK98919/figure/ml210.fu27/?report=objectonly" target="object"><sup>1</sup>HNMR Spectrum (300 MHz, CDCl<sub>3</sub>) of Analog CID 5307265</a></p><p id="ml210.fu28"><a href="/books/NBK98919/figure/ml210.fu28/?report=objectonly" target="object">UPLC Chromatogram of Analog CID 5307265</a></p><p id="ml210.fu29"><a href="/books/NBK98919/figure/ml210.fu29/?report=objectonly" target="object"><sup>1</sup>HNMR Spectrum (300 MHz, CDCl<sub>3</sub>) of Analog CID 49766531</a></p><p id="ml210.fu30"><a href="/books/NBK98919/figure/ml210.fu30/?report=objectonly" target="object">UPLC Chromatogram of Analog CID 49766531</a></p><p id="ml210.fu31"><a href="/books/NBK98919/figure/ml210.fu31/?report=objectonly" target="object"><sup>1</sup>HNMR Spectrum (300 MHz, CDCl<sub>3</sub>) of Analog CID 18271554</a></p><p id="ml210.fu32"><a href="/books/NBK98919/figure/ml210.fu32/?report=objectonly" target="object">UPLC Chromatogram of Analog CID 18271554</a></p><p id="ml210.fu33"><a href="/books/NBK98919/figure/ml210.fu33/?report=objectonly" target="object"><sup>1</sup>HNMR Spectrum (300 MHz, CDCl<sub>3</sub>) of Analog CID 49766550</a></p><p id="ml210.fu34"><a href="/books/NBK98919/figure/ml210.fu34/?report=objectonly" target="object">UPLC Chromatogram of Analog CID 49766550</a></p><p id="ml210.fu35"><a href="/books/NBK98919/figure/ml210.fu35/?report=objectonly" target="object"><sup>1</sup>HNMR Spectrum (300 MHz, CDCl<sub>3</sub>) of Analog CID 49786173</a></p><p id="ml210.fu36"><a href="/books/NBK98919/figure/ml210.fu36/?report=objectonly" target="object">UPLC Chromatogram of Analog CID 49786173</a></p><p id="ml210.fu37"><a href="/books/NBK98919/figure/ml210.fu37/?report=objectonly" target="object"><sup>1</sup>HNMR Spectrum (300 MHz, CDCl<sub>3</sub>) of Analog CID 9337176</a></p><p id="ml210.fu38"><a href="/books/NBK98919/figure/ml210.fu38/?report=objectonly" target="object">UPLC Chromatogram of Analog CID 9337176</a></p><p id="ml210.fu39"><a href="/books/NBK98919/figure/ml210.fu39/?report=objectonly" target="object"><sup>1</sup>HNMR Spectrum (300 MHz, CDCl<sub>3</sub>) of Analog CID 16188083</a></p><p id="ml210.fu40"><a href="/books/NBK98919/figure/ml210.fu40/?report=objectonly" target="object">LCMS Chromatogram of Analog CID 16188083</a></p><p id="ml210.fu41"><a href="/books/NBK98919/figure/ml210.fu41/?report=objectonly" target="object"><sup>1</sup>HNMR Spectrum (300 MHz, CDCl<sub>3</sub>) of Analog CID 16189308</a></p><p id="ml210.fu42"><a href="/books/NBK98919/figure/ml210.fu42/?report=objectonly" target="object">LCMS Chromatogram of Analog CID 16189308</a></p><p id="ml210.fu43"><a href="/books/NBK98919/figure/ml210.fu43/?report=objectonly" target="object"><sup>1</sup>HNMR Spectrum (300 MHz, CDCl<sub>3</sub>) of Analog CID 46255915</a></p><p id="ml210.fu44"><a href="/books/NBK98919/figure/ml210.fu44/?report=objectonly" target="object">LCMS Chromatogram of Analog CID 46255915</a></p><p id="ml210.fu45"><a href="/books/NBK98919/figure/ml210.fu45/?report=objectonly" target="object"><sup>1</sup>HNMR Spectrum (300 MHz, CDCl<sub>3</sub>) of Analog CID 20864743</a></p><p id="ml210.fu46"><a href="/books/NBK98919/figure/ml210.fu46/?report=objectonly" target="object">LCMS Chromatogram of Analog CID 20864743</a></p><p id="ml210.fu47"><a href="/books/NBK98919/figure/ml210.fu47/?report=objectonly" target="object"><sup>1</sup>HNMR Spectrum (300 MHz, CDCl<sub>3</sub>) of Analog CID 15945452</a></p><p id="ml210.fu48"><a href="/books/NBK98919/figure/ml210.fu48/?report=objectonly" target="object">UPLC Chromatogram of Analog CID 15945452</a></p><p id="ml210.fu49"><a href="/books/NBK98919/figure/ml210.fu49/?report=objectonly" target="object"><sup>1</sup>HNMR Spectrum (300 MHz, CDCl<sub>3</sub>) of Analog CID 49766520</a></p><p id="ml210.fu50"><a href="/books/NBK98919/figure/ml210.fu50/?report=objectonly" target="object">UPLC Chromatogram of Analog CID 49766520</a></p><p id="ml210.fu51"><a href="/books/NBK98919/figure/ml210.fu51/?report=objectonly" target="object"><sup>1</sup>HNMR Spectrum (300 MHz, CDCl<sub>3</sub>) of Analog CID 49766541</a></p><p id="ml210.fu52"><a href="/books/NBK98919/figure/ml210.fu52/?report=objectonly" target="object">UPLC Chromatogram of Analog CID 49766541</a></p><p id="ml210.fu53"><a href="/books/NBK98919/figure/ml210.fu53/?report=objectonly" target="object"><sup>1</sup>HNMR Spectrum (300 MHz, CDCl<sub>3</sub>) of Analog CID 49766542</a></p><p id="ml210.fu54"><a href="/books/NBK98919/figure/ml210.fu54/?report=objectonly" target="object">UPLC Chromatogram of Analog CID 49766542</a></p><p id="ml210.fu55"><a href="/books/NBK98919/figure/ml210.fu55/?report=objectonly" target="object"><sup>1</sup>HNMR Spectrum (300 MHz, CDCl<sub>3</sub>) of Analog CID 49766547</a></p><p id="ml210.fu56"><a href="/books/NBK98919/figure/ml210.fu56/?report=objectonly" target="object">UPLC Chromatogram of Analog CID 49766547</a></p><p id="ml210.fu57"><a href="/books/NBK98919/figure/ml210.fu57/?report=objectonly" target="object"><sup>1</sup>HNMR Spectrum (300 MHz, CDCl<sub>3</sub>) of Analog CID 49766518</a></p><p id="ml210.fu58"><a href="/books/NBK98919/figure/ml210.fu58/?report=objectonly" target="object">UPLC Chromatogram of Analog CID 49766518</a></p><p id="ml210.fu59"><a href="/books/NBK98919/figure/ml210.fu59/?report=objectonly" target="object"><sup>1</sup>HNMR Spectrum (300 MHz, CDCl<sub>3</sub>) of Analog CID 49766549</a></p><p id="ml210.fu60"><a href="/books/NBK98919/figure/ml210.fu60/?report=objectonly" target="object">UPLC Chromatogram of Analog CID 49766549</a></p><p id="ml210.fu61"><a href="/books/NBK98919/figure/ml210.fu61/?report=objectonly" target="object"><sup>1</sup>HNMR Spectrum (300 MHz, CDCl<sub>3</sub>) of Analog CID 49766530</a></p><p id="ml210.fu62"><a href="/books/NBK98919/figure/ml210.fu62/?report=objectonly" target="object">UPLC Chromatogram of Analog CID 49766530</a></p><p id="ml210.fu63"><a href="/books/NBK98919/figure/ml210.fu63/?report=objectonly" target="object"><sup>1</sup>HNMR Spectrum (300 MHz, CDCl<sub>3</sub>) of Analog CID 49766534</a></p><p id="ml210.fu64"><a href="/books/NBK98919/figure/ml210.fu64/?report=objectonly" target="object">UPLC Chromatogram of Analog CID49766534</a></p><p id="ml210.fu65"><a href="/books/NBK98919/figure/ml210.fu65/?report=objectonly" target="object"><sup>1</sup>HNMR Spectrum (300 MHz, CDCl<sub>3</sub>) of Analog CID 29218174</a></p><p id="ml210.fu66"><a href="/books/NBK98919/figure/ml210.fu66/?report=objectonly" target="object">UPLC Chromatogram of Analog CID 29218174</a></p><p id="ml210.fu67"><a href="/books/NBK98919/figure/ml210.fu67/?report=objectonly" target="object"><sup>1</sup>HNMR Spectrum (300 MHz, CDCl<sub>3</sub>) of Analog CID 49786174</a></p><p id="ml210.fu68"><a href="/books/NBK98919/figure/ml210.fu68/?report=objectonly" target="object">UPLC Chromatogram of Analog CID 49786174</a></p><p id="ml210.fu69"><a href="/books/NBK98919/figure/ml210.fu69/?report=objectonly" target="object"><sup>1</sup>HNMR Spectrum (300 MHz, CDCl<sub>3</sub>) of Analog CID 49786176</a></p><p id="ml210.fu70"><a href="/books/NBK98919/figure/ml210.fu70/?report=objectonly" target="object">UPLC Chromatogram of Analog CID 49786176</a></p><p id="ml210.fu71"><a href="/books/NBK98919/figure/ml210.fu71/?report=objectonly" target="object"><sup>1</sup>HNMR Spectrum (300 MHz, CDCl<sub>3</sub>) of Analog CID 49786172</a></p><p id="ml210.fu72"><a href="/books/NBK98919/figure/ml210.fu72/?report=objectonly" target="object">UPLC Chromatogram of Analog CID 49786172</a></p><p id="ml210.fu73"><a href="/books/NBK98919/figure/ml210.fu73/?report=objectonly" target="object"><sup>1</sup>HNMR Spectrum (300 MHz, CDCl<sub>3</sub>) of Analog CID 49786175</a></p><p id="ml210.fu74"><a href="/books/NBK98919/figure/ml210.fu74/?report=objectonly" target="object">UPLC Chromatogram of Analog CID 49786175</a></p></div></div><div id="ml210.app4"><h2 id="_ml210_app4_">Appendix D. Compounds Submitted to BioFocus</h2><p id="ml210.t11"><a href="/books/NBK98919/table/ml210.t11/?report=objectonly" target="object">Table A2. Probe and Analog Information</a></p></div><div id="bk_toc_contnr"></div></div></div>
|
||
<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: NBK98919</span><span class="label">PMID: <a href="https://pubmed.ncbi.nlm.nih.gov/22834036" title="PubMed record of this page" ref="pagearea=meta&targetsite=entrez&targetcat=link&targettype=pubmed">22834036</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/ml211/" title="Previous page in this title">< Prev</a><a class="active page_link next" href="/books/n/mlprobe/ml209/" title="Next page in this title">Next ></a></div></div></div></div>
|
||
|
||
</div>
|
||
|
||
<!-- Custom content below content -->
|
||
<div class="col4">
|
||
|
||
</div>
|
||
|
||
|
||
<!-- Book content -->
|
||
|
||
<!-- Custom contetnt below bottom nav -->
|
||
<div class="col5">
|
||
|
||
</div>
|
||
</div>
|
||
|
||
<div id="rightcolumn" class="four_col col last">
|
||
<!-- Custom content above discovery portlets -->
|
||
<div class="col6">
|
||
<div id="ncbi_share_book"><a href="#" class="ncbi_share" data-ncbi_share_config="popup:false,shorten:true" ref="id=NBK98919&db=books">Share</a></div>
|
||
|
||
</div>
|
||
<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/NBK98919/?report=reader">PubReader</a></li><li><a href="/books/NBK98919/?report=printable">Print View</a></li><li><a data-jig="ncbidialog" href="#_ncbi_dlg_citbx_NBK98919" data-jigconfig="width:400,modal:true">Cite this Page</a><div id="_ncbi_dlg_citbx_NBK98919" style="display:none" title="Cite this Page"><div class="bk_tt">Bittker JA, Weiwer M, Lewis T, et al. Screen for RAS-Selective Lethal Compounds and VDAC Ligands - Probe 2. 2011 Feb 10 [Updated 2011 Dec 12]. 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/NBK98919/pdf/Bookshelf_NBK98919.pdf">PDF version of this page</a> (2.3M)</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="#ml210.s1" ref="log$=inpage&link_id=inpage">Probe Structure & Characteristics</a></li><li><a href="#ml210.s2" ref="log$=inpage&link_id=inpage">Recommendations for scientific use of the probe</a></li><li><a href="#ml210.s3" ref="log$=inpage&link_id=inpage">Introduction</a></li><li><a href="#ml210.s4" ref="log$=inpage&link_id=inpage">Materials and Methods</a></li><li><a href="#ml210.s20" ref="log$=inpage&link_id=inpage">Results</a></li><li><a href="#ml210.s28" ref="log$=inpage&link_id=inpage">Discussion</a></li><li><a href="#ml210.s32" ref="log$=inpage&link_id=inpage">References</a></li><li><a href="#ml210.app1" ref="log$=inpage&link_id=inpage">Summary of Completed Assays and AIDs</a></li><li><a href="#ml210.app2" ref="log$=inpage&link_id=inpage">Detailed Assay Protocols</a></li><li><a href="#ml210.app3" ref="log$=inpage&link_id=inpage">NMR and LC Data of Probe and Analogs</a></li><li><a href="#ml210.app4" ref="log$=inpage&link_id=inpage">Compounds Submitted to BioFocus</a></li></ul></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Related information</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="discovery_db_links" id="Shutter"></a></div><div class="portlet_content"><ul><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pmc&DbFrom=books&Cmd=Link&LinkName=books_pmc_refs&IdsFromResult=2961564" ref="log$=recordlinks">PMC</a><div class="brieflinkpop offscreen_noflow">PubMed Central citations</div></li><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pcassay&DbFrom=books&Cmd=Link&LinkName=books_pcassay_probe&IdsFromResult=2961564" ref="log$=recordlinks">PubChem BioAssay for Chemical Probe</a><div class="brieflinkpop offscreen_noflow">PubChem BioAssay records reporting screening data for the development of the chemical probe(s) described in this book chapter</div></li><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pcsubstance&DbFrom=books&Cmd=Link&LinkName=books_pcsubstance&IdsFromResult=2961564" ref="log$=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=2961564" ref="log$=recordlinks">PubMed</a><div class="brieflinkpop offscreen_noflow">Links to PubMed</div></li></ul></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Similar articles in PubMed</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="PBooksDiscovery_RA" id="Shutter"></a></div><div class="portlet_content"><ul><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/21634081" ref="ordinalpos=1&linkpos=1&log$=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> Screen for RAS-Selective Lethal Compounds and VDAC Ligands - Probe 1.</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> Screen for RAS-Selective Lethal Compounds and VDAC Ligands - Probe 1.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Bittker JA, Weiwer M, Shimada K, Yang WS, MacPherson L, Dandapani S, Munoz B, Palmer M, Stockwell BR, 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/22297109" ref="ordinalpos=1&linkpos=2&log$=relatedarticles&logdbfrom=pubmed">Development of small-molecule probes that selectively kill cells induced to express mutant RAS.</a><span class="source">[Bioorg Med Chem Lett. 2012]</span><div class="brieflinkpop offscreen_noflow">Development of small-molecule probes that selectively kill cells induced to express mutant RAS.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Weïwer M, Bittker JA, Lewis TA, Shimada K, Yang WS, MacPherson L, Dandapani S, Palmer M, Stockwell BR, Schreiber SL, et al. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Bioorg Med Chem Lett. 2012 Feb 15; 22(4):1822-6. Epub 2011 Sep 28.</em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/23658966" ref="ordinalpos=1&linkpos=3&log$=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 a Selective Small-Molecule Inhibitor of Breast Cancer Stem Cells - Probe 1.</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 a Selective Small-Molecule Inhibitor of Breast Cancer Stem Cells - Probe 1.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Carmody L, Germain A, Morgan B, VerPlank L, Fernandez C, Forbeck E, Ting A, Feng Y, Perez J, Dandapani S, 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/23762951" ref="ordinalpos=1&linkpos=4&log$=relatedreviews&logdbfrom=pubmed"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> Identification of a Selective Small-Molecule Inhibitor of Breast Cancer Stem Cells—Probe 2.</a><span class="source">[Probe Reports from the NIH Mol...]</span><div class="brieflinkpop offscreen_noflow"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> Identification of a Selective Small-Molecule Inhibitor of Breast Cancer Stem Cells—Probe 2.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Carmody LC, Germain A, Morgan B, VerPlank L, Fernandez C, Feng Y, Perez JR, Dandapani S, Munoz B, Palmer M, et al. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Probe Reports from the NIH Molecular Libraries Program. 2010</em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/23762939" ref="ordinalpos=1&linkpos=5&log$=relatedreviews&logdbfrom=pubmed"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> Identification of a Selective Small-Molecule Inhibitor of Breast Cancer Stem Cells - 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 a Selective Small-Molecule Inhibitor of Breast Cancer Stem Cells - 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 L, Germain A, Morgan B, VerPlank L, Fernandez C, Feng Y, Perez J, Dandapani S, Munoz B, Palmer M, et al. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Probe Reports from the NIH Molecular Libraries Program. 2010</em></div></div></li></ul><a class="seemore" href="/sites/entrez?db=pubmed&cmd=link&linkname=pubmed_pubmed_reviews&uid=22834036" ref="ordinalpos=1&log$=relatedreviews_seeall&logdbfrom=pubmed">See reviews...</a><a class="seemore" href="/sites/entrez?db=pubmed&cmd=link&linkname=pubmed_pubmed&uid=22834036" ref="ordinalpos=1&log$=relatedarticles_seeall&logdbfrom=pubmed">See all...</a></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Recent Activity</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="recent_activity" id="Shutter"></a></div><div class="portlet_content"><div xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" id="HTDisplay" class=""><div class="action"><a href="javascript:historyDisplayState('ClearHT')">Clear</a><a href="javascript:historyDisplayState('HTOff')" class="HTOn">Turn Off</a><a href="javascript:historyDisplayState('HTOn')" class="HTOff">Turn On</a></div><ul id="activity"><li class="ra_rcd ralinkpopper two_line"><a class="htb ralinkpopperctrl" ref="log$=activity&linkpos=1" href="/portal/utils/pageresolver.fcgi?recordid=67d66bc9cde49f3df7dbd6d2">Screen for RAS-Selective Lethal Compounds and VDAC Ligands - Probe 2 - Probe Rep...</a><div class="ralinkpop offscreen_noflow">Screen for RAS-Selective Lethal Compounds and VDAC Ligands - Probe 2 - Probe Reports from the NIH Molecular Libraries Program<div class="brieflinkpopdesc"></div></div><div class="tertiary"></div></li><li class="ra_rcd ralinkpopper two_line"><a class="htb ralinkpopperctrl" ref="log$=activity&linkpos=2" href="/portal/utils/pageresolver.fcgi?recordid=67d66bc7cde49f3df7dbd396">Optimization and characterization of a triazole urea dual inhibitor for lysophos...</a><div class="ralinkpop offscreen_noflow">Optimization and characterization of a triazole urea dual inhibitor for lysophospholipase 1 (LYPLA1) and lysophospholipase 2 (LYPLA2) - Probe Reports from the NIH Molecular Libraries Program<div class="brieflinkpopdesc"></div></div><div class="tertiary"></div></li><li class="ra_rcd ralinkpopper two_line"><a class="htb ralinkpopperctrl" ref="log$=activity&linkpos=3" href="/portal/utils/pageresolver.fcgi?recordid=67d66bc6cde49f3df7dbce60">Identification of small molecules that selectively inhibit fluconazole-resistant...</a><div class="ralinkpop offscreen_noflow">Identification of small molecules that selectively inhibit fluconazole-resistant Candida albicans in the presence of fluconazole but not in its absence - Probe 2 - Probe Reports from the NIH Molecular Libraries Program<div class="brieflinkpopdesc"></div></div><div class="tertiary"></div></li><li class="ra_rcd ralinkpopper two_line"><a class="htb ralinkpopperctrl" ref="log$=activity&linkpos=4" href="/portal/utils/pageresolver.fcgi?recordid=67d66bc584f3725e592cb628">A small molecule activator of KCNQ2 and KCNQ4 channels - Probe Reports from the ...</a><div class="ralinkpop offscreen_noflow">A small molecule activator of KCNQ2 and KCNQ4 channels - Probe Reports from the NIH Molecular Libraries Program<div class="brieflinkpopdesc"></div></div><div class="tertiary"></div></li><li class="ra_rcd ralinkpopper two_line"><a class="htb ralinkpopperctrl" ref="log$=activity&linkpos=5" href="/portal/utils/pageresolver.fcgi?recordid=67d66bc467c23b31e0b28e66">Discovery of Inhibitors of Anti-Apoptotic Protein A1 - Probe Reports from the NI...</a><div class="ralinkpop offscreen_noflow">Discovery of Inhibitors of Anti-Apoptotic Protein A1 - Probe Reports from the NIH Molecular Libraries Program<div class="brieflinkpopdesc"></div></div><div class="tertiary"></div></li></ul><p class="HTOn">Your browsing activity is empty.</p><p class="HTOff">Activity recording is turned off.</p><p id="turnOn" class="HTOff"><a href="javascript:historyDisplayState('HTOn')">Turn recording back on</a></p><a class="seemore" href="/sites/myncbi/recentactivity">See more...</a></div></div></div>
|
||
|
||
<!-- Custom content below discovery portlets -->
|
||
<div class="col7">
|
||
|
||
</div>
|
||
</div>
|
||
</div>
|
||
|
||
<!-- Custom content after all -->
|
||
<div class="col8">
|
||
|
||
</div>
|
||
<div class="col9">
|
||
|
||
</div>
|
||
|
||
<script type="text/javascript" src="/corehtml/pmc/js/jquery.scrollTo-1.4.2.js"></script>
|
||
<script type="text/javascript">
|
||
(function($){
|
||
$('.skiplink').each(function(i, item){
|
||
var href = $($(item).attr('href'));
|
||
href.attr('tabindex', '-1').addClass('skiptarget'); // ensure the target can receive focus
|
||
$(item).on('click', function(event){
|
||
event.preventDefault();
|
||
$.scrollTo(href, 0, {
|
||
onAfter: function(){
|
||
href.focus();
|
||
}
|
||
});
|
||
});
|
||
});
|
||
})(jQuery);
|
||
</script>
|
||
</div>
|
||
<div class="bottom">
|
||
|
||
<div id="NCBIFooter_dynamic">
|
||
<!--<component id="Breadcrumbs" label="breadcrumbs"/>
|
||
<component id="Breadcrumbs" label="helpdesk"/>-->
|
||
|
||
</div>
|
||
|
||
<div class="footer" id="footer">
|
||
<section class="icon-section">
|
||
<div id="icon-section-header" class="icon-section_header">Follow NCBI</div>
|
||
<div class="grid-container container">
|
||
<div class="icon-section_container">
|
||
<a class="footer-icon" id="footer_twitter" href="https://twitter.com/ncbi" aria-label="Twitter"><svg xmlns="http://www.w3.org/2000/svg" data-name="Layer 1" viewBox="0 0 300 300">
|
||
<defs>
|
||
<style>
|
||
.cls-11 {
|
||
fill: #737373;
|
||
}
|
||
</style>
|
||
</defs>
|
||
<title>Twitter</title>
|
||
<path class="cls-11" d="M250.11,105.48c-7,3.14-13,3.25-19.27.14,8.12-4.86,8.49-8.27,11.43-17.46a78.8,78.8,0,0,1-25,9.55,39.35,39.35,0,0,0-67,35.85,111.6,111.6,0,0,1-81-41.08A39.37,39.37,0,0,0,81.47,145a39.08,39.08,0,0,1-17.8-4.92c0,.17,0,.33,0,.5a39.32,39.32,0,0,0,31.53,38.54,39.26,39.26,0,0,1-17.75.68,39.37,39.37,0,0,0,36.72,27.3A79.07,79.07,0,0,1,56,223.34,111.31,111.31,0,0,0,116.22,241c72.3,0,111.83-59.9,111.83-111.84,0-1.71,0-3.4-.1-5.09C235.62,118.54,244.84,113.37,250.11,105.48Z">
|
||
</path>
|
||
</svg></a>
|
||
<a class="footer-icon" id="footer_facebook" href="https://www.facebook.com/ncbi.nlm" aria-label="Facebook"><svg xmlns="http://www.w3.org/2000/svg" data-name="Layer 1" viewBox="0 0 300 300">
|
||
<title>Facebook</title>
|
||
<path class="cls-11" d="M210.5,115.12H171.74V97.82c0-8.14,5.39-10,9.19-10h27.14V52l-39.32-.12c-35.66,0-42.42,26.68-42.42,43.77v19.48H99.09v36.32h27.24v109h45.41v-109h35Z">
|
||
</path>
|
||
</svg></a>
|
||
<a class="footer-icon" id="footer_linkedin" href="https://www.linkedin.com/company/ncbinlm" aria-label="LinkedIn"><svg xmlns="http://www.w3.org/2000/svg" data-name="Layer 1" viewBox="0 0 300 300">
|
||
<title>LinkedIn</title>
|
||
<path class="cls-11" d="M101.64,243.37H57.79v-114h43.85Zm-22-131.54h-.26c-13.25,0-21.82-10.36-21.82-21.76,0-11.65,8.84-21.15,22.33-21.15S101.7,78.72,102,90.38C102,101.77,93.4,111.83,79.63,111.83Zm100.93,52.61A17.54,17.54,0,0,0,163,182v61.39H119.18s.51-105.23,0-114H163v13a54.33,54.33,0,0,1,34.54-12.66c26,0,44.39,18.8,44.39,55.29v58.35H198.1V182A17.54,17.54,0,0,0,180.56,164.44Z">
|
||
</path>
|
||
</svg></a>
|
||
<a class="footer-icon" id="footer_github" href="https://github.com/ncbi" aria-label="GitHub"><svg xmlns="http://www.w3.org/2000/svg" data-name="Layer 1" viewBox="0 0 300 300">
|
||
<defs>
|
||
<style>
|
||
.cls-11,
|
||
.cls-12 {
|
||
fill: #737373;
|
||
}
|
||
|
||
.cls-11 {
|
||
fill-rule: evenodd;
|
||
}
|
||
</style>
|
||
</defs>
|
||
<title>GitHub</title>
|
||
<path class="cls-11" d="M151.36,47.28a105.76,105.76,0,0,0-33.43,206.1c5.28,1,7.22-2.3,7.22-5.09,0-2.52-.09-10.85-.14-19.69-29.42,6.4-35.63-12.48-35.63-12.48-4.81-12.22-11.74-15.47-11.74-15.47-9.59-6.56.73-6.43.73-6.43,10.61.75,16.21,10.9,16.21,10.9,9.43,16.17,24.73,11.49,30.77,8.79,1-6.83,3.69-11.5,6.71-14.14C108.57,197.1,83.88,188,83.88,147.51a40.92,40.92,0,0,1,10.9-28.39c-1.1-2.66-4.72-13.42,1-28,0,0,8.88-2.84,29.09,10.84a100.26,100.26,0,0,1,53,0C198,88.3,206.9,91.14,206.9,91.14c5.76,14.56,2.14,25.32,1,28a40.87,40.87,0,0,1,10.89,28.39c0,40.62-24.74,49.56-48.29,52.18,3.79,3.28,7.17,9.71,7.17,19.58,0,14.15-.12,25.54-.12,29,0,2.82,1.9,6.11,7.26,5.07A105.76,105.76,0,0,0,151.36,47.28Z">
|
||
</path>
|
||
<path class="cls-12" d="M85.66,199.12c-.23.52-1.06.68-1.81.32s-1.2-1.06-.95-1.59,1.06-.69,1.82-.33,1.21,1.07.94,1.6Zm-1.3-1">
|
||
</path>
|
||
<path class="cls-12" d="M90,203.89c-.51.47-1.49.25-2.16-.49a1.61,1.61,0,0,1-.31-2.19c.52-.47,1.47-.25,2.17.49s.82,1.72.3,2.19Zm-1-1.08">
|
||
</path>
|
||
<path class="cls-12" d="M94.12,210c-.65.46-1.71,0-2.37-.91s-.64-2.07,0-2.52,1.7,0,2.36.89.65,2.08,0,2.54Zm0,0"></path>
|
||
<path class="cls-12" d="M99.83,215.87c-.58.64-1.82.47-2.72-.41s-1.18-2.06-.6-2.7,1.83-.46,2.74.41,1.2,2.07.58,2.7Zm0,0">
|
||
</path>
|
||
<path class="cls-12" d="M107.71,219.29c-.26.82-1.45,1.2-2.64.85s-2-1.34-1.74-2.17,1.44-1.23,2.65-.85,2,1.32,1.73,2.17Zm0,0">
|
||
</path>
|
||
<path class="cls-12" d="M116.36,219.92c0,.87-1,1.59-2.24,1.61s-2.29-.68-2.3-1.54,1-1.59,2.26-1.61,2.28.67,2.28,1.54Zm0,0">
|
||
</path>
|
||
<path class="cls-12" d="M124.42,218.55c.15.85-.73,1.72-2,1.95s-2.37-.3-2.52-1.14.73-1.75,2-2,2.37.29,2.53,1.16Zm0,0"></path>
|
||
</svg></a>
|
||
<a class="footer-icon" id="footer_blog" href="https://ncbiinsights.ncbi.nlm.nih.gov/" aria-label="Blog">
|
||
<svg xmlns="http://www.w3.org/2000/svg" id="Layer_1" data-name="Layer 1" viewBox="0 0 40 40">
|
||
<defs><style>.cls-1{fill:#737373;}</style></defs>
|
||
<title>NCBI Insights Blog</title>
|
||
<path class="cls-1" d="M14,30a4,4,0,1,1-4-4,4,4,0,0,1,4,4Zm11,3A19,19,0,0,0,7.05,15a1,1,0,0,0-1,1v3a1,1,0,0,0,.93,1A14,14,0,0,1,20,33.07,1,1,0,0,0,21,34h3a1,1,0,0,0,1-1Zm9,0A28,28,0,0,0,7,6,1,1,0,0,0,6,7v3a1,1,0,0,0,1,1A23,23,0,0,1,29,33a1,1,0,0,0,1,1h3A1,1,0,0,0,34,33Z"></path>
|
||
</svg>
|
||
</a>
|
||
</div>
|
||
</div>
|
||
</section>
|
||
|
||
<section class="container-fluid bg-primary">
|
||
<div class="container pt-5">
|
||
<div class="row mt-3">
|
||
<div class="col-lg-3 col-12">
|
||
<p><a class="text-white" href="https://www.nlm.nih.gov/socialmedia/index.html">Connect with NLM</a></p>
|
||
<ul class="list-inline social_media">
|
||
<li class="list-inline-item"><a href="https://twitter.com/NLM_NIH" aria-label="Twitter" target="_blank" rel="noopener noreferrer"><svg xmlns="http://www.w3.org/2000/svg" xmlns:xlink="http://www.w3.org/1999/xlink" version="1.1" x="0px" y="0px" viewBox="0 0 249 249" style="enable-background:new 0 0 249 249;" xml:space="preserve">
|
||
<style type="text/css">
|
||
.st20 {
|
||
fill: #FFFFFF;
|
||
}
|
||
|
||
.st30 {
|
||
fill: none;
|
||
stroke: #FFFFFF;
|
||
stroke-width: 8;
|
||
stroke-miterlimit: 10;
|
||
}
|
||
</style>
|
||
<title>Twitter</title>
|
||
<g>
|
||
<g>
|
||
<g>
|
||
<path class="st20" d="M192.9,88.1c-5,2.2-9.2,2.3-13.6,0.1c5.7-3.4,6-5.8,8.1-12.3c-5.4,3.2-11.4,5.5-17.6,6.7 c-10.5-11.2-28.1-11.7-39.2-1.2c-7.2,6.8-10.2,16.9-8,26.5c-22.3-1.1-43.1-11.7-57.2-29C58,91.6,61.8,107.9,74,116 c-4.4-0.1-8.7-1.3-12.6-3.4c0,0.1,0,0.2,0,0.4c0,13.2,9.3,24.6,22.3,27.2c-4.1,1.1-8.4,1.3-12.5,0.5c3.6,11.3,14,19,25.9,19.3 c-11.6,9.1-26.4,13.2-41.1,11.5c12.7,8.1,27.4,12.5,42.5,12.5c51,0,78.9-42.2,78.9-78.9c0-1.2,0-2.4-0.1-3.6 C182.7,97.4,189.2,93.7,192.9,88.1z"></path>
|
||
</g>
|
||
</g>
|
||
<circle class="st30" cx="124.4" cy="128.8" r="108.2"></circle>
|
||
</g>
|
||
</svg></a></li>
|
||
<li class="list-inline-item"><a href="https://www.facebook.com/nationallibraryofmedicine" aria-label="Facebook" rel="noopener noreferrer" target="_blank">
|
||
<svg xmlns="http://www.w3.org/2000/svg" xmlns:xlink="http://www.w3.org/1999/xlink" version="1.1" x="0px" y="0px" viewBox="0 0 249 249" style="enable-background:new 0 0 249 249;" xml:space="preserve">
|
||
<style type="text/css">
|
||
.st10 {
|
||
fill: #FFFFFF;
|
||
}
|
||
|
||
.st110 {
|
||
fill: none;
|
||
stroke: #FFFFFF;
|
||
stroke-width: 8;
|
||
stroke-miterlimit: 10;
|
||
}
|
||
</style>
|
||
<title>Facebook</title>
|
||
<g>
|
||
<g>
|
||
<path class="st10" d="M159,99.1h-24V88.4c0-5,3.3-6.2,5.7-6.2h16.8V60l-24.4-0.1c-22.1,0-26.2,16.5-26.2,27.1v12.1H90v22.5h16.9 v67.5H135v-67.5h21.7L159,99.1z"></path>
|
||
</g>
|
||
</g>
|
||
<circle class="st110" cx="123.6" cy="123.2" r="108.2"></circle>
|
||
</svg>
|
||
</a></li>
|
||
<li class="list-inline-item"><a href="https://www.youtube.com/user/NLMNIH" aria-label="Youtube" target="_blank" rel="noopener noreferrer"><svg xmlns="http://www.w3.org/2000/svg" xmlns:xlink="http://www.w3.org/1999/xlink" version="1.1" x="0px" y="0px" viewBox="0 0 249 249" style="enable-background:new 0 0 249 249;" xml:space="preserve">
|
||
<title>Youtube</title>
|
||
<style type="text/css">
|
||
.st4 {
|
||
fill: none;
|
||
stroke: #FFFFFF;
|
||
stroke-width: 8;
|
||
stroke-miterlimit: 10;
|
||
}
|
||
|
||
.st5 {
|
||
fill: #FFFFFF;
|
||
}
|
||
</style>
|
||
<circle class="st4" cx="124.2" cy="123.4" r="108.2"></circle>
|
||
<g transform="translate(0,-952.36218)">
|
||
<path class="st5" d="M88.4,1037.4c-10.4,0-18.7,8.3-18.7,18.7v40.1c0,10.4,8.3,18.7,18.7,18.7h72.1c10.4,0,18.7-8.3,18.7-18.7 v-40.1c0-10.4-8.3-18.7-18.7-18.7H88.4z M115.2,1058.8l29.4,17.4l-29.4,17.4V1058.8z"></path>
|
||
</g>
|
||
</svg></a></li>
|
||
</ul>
|
||
</div>
|
||
<div class="col-lg-3 col-12">
|
||
<p class="address_footer text-white">National Library of Medicine<br />
|
||
<a href="https://www.google.com/maps/place/8600+Rockville+Pike,+Bethesda,+MD+20894/@38.9959508,-77.101021,17z/data=!3m1!4b1!4m5!3m4!1s0x89b7c95e25765ddb:0x19156f88b27635b8!8m2!3d38.9959508!4d-77.0988323" class="text-white" target="_blank" rel="noopener noreferrer">8600 Rockville Pike<br />
|
||
Bethesda, MD 20894</a></p>
|
||
</div>
|
||
<div class="col-lg-3 col-12 centered-lg">
|
||
<p><a href="https://www.nlm.nih.gov/web_policies.html" class="text-white">Web Policies</a><br />
|
||
<a href="https://www.nih.gov/institutes-nih/nih-office-director/office-communications-public-liaison/freedom-information-act-office" class="text-white">FOIA</a><br />
|
||
<a href="https://www.hhs.gov/vulnerability-disclosure-policy/index.html" class="text-white" id="vdp">HHS Vulnerability Disclosure</a></p>
|
||
</div>
|
||
<div class="col-lg-3 col-12 centered-lg">
|
||
<p><a class="supportLink text-white" href="https://support.nlm.nih.gov/">Help</a><br />
|
||
<a href="https://www.nlm.nih.gov/accessibility.html" class="text-white">Accessibility</a><br />
|
||
<a href="https://www.nlm.nih.gov/careers/careers.html" class="text-white">Careers</a></p>
|
||
</div>
|
||
</div>
|
||
<div class="row">
|
||
<div class="col-lg-12 centered-lg">
|
||
<nav class="bottom-links">
|
||
<ul class="mt-3">
|
||
<li>
|
||
<a class="text-white" href="//www.nlm.nih.gov/">NLM</a>
|
||
</li>
|
||
<li>
|
||
<a class="text-white" href="https://www.nih.gov/">NIH</a>
|
||
</li>
|
||
<li>
|
||
<a class="text-white" href="https://www.hhs.gov/">HHS</a>
|
||
</li>
|
||
<li>
|
||
<a class="text-white" href="https://www.usa.gov/">USA.gov</a>
|
||
</li>
|
||
</ul>
|
||
</nav>
|
||
</div>
|
||
</div>
|
||
</div>
|
||
</section>
|
||
<script type="text/javascript" src="/portal/portal3rc.fcgi/rlib/js/InstrumentOmnitureBaseJS/InstrumentNCBIConfigJS/InstrumentNCBIBaseJS/InstrumentPageStarterJS.js?v=1"> </script>
|
||
<script type="text/javascript" src="/portal/portal3rc.fcgi/static/js/hfjs2.js"> </script>
|
||
</div>
|
||
</div>
|
||
</div>
|
||
<!--/.page-->
|
||
</div>
|
||
<!--/.wrap-->
|
||
</div><!-- /.twelve_col -->
|
||
</div>
|
||
<!-- /.grid -->
|
||
|
||
<span class="PAFAppResources"></span>
|
||
|
||
<!-- BESelector tab -->
|
||
|
||
|
||
|
||
<noscript><img alt="statistics" src="/stat?jsdisabled=true&ncbi_db=books&ncbi_pdid=book-part&ncbi_acc=NBK98919&ncbi_domain=mlprobe&ncbi_report=record&ncbi_type=fulltext&ncbi_objectid=&ncbi_pcid=/NBK98919/&ncbi_pagename=Screen for RAS-Selective Lethal Compounds and VDAC Ligands - Probe 2 - Probe Reports from the NIH Molecular Libraries Program - NCBI Bookshelf&ncbi_bookparttype=chapter&ncbi_app=bookshelf" /></noscript>
|
||
|
||
|
||
<!-- usually for JS scripts at page bottom -->
|
||
<!--<component id="PageFixtures" label="styles"></component>-->
|
||
|
||
|
||
<!-- CE8B5AF87C7FFCB1_0191SID /projects/books/PBooks@9.11 portal105 v4.1.r689238 Tue, Oct 22 2024 16:10:51 -->
|
||
<span id="portal-csrf-token" style="display:none" data-token="CE8B5AF87C7FFCB1_0191SID"></span>
|
||
|
||
<script type="text/javascript" src="//static.pubmed.gov/portal/portal3rc.fcgi/4216699/js/3879255/4121861/3501987/4008961/3893018/3821238/4062932/4209313/4212053/4076480/3921943/3400083/3426610.js" snapshot="books"></script></body>
|
||
</html> |