484 lines
No EOL
114 KiB
HTML
484 lines
No EOL
114 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="NBK259186" /><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="/NBK259186/" /><meta name="ncbi_pagename" content="Discovery of ML323 as a Novel Inhibitor of the USP1/UAF1 Deubiquitinase Complex - 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>Discovery of ML323 as a Novel Inhibitor of the USP1/UAF1 Deubiquitinase Complex - 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="Discovery of ML323 as a Novel Inhibitor of the USP1/UAF1 Deubiquitinase Complex" /><meta name="citation_publisher" content="National Center for Biotechnology Information (US)" /><meta name="citation_date" content="2014/09/18" /><meta name="citation_author" content="Thomas S. Dexheimer" /><meta name="citation_author" content="Andrew S. Rosenthal" /><meta name="citation_author" content="Qin Liang" /><meta name="citation_author" content="Junjun Chen" /><meta name="citation_author" content="Mark A. Villamil" /><meta name="citation_author" content="Edward H. Kerns" /><meta name="citation_author" content="Anton Simeonov" /><meta name="citation_author" content="Ajit Jadhav" /><meta name="citation_author" content="Zhihao Zhuang" /><meta name="citation_author" content="David J. Maloney" /><meta name="citation_pmid" content="25506968" /><meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK259186/" /><link rel="schema.DC" href="http://purl.org/DC/elements/1.0/" /><meta name="DC.Title" content="Discovery of ML323 as a Novel Inhibitor of the USP1/UAF1 Deubiquitinase Complex" /><meta name="DC.Type" content="Text" /><meta name="DC.Publisher" content="National Center for Biotechnology Information (US)" /><meta name="DC.Contributor" content="Thomas S. Dexheimer" /><meta name="DC.Contributor" content="Andrew S. Rosenthal" /><meta name="DC.Contributor" content="Qin Liang" /><meta name="DC.Contributor" content="Junjun Chen" /><meta name="DC.Contributor" content="Mark A. Villamil" /><meta name="DC.Contributor" content="Edward H. Kerns" /><meta name="DC.Contributor" content="Anton Simeonov" /><meta name="DC.Contributor" content="Ajit Jadhav" /><meta name="DC.Contributor" content="Zhihao Zhuang" /><meta name="DC.Contributor" content="David J. Maloney" /><meta name="DC.Date" content="2014/09/18" /><meta name="DC.Identifier" content="https://www.ncbi.nlm.nih.gov/books/NBK259186/" /><meta name="description" content="Advances in pharmacological approaches to target the ubiquitin–proteasome system have revealed several potential new molecular targets within the ubiquitin machinery. Likewise, the functional consequences of ubiquitination and deubiquitination have recently been linked to a wide variety of critical biological processes well beyond just protein disposal [1]. For example, the deubiquitining enzyme, ubiquitin-specific protease 1 (USP1), in association with its WD40 binding partner, UAF1 (USP1-associated factor 1), is a known regulator of DNA damage response and has been suggested as a promising target to improve the efficacy of the commonly used DNA damaging drugs by modulating the cancer cells' ability to repair or tolerate DNA lesions [2-7]. To further evaluate the therapeutic potential of targeting the USP1/UAF1 deubiquitinase complex, we conducted a quantitative high-throughput screen and a subsequent medicinal chemistry optimization campaign in pursuit of small molecules that inhibit USP1/UAF1. Herein, we describe the discovery and optimization of ML323, a probe molecule that displays reversible, nanomolar inhibitory activity and excellent selectivity toward USP1/UAF1. In addition, ML323 potentiates the cytotoxicity of cisplatin and increases endogenous monoubiquitination levels of both PCNA and FANCD2, two known cellular targets of USP1/UAF1 [8]. Lastly, ML323 possesses a promising in vitro ADME profile, suggesting its suitability for further testing in PK/PD studies." /><meta name="og:title" content="Discovery of ML323 as a Novel Inhibitor of the USP1/UAF1 Deubiquitinase Complex" /><meta name="og:type" content="book" /><meta name="og:description" content="Advances in pharmacological approaches to target the ubiquitin–proteasome system have revealed several potential new molecular targets within the ubiquitin machinery. Likewise, the functional consequences of ubiquitination and deubiquitination have recently been linked to a wide variety of critical biological processes well beyond just protein disposal [1]. For example, the deubiquitining enzyme, ubiquitin-specific protease 1 (USP1), in association with its WD40 binding partner, UAF1 (USP1-associated factor 1), is a known regulator of DNA damage response and has been suggested as a promising target to improve the efficacy of the commonly used DNA damaging drugs by modulating the cancer cells' ability to repair or tolerate DNA lesions [2-7]. To further evaluate the therapeutic potential of targeting the USP1/UAF1 deubiquitinase complex, we conducted a quantitative high-throughput screen and a subsequent medicinal chemistry optimization campaign in pursuit of small molecules that inhibit USP1/UAF1. Herein, we describe the discovery and optimization of ML323, a probe molecule that displays reversible, nanomolar inhibitory activity and excellent selectivity toward USP1/UAF1. In addition, ML323 potentiates the cytotoxicity of cisplatin and increases endogenous monoubiquitination levels of both PCNA and FANCD2, two known cellular targets of USP1/UAF1 [8]. Lastly, ML323 possesses a promising in vitro ADME profile, suggesting its suitability for further testing in PK/PD studies." /><meta name="og:url" content="https://www.ncbi.nlm.nih.gov/books/NBK259186/" /><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/ml323/" /><link rel="canonical" href="https://www.ncbi.nlm.nih.gov/books/NBK259186/" /><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="/core/mathjax/2.7.9/MathJax.js?config=/corehtml/pmc/js/mathjax-config-classic.3.4.js"></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="CE8D2BBB7D65402100000000010600E7.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="#__NBK259186_dtls__">Show details</a><div style="display:none" class="ui-widget" id="__NBK259186_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/ml328/" title="Previous page in this title">< Prev</a><a class="active page_link next" href="/books/n/mlprobe/ml327/" 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="_NBK259186_"><span class="title" itemprop="name">Discovery of ML323 as a Novel Inhibitor of the USP1/UAF1 Deubiquitinase Complex</span></h1><p class="contrib-group"><span itemprop="author">Thomas S. Dexheimer</span>, <span itemprop="author">Andrew S. Rosenthal</span>, <span itemprop="author">Qin Liang</span>, <span itemprop="author">Junjun Chen</span>, <span itemprop="author">Mark A. Villamil</span>, <span itemprop="author">Edward H. Kerns</span>, <span itemprop="author">Anton Simeonov</span>, <span itemprop="author">Ajit Jadhav</span>, <span itemprop="author">Zhihao Zhuang</span>, and <span itemprop="author">David J. Maloney</span>.</p><a data-jig="ncbitoggler" href="#__NBK259186_ai__" style="border:0;text-decoration:none">Author Information and Affiliations</a><div style="display:none" class="ui-widget" id="__NBK259186_ai__"><p class="contrib-group"><h4>Authors</h4><span itemprop="author">Thomas S. Dexheimer</span>,<sup>1</sup><sup>,<a href="#ml323.fn1" class="bk_pop">#</a></sup> <span itemprop="author">Andrew S. Rosenthal</span>,<sup>1</sup><sup>,<a href="#ml323.fn1" class="bk_pop">#</a></sup> <span itemprop="author">Qin Liang</span>,<sup>2</sup><sup>,<a href="#ml323.fn1" class="bk_pop">#</a></sup> <span itemprop="author">Junjun Chen</span>,<sup>2</sup> <span itemprop="author">Mark A. Villamil</span>,<sup>2</sup> <span itemprop="author">Edward H. Kerns</span>,<sup>1</sup> <span itemprop="author">Anton Simeonov</span>,<sup>1</sup> <span itemprop="author">Ajit Jadhav</span>,<sup>1</sup> <span itemprop="author">Zhihao Zhuang</span>,<sup>2</sup> and <span itemprop="author">David J. Maloney</span><sup>1</sup><sup>,*</sup>.</p><h4>Affiliations</h4><div class="affiliation"><sup>1</sup>
|
||
NIH Chemical Genomics Center, National Center for Advancing Translational Sciences, 9800 Medical Center Drive, Rockville, MD, 20850</div><div class="affiliation"><sup>2</sup>
|
||
Department of Chemistry and Biochemistry, 214A Drake Hall, University of Delaware, Newark, DE, 19716, USA</div><div class="affiliation">
|
||
<sup>*</sup> To whom correspondence should be addressed: Email:
|
||
<a href="mailto:dev@null" data-email="vog.hin.liam@dyenolam" class="oemail">vog.hin.liam@dyenolam</a></div></div><p class="small">Received: <span itemprop="datePublished">October 23, 2012</span>; Last Update: <span itemprop="dateModified">September 18, 2014</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>Advances in pharmacological approaches to target the ubiquitin–proteasome system have revealed several potential new molecular targets within the ubiquitin machinery. Likewise, the functional consequences of ubiquitination and deubiquitination have recently been linked to a wide variety of critical biological processes well beyond just protein disposal [<a class="bk_pop" href="#ml323.r1">1</a>]. For example, the deubiquitining enzyme, ubiquitin-specific protease 1 (USP1), in association with its WD40 binding partner, UAF1 (USP1-associated factor 1), is a known regulator of DNA damage response and has been suggested as a promising target to improve the efficacy of the commonly used DNA damaging drugs by modulating the cancer cells' ability to repair or tolerate DNA lesions [<a class="bk_pop" href="#ml323.r2">2</a>-<a class="bk_pop" href="#ml323.r7">7</a>]. To further evaluate the therapeutic potential of targeting the USP1/UAF1 deubiquitinase complex, we conducted a quantitative high-throughput screen and a subsequent medicinal chemistry optimization campaign in pursuit of small molecules that inhibit USP1/UAF1. Herein, we describe the discovery and optimization of <a href="/pcsubstance/?term=ML323[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML323</a>, a probe molecule that displays reversible, nanomolar inhibitory activity and excellent selectivity toward USP1/UAF1. In addition, <a href="/pcsubstance/?term=ML323[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML323</a> potentiates the cytotoxicity of cisplatin and increases endogenous monoubiquitination levels of both PCNA and FANCD2, two known cellular targets of USP1/UAF1 [<a class="bk_pop" href="#ml323.r8">8</a>]. Lastly, <a href="/pcsubstance/?term=ML323[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML323</a> possesses a promising <i>in vitro</i> ADME profile, suggesting its suitability for further testing in PK/PD studies.</p></div><div class="h2"></div><p><b>Assigned Assay Grant #:</b> R03 DA030552</p><p><b>Screening Center Name & PI:</b> NIH Chemical Genomics Center, Christopher P. Austin</p><p><b>Chemistry Center Name & PI:</b> NIH Chemical Genomics Center, Christopher P. Austin</p><p><b>Assay Submitter & Institution:</b> Zhihao Zhuang, Department of Chemistry and Biochemistry, University of Delaware</p><p><b>PubChem Summary Bioassay Identifier (AID):</b>
|
||
<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504878" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">504878</a></p><div id="ml323.s1"><h2 id="_ml323_s1_">Probe Structure & Characteristics</h2><div id="ml323.f1" class="figure"><div class="graphic"><img src="/books/NBK259186/bin/ml323f1.jpg" alt="Image ml323f1" /></div></div><div id="ml323.t1" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK259186/table/ml323.t1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml323.t1_lrgtbl__"><table><thead><tr><th id="hd_h_ml323.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">CID/ML#</th><th id="hd_h_ml323.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Target Name</th><th id="hd_h_ml323.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">IC50/EC50 (nM) [SID, AID]</th><th id="hd_h_ml323.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Anti-target Name(s)</th><th id="hd_h_ml323.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">IC50/EC50 (mM) [SID, AID]</th><th id="hd_h_ml323.t1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Fold Selective</th><th id="hd_h_ml323.t1_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Secondary Assay(s) Name: IC50/EC50 (nM) [SID, AID]</th></tr></thead><tbody><tr><td headers="hd_h_ml323.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">CID 60167849/<a href="/pcsubstance/?term=ML323[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML323</a></td><td headers="hd_h_ml323.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">USP1/UAF1</td><td headers="hd_h_ml323.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">76 nM (Ub-Rho) [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/44116952" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 44116952</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651605" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651605</a>]</td><td headers="hd_h_ml323.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">USP2<br /><br />USP5<br /><br />USP7<br /><br />USP8<br /><br />USP46/UAF1</td><td headers="hd_h_ml323.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">>114 mM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/144116952" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 144116952</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651605" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651626</a>];<br />>114 mM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/144116952" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 144116952</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651605" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651625</a>];<br />>114 mM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/144116952" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 144116952</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651605" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651624</a>];<br />>114 mM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/144116952" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 144116952</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651605" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651623</a>];<br />>114 mM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/144116952" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 144116952</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651605" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651622</a>];</td><td headers="hd_h_ml323.t1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">>1500<br /><br />>1500<br /><br />>1500<br /><br />>1500<br /><br />>1500</td><td headers="hd_h_ml323.t1_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">174 nM (Di-Ub) [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/144116952" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 144116952</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651621" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651621</a>]</td></tr></tbody></table></div></div></div><div id="ml323.s2"><h2 id="_ml323_s2_">1. Recommendations for Scientific Use of the Probe</h2><p><a href="/pcsubstance/?term=ML323[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML323</a>, with its ability to potently and selectively inhibit USP1/UAF1 both <i>in vitro</i> and in a cellular context, enables biologists the evaluation of the USP1/UAF1 deubiquitinase complex as a target for therapeutic development. Ubiquitin-specific Protease 1 (USP1) is a deubiquitinating (DUB) enzyme implicated in DNA damage response through modulation of the ubiquitination levels of key proteins, PCNA and FANCD2, which play important roles in translesion synthesis and Fanconi anemia pathways, respectively [<a class="bk_pop" href="#ml323.r8">8</a>]. Subsequent secondary and tertiary assay results also suggest that <a href="/pcsubstance/?term=ML323[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML323</a> inhibitor may be used in combination with known DNA damaging agents, such as cisplatin, to potentiate the cytotoxicity [<a class="bk_pop" href="#ml323.r9">9</a>]. In addition, <a href="/pcsubstance/?term=ML323[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML323</a> addresses the limitations exhibited by the current probes. GW7647 and Pimozide, the first annotated USP1 inhibitors, display fairly promiscuous PubChem activity profile with a 9.9% and 12.0% hit rate respectively; while another compound C527 (Publication no. WO/2011/137320) also display limited selectivity across the deubiquitinase families (USP12/46, IC<sub>50</sub> = 5.97 µM; USP5, IC<sub>50</sub> = 1.65 µM; UCL-H3, IC<sub>50</sub> = 2.18 µM). Therefore, given its favorable biochemical and physicochemical attributes, <a href="/pcsubstance/?term=ML323[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML323</a> can be used by the research community to interrogate USP1 biology <i>in vivo</i>.</p></div><div id="ml323.s3"><h2 id="_ml323_s3_">2. Materials and Methods</h2><p><b>General Methods for Chemistry</b>. All air or moisture sensitive reactions were performed under positive pressure of nitrogen with oven-dried glassware. Anhydrous solvents such as dichloromethane, <i>N,N</i>-dimethylforamide (DMF), acetonitrile, methanol and triethylamine were purchased from Sigma-Aldrich. Preparative purification was performed on a Waters semi-preparative HPLC system. The column used was a Phenomenex Luna C18 (5 micron, 30 × 75 mm) at a flow rate of 45 mL/min. The mobile phase consisted of acetonitrile and water (each containing 0.1% trifluoroacetic acid). A gradient of 10% to 50% acetonitrile over 8 minutes was used during the purification. Fraction collection was triggered by UV detection (220 nm). Analytical analysis was performed on an Agilent LC/MS (Agilent Technologies, Santa Clara, CA). Method 1: A 7 minute gradient of 4% to 100% Acetonitrile (containing 0.025% trifluoroacetic acid) in water (containing 0.05% trifluoroacetic acid) was used with an 8 minute run time at a flow rate of 1 mL/min. A Phenomenex Luna C18 column (3 micron, 3 × 75 mm) was used at a temperature of 50 °C. Method 2: A 3 minute gradient of 4% to 100% Acetonitrile (containing 0.025% trifluoroacetic acid) in water (containing 0.05% trifluoroacetic acid) was used with a 4.5 minute run time at a flow rate of 1 mL/min. A Phenomenex Gemini Phenyl column (3 micron, 3 × 100 mm) was used at a temperature of 50° C. Purity determination was performed using an Agilent Diode Array Detector for both Method 1 and Method 2. Mass determination was performed using an Agilent 6130 mass spectrometer with electrospray ionization in the positive mode. <sup>1</sup>H NMR spectra were recorded on Varian 400 MHz spectrometers. Chemical shifts are reported in ppm with undeuterated solvent (DMSO-d<sub>6</sub> at 2.49 ppm) as internal standard for DMSO-d<sub>6</sub> solutions. All of the analogs tested in the biological assays have purity greater than 95%, based on both analytical methods. High resolution mass spectrometry was recorded on Agilent 6210 Time-of-Flight LC/MS system. Confirmation of molecular formula was accomplished using electrospray ionization in the positive mode with the Agilent Masshunter software (version B.02).</p><p><b>Cell lines and culture conditions.</b> The human non-small cell lung cancer (NSCLC) H596 was cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) at 37 °C and 5% CO<sub>2</sub>. The human embryonic kidney 293T (HEK293T), human osteosarcoma U2OS, and human colon cancer HCT116 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS at 37 °C and 5% CO<sub>2</sub>. All culture mediums contain 100 units/mL penicillin and 0.1 mg/mL streptomycin as antibiotics.</p><p><b>Reagents and antibodies sources.</b> Ubiquitin-rhodamine110, K63-linked diubiquitin, full length USP7 (HAUSP), USP5 (Isopeptidase T), USP8, and the catalytic domain of USP2 (a. a. 259 – 605) were purchased from Boston Biochem. USP1/UAF1 and USP46/UAF1 complexes were generated as previously described [<a class="bk_pop" href="#ml323.r9">9</a>]. Both human anti-PCNA and anti-FANCD2 antibodies were from Santa Cruz. USP1 antibody was from Abcam. HA-tag and HRP-conjugated anti-mouse antibodies were from Sigma-Aldrich. HRP-conjugated anti-rabbit secondary antibody was from Bio-Rad.</p><div id="ml323.s4"><h3>2.1. Assays</h3><p><b>USP1/UAF1 qHTS assay (BioAssay <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651605" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651605</a>).</b> USP1/UAF1 activity was monitored in a fluorometric assay using ubiquitin-rhodamine110 (<a class="figpopup" href="/books/NBK259186/table/ml323.t2/?report=objectonly" target="object" rid-figpopup="figml323t2" rid-ob="figobml323t2">Table 1</a>). Enzymatic reactions were conducted in an assay buffer composed of 50 mM HEPES, pH 7.8, 0.5 mM EDTA, 100 mM NaCl, 1 mM TCEP, 0.1 mg/mL BSA, and 0.01% Tween-20 that contained 1 nM USP1/UAF1. Each individual compound was tested at five concentrations in the range of 0.46 to 115 µM. The plates were incubated for 15 min to attain equilibrium, and then the enzymatic reaction was initiated by dispensing 150 nM Ub-Rho solution. The plates were directly transferred to a ViewLux high-throughput CCD imager (PerkinElmer), where kinetic measurements of rhodamine fluorescence were acquired using a 480 nm excitation/540 nm emission filter set. The change in fluorescence intensity over a 5-minute reaction period (typically associated with less than 10% substrate conversion) was normalized against no-inhibitor and no-enzyme controls and the resulting percent inhibition data were fitted to a sigmoidal dose response using a four-parameter Hill equation. All screening operations were performed on a fully integrated robotic system (Kalypsys Inc., San Diego, CA) as described elsewhere. Plates containing DMSO only were included approximately every 50 plates throughout the screen to monitor any systematic trend in the assay signal associated with reagent dispenser variation or decrease in enzyme specific activity.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml323t2"><a href="/books/NBK259186/table/ml323.t2/?report=objectonly" target="object" title="Table 1" class="img_link icnblk_img figpopup" rid-figpopup="figml323t2" rid-ob="figobml323t2"><img class="small-thumb" src="/books/NBK259186/table/ml323.t2/?report=thumb" src-large="/books/NBK259186/table/ml323.t2/?report=previmg" alt="Table 1. USP1/UAF1 qHTS protocol." /></a><div class="icnblk_cntnt"><h4 id="ml323.t2"><a href="/books/NBK259186/table/ml323.t2/?report=objectonly" target="object" rid-ob="figobml323t2">Table 1</a></h4><p class="float-caption no_bottom_margin">USP1/UAF1 qHTS protocol. </p></div></div><p><b>K63-linked diubiquitin gel assay (BioAssay <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651621" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651621</a> [USP1/UAF1], <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651622" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651622</a> [USP46/UAF1], <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651623" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651623</a> [USP8], <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651624" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651624</a> [USP7], <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651625" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651625</a> [USP5], and <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651626" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651626</a> [USP]).</b> Inhibitors at varied concentrations were added to the assay containing USP at appropriate concentration (150 nM USP1/UAF1, 30 nM USP2, 15 nM USP5, 7.5 nM USP7, 255 nM USP8, and 600 nM USP46/UAF1) and 3 µM K63-linked diubiquitin in a buffer containing 50 mM HEPES pH 7.8, 0.1 mg/mL BSA, 0.5 mM EDTA and 1 mM DTT. After 1 hour at 37 °C, the reaction was quenched by the addition of Laemmli sample buffer. The reaction products were then separated on a 20% denaturing SDS-PAGE gel and stained with Coomassie Blue. The intensity of the individual diubiquitin and monoubiquitin bands were quantified using Quantity One software (Bio-Rad). The enzyme activity was normalized against no-inhibitor control and plotted against inhibitor concentration. The IC<sub>50</sub> value was determined by fitting the dose response curve to the equation below using GraphPad Prism (GraphPad Software), where <i>X</i> = Log[inhibitor]; <i>Y</i> is the enzyme activity in percent relative to control.</p><div class="pmc_disp_formula whole_rhythm clearfix" id="ml323.eq1"><div class="inline_block pmc_inline_block pmc_va_middle pmc_hide_overflow twelve_col">
|
||
<math id="ml323.m1" display="block"><mrow><mi>Y</mi><mo>=</mo><msub><mi>Y</mi><mn>0</mn></msub><mo>+</mo><mfrac><mrow><msub><mi>Y</mi><mtext mathvariant="italic">max</mtext></msub><mo>−</mo><msub><mi>Y</mi><mn>0</mn></msub></mrow><mrow><mn>1</mn><mo>+</mo><msup><mrow><mn>10</mn></mrow><mrow><mi>X</mi><mo>−</mo><mtext mathvariant="italic">Log</mtext><mspace width="0.2em"></mspace><mi>I</mi><msub><mi>C</mi><mrow><mn>50</mn></mrow></msub></mrow></msup></mrow></mfrac></mrow></math></div><div class="inline_block pmc_inline_block pmc_va_middle pmc_hide_overflow last bk_equ_label "><div><span class="nowrap"></span></div></div></div><p><b>Ki determination using diubiquitin gel assay.</b> The inhibition assay solution contained 150 nM USP1/UAF1, 7 – 80 µM K63-linked diubiquitin, and <a href="/pcsubstance/?term=ML323[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML323</a> at varied concentrations in a buffer containing 50 mM HEPES, pH 7.8, 0.1 mg/mL BSA, 0.5 mM EDTA, and 1 mM DTT. The reaction was incubated for 10 – 90 minutes at 37 °C, and then quenched by the addition of Laemmli sample buffer at the given time point. The reaction products were separated on a 20% denaturing SDS-PAGE gel and stained with Coomassie Blue. The intensity of the individual diubiquitin and monoubiquitin bands were quantified using Quantity One 4.3.1 (Biorad, Hercules, CA). The percentage of the conversion was determined and used to calculate the reaction rate. The Lineweaver-Burk plot was obtained by plotting 1/v against 1/[di-Ub] at four different inhibitor concentrations. For noncompetitive inhibition, Ki was determined by fitting the data to the equations shown below using Origin 8 (OriginLab Corp., Northampton, MA).</p><div class="pmc_disp_formula whole_rhythm clearfix" id="ml323.eq2"><div class="inline_block pmc_inline_block pmc_va_middle pmc_hide_overflow twelve_col">
|
||
<math id="ml323.m2" display="block"><mrow><mtable><mtr><mtd><mrow><mfrac><mn>1</mn><mi>υ</mi></mfrac><mo>=</mo><mrow><mo>[</mo><mrow><mfrac><mrow><msub><mi>K</mi><mi>m</mi></msub></mrow><mrow><msub><mi>V</mi><mtext mathvariant="italic">max</mtext></msub></mrow></mfrac><mrow><mo>(</mo><mrow><mfrac><mn>1</mn><mrow><mo>[</mo><mi>S</mi><mo>]</mo></mrow></mfrac></mrow><mo>)</mo></mrow><mo>+</mo><mfrac><mn>1</mn><mrow><msub><mi>V</mi><mtext mathvariant="italic">max</mtext></msub></mrow></mfrac></mrow><mo>]</mo></mrow><mspace width="0.2em"></mspace><mrow><mo>(</mo><mrow><mn>1</mn><mo>+</mo><mfrac><mrow><mo stretchy="false">[</mo><mi>I</mi><mo stretchy="false">]</mo></mrow><mrow><msub><mi>K</mi><mi>i</mi></msub></mrow></mfrac></mrow><mo>)</mo></mrow></mrow></mtd></mtr><mtr><mtd><mrow><mtext>Slope</mtext><mo>=</mo><mfrac><mrow><msub><mi>K</mi><mi>m</mi></msub></mrow><mrow><msub><mi>V</mi><mtext mathvariant="italic">max</mtext></msub></mrow></mfrac><mspace width="0.2em"></mspace><mrow><mo>(</mo><mrow><mn>1</mn><mo>+</mo><mfrac><mrow><mo stretchy="false">[</mo><mi>I</mi><mo stretchy="false">]</mo></mrow><mrow><msub><mi>K</mi><mi>i</mi></msub></mrow></mfrac></mrow><mo>)</mo></mrow></mrow></mtd></mtr><mtr><mtd><mrow><mtext>Intercept</mtext><mo>=</mo><mfrac><mn>1</mn><mrow><msub><mi>V</mi><mtext mathvariant="italic">max</mtext></msub></mrow></mfrac><mspace width="0.2em"></mspace><mrow><mo>(</mo><mrow><mn>1</mn><mo>+</mo><mfrac><mrow><mo stretchy="false">[</mo><mi>I</mi><mo stretchy="false">]</mo></mrow><mrow><msub><mi>K</mi><mi>i</mi></msub></mrow></mfrac></mrow><mo>)</mo></mrow></mrow></mtd></mtr></mtable></mrow></math></div><div class="inline_block pmc_inline_block pmc_va_middle pmc_hide_overflow last bk_equ_label "><div><span class="nowrap"></span></div></div></div><p><b>Reversibility assay (BioAssay AIDxxx).</b> The compound <a href="/pcsubstance/?term=ML323[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML323</a> at a concentration of ten times its IC<sub>50</sub> value was pre-incubated with 10 µM USP1/UAF1 at room temperature for 15 min. This solution was then diluted by 100-fold in assay buffer containing 50 mM HEPES, pH 7.8, 0.1 mg/mL BSA, 0.5 mM EDTA, and 1 mM DTT and incubated at room temperature for an additional 15 min. Next, 20 µM K63-linked diubiquitin was added into the solution and incubated at 37 °C for 10 min. Laemmli sample buffer was added to quench the reaction. The reaction products were then separated on a 20% denaturing SDS-PAGE gel and stained with Coomassie Blue. USP1/UAF1 incubated with DMSO was treated as 100% USP1/UAF1 activity. Rottlerin was utilized as an irreversible control inhibitor [<a class="bk_pop" href="#ml323.r9">9</a>].</p><p><b>Inhibition of the cellular activity of USP1/UAF1.</b> HEK293T or H596 cells were plated in 10 cm dishes and synchronized in S-phase with the double-thymidine block method as described previously [<a class="bk_pop" href="#ml323.r9">9</a>]. Then cells were treated with 100 µM cisplatin, 30 µM <a href="/pcsubstance/?term=ML323[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML323</a>, or 100 µM cisplatin plus 30 µM <a href="/pcsubstance/?term=ML323[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML323</a>. Negative control cells were treated with an equal volume of DMSO and saline (0.9% NaCl). After 6 hr at 37 °C, cells were harvested and lysed. Cell extracts were separated on SDS-PAGE and transferred to nitrocellulose membrane. The membranes were then blotted with PCNA antibody or FANCD2 antibody, followed by incubation with HRP-conjugated anti-mouse secondary antibody. Images were quantified in FluorChem Q software (Imgen technologies). Alternatively, HCT116 cells were plated in 6-well plates and allowed to adhere overnight. Cells were treated with the indicated concentrations of <a href="/pcsubstance/?term=ML323[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML323</a> and were incubated for an additional 6 hr. Western blot analysis using anti-PCNA antibody was performed as described above.</p><p><b>Cytoxicity and clonogenicity assays.</b> For cytotoxicity assays, 5 × 10<sup>3</sup> H596 cells were seeded in each well of the 96-well plate and grown for 24 hr in humidified incubator. Cells were initially treated with cisplatin or <a href="/pcsubstance/?term=ML323[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML323</a> individually for 48 hr to determine the EC<sub>50</sub> of each compound. In the combination assay, cisplatin was dissolved in saline solution and <a href="/pcsubstance/?term=ML323[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML323</a> was dissolved in DMSO. Equal volume of above solutions was added to each well and cells were incubated for 48 hr. Cells treated with equal volume of DMSO and saline were used as control and designated as 100% viability. The cell viability was measured by CCK-8 solution (Dojindo Molecular Technologies, Inc) as previously described [<a class="bk_pop" href="#ml323.r9">9</a>]. For the colony formation assay, H596 cells were seeded at the density of 200 cells per well in 6-well plates and grown overnight in humidified incubator. The medium was replaced with growth medium containing cisplatin and <a href="/pcsubstance/?term=ML323[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML323</a> at different concentrations individually or in combination. After 48 hr treatment, the medium was replaced with fresh growth medium and the plates were incubated for an additional 7-8 days to allow for colony formation. Cells were then fixed with methanol and stained with 0.1% crystal violet (Sigma-Aldrich). The number of colonies consisting of 50 or more cells was scored. Cells treated with equal volume of DMSO and saline were used as a control and designated as 100%. Colony numbers were determined from triplicate plates. Dose-response curves for cytotoxicity and colony formation assays were generated by using GraphPad Prism and analyzed using CalcuSyn (Biosoft) to calculate the combination index (CI), which was determined for the fraction of cells affected [<a class="bk_pop" href="#ml323.r10">10</a>] following addition of the fixed ratios of cisplatin and <a href="/pcsubstance/?term=ML323[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML323</a>.</p></div><div id="ml323.s5"><h3>2.2. Probe Chemical Characterization</h3><div id="ml323.f2" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Image%20ml323f2&p=BOOKS&id=259186_ml323f2.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img src="/books/NBK259186/bin/ml323f2.jpg" alt="Image ml323f2" class="tileshop" title="Click on image to zoom" /></a></div><div class="caption"><p>*Purity >98% as determined by LC/MS and <sup>1</sup>H NMR analyses</p></div></div><p><b><i>N</i>-(4-(1H-1,2,3-triazol-1-yl)benzyl)-5-methyl-2-(2-isopropylphenyl)pyrimidin-4-amine (NCGC00262306-01):</b> LC-MS Retention Time: t<sub>1</sub> (Method 1) = 2.938 min and t<sub>2</sub> (Method 2) = 1.756 min; <sup>1</sup>H NMR (400 MHz, CDCl<sub>3</sub>) δ 8.82 (dd, <i>J</i> = 7.9, 3.4 Hz, 1H), 8.02 – 7.93 (m, 2H), 7.76 (d, <i>J</i> = 1.4 Hz, 1H), 7.64 – 7.56 (m, 2H), 7.51 – 7.33 (m, 5H), 7.25 – 7.17 (m, 1H), 4.88 (d, <i>J</i> = 5.9 Hz, 2H), 3.19 (p, <i>J</i> = 6.8 Hz, 1H), 2.22 (s, 3H), 1.10 (d, <i>J</i> = 1.2 Hz, 3H), 1.09 – 1.07 (m, 4H); <sup>13</sup>C NMR (100 MHz, CDCl<sub>3</sub>) δ 162.02, 160.18, 148.00, 140.63, 138.03, 136.14, 134.35, 134.28, 131.89, 130.34, 129.25, 129.00, 126.61, 126.04, 122.04, 121.99, 120.74, 113.81, 44.62, 29.43, 23.95, 13.71, 13.67; HRMS (ESI) <i>m/z</i> (M+H)<sup>+</sup> calcd. for C<sub>23</sub>H<sub>25</sub>N<sub>6</sub>, 385.2135; found 385.2146.</p><div id="ml323.f3" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%201.%20Chemical%20stability%20of%20ML323%20measured%20as%20percent%20composition%20of%20probe%20molecule%20in%20aqueous%20solution%20at%20room%20temperature%20in%20(A)%20DPBS%20pH%207.4%20buffer%3B%20(B)%20USP1%2FUAF1%20assay%20buffer%20at%20pH%207.8%20(50%20mM%20HEPES%2C%200.5%20mM%20EDTA%2C%20100%20mM%20NaCl%2C%201%20mM%20TCEP%2C%200.1%20mg%2FmL%20BSA%2C%20and%200.01%25%20Tween-20)%3B%20(C)%20USP1%2FUAF1%20assay%20buffer%20at%20pH%202%20and%20(D)%20at%20pH%209%20buffer.&p=BOOKS&id=259186_ml323f3.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img src="/books/NBK259186/bin/ml323f3.jpg" alt="Figure 1. Chemical stability of ML323 measured as percent composition of probe molecule in aqueous solution at room temperature in (A) DPBS pH 7.4 buffer; (B) USP1/UAF1 assay buffer at pH 7.8 (50 mM HEPES, 0.5 mM EDTA, 100 mM NaCl, 1 mM TCEP, 0.1 mg/mL BSA, and 0.01% Tween-20); (C) USP1/UAF1 assay buffer at pH 2 and (D) at pH 9 buffer." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 1</span><span class="title">Chemical stability of ML323 measured as percent composition of probe molecule in aqueous solution at room temperature in (A) DPBS pH 7.4 buffer; (B) USP1/UAF1 assay buffer at pH 7.8 (50 mM HEPES, 0.5 mM EDTA, 100 mM NaCl, 1 mM TCEP, 0.1 mg/mL BSA, and 0.01% Tween-20); (C) USP1/UAF1 assay buffer at pH 2 and (D) at pH 9 buffer</span></h3></div><div id="ml323.t3" class="table"><h3><span class="label">Table 2</span><span class="title">List of the USP1/UAF1 deubiquitinase complex inhibitor probe ML323 and related analogs and their corresponding identification numbers</span></h3><div class="caption"><p>Their corresponding molecular structures are shown in <a class="figpopup" href="/books/NBK259186/figure/ml323.f4/?report=objectonly" target="object" rid-figpopup="figml323f4" rid-ob="figobml323f4">Figure 2</a>.</p></div><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK259186/table/ml323.t3/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml323.t3_lrgtbl__"><table><thead><tr><th id="hd_h_ml323.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Internal ID</th><th id="hd_h_ml323.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS ID</th><th id="hd_h_ml323.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">SID</th><th id="hd_h_ml323.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CID</th><th id="hd_h_ml323.t3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">ML #</th><th id="hd_h_ml323.t3_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Type</th><th id="hd_h_ml323.t3_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Source</th></tr></thead><tbody><tr><td headers="hd_h_ml323.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>NCGC00262306</b></td><td headers="hd_h_ml323.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS004556019</td><td headers="hd_h_ml323.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/144116952" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">144116952</a></td><td headers="hd_h_ml323.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">60167849</td><td headers="hd_h_ml323.t3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="/pcsubstance/?term=ML323[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML323</a></td><td headers="hd_h_ml323.t3_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Probe</td><td headers="hd_h_ml323.t3_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC</td></tr><tr><td headers="hd_h_ml323.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>NCGC00262647</b></td><td headers="hd_h_ml323.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS004556020</td><td headers="hd_h_ml323.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/114116970" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">114116970</a></td><td headers="hd_h_ml323.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">60167812</td><td headers="hd_h_ml323.t3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml323.t3_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Analog</td><td headers="hd_h_ml323.t3_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC</td></tr><tr><td headers="hd_h_ml323.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>NCGC00253549</b></td><td headers="hd_h_ml323.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS004556023</td><td headers="hd_h_ml323.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/144116927" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">144116927</a></td><td headers="hd_h_ml323.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">60167883</td><td headers="hd_h_ml323.t3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml323.t3_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Analog</td><td headers="hd_h_ml323.t3_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC</td></tr><tr><td headers="hd_h_ml323.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>NCGC00253892</b></td><td headers="hd_h_ml323.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS004556022</td><td headers="hd_h_ml323.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/144116950" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">144116950</a></td><td headers="hd_h_ml323.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">60167929</td><td headers="hd_h_ml323.t3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml323.t3_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Analog</td><td headers="hd_h_ml323.t3_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC</td></tr><tr><td headers="hd_h_ml323.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>NCGC00012848</b></td><td headers="hd_h_ml323.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS004556021</td><td headers="hd_h_ml323.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/104223745" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">104223745</a></td><td headers="hd_h_ml323.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">3235425</td><td headers="hd_h_ml323.t3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml323.t3_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Analog</td><td headers="hd_h_ml323.t3_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC</td></tr><tr><td headers="hd_h_ml323.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>NCGC00253883</b></td><td headers="hd_h_ml323.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS004556024</td><td headers="hd_h_ml323.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/144116940" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">144116940</a></td><td headers="hd_h_ml323.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">60167905</td><td headers="hd_h_ml323.t3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml323.t3_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Analog</td><td headers="hd_h_ml323.t3_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC</td></tr></tbody></table></div></div><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml323f4" co-legend-rid="figlgndml323f4"><a href="/books/NBK259186/figure/ml323.f4/?report=objectonly" target="object" title="Figure 2" class="img_link icnblk_img figpopup" rid-figpopup="figml323f4" rid-ob="figobml323f4"><img class="small-thumb" src="/books/NBK259186/bin/ml323f4.gif" src-large="/books/NBK259186/bin/ml323f4.jpg" alt="Figure 2. Structures of ML323 and five other analogs that have been submitted to the MLSMR." /></a><div class="icnblk_cntnt" id="figlgndml323f4"><h4 id="ml323.f4"><a href="/books/NBK259186/figure/ml323.f4/?report=objectonly" target="object" rid-ob="figobml323f4">Figure 2</a></h4><p class="float-caption no_bottom_margin">Structures of ML323 and five other analogs that have been submitted to the MLSMR. </p></div></div></div><div id="ml323.s6"><h3>2.3. Probe Preparation</h3><p>Preparation of N-(4-(1H-1,2,3-triazol-1-yl)benzyl)-5-methyl-2-(2-isopropylphenyl)pyrimidin-4-amine (<a href="/pcsubstance/?term=ML323[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML323</a>) is a multi-step process illustrated in <a class="figpopup" href="/books/NBK259186/figure/ml323.f5/?report=objectonly" target="object" rid-figpopup="figml323f5" rid-ob="figobml323f5">Scheme 1</a> and summarized below.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml323f5" co-legend-rid="figlgndml323f5"><a href="/books/NBK259186/figure/ml323.f5/?report=objectonly" target="object" title="Scheme 1" class="img_link icnblk_img figpopup" rid-figpopup="figml323f5" rid-ob="figobml323f5"><img class="small-thumb" src="/books/NBK259186/bin/ml323f5.gif" src-large="/books/NBK259186/bin/ml323f5.jpg" alt="Scheme 1. Synthetic route to ML323." /></a><div class="icnblk_cntnt" id="figlgndml323f5"><h4 id="ml323.f5"><a href="/books/NBK259186/figure/ml323.f5/?report=objectonly" target="object" rid-ob="figobml323f5">Scheme 1</a></h4><p class="float-caption no_bottom_margin">Synthetic route to ML323. </p></div></div><ol class="upper-alpha"><li class="half_rhythm"><div>A solution of 2,4-dichloro-5-methylpyrimidine and 4-iodobenzylamine, HCl were dissolved in chloroform. Triethylamine was added and the sealed tube was heated at 60 ºC overnight. The reaction was purified directly by flash column chromatography (gradient elution with 0→100% ethyl acetate/hexane) to yield pure 2-chloro-5-methyl-N-(4-iodobenzyl)pyrimidin-4-amine.</div></li><li class="half_rhythm"><div>The requisite phenyl iodide was combined in a sealed tube with L-proline, sodium ascorbate, NaN<sub>3</sub>, Cu(II)SO<sub>4</sub>, K<sub>2</sub>CO<sub>3</sub> and propiolic acid in dimethyl sulfoxide (DMSO) and water. The mixture was heated at 65 ºC overnight. The resulting mixture was quenched with a saturated aqueous ammonium chloride (NH<sub>4</sub>Cl) solution and extracted with ethyl acetate. The organic layer was washed with brine, dried with magnesium sulfate (MgSO<sub>4</sub>), and concentrated <i>in vacuo</i>. The crude product was isolated by flash column chromatography (gradient elution with 0→100% ethyl acetate/hexane) to yield pure triazole.</div></li><li class="half_rhythm"><div>The methylpyrimidine triazole derivative was combined with 2-isopropylphenylboronic acid, Palladium-tetrakis triphenylphosphine (Pd(PPh<sub>3</sub>)<sub>4</sub>), and sodium carbonate in dimethylformamide (DMF). The reaction was heated at 150 ºC for 15 min in a Biotage Initiator<sup>®</sup> microwave reactor. The resulting mixture was filtered over Celite and purified by HPLC using gradient 20-100% acetonitrile with 0.1% trifluoroacetic acid (TFA) in water with 0.1% TFA to yield pure N-(4-(1H-1,2,3-triazol-1-yl)benzyl)-5-methyl-2-(2-isopropylphenyl)pyrimidin-4-amine (<a href="/pcsubstance/?term=ML323[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML323</a>) as a TFA salt after lyophilization.</div></li></ol></div></div><div id="ml323.s7"><h2 id="_ml323_s7_">3. Results</h2><div id="ml323.s8"><h3>3.1. Dose Response Curves for Probe</h3><div id="ml323.f6" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%203.%20Dose%20response%20activity%20of%20ML323%20(CID%2060167849)%20in%20(A)%20SDS-PAGE%20gel%20analysis%20of%20K63-linked%20diubiquitin%20cleavage%20by%20USP1%2FUAF1%20and%20(B)%20the%20Ub-rhodamine%20qHTS%20assay%20(green%20circles)%20and%20the%20K63-linked%20diubiquitin%20gel%20assay%20(yellow%20circles).&p=BOOKS&id=259186_ml323f6.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img src="/books/NBK259186/bin/ml323f6.jpg" alt="Figure 3. Dose response activity of ML323 (CID 60167849) in (A) SDS-PAGE gel analysis of K63-linked diubiquitin cleavage by USP1/UAF1 and (B) the Ub-rhodamine qHTS assay (green circles) and the K63-linked diubiquitin gel assay (yellow circles)." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 3</span><span class="title">Dose response activity of ML323 (CID 60167849) in (A) SDS-PAGE gel analysis of K63-linked diubiquitin cleavage by USP1/UAF1 and (B) the Ub-rhodamine qHTS assay (green circles) and the K63-linked diubiquitin gel assay (yellow circles)</span></h3><div class="caption"><p>Results showed dose-dependent inhibition of USP1/UAF1 activity by <a href="/pcsubstance/?term=ML323[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML323</a> in all three assays.</p></div></div></div><div id="ml323.s9"><h3>3.2. Cellular Activity</h3><div id="ml323.f7" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%204.%20Activity%20profile%20of%20the%20probe%20ML323%20showing%20treatment%20of%20human%20cells%20by%20USP1%2FUAF1%20inhibitors%20led%20to%20the%20increase%20in%20PCNA%20and%20FANCD2%20monoubiquitination.&p=BOOKS&id=259186_ml323f7.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img src="/books/NBK259186/bin/ml323f7.jpg" alt="Figure 4. Activity profile of the probe ML323 showing treatment of human cells by USP1/UAF1 inhibitors led to the increase in PCNA and FANCD2 monoubiquitination." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 4</span><span class="title">Activity profile of the probe ML323 showing treatment of human cells by USP1/UAF1 inhibitors led to the increase in PCNA and FANCD2 monoubiquitination</span></h3><div class="caption"><p>HEK293T (<b>A</b> and <b>B</b>) and H596 (<b>C</b> and <b>D</b>) cells were treated with 100 µM cisplatin (lane 2), 30 µM <a href="/pcsubstance/?term=ML323[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML323</a> (lane 4), or in combination (lane 3). Cell lysates were analyzed by Western blotting using anti-PCNA antibody (<b>A</b> and <b>C</b>) or anti-FANCD2 antibody (<b>B</b> and <b>D</b>). The percentages of monoubiquitinated PCNA or FANCD2 were reported below each blot. <b>E</b>) Dose-dependent increase in Ub-PCNA in HCT116 cells treated with <a href="/pcsubstance/?term=ML323[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML323</a>.</p></div></div><div id="ml323.f8" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%205.%20Cytotoxicity%20of%20cisplatin%20and%20ML323%20tested%20as%20a%20single%20agent%20or%20in%20combination%20on%20cisplatin-resistant%20NSCLC%20cells%20(H596).&p=BOOKS&id=259186_ml323f8.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img src="/books/NBK259186/bin/ml323f8.jpg" alt="Figure 5. Cytotoxicity of cisplatin and ML323 tested as a single agent or in combination on cisplatin-resistant NSCLC cells (H596)." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 5</span><span class="title">Cytotoxicity of cisplatin and ML323 tested as a single agent or in combination on cisplatin-resistant NSCLC cells (H596)</span></h3><div class="caption"><p>(<b>A</b>) Cytotoxicity of cisplatin alone (<i>green diamond</i>), <a href="/pcsubstance/?term=ML323[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML323</a> (<i>blue triangle</i>), or a combination of cisplatin and <a href="/pcsubstance/?term=ML323[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML323</a> at ratios of 1:1 (<i>red circle</i>), 1:4 (<i>black square</i>) in CCK assay and (<b>C</b>) colony formation assay. (<b>B</b>) Combination index analysis of the synergistic interaction of cisplatin and (D) <a href="/pcsubstance/?term=ML323[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML323</a> in CCK assay and colony formation assay. Ratios of 1:1 and 1:4 were shown as <i>black triangles</i> and <i>red circles</i>, respectively. The dashed horizontal line represents a combination index = 1. Results indicated the synergistic effect of the probe <a href="/pcsubstance/?term=ML323[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML323</a> with cisplatin against H596 cell line.</p></div></div><div id="ml323.f9" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%206.%20Evaluation%20of%20the%20probe%20ML323%20in%20SDS-PAGE%20gel%20assays%20of%20the%20cleavage%20of%20K63-linked%20diubiquitin%20by%20(A)%20USP2%2C%20(B)%20USP5%2C%20(C)%20USP7%2C%20(D)%20USP8%2C%20and%20(E)%20USP46%2FUAF1%20in%20the%20presence%20of%20ML323.&p=BOOKS&id=259186_ml323f9.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img src="/books/NBK259186/bin/ml323f9.jpg" alt="Figure 6. Evaluation of the probe ML323 in SDS-PAGE gel assays of the cleavage of K63-linked diubiquitin by (A) USP2, (B) USP5, (C) USP7, (D) USP8, and (E) USP46/UAF1 in the presence of ML323." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 6</span><span class="title">Evaluation of the probe ML323 in SDS-PAGE gel assays of the cleavage of K63-linked diubiquitin by (<b>A</b>) USP2, (<b>B</b>) USP5, (<b>C</b>) USP7, (<b>D</b>) USP8, and (<b>E</b>) USP46/UAF1 in the presence of ML323.</span></h3><div class="caption"><p>Graphical representation (<b>F</b>) of the percent inhibition of the different USPs (A-E) by <a href="/pcsubstance/?term=ML323[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML323</a> showed inhibitory effect only against USP1/UAF1.</p></div></div></div><div id="ml323.s10"><h3>3.3. Profiling Assays</h3><div id="ml323.f10" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%207.%20Graphical%20representation%20of%20the%20kinase%20profiling%20study%20for%20ML323%20conducted%20by%20DiscoveRx%20(KINOMEscan%02122)%20on%20a%20panel%20of%20451%20kinases.&p=BOOKS&id=259186_ml323f10.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img src="/books/NBK259186/bin/ml323f10.jpg" alt="Figure 7. Graphical representation of the kinase profiling study for ML323 conducted by DiscoveRx (KINOMEscan™) on a panel of 451 kinases." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 7</span><span class="title">Graphical representation of the kinase profiling study for ML323 conducted by DiscoveRx (KINOMEscan™) on a panel of 451 kinases</span></h3><div class="caption"><p>The profiling study was done using the testing concentrations of 10 µM. See <a href="http://www.discoverx.com" ref="pagearea=body&targetsite=external&targetcat=link&targettype=uri">www.discoverx.com</a> for details on profiling assays.</p></div></div><div id="ml323.t4" class="table"><h3><span class="label">Table 3</span><span class="title">ADME profile for ML323</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK259186/table/ml323.t4/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml323.t4_lrgtbl__"><table class="no_margin"><thead><tr><th id="hd_h_ml323.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Compound</th><th id="hd_h_ml323.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">PBS buffer (pH 7.4) Solubility (µM)<sup><a class="bk_pop" href="#ml323.tfn1">a</a></sup></th><th id="hd_h_ml323.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Log D (pH 7.4)<sup><a class="bk_pop" href="#ml323.tfn2">b</a></sup></th><th id="hd_h_ml323.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Rat Liver Microsome Stability (T<sub>1/2</sub>)<sup><a class="bk_pop" href="#ml323.tfn3">c</a></sup></th><th id="hd_h_ml323.t4_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Caco-2 (A→B) P<sub>app</sub> (10<sup>-6</sup> cm/s)<sup><a class="bk_pop" href="#ml323.tfn4">d</a></sup></th><th id="hd_h_ml323.t4_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Caco-2 (B→A) P<sub>app</sub> (10<sup>-6</sup> cm/s)<sup><a class="bk_pop" href="#ml323.tfn4">d</a></sup></th><th id="hd_h_ml323.t4_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Efflux Ratio</th><th id="hd_h_ml323.t4_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">PBS buffer (pH 7.4) Stability (48h)<sup><a class="bk_pop" href="#ml323.tfn4">d</a></sup></th><th id="hd_h_ml323.t4_1_1_1_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Mouse Plasma Stability (2h)<sup><a class="bk_pop" href="#ml323.tfn4">d</a></sup></th></tr></thead><tbody><tr><td headers="hd_h_ml323.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="/pcsubstance/?term=ML323[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML323</a></td><td headers="hd_h_ml323.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>86</b></td><td headers="hd_h_ml323.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">1.97</td><td headers="hd_h_ml323.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>12 min</b></td><td headers="hd_h_ml323.t4_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>22.98</b></td><td headers="hd_h_ml323.t4_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>20.78</b></td><td headers="hd_h_ml323.t4_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>0.90</b></td><td headers="hd_h_ml323.t4_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>99%</b></td><td headers="hd_h_ml323.t4_1_1_1_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>99%</b></td></tr></tbody></table></div><div><div><dl class="temp-labeled-list small"><dt>a</dt><dd><div id="ml323.tfn1"><p class="no_margin">Study was conducted by Pharmaron Inc. and was determined by LC/MS/MS.</p></div></dd><dt>b</dt><dd><div id="ml323.tfn2"><p class="no_margin">Study was conducted by Analiza Inc. using the scaled-down shake flask lipophilicity method (see: <a href="http://www.analiza.com" ref="pagearea=body&targetsite=external&targetcat=link&targettype=uri">www<wbr style="display:inline-block"></wbr>.analiza.com</a>).</p></div></dd><dt>c</dt><dd><div id="ml323.tfn3"><p class="no_margin">The stability in the presence of NADPH was also assessed. The probe compound showed no degradation without NADPH present over a 1 hour period.</p></div></dd><dt>d</dt><dd><div id="ml323.tfn4"><p class="no_margin">Studies were conducted at Pharmaron Inc. Results showed that <a href="/pcsubstance/?term=ML323[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML323</a> has good kinetic solubility and bioavailability indicated by a favorable results in solubility, Log D, and Caco-2 permeability.</p></div></dd></dl></div></div></div></div></div><div id="ml323.s11"><h2 id="_ml323_s11_">4. Discussion</h2><div id="ml323.s12"><h3>4.1. Comparison to Existing Art and How the New Probe is an Improvement</h3><p>A minimal number of USP1 inhibitors have been described to date. The primary compounds arising from our pilot screening efforts constitute two known bioactive molecules GW7647 and Pimozide. Both compounds possess reasonable selectivity for USP1/UAF1 (<a class="figpopup" href="/books/NBK259186/table/ml323.t5/?report=objectonly" target="object" rid-figpopup="figml323t5" rid-ob="figobml323t5">Table 4</a>) and have been shown to inactivate USP1 as well as potentiate the effects of cisplatin in cells [<a class="bk_pop" href="#ml323.r9">9</a>]. GW7647 and Pimozide facilitated proof-of-principle studies for targeting USP1, but both are limited by potency and off-target pharmacology, particularly because these compounds have annotated activity against unrelated targets. The other, non-NCGC identified, small molecule inhibitor of USP1 listed in the Prior Art is C527, which was reported by D'Andrea <i>et al</i>. in a recent patent application (Publication No. WO/2011/137320). C527 sensitizes cells to both the crosslinking agent, mitomycin C, and the topoisomerase I inhibitor, camptothecin. Although it is more potent than the previously mentioned Prior Art, C527 also has reduced selectivity compared to <a href="/pcsubstance/?term=ML323[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML323</a>, showing low micromolar inhibition of related USPs (<a class="figpopup" href="/books/NBK259186/table/ml323.t5/?report=objectonly" target="object" rid-figpopup="figml323t5" rid-ob="figobml323t5">Table 4</a>) as well as dissimilar DUBs (i.e. UCL-H1 and UCL-H3). With respect to potency against USP1/UAF1, <a href="/pcsubstance/?term=ML323[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML323</a> displayed a 5- to 10-fold increase over C527, depending on assay format. Moreover, <a href="/pcsubstance/?term=ML323[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML323</a> showed no activity against other related USPs compared to current prior art (<a class="figpopup" href="/books/NBK259186/table/ml323.t5/?report=objectonly" target="object" rid-figpopup="figml323t5" rid-ob="figobml323t5">Table 4</a>).</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml323t5"><a href="/books/NBK259186/table/ml323.t5/?report=objectonly" target="object" title="Table 4" class="img_link icnblk_img figpopup" rid-figpopup="figml323t5" rid-ob="figobml323t5"><img class="small-thumb" src="/books/NBK259186/table/ml323.t5/?report=thumb" src-large="/books/NBK259186/table/ml323.t5/?report=previmg" alt="Table 4. Comparison of ML323 to previously identified USP1 inhibitors." /></a><div class="icnblk_cntnt"><h4 id="ml323.t5"><a href="/books/NBK259186/table/ml323.t5/?report=objectonly" target="object" rid-ob="figobml323t5">Table 4</a></h4><p class="float-caption no_bottom_margin">Comparison of ML323 to previously identified USP1 inhibitors. </p></div></div></div></div><div id="ml323.s13"><h2 id="_ml323_s13_">5. References</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="ml323.r1">Pickart CM. Mechanisms underlying ubiquitination. <span><span class="ref-journal">Annu Rev Biochem. </span>2001;<span class="ref-vol">70</span>:503–533.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/11395416" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 11395416</span></a>]</div></dd><dt>2.</dt><dd><div class="bk_ref" id="ml323.r2">Bergink S, Jentsch S. Principles of ubiquitin and SUMO modifications in DNA repair. <span><span class="ref-journal">Nature. </span>2009;<span class="ref-vol">458</span>(7237):461–467.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/19325626" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19325626</span></a>]</div></dd><dt>3.</dt><dd><div class="bk_ref" id="ml323.r3">Conaway RC, Brower CS, Conaway JW. Emerging roles of ubiquitin in transcription regulation. <span><span class="ref-journal">Science. </span>2002;<span class="ref-vol">296</span>(5571):1254–1258.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/12016299" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 12016299</span></a>]</div></dd><dt>4.</dt><dd><div class="bk_ref" id="ml323.r4">Mukhopadhyay D, Riezman H. Proteasome-independent functions of ubiquitin in endocytosis and signaling. <span><span class="ref-journal">Science. </span>2007;<span class="ref-vol">315</span>(5809):201–205.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/17218518" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17218518</span></a>]</div></dd><dt>5.</dt><dd><div class="bk_ref" id="ml323.r5">Nakayama KI, Nakayama K. Ubiquitin ligases: cell-cycle control and cancer. <span><span class="ref-journal">Nat Rev Cancer. </span>2006;<span class="ref-vol">6</span>(5):369–81.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/16633365" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 16633365</span></a>]</div></dd><dt>6.</dt><dd><div class="bk_ref" id="ml323.r6">McConkey DJ, Zhu K. Mechanisms of proteasome inhibitor action and resistance in cancer. <span><span class="ref-journal">Drug Resist Updat. </span>2008;<span class="ref-vol">11</span>(4-5):164–179.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/18818117" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18818117</span></a>]</div></dd><dt>7.</dt><dd><div class="bk_ref" id="ml323.r7">Singhal S, Taylor MC, Baker RT. Deubiquitylating enzymes and disease. <span><span class="ref-journal">BMC Biochem. </span>2008;<span class="ref-vol">9</span>(Suppl 1):S3.</span> [<a href="/pmc/articles/PMC2582804/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2582804</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/19007433" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19007433</span></a>]</div></dd><dt>8.</dt><dd><div class="bk_ref" id="ml323.r8">Nijman S, Huang T, Dirac A, Brummelkamp T, Kerkhoven R, D'Andrea A, Bernards R. The Deubiquitinating Enzyme USP1 Regulates the Fanconi Anemia Pathway. <span><span class="ref-journal">Mol Cell. </span>2005;<span class="ref-vol">17</span>(3):331–339.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/15694335" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 15694335</span></a>]</div></dd><dt>9.</dt><dd><div class="bk_ref" id="ml323.r9">Chen J, et al. Selective and Cell – Active Inhibitors of the USP1/ UAF1 Deubiquitinase Complex Reverse Cisplatin Resistance in Non-small Cell Lung Cancer Cells. <span><span class="ref-journal">Chemistry & biology. </span>2011;<span class="ref-vol">18</span>(11):1390–400.</span> [<a href="/pmc/articles/PMC3344384/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3344384</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/22118673" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 22118673</span></a>]</div></dd><dt>10.</dt><dd><div class="bk_ref" id="ml323.r10">McGovern SL, et al. A specific mechanism of nonspecific inhibition. <span><span class="ref-journal">J Med Chem. </span>2003;<span class="ref-vol">46</span>(20):4265–72.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/13678405" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 13678405</span></a>]</div></dd></dl></div><div><dl class="temp-labeled-list small"><dt>#</dt><dd><div id="ml323.fn1"><p class="no_top_margin">These authors contributed equally to this report</p></div></dd></dl></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: NBK259186</span><span class="label">PMID: <a href="https://pubmed.ncbi.nlm.nih.gov/25506968" title="PubMed record of this page" ref="pagearea=meta&targetsite=entrez&targetcat=link&targettype=pubmed">25506968</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/ml328/" title="Previous page in this title">< Prev</a><a class="active page_link next" href="/books/n/mlprobe/ml327/" 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=NBK259186&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/NBK259186/?report=reader">PubReader</a></li><li><a href="/books/NBK259186/?report=printable">Print View</a></li><li><a data-jig="ncbidialog" href="#_ncbi_dlg_citbx_NBK259186" data-jigconfig="width:400,modal:true">Cite this Page</a><div id="_ncbi_dlg_citbx_NBK259186" style="display:none" title="Cite this Page"><div class="bk_tt">Dexheimer TS, Rosenthal AS, Liang Q, et al. Discovery of ML323 as a Novel Inhibitor of the USP1/UAF1 Deubiquitinase Complex. 2012 Oct 23 [Updated 2014 Sep 18]. 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></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="#ml323.s1" ref="log$=inpage&link_id=inpage">Probe Structure & Characteristics</a></li><li><a href="#ml323.s2" ref="log$=inpage&link_id=inpage">Recommendations for Scientific Use of the Probe</a></li><li><a href="#ml323.s3" ref="log$=inpage&link_id=inpage">Materials and Methods</a></li><li><a href="#ml323.s7" ref="log$=inpage&link_id=inpage">Results</a></li><li><a href="#ml323.s11" ref="log$=inpage&link_id=inpage">Discussion</a></li><li><a href="#ml323.s13" ref="log$=inpage&link_id=inpage">References</a></li></ul></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Related information</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="discovery_db_links" id="Shutter"></a></div><div class="portlet_content"><ul><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pmc&DbFrom=books&Cmd=Link&LinkName=books_pmc_refs&IdsFromResult=3318568" ref="log$=recordlinks">PMC</a><div class="brieflinkpop offscreen_noflow">PubMed Central citations</div></li><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pcsubstance&DbFrom=books&Cmd=Link&LinkName=books_pcsubstance&IdsFromResult=3318568" ref="log$=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=3318568" ref="log$=recordlinks">PubMed</a><div class="brieflinkpop offscreen_noflow">Links to PubMed</div></li></ul></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Similar articles in PubMed</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="PBooksDiscovery_RA" id="Shutter"></a></div><div class="portlet_content"><ul><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/25229643" ref="ordinalpos=1&linkpos=1&log$=relatedarticles&logdbfrom=pubmed">Synthesis and structure-activity relationship studies of N-benzyl-2-phenylpyrimidin-4-amine derivatives as potent USP1/UAF1 deubiquitinase inhibitors with anticancer activity against nonsmall cell lung cancer.</a><span class="source">[J Med Chem. 2014]</span><div class="brieflinkpop offscreen_noflow">Synthesis and structure-activity relationship studies of N-benzyl-2-phenylpyrimidin-4-amine derivatives as potent USP1/UAF1 deubiquitinase inhibitors with anticancer activity against nonsmall cell lung cancer.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Dexheimer TS, Rosenthal AS, Luci DK, Liang Q, Villamil MA, Chen J, Sun H, Kerns EH, Simeonov A, Jadhav A, et al. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">J Med Chem. 2014 Oct 9; 57(19):8099-110. Epub 2014 Sep 17.</em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/26758085" ref="ordinalpos=1&linkpos=2&log$=relatedarticles&logdbfrom=pubmed">Mutations in the 'Fingers' subdomain of the deubiquitinase USP1 modulate its function and activity.</a><span class="source">[FEBS J. 2016]</span><div class="brieflinkpop offscreen_noflow">Mutations in the 'Fingers' subdomain of the deubiquitinase USP1 modulate its function and activity.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Olazabal-Herrero A, García-Santisteban I, Rodríguez JA. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">FEBS J. 2016 Mar; 283(5):929-46. Epub 2016 Feb 3.</em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/21482670" ref="ordinalpos=1&linkpos=3&log$=relatedarticles&logdbfrom=pubmed">The USP1/UAF1 complex promotes double-strand break repair through homologous recombination.</a><span class="source">[Mol Cell Biol. 2011]</span><div class="brieflinkpop offscreen_noflow">The USP1/UAF1 complex promotes double-strand break repair through homologous recombination.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Murai J, Yang K, Dejsuphong D, Hirota K, Takeda S, D'Andrea AD. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Mol Cell Biol. 2011 Jun; 31(12):2462-9. Epub 2011 Apr 11.</em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/23797609" ref="ordinalpos=1&linkpos=4&log$=relatedreviews&logdbfrom=pubmed"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> The WD40-repeat protein-containing deubiquitinase complex: catalysis, regulation, and potential for therapeutic intervention.</a><span class="source">[Cell Biochem Biophys. 2013]</span><div class="brieflinkpop offscreen_noflow"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> The WD40-repeat protein-containing deubiquitinase complex: catalysis, regulation, and potential for therapeutic intervention.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Villamil MA, Liang Q, Zhuang Z. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Cell Biochem Biophys. 2013 Sep; 67(1):111-26. </em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/28302046" ref="ordinalpos=1&linkpos=5&log$=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> The Role of the Complex USP1/WDR48 in Differentiation and Proliferation Processes in Cancer Stem Cells.</a><span class="source">[Curr Stem Cell Res Ther. 2017]</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> The Role of the Complex USP1/WDR48 in Differentiation and Proliferation Processes in Cancer Stem Cells.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Goncalves JM, Cordeiro MMR, Rivero ERC. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Curr Stem Cell Res Ther. 2017; 12(5):416-422. </em></div></div></li></ul><a class="seemore" href="/sites/entrez?db=pubmed&cmd=link&linkname=pubmed_pubmed_reviews&uid=25506968" ref="ordinalpos=1&log$=relatedreviews_seeall&logdbfrom=pubmed">See reviews...</a><a class="seemore" href="/sites/entrez?db=pubmed&cmd=link&linkname=pubmed_pubmed&uid=25506968" ref="ordinalpos=1&log$=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$=activity&linkpos=1" href="/portal/utils/pageresolver.fcgi?recordid=67d66b5f84f3725e592b409d">Discovery of ML323 as a Novel Inhibitor of the USP1/UAF1 Deubiquitinase Complex ...</a><div class="ralinkpop offscreen_noflow">Discovery of ML323 as a Novel Inhibitor of the USP1/UAF1 Deubiquitinase Complex - Probe Reports from the NIH Molecular Libraries Program<div class="brieflinkpopdesc"></div></div><div class="tertiary"></div></li><li class="ra_rcd ralinkpopper two_line"><a class="htb ralinkpopperctrl" ref="log$=activity&linkpos=2" href="/portal/utils/pageresolver.fcgi?recordid=67d66b5e2f30673f7bf61ad1">ML328: A Novel Dual Inhibitor of Bacterial AddAB and RecBCD Helicase-nuclease DN...</a><div class="ralinkpop offscreen_noflow">ML328: A Novel Dual Inhibitor of Bacterial AddAB and RecBCD Helicase-nuclease DNA Repair Enzymes - 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$=activity&linkpos=3" href="/portal/utils/pageresolver.fcgi?recordid=67d66b5d67c23b31e0b113cc">A Small Molecule Inhibitor of the MITF Molecular Pathway - Probe Reports from th...</a><div class="ralinkpop offscreen_noflow">A Small Molecule Inhibitor of the MITF Molecular Pathway - Probe Reports from the NIH Molecular Libraries Program<div class="brieflinkpopdesc"></div></div><div class="tertiary"></div></li><li class="ra_rcd ralinkpopper two_line"><a class="htb ralinkpopperctrl" ref="log$=activity&linkpos=4" href="/portal/utils/pageresolver.fcgi?recordid=67d66b5c2f30673f7bf613b7">Cardioprotective inhibitors of reperfusion injury - Probe Reports from the NIH M...</a><div class="ralinkpop offscreen_noflow">Cardioprotective inhibitors of reperfusion injury - 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$=activity&linkpos=5" href="/portal/utils/pageresolver.fcgi?recordid=67d66b5a84f3725e592b34e7">Identification of a glycine sulfonamide based non-MPEP site positive allosteric ...</a><div class="ralinkpop offscreen_noflow">Identification of a glycine sulfonamide based non-MPEP site positive allosteric potentiator (PAM) of mGlu5 - Probe Reports from the NIH Molecular Libraries Program<div class="brieflinkpopdesc"></div></div><div class="tertiary"></div></li></ul><p class="HTOn">Your browsing activity is empty.</p><p class="HTOff">Activity recording is turned off.</p><p id="turnOn" class="HTOff"><a href="javascript:historyDisplayState('HTOn')">Turn recording back on</a></p><a class="seemore" href="/sites/myncbi/recentactivity">See more...</a></div></div></div>
|
||
|
||
<!-- Custom content below discovery portlets -->
|
||
<div class="col7">
|
||
|
||
</div>
|
||
</div>
|
||
</div>
|
||
|
||
<!-- Custom content after all -->
|
||
<div class="col8">
|
||
|
||
</div>
|
||
<div class="col9">
|
||
|
||
</div>
|
||
|
||
<script type="text/javascript" src="/corehtml/pmc/js/jquery.scrollTo-1.4.2.js"></script>
|
||
<script type="text/javascript">
|
||
(function($){
|
||
$('.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=NBK259186&ncbi_domain=mlprobe&ncbi_report=record&ncbi_type=fulltext&ncbi_objectid=&ncbi_pcid=/NBK259186/&ncbi_pagename=Discovery of ML323 as a Novel Inhibitor of the USP1/UAF1 Deubiquitinase Complex - 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 portal106 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> |