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<title>Discovery and Development of Small Molecules That Reduce PNC Prevalence - Probe Reports from the NIH Molecular Libraries Program - NCBI Bookshelf</title>
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<meta name="robots" content="INDEX,FOLLOW,NOARCHIVE" /><meta name="citation_inbook_title" content="Probe Reports from the NIH Molecular Libraries Program [Internet]" /><meta name="citation_title" content="Discovery and Development of Small Molecules That Reduce PNC Prevalence" /><meta name="citation_publisher" content="National Center for Biotechnology Information (US)" /><meta name="citation_date" content="2010" /><meta name="citation_author" content="Kevin Frankowski" /><meta name="citation_author" content="Samarjit Patnaik" /><meta name="citation_author" content="Frank Schoenen" /><meta name="citation_author" content="Sui Huang" /><meta name="citation_author" content="John Norton" /><meta name="citation_author" content="Chen Wang" /><meta name="citation_author" content="Steve Titus" /><meta name="citation_author" content="Marc Ferrer" /><meta name="citation_author" content="Wei Zheng" /><meta name="citation_author" content="Noel Southall" /><meta name="citation_author" content="Victor W. Day" /><meta name="citation_author" content="Jeffrey Aubé" /><meta name="citation_author" content="Juan Jose Marugan" /><meta name="citation_pmid" content="23762948" /><meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK143533/" /><link rel="schema.DC" href="http://purl.org/DC/elements/1.0/" /><meta name="DC.Title" content="Discovery and Development of Small Molecules That Reduce PNC Prevalence" /><meta name="DC.Type" content="Text" /><meta name="DC.Publisher" content="National Center for Biotechnology Information (US)" /><meta name="DC.Contributor" content="Kevin Frankowski" /><meta name="DC.Contributor" content="Samarjit Patnaik" /><meta name="DC.Contributor" content="Frank Schoenen" /><meta name="DC.Contributor" content="Sui Huang" /><meta name="DC.Contributor" content="John Norton" /><meta name="DC.Contributor" content="Chen Wang" /><meta name="DC.Contributor" content="Steve Titus" /><meta name="DC.Contributor" content="Marc Ferrer" /><meta name="DC.Contributor" content="Wei Zheng" /><meta name="DC.Contributor" content="Noel Southall" /><meta name="DC.Contributor" content="Victor W. Day" /><meta name="DC.Contributor" content="Jeffrey Aubé" /><meta name="DC.Contributor" content="Juan Jose Marugan" /><meta name="DC.Date" content="2010" /><meta name="DC.Identifier" content="https://www.ncbi.nlm.nih.gov/books/NBK143533/" /><meta name="description" content="The perinucleolar compartment (PNC) is a subnuclear body found at the periphery of the nucleolus in the nucleus. Its function is not completely known yet, but it is a highly dynamic body that is enriched with Ribonucleic acid (RNA) transcripts and RNA-binding proteins. This compartment is highly prevalent in metastatic tumors and metastatically transformed cancer cell lines, making it a potential pan marker for metastatic progression. A high throughput, high content assay was developed to identify novel small molecules that reduce the prevalence of this metastasis biomarker in cancer cells. We have identified and further optimized a potent pyrrolopyrimidine series that reduces PNC prevalence in PC3M cells at submicromolar concentrations, where the cell viability is not affected. These compounds also show dose-dependent inhibition of migration and anchorage-independent growth in invasion and soft-agar assays, respectively. The probe candidate, ML246, has drug-like physical properties and displays promising protein kinase, making it an ideal in vivo tool for establishing the link between the PNC and the metastatic transformation of tumor cells. ML246 will serve as a pivotal first step to further research the function of the PNC and its role in cancer metastasis." /><meta name="og:title" content="Discovery and Development of Small Molecules That Reduce PNC Prevalence" /><meta name="og:type" content="book" /><meta name="og:description" content="The perinucleolar compartment (PNC) is a subnuclear body found at the periphery of the nucleolus in the nucleus. Its function is not completely known yet, but it is a highly dynamic body that is enriched with Ribonucleic acid (RNA) transcripts and RNA-binding proteins. This compartment is highly prevalent in metastatic tumors and metastatically transformed cancer cell lines, making it a potential pan marker for metastatic progression. A high throughput, high content assay was developed to identify novel small molecules that reduce the prevalence of this metastasis biomarker in cancer cells. We have identified and further optimized a potent pyrrolopyrimidine series that reduces PNC prevalence in PC3M cells at submicromolar concentrations, where the cell viability is not affected. These compounds also show dose-dependent inhibition of migration and anchorage-independent growth in invasion and soft-agar assays, respectively. The probe candidate, ML246, has drug-like physical properties and displays promising protein kinase, making it an ideal in vivo tool for establishing the link between the PNC and the metastatic transformation of tumor cells. ML246 will serve as a pivotal first step to further research the function of the PNC and its role in cancer metastasis." /><meta name="og:url" content="https://www.ncbi.nlm.nih.gov/books/NBK143533/" /><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/ml246/" /><link rel="canonical" href="https://www.ncbi.nlm.nih.gov/books/NBK143533/" /><link rel="stylesheet" href="/corehtml/pmc/css/figpopup.css" type="text/css" media="screen" /><link rel="stylesheet" href="/corehtml/pmc/css/bookshelf/2.26/css/books.min.css" type="text/css" /><link rel="stylesheet" href="/corehtml/pmc/css/bookshelf/2.26/css/books_print.min.css" type="text/css" media="print" /><style type="text/css">p a.figpopup{display:inline !important} .bk_tt {font-family: monospace} .first-line-outdent .bk_ref {display: inline} .body-content h2, .body-content .h2 {border-bottom: 1px solid #97B0C8} .body-content h2.inline {border-bottom: none} a.page-toc-label , .jig-ncbismoothscroll a {text-decoration:none;border:0 !important} .temp-labeled-list .graphic {display:inline-block !important} .temp-labeled-list img{width:100%}</style><script type="text/javascript" src="/corehtml/pmc/js/jquery.hoverIntent.min.js"> </script><script type="text/javascript" src="/corehtml/pmc/js/common.min.js?_=3.18"> </script><script type="text/javascript" src="/corehtml/pmc/js/large-obj-scrollbars.min.js"> </script><script type="text/javascript">window.name="mainwindow";</script><script type="text/javascript" src="/corehtml/pmc/js/bookshelf/2.26/book-toc.min.js"> </script><script type="text/javascript" src="/corehtml/pmc/js/bookshelf/2.26/books.min.js"> </script><meta name="book-collection" content="NONE" />
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<div class="pre-content"><div><div class="bk_prnt"><p class="small">NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health.</p><p>Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-. </p></div><div class="iconblock clearfix whole_rhythm no_top_margin bk_noprnt"><a class="img_link icnblk_img" title="Table of Contents Page" href="/books/n/mlprobe/"><img class="source-thumb" src="/corehtml/pmc/pmcgifs/bookshelf/thumbs/th-mlprobe-lrg.png" alt="Cover of Probe Reports from the NIH Molecular Libraries Program" height="100px" width="80px" /></a><div class="icnblk_cntnt eight_col"><h2>Probe Reports from the NIH Molecular Libraries Program [Internet].</h2><a data-jig="ncbitoggler" href="#__NBK143533_dtls__">Show details</a><div style="display:none" class="ui-widget" id="__NBK143533_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/ml247/" title="Previous page in this title">< Prev</a><a class="active page_link next" href="/books/n/mlprobe/ml245/" title="Next page in this title">Next ></a></div></div></div></div></div>
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<div class="main-content lit-style" itemscope="itemscope" itemtype="http://schema.org/CreativeWork"><div class="meta-content fm-sec"><h1 id="_NBK143533_"><span class="title" itemprop="name">Discovery and Development of Small Molecules That Reduce PNC Prevalence</span></h1><p class="contrib-group"><span itemprop="author">Kevin Frankowski</span>, <span itemprop="author">Samarjit Patnaik</span>, <span itemprop="author">Frank Schoenen</span>, <span itemprop="author">Sui Huang</span>, <span itemprop="author">John Norton</span>, <span itemprop="author">Chen Wang</span>, <span itemprop="author">Steve Titus</span>, <span itemprop="author">Marc Ferrer</span>, <span itemprop="author">Wei Zheng</span>, <span itemprop="author">Noel Southall</span>, <span itemprop="author">Victor W. Day</span>, <span itemprop="author">Jeffrey Aubé</span>, and <span itemprop="author">Juan Jose Marugan</span>.</p><a data-jig="ncbitoggler" href="#__NBK143533_ai__" style="border:0;text-decoration:none">Author Information and Affiliations</a><div style="display:none" class="ui-widget" id="__NBK143533_ai__"><p class="contrib-group"><h4>Authors</h4><span itemprop="author">Kevin Frankowski</span>,<sup>a</sup> <span itemprop="author">Samarjit Patnaik</span>,<sup>b</sup> <span itemprop="author">Frank Schoenen</span>,<sup>a</sup> <span itemprop="author">Sui Huang</span>,<sup>c</sup> <span itemprop="author">John Norton</span>,<sup>c</sup> <span itemprop="author">Chen Wang</span>,<sup>c</sup> <span itemprop="author">Steve Titus</span>,<sup>b</sup> <span itemprop="author">Marc Ferrer</span>,<sup>b</sup> <span itemprop="author">Wei Zheng</span>,<sup>b</sup> <span itemprop="author">Noel Southall</span>,<sup>b</sup> <span itemprop="author">Victor W. Day</span>,<sup>d</sup> <span itemprop="author">Jeffrey Aubé</span>,<sup>a</sup><sup>,*</sup> and <span itemprop="author">Juan Jose Marugan</span><sup>b</sup><sup>,*</sup>.</p><h4>Affiliations</h4><div class="affiliation"><sup>a</sup>
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Specialized Chemistry Center, The University of Kansas, 2034 Becker Drive, Lawrence, KS 66047.</div><div class="affiliation"><sup>b</sup>
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NIH Chemical Genomics Center, National Human Genome Research Institute, National Institutes of Health, 9800 Medical Center Drive, Rockville, MD, 20850.</div><div class="affiliation"><sup>c</sup>
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Department of Cell and Molecular Biology, Northwestern University, Chicago, Illinois.</div><div class="affiliation"><sup>d</sup>
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X-ray Crystallography Laboratory, University of Kansas, 1251 Wescoe Hall Drive, Lawrence, Kansas 66045.</div><div class="affiliation">
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<sup>*</sup> To whom correspondence should be addressed: Email:
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<a href="mailto:dev@null" data-email="ude.uk@ebuaj" class="oemail">ude.uk@ebuaj</a> and
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<span class="before-email-separator">; </span><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="vog.hin.liam@jnaguram" class="oemail">vog.hin.liam@jnaguram</a></div></div></div><div class="jig-ncbiinpagenav body-content whole_rhythm" data-jigconfig="allHeadingLevels: ['h2'],smoothScroll: false" itemprop="text"><div id="_abs_rndgid_" itemprop="description"><p>The perinucleolar compartment (PNC) is a subnuclear body found at the periphery of the nucleolus in the nucleus. Its function is not completely known yet, but it is a highly dynamic body that is enriched with Ribonucleic acid (RNA) transcripts and RNA-binding proteins. This compartment is highly prevalent in metastatic tumors and metastatically transformed cancer cell lines, making it a potential pan marker for metastatic progression. A high throughput, high content assay was developed to identify novel small molecules that reduce the prevalence of this metastasis biomarker in cancer cells. We have identified and further optimized a potent pyrrolopyrimidine series that reduces PNC prevalence in PC3M cells at submicromolar concentrations, where the cell viability is not affected. These compounds also show dose-dependent inhibition of migration and anchorage-independent growth in invasion and soft-agar assays, respectively. The probe candidate, <a href="/pcsubstance/?term=ML246[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML246</a>, has drug-like physical properties and displays promising protein kinase, making it an ideal <i>in vivo</i> tool for establishing the link between the PNC and the metastatic transformation of tumor cells. <a href="/pcsubstance/?term=ML246[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML246</a> will serve as a pivotal first step to further research the function of the PNC and its role in cancer metastasis.</p></div><div class="h2"></div><p><b>Assigned Assay Grant #:</b> R03MH082371</p><p><b>Screening Center Name & PI:</b> NIH Chemical Genomics Center & Christopher P. Austin</p><p><b>Chemistry Center Name & PI:</b> Kansas University & Jeffrey Aubé; NIH Chemical Genomics Center & Christopher P. Austin</p><p><b>Assay Submitter & Institution:</b> Sui Huang, Northwestern University</p><p><b>PubChem Summary Bioassay Identifier (AID):</b>
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<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2431" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2431</a></p><div id="ml246.s1"><h2 id="_ml246_s1_">Probe Structure & Characteristics</h2><div id="ml246.fu1" class="figure"><div class="graphic"><img src="/books/NBK143533/bin/ml246fu1.jpg" alt="Image ml246fu1" /></div></div><div id="ml246.tu1" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK143533/table/ml246.tu1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml246.tu1_lrgtbl__"><table><thead><tr><th id="hd_h_ml246.tu1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CID/ML#</th><th id="hd_h_ml246.tu1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Target Name</th><th id="hd_h_ml246.tu1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">IC<sub>50</sub>/EC<sub>50</sub> (nM) [SID, AID]</th><th id="hd_h_ml246.tu1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Anti-target Name(s)</th><th id="hd_h_ml246.tu1_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">IC<sub>50</sub>/EC<sub>50</sub> (μM) [SID, AID]</th><th id="hd_h_ml246.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Fold Selective</th><th id="hd_h_ml246.tu1_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Secondary Assay(s) Name: IC<sub>50</sub>/EC<sub>50</sub> (nM) [SID, AID]</th></tr></thead><tbody><tr><td headers="hd_h_ml246.tu1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CID 50985821/<a href="/pcsubstance/?term=ML246[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML246</a></td><td headers="hd_h_ml246.tu1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">PNC</td><td headers="hd_h_ml246.tu1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">PNC Reduction (PC3M cells): 397 nM IC<sub>50</sub> [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/117695978" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 117695978</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/540360" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 540360</a>]</td><td headers="hd_h_ml246.tu1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Cytotoxicity (ATP assay, PC3M cells)</td><td headers="hd_h_ml246.tu1_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">9960 nM IC<sub>50</sub> [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/117695978" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 117695978</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/588740" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 588740</a>]</td><td headers="hd_h_ml246.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">25-fold</td><td headers="hd_h_ml246.tu1_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Migration: 3.16 μM IC<sub>50</sub> [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/117695978" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 117695978</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/588739" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 588739</a>]</td></tr></tbody></table></div></div></div><div id="ml246.s2"><h2 id="_ml246_s2_">1. Recommendations for Scientific Use of the Probe</h2><p>The prevention and treatment of cancer metastasis is a prominent unmet medical need. Several molecular targets have been identified and explored for inhibition of the metastatic process, but these approaches have not yet yielded success in the clinic. The perinucleolar compartment (PNC) is a multicomponent subnuclear structure that is highly prevalent in metastatic tumors. [<a class="bk_pop" href="#ml246.r1">1</a>, <a class="bk_pop" href="#ml246.r2">2</a>] Here, we present the discovery of a probe molecule that is able to disrupt the assembly of PNC in a metastatically transformed prostate cancer cell line without overt cytotoxicity. This structural class also impacts <i>in vitro</i> migration of tumor cells, making it useful for advanced cell-based studies. The probe displays promising <i>in vitro</i> ADME properties and <i>in vivo</i> mouse pharmacokinetics, making it an ideal candidate for understanding the therapeutic value of PNC disruption as a novel approach towards combating metastasis in cancer treatment.</p></div><div id="ml246.s3"><h2 id="_ml246_s3_">2. Materials and Methods</h2><p>The reagents and solvents used were commercial anhydrous grade and used without further purification. The column chromatography was carried out over silica gel (100–200 mesh). <sup>1</sup>H NMR spectra were recorded with a Bruker 400 MHz spectrometer from solutions in CDCl<sub>3</sub> and DMSO-d<sub>6</sub>. Chemical shifts in <sup>1</sup>H NMR spectra are reported in parts per million (ppm, δ) downfield from the internal standard Me<sub>4</sub>Si (TMS, δ = 0 ppm). Chemical shifts in <sup>13</sup>C NMR spectra are reported in parts per million (ppm, δ) calibrated from residual CHCl<sub>3</sub> (δ = 77.0 ppm) signal and are reported using an APT pulse sequence displaying methyl and methine (CH<sub>3</sub> and CH) signals as down, and quaternary and methylene (C and CH<sub>2</sub>) signals as up. Molecular weight confirmation was performed using an Agilent 6224 Time-Of-Flight Mass Spectrometer (TOF, Agilent Technologies, Santa Clara, CA). A 3 minute gradient from 5 to 100% Acetonitrile in water (0.03% formic acid) was used with a 5.1 minute run time at a flow rate of 0.4 mL/min. A Waters Atlantis T3 C18 column (1.8 micron, 2.1 × 50 mm) was used at a temperature of 25 °C. Confirmation of molecular formula was confirmed using electrospray ionization in the positive mode with the Agilent Masshunter software (version B.02).</p><div id="ml246.s4"><h3>2.1. Assays</h3><div id="ml246.s5"><h4>High Content Assay for PNC Detection</h4><p>The quantitative output for this assay is the reduction of PNC prevalence. The PNC can be detected in living cells by the expression of a green fluorescent protein (GFP) tagged to the PNC localized protein, PTB, as shown in <a class="figpopup" href="/books/NBK143533/figure/ml246.f1/?report=objectonly" target="object" rid-figpopup="figml246f1" rid-ob="figobml246f1">Figure 1</a>. We have created a PC3M cell line that stably expresses GFP-PTB to mark PNCs. This method eliminates the need for immunofluorescent staining. Previous studies demonstrate that the fusion proteins behave similarly to their endogenous counterparts: transient and stable over-expression of the fusion protein did not have detectable adverse effects on cell morphology or cell growth.[<a class="bk_pop" href="#ml246.r3">3</a>] After treatment, cells are fixed and the nuclei are counterstained with Hoechst 33342 dye; the cells are then ready for analysis.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml246f1" co-legend-rid="figlgndml246f1"><a href="/books/NBK143533/figure/ml246.f1/?report=objectonly" target="object" title="Figure 1" class="img_link icnblk_img figpopup" rid-figpopup="figml246f1" rid-ob="figobml246f1"><img class="small-thumb" src="/books/NBK143533/bin/ml246f1.gif" src-large="/books/NBK143533/bin/ml246f1.jpg" alt="Figure 1. PTB-GFP PC-3M cells." /></a><div class="icnblk_cntnt" id="figlgndml246f1"><h4 id="ml246.f1"><a href="/books/NBK143533/figure/ml246.f1/?report=objectonly" target="object" rid-ob="figobml246f1">Figure 1</a></h4><p class="float-caption no_bottom_margin">PTB-GFP PC-3M cells. PNCs are detected as intense, punctuated fluorescence dots at the perinucleolar regions, as indicated with the arrows. </p></div></div><p>The IN Cell Analyzer 1000 automated fluorescent imaging system (GE Healthcare, Piscataway, NJ) was used for automated image acquisition. Images were acquired with a 20× objective using a 475/20nm excitation filter, a 535/50 nm HQ emission filter, a Q505LP dichroic filter, and an exposure time of 100 to 150 ms (adjusted to obtain a dynamic range of ~200 to 1750), with no camera binning. The instrument acquired images of each well in a 1536-well plate with a laser-based autofocus system. To score PNC prevalence in a high-content throughput, we used the Multi-Target Analysis (MTA) algorithm (GE Healthcare, Investigator v3.5) to identify individual cells and granules (PNCs) within these cells. The nucleus was segmented via a region growing method (50 μm<sup>2</sup> minimum area) with light shading and noise removal to allow “touching” nuclei to be separated. Granules in the nucleus (PNCs) were segmented using a multiscale top hat method, which measures granules of 1 to 2 μm in size and used a smart masking method to identify the boundaries of each segmented granule. The algorithm was optimized and validated using positive and negative controls (50 μM camptothecin and DMSO, respectively). In particular, the MTA algorithm allows for the identification of multiple subcellular compartments and organelles (or granules) within those compartments. In this instance, we took advantage of the algorithm’s capability to identify objects within the same color channel that only differ in size or fluorescent intensity. Also, the algorithm allows for building complex hierarchical classification systems, using output measures within the algorithm to filter and define subpopulations. For this particular assay, PNC-positive cells were scored when 1 to 3 PNC granules were detected per nucleus. Cells that contained 0 granules were scored as PNC negative, and cells with >3 granules were assumed to be false positives (very few cells have more than 3 PNCs in one focus plane), and were also scored as PNC negative.</p><div id="ml246.tu2" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK143533/table/ml246.tu2/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml246.tu2_lrgtbl__"><table><thead><tr><th id="hd_h_ml246.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Sequence</th><th id="hd_h_ml246.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Parameter</th><th id="hd_h_ml246.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Value</th><th id="hd_h_ml246.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Description</th></tr></thead><tbody><tr><td headers="hd_h_ml246.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>1</b></td><td headers="hd_h_ml246.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Cells</td><td headers="hd_h_ml246.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">5 μL</td><td headers="hd_h_ml246.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">750–1000 cells/well</td></tr><tr><td headers="hd_h_ml246.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>2</b></td><td headers="hd_h_ml246.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Time</td><td headers="hd_h_ml246.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">4 hrs</td><td headers="hd_h_ml246.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Incubate at 37 ºC and 5% CO<sub>2</sub></td></tr><tr><td headers="hd_h_ml246.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>3</b></td><td headers="hd_h_ml246.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Reagent</td><td headers="hd_h_ml246.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">23 nL</td><td headers="hd_h_ml246.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Compound library, camptothecin as control</td></tr><tr><td headers="hd_h_ml246.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>4</b></td><td headers="hd_h_ml246.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Time</td><td headers="hd_h_ml246.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">16 hrs</td><td headers="hd_h_ml246.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Incubate at 37 ºC and 5% CO<sub>2</sub></td></tr><tr><td headers="hd_h_ml246.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>5</b></td><td headers="hd_h_ml246.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Reagent</td><td headers="hd_h_ml246.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">4 μL</td><td headers="hd_h_ml246.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Fixation step with 6% EM grade paraformaldehyde and 0.1% Triton X-100</td></tr><tr><td headers="hd_h_ml246.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>6</b></td><td headers="hd_h_ml246.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Time</td><td headers="hd_h_ml246.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">20 min</td><td headers="hd_h_ml246.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">RT incubation</td></tr><tr><td headers="hd_h_ml246.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>7</b></td><td headers="hd_h_ml246.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Wash</td><td headers="hd_h_ml246.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">5 μL</td><td headers="hd_h_ml246.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Liquid was aspirated and 5μL of PBS was added</td></tr><tr><td headers="hd_h_ml246.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>8</b></td><td headers="hd_h_ml246.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Wash</td><td headers="hd_h_ml246.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">5 μL</td><td headers="hd_h_ml246.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Liquid was aspirated and 5μL of PBS was added</td></tr><tr><td headers="hd_h_ml246.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>9</b></td><td headers="hd_h_ml246.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Reagent</td><td headers="hd_h_ml246.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">5 μL</td><td headers="hd_h_ml246.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Staining with PBS containing 1 μg/mL Hoechst 33342</td></tr><tr><td headers="hd_h_ml246.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>10</b></td><td headers="hd_h_ml246.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Detector</td><td headers="hd_h_ml246.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Fluorescence</td><td headers="hd_h_ml246.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">IN Cell 1000, 20 × objective</td></tr></tbody></table></div></div></div><div id="ml246.s6"><h4>BellBrooks Labs<sup>®</sup> Tumor Cell Migration Assay</h4><p>High-content tumor cell migration assays in 3-dimensional extracellular matrices are powerful tools for modeling and understanding the biology of this critical step in the process of metastasis. However, most of the available methods are not amenable to the throughput required by studies of comparative pharmacology or small scale screening. For this reason, we worked with BellBrook Labs<sup>®</sup> to test our compounds in their automated high-content tumor cell invasion assays (<a class="figpopup" href="/books/NBK143533/figure/ml246.f7/?report=objectonly" target="object" rid-figpopup="figml246f7" rid-ob="figobml246f7">Figure 7</a>).[<a class="bk_pop" href="#ml246.r4">4</a>] A standard screening-sized plate with an array of embedded microchannels was designed and constructed from common thermoplastics.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml246f7" co-legend-rid="figlgndml246f7"><a href="/books/NBK143533/figure/ml246.f7/?report=objectonly" target="object" title="Figure 7" class="img_link icnblk_img figpopup" rid-figpopup="figml246f7" rid-ob="figobml246f7"><img class="small-thumb" src="/books/NBK143533/bin/ml246f7.gif" src-large="/books/NBK143533/bin/ml246f7.jpg" alt="Figure 7. A) Brief outline of BellBrook Labs® assay." /></a><div class="icnblk_cntnt" id="figlgndml246f7"><h4 id="ml246.f7"><a href="/books/NBK143533/figure/ml246.f7/?report=objectonly" target="object" rid-ob="figobml246f7">Figure 7</a></h4><p class="float-caption no_bottom_margin"><i>A</i>) Brief outline of BellBrook Labs<sup>®</sup> assay. <i>B</i>) Invasion dose response curves for ML246 and an inactive analog with PC3M cells. </p></div></div><p>PC3M cells were tested for invasion through 3D fibrillar collagen in the Iuvo Single Microchannel Plate, in the presence of varying levels of test compounds. Channels were prefilled with 820 nL of 3-dimensional type I collagen at 1 mg/mL, through the input port. Following gelation, 2,000 PC3M cells were seeded into the output port using growth media (RPMI + 10% FBS with antibiotics) in a volume of 5 μL. Cells were incubated in a 37 ºC incubator inside a humidified container to control evaporation (Bioassay dish, Corning). Media, including test compounds, was changed daily for 5 days. At the end of the assay, cells were fixed and stained with Hoechst 33342, then imaged with 4× objective under epifluorescence. Under these conditions, we can reliably identify cells across the 140 μm height range of the microchannel. Test compounds were serially diluted by a factor of 3 to produce 10 concentrations ranging from 50 or 100 μM to 2.5 nM. All assays were conducted in the presence of 0.1% DMSO. Four replicates were performed for all test concentrations. Each plate had 4 dose response curves, as well as 16 channels with no compound and 16 channels with 50 μM blebbistatin (positive control). Analysis was done by automatically cropping each image at the right edge of the channel and counting cells via the ‘count nuclei’ function on Metamorph (Molecular Devices). Non-linear regression analysis was performed with GraphPad Prism.</p></div><div id="ml246.s7"><h4>Soft Agar Assay</h4><p><i>In vitro</i> cellular transformation detection assays are semi-quantitative and measure the morphological transformation of cell colonies modulated by chemicals. This transformation is associated with certain phenotypic changes, such as loss of contact inhibition (cells can grow over one another) and anchorage independence (cells form colonies in soft agar). Anchorage independence can be described in the light of primary fibroblasts and many other cell lines (e.g. BALB/c3T3, NIH-3T3, etc.) that must attach to a solid surface before they can divide. They fail to grow when suspended in a viscous fluid or gel (e.g. agar or agarose); however, when these cell lines are transformed, they are able to grow in a viscous fluid or gel and become anchorage-independent. This process, by which these phenotypic changes occur, is assumed to be closely related; hence, it is a good predictor of <i>in vivo</i> carcinogenesis.</p><p>PC3M cells were gently suspended in 0.35% agar in serum/antibiotic free RPMI supplemented with 10% FBS, 1% penicillin-streptomycin, and compounds whose concentrations was based on the EC<sub>10</sub> and EC<sub>20</sub>. Then, 5000 cells/mL per well were seeded onto 1:1 mixture of 1% agar RPMI containing supplements into 6-well plates. After the top layer had dried, the agarose was overlaid with 3 mL RPMI +10% FBS and drug concentration at EC<sub>10</sub> or EC<sub>20</sub>. The overlaying media was changed twice per week. After 14 and 21 days, the number and size of colonies were visualized and measured, using 40× and 20× objectives. Triplicate wells were used for every treatment condition, and the same field was used for analysis and then averaged. After counting and measuring the colonies, the soft agar cell plates were incubated for another 21 days to observe a phenotype.</p></div><div id="ml246.s8"><h4>ATP Quantitation Assay (viability assay)</h4><p>We conducted this follow-up assay to measure the effect of compounds on cell health by measuring ATP levels (ATPLite™). ATPLite™ is an Adenosine TriPhosphate (ATP) monitoring system based on firefly (<i>Photinus pyralis</i>) luciferase. The level of ATP in a metabolically active cell is a general marker for its viability. ATP levels are often reduced during necrosis or apoptosis. In this assay, the luciferase enzyme catalyzes the conversion of the added substrate D-luciferin to oxyluciferin and light with ATP. Thus, the emitted light is proportional to the ATP concentration. For this assay, the highly metastatic PC3M reporter cell line stably expressing the PTB-GFP was provided by Professor Sui Huang of Northwestern University. The media and cell culture reagents were purchased from Invitrogen (Carlsbad, CA), ATPLite™ came from PerkinElmer.</p><div id="ml246.tu3" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK143533/table/ml246.tu3/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml246.tu3_lrgtbl__"><table><thead><tr><th id="hd_h_ml246.tu3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Sequence</th><th id="hd_h_ml246.tu3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Parameter</th><th id="hd_h_ml246.tu3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Value</th><th id="hd_h_ml246.tu3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Description</th></tr></thead><tbody><tr><td headers="hd_h_ml246.tu3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>1</b></td><td headers="hd_h_ml246.tu3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Cells</td><td headers="hd_h_ml246.tu3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">5 μL</td><td headers="hd_h_ml246.tu3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">2000 cells/well</td></tr><tr><td headers="hd_h_ml246.tu3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>2</b></td><td headers="hd_h_ml246.tu3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Time</td><td headers="hd_h_ml246.tu3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">4 hrs</td><td headers="hd_h_ml246.tu3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Incubate at 37 ºC and 5% CO<sub>2</sub></td></tr><tr><td headers="hd_h_ml246.tu3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>3</b></td><td headers="hd_h_ml246.tu3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Reagent</td><td headers="hd_h_ml246.tu3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">23 nL</td><td headers="hd_h_ml246.tu3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Compound library, camptothecin as control</td></tr><tr><td headers="hd_h_ml246.tu3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>4</b></td><td headers="hd_h_ml246.tu3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Time</td><td headers="hd_h_ml246.tu3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">24 hrs</td><td headers="hd_h_ml246.tu3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Incubate at 37 ºC and 5% CO<sub>2</sub></td></tr><tr><td headers="hd_h_ml246.tu3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>5</b></td><td headers="hd_h_ml246.tu3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Reagent</td><td headers="hd_h_ml246.tu3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">3 μL</td><td headers="hd_h_ml246.tu3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">ATPLite</td></tr><tr><td headers="hd_h_ml246.tu3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>6</b></td><td headers="hd_h_ml246.tu3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Time</td><td headers="hd_h_ml246.tu3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">20 min</td><td headers="hd_h_ml246.tu3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">RT incubation</td></tr><tr><td headers="hd_h_ml246.tu3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>7</b></td><td headers="hd_h_ml246.tu3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Centrifuge</td><td headers="hd_h_ml246.tu3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">1 min</td><td headers="hd_h_ml246.tu3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">1500 RPM</td></tr><tr><td headers="hd_h_ml246.tu3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>8</b></td><td headers="hd_h_ml246.tu3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Detector</td><td headers="hd_h_ml246.tu3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Luminescence</td><td headers="hd_h_ml246.tu3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">ViewLux</td></tr></tbody></table></div></div></div><div id="ml246.s9"><h4>PC3M Caspase 3/7 Activation Assay</h4><p>This assay was used as an orthogonal measurement of cytotoxicity of the compounds that reduced PNC prevalence. The activation of caspases is a pivotal step during apoptosis, because these cysteine-dependent, aspartate-directed proteases enable the systematic structural disassembly of dying cells. Their activation is typically triggered by a variety of stimuli, including growth factor withdrawal, exposure to radiation or chemotherapeutic agents, or initiation of the Fas/Apo-1 receptor-mediated cell death process. The caspase-3/7 substrate rhodamine 110, bis-(N-CBZL- aspartyl-L-glutamyl-L-valyl-L-aspartic acid amide; Z-DEVD-R110) is a pro-fluorescent substrate. To perform the Apo-ONE® Homogeneous caspase-3/7 Assay, the buffer and substrate are mixed and added to the sample. Upon sequential cleavage and removal of the DEVD peptides by caspase-3/7 activity and excitation at 499 nm, the rhodamine 110 leaving group becomes intensely fluorescent. The emission maximum is 521 nm.</p><p>The media and cell culture reagents were purchased from Invitrogen (Carlsbad, CA), and Caspase Glo 3/7 came from Promega. The highly metastatic PC3M-GFP reporter cell line was provided by Professor Sui Huang of Northwestern University.</p><div id="ml246.tu4" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK143533/table/ml246.tu4/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml246.tu4_lrgtbl__"><table><thead><tr><th id="hd_h_ml246.tu4_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Sequence</th><th id="hd_h_ml246.tu4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Parameter</th><th id="hd_h_ml246.tu4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Value</th><th id="hd_h_ml246.tu4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Description</th></tr></thead><tbody><tr><td headers="hd_h_ml246.tu4_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>1</b></td><td headers="hd_h_ml246.tu4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Cells</td><td headers="hd_h_ml246.tu4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">5 μL</td><td headers="hd_h_ml246.tu4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">2000 cells/well</td></tr><tr><td headers="hd_h_ml246.tu4_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>2</b></td><td headers="hd_h_ml246.tu4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Time</td><td headers="hd_h_ml246.tu4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">4 hrs</td><td headers="hd_h_ml246.tu4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Incubate at 37 ºC and 5% CO<sub>2</sub></td></tr><tr><td headers="hd_h_ml246.tu4_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>3</b></td><td headers="hd_h_ml246.tu4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Reagent</td><td headers="hd_h_ml246.tu4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">23 nL</td><td headers="hd_h_ml246.tu4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Compound library, camptothecin as control</td></tr><tr><td headers="hd_h_ml246.tu4_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>4</b></td><td headers="hd_h_ml246.tu4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Time</td><td headers="hd_h_ml246.tu4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">24 hrs</td><td headers="hd_h_ml246.tu4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Incubate at 37 ºC and 5% CO<sub>2</sub></td></tr><tr><td headers="hd_h_ml246.tu4_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>5</b></td><td headers="hd_h_ml246.tu4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Reagent</td><td headers="hd_h_ml246.tu4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">3 μL</td><td headers="hd_h_ml246.tu4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Caspase Glo 3/7</td></tr><tr><td headers="hd_h_ml246.tu4_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>6</b></td><td headers="hd_h_ml246.tu4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Time</td><td headers="hd_h_ml246.tu4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">5 min</td><td headers="hd_h_ml246.tu4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">RT incubation</td></tr><tr><td headers="hd_h_ml246.tu4_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>7</b></td><td headers="hd_h_ml246.tu4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Centrifuge</td><td headers="hd_h_ml246.tu4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">1 min</td><td headers="hd_h_ml246.tu4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">1500 RPM</td></tr><tr><td headers="hd_h_ml246.tu4_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>8</b></td><td headers="hd_h_ml246.tu4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Detector</td><td headers="hd_h_ml246.tu4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Luminescence</td><td headers="hd_h_ml246.tu4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">ViewLux</td></tr></tbody></table></div></div></div><div id="ml246.s10"><h4>PicoGreen Assay</h4><p>The pilot screen for PNC prevalence reduction revealed several small molecules that acted as PNC reducers by intercalating DNA. Therefore, we conducted a DNA intercalation displacement assay on the lead candidates in order to exclude compounds with this mechanism of action. PicoGreen is a DNA intercalator that becomes excitable at 488 nm and emits at 520 nm when bound to DNA. Compounds that displaced the dye decreased fluorescence intensity and were considered DNA intercalators/displacers. In order to rule out potential fluorescence artifacts, a measurement of compound plus DNA only was also calculated.</p><div id="ml246.tu5" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK143533/table/ml246.tu5/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml246.tu5_lrgtbl__"><table><thead><tr><th id="hd_h_ml246.tu5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Sequence</th><th id="hd_h_ml246.tu5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Parameter</th><th id="hd_h_ml246.tu5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Value</th><th id="hd_h_ml246.tu5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Description</th></tr></thead><tbody><tr><td headers="hd_h_ml246.tu5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>1</b></td><td headers="hd_h_ml246.tu5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Reagent</td><td headers="hd_h_ml246.tu5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">2 μL</td><td headers="hd_h_ml246.tu5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Supercoiled plasmid (pBR322) at 200 ng/mL in TE</td></tr><tr><td headers="hd_h_ml246.tu5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>3</b></td><td headers="hd_h_ml246.tu5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Reagent</td><td headers="hd_h_ml246.tu5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">23 nL</td><td headers="hd_h_ml246.tu5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Compound library, camptothecin as control</td></tr><tr><td headers="hd_h_ml246.tu5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>4</b></td><td headers="hd_h_ml246.tu5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Time</td><td headers="hd_h_ml246.tu5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">30 min</td><td headers="hd_h_ml246.tu5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">RT incubation</td></tr><tr><td headers="hd_h_ml246.tu5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>5</b></td><td headers="hd_h_ml246.tu5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Detector</td><td headers="hd_h_ml246.tu5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Fluorescence</td><td headers="hd_h_ml246.tu5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Baseline signal on Envision Ex488/Em520</td></tr><tr><td headers="hd_h_ml246.tu5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>6</b></td><td headers="hd_h_ml246.tu5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Reagent</td><td headers="hd_h_ml246.tu5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">2 μL</td><td headers="hd_h_ml246.tu5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">PicoGreen in TE</td></tr><tr><td headers="hd_h_ml246.tu5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>7</b></td><td headers="hd_h_ml246.tu5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Time</td><td headers="hd_h_ml246.tu5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">10 min</td><td headers="hd_h_ml246.tu5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Incubate at 37 ºC</td></tr><tr><td headers="hd_h_ml246.tu5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>8</b></td><td headers="hd_h_ml246.tu5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Detector</td><td headers="hd_h_ml246.tu5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Fluorescence</td><td headers="hd_h_ml246.tu5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Envision Ex488/Em520</td></tr></tbody></table></div></div></div></div><div id="ml246.s11"><h3>2.2. Probe Chemical Characterization</h3><div id="ml246.fu2" class="figure bk_fig"><div class="graphic"><img src="/books/NBK143533/bin/ml246fu2.jpg" alt="Probe Characterization of ML246." /></div><h3><span class="title">Probe Characterization of ML246</span></h3><div class="caption"><p><sup>*</sup>Purity 100% as determined by LC/MS and <sup>1</sup>H NMR analyses</p></div></div><p><b><i>Trans</i></b><b>-4-(7-benzyl-4-imino-5,6-diphenyl-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-3-yl) cyclohexanol</b>: LC/MS (Agilent system) Retention time t<sub>1</sub> (long) = 2.73 min; <sup>1</sup>H NMR (400MHz, DMSO-<i>d</i><sub><i>6</i></sub>) δ1.66 (m, 4 H), 2.13 (d, <i>J</i> = 8.8 Hz, 4 H), 3.70 (m, 1 H), 5.14 (m, 1 H), 5.28 (s, 2 H), 6.45 (br s, 1 H),6.95 (m, 2 H), 7.04 (d, <i>J</i> = 6.8 Hz, 2 H), 7.18–7.26 (complex, 11 H), 7.80 (s, 1 H); HRMS (ESI) m/z calculated for C<sub>31</sub>H<sub>31</sub>N<sub>4</sub>O [M + H]<sup>+</sup> 475.2498 found 475.2492.</p><div id="ml246.f2" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%202.%20LC%2FMS%20chromatogram%20of%20probe.&p=BOOKS&id=143533_ml246f2.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/NBK143533/bin/ml246f2.jpg" alt="Figure 2. LC/MS chromatogram of probe." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 2</span><span class="title">LC/MS chromatogram of probe</span></h3></div><div id="ml246.f3" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%203.%201H%20NMR%20and%2013C%20APT%20NMR%20spectra%20of%20probe.&p=BOOKS&id=143533_ml246f3.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/NBK143533/bin/ml246f3.jpg" alt="Figure 3. 1H NMR and 13C APT NMR spectra of probe." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 3</span><span class="title"><sup>1</sup>H NMR and <sup>13</sup>C APT NMR spectra of probe</span></h3></div><div id="ml246.f4" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%204.%20A%20Stability%20of%20ML246%20in%20D-PBS%20pH%207.&p=BOOKS&id=143533_ml246f4.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/NBK143533/bin/ml246f4.jpg" alt="Figure 4. A Stability of ML246 in D-PBS pH 7." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 4</span></h3><div class="caption"><p><b>A</b> Stability of <a href="/pcsubstance/?term=ML246[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML246</a> in D-PBS pH 7.4 PBS at room temperature evaluated by LC/MS/MS. <b>B</b>. Stability of <a href="/pcsubstance/?term=ML246[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML246</a> in mouse plasma at room temperature evaluated by LC/MS/MS.</p></div></div><div id="ml246.t1" class="table"><h3><span class="label">Table 1</span><span class="title">Summary of Key Properties for ML246</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK143533/table/ml246.t1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml246.t1_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml246.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Compound</th><th id="hd_h_ml246.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Aqueous Kinetic Solubility (μM)</th><th id="hd_h_ml246.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Stability in PBS buffer (% remaining at 48 hours)</th><th id="hd_h_ml246.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Stability in mouse plasma (% remaining at 2 hours)</th></tr></thead><tbody><tr><td headers="hd_h_ml246.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="/pcsubstance/?term=ML246[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML246</a></td><td headers="hd_h_ml246.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">>150.0</td><td headers="hd_h_ml246.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">98</td><td headers="hd_h_ml246.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">98</td></tr></tbody></table></div></div><div id="ml246.t2" class="table"><h3><span class="label">Table 2</span><span class="title">Probe and analog submissions to the MLSMR (Evotec) for Identification of Compounds that Reduce PNC Prevalence</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK143533/table/ml246.t2/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml246.t2_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml246.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Internal ID</th><th id="hd_h_ml246.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">MLS ID</th><th id="hd_h_ml246.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">SID</th><th id="hd_h_ml246.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">CID</th><th id="hd_h_ml246.t2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">ML#</th><th id="hd_h_ml246.t2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Type</th><th id="hd_h_ml246.t2_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Source</th></tr></thead><tbody><tr><td headers="hd_h_ml246.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00247785</td><td headers="hd_h_ml246.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS003678589</td><td headers="hd_h_ml246.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/117695978" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 117695978</a></td><td headers="hd_h_ml246.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CID 50985821</td><td headers="hd_h_ml246.t2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="/pcsubstance/?term=ML246[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML246</a></td><td headers="hd_h_ml246.t2_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Probe</td><td headers="hd_h_ml246.t2_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Kansas</td></tr><tr><td headers="hd_h_ml246.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00244845</td><td headers="hd_h_ml246.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS003678590</td><td headers="hd_h_ml246.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/116933572" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 116933572</a></td><td headers="hd_h_ml246.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CID 50985817</td><td headers="hd_h_ml246.t2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml246.t2_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Analog</td><td headers="hd_h_ml246.t2_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Kansas</td></tr><tr><td headers="hd_h_ml246.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00247790</td><td headers="hd_h_ml246.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS003678591</td><td headers="hd_h_ml246.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/117695983" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 117695983</a></td><td headers="hd_h_ml246.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CID 51035471</td><td headers="hd_h_ml246.t2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml246.t2_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Analog</td><td headers="hd_h_ml246.t2_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Kansas</td></tr><tr><td headers="hd_h_ml246.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS000556915</td><td headers="hd_h_ml246.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS003678592</td><td headers="hd_h_ml246.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/113635282" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 113635282</a></td><td headers="hd_h_ml246.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CID 5152963</td><td headers="hd_h_ml246.t2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml246.t2_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Analog</td><td headers="hd_h_ml246.t2_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Kansas</td></tr><tr><td headers="hd_h_ml246.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00247775</td><td headers="hd_h_ml246.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS003678593</td><td headers="hd_h_ml246.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/117695968" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 117695968</a></td><td headers="hd_h_ml246.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CID 51035453</td><td headers="hd_h_ml246.t2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml246.t2_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Analog</td><td headers="hd_h_ml246.t2_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Kansas</td></tr><tr><td headers="hd_h_ml246.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00244849</td><td headers="hd_h_ml246.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS003678594</td><td headers="hd_h_ml246.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/116933577" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 116933577</a></td><td headers="hd_h_ml246.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CID 50985820</td><td headers="hd_h_ml246.t2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml246.t2_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Analog</td><td headers="hd_h_ml246.t2_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Kansas</td></tr></tbody></table></div></div><div id="ml246.f5" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%205.%20Structures%20of%20compounds%20submitted%20to%20the%20MLSMR%20compound%20repository.&p=BOOKS&id=143533_ml246f5.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/NBK143533/bin/ml246f5.jpg" alt="Figure 5. Structures of compounds submitted to the MLSMR compound repository." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 5</span><span class="title">Structures of compounds submitted to the MLSMR compound repository</span></h3></div></div><div id="ml246.s12"><h3>2.3. Probe Preparation</h3><div id="ml246.f10" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Scheme%201.%20Reagents%20and%20conditions.&p=BOOKS&id=143533_ml246f10.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/NBK143533/bin/ml246f10.jpg" alt="Scheme 1. Reagents and conditions." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Scheme 1</span><span class="title">Reagents and conditions</span></h3><div class="caption"><p>(a) benzyl amine, zinc chloride (cat), neat; (b) malononitrile, DMF (46%, two steps); (c) triethylorthoformate, neat (64%); (d) <i>trans</i>-4-aminocyclohexanol hydrochloride, MeOH (34%).</p></div></div><div id="ml246.fu3" class="figure bk_fig"><div class="graphic"><img src="/books/NBK143533/bin/ml246fu3.jpg" alt="Preparation of trans-4-(7-benzyl-4-imino-5,6-diphenyl-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-3-yl) cyclohexanol (ML246)." /></div><h3><span class="title">Preparation of t<i>rans</i>-4-(7-benzyl-4-imino-5,6-diphenyl-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-3-yl) cyclohexanol (ML246)</span></h3></div><p><b>2-Amino-1-benzyl-4,5-diphenyl-1H-pyrrole-3-carbonitrile (C)</b>. A modified Voigt reaction/Knoevenagel condensation sequence was carried out using the procedure of Roth and Eger.[<a class="bk_pop" href="#ml246.r5">5</a>] Thus, benzoin <b>A</b> (2.19 g, 10.3 mmol), benzylamine (1.66 g, 15.5 mmol, 1.5 equiv.), and zinc chloride (0.10 g, 0.73 mmol, 0.07 equiv.) were heated at reflux for 3 hours and the mixture was removed from the oil bath. Malononitrile (1.35 g, 20.6 mmol, 2.0 equiv.) in DMF (3 mL) was added to the mixture, which was still warm. The reaction mixture was allowed to cool to room temperature and stirred for 16 hrs, affording the crude pyrrole as a dark brown solid. The solid was partitioned between water and CH<sub>2</sub>Cl<sub>2</sub>, and the aqueous layer was extracted with additional CH<sub>2</sub>Cl<sub>2</sub> (2×50 mL). The combined organics were dried with Na<sub>2</sub>SO<sub>4</sub>, and the solvent removed <i>in vacuo</i> to afford the previously reported pyrrole product <b>C</b>[<a class="bk_pop" href="#ml246.r6">6</a>] as a light brown solid (1.67 g, 4.78 mmol, 46% yield), which was used without further purification.</p><div id="ml246.fu4" class="figure"><div class="graphic"><img src="/books/NBK143533/bin/ml246fu4.jpg" alt="Image ml246fu4" /></div></div><p><b>(E)-Ethyl N-(1-benzyl-3-cyano-4,5-diphenyl-1H-pyrrol-2-yl)formimidate (D)</b>. 2-Amino-1-benzyl-4,5-diphenyl-1H-pyrrole-3-carbonitrile <b>C</b> (1.07 g, 3.06 mmol) and triethylorthoformate (4.54 g, 30.6 mmol, 10 equiv.) were heated at 75 °C for 14 hrs, and the excess triethylorthoformate was removed <i>in vacuo</i>. The residue was dissolved in a minimum of CH<sub>2</sub>Cl<sub>2</sub>, adsorbed onto celite, and chromatographed on silica to afford the previously reported formimidate product[<a class="bk_pop" href="#ml246.r6">6</a>]<b>D</b> as a tan solid (0.80 g, 1.97 mmol, 64% yield).</p><div id="ml246.fu5" class="figure"><div class="graphic"><img src="/books/NBK143533/bin/ml246fu5.jpg" alt="Image ml246fu5" /></div></div><p><b><i>trans</i></b><b>-4-(7-Benzyl-4-imino-5,6-diphenyl-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-3-yl)cyclohexanol (CID 5098582, <a href="/pcsubstance/?term=ML246[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML246</a>)</b>. A solution of the formimidate <b>D</b> (40 mg, 0.099 mmol) and <i>trans</i>-4-aminocyclohexanol hydrochloride (23 mg, 0.15 mmol, 1.5 equiv) in MeOH (1.5 mL) was heated in a reaction vial at 60 ºC for 17 hrs, and then cooled to room temperature. Evaporation of the solvent and purification of the residue by mass-directed preparative reverse-phase HPLC afforded the pyrolopyrimidine product, <a href="/pcsubstance/?term=ML246[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML246</a>, as a tan solid (16 mg, 0.034 mmol, 34% yield).</p></div></div><div id="ml246.s13"><h2 id="_ml246_s13_">3. Results</h2><div id="ml246.s14"><h3>3.1. Dose Response Curves for Probe</h3><div id="ml246.f6" class="figure bk_fig"><div class="graphic"><img src="/books/NBK143533/bin/ml246f6.jpg" alt="Figure 6. PNC reduction dose response curve for ML246." /></div><h3><span class="label">Figure 6</span><span class="title">PNC reduction dose response curve for ML246</span></h3></div></div><div id="ml246.s15"><h3>3.2. Cellular Activity</h3><p>After activity optimization in the PNC assay, it was necessary to examine if these compounds had an effect on migration. <a class="figpopup" href="/books/NBK143533/table/ml246.t3/?report=objectonly" target="object" rid-figpopup="figml246t3" rid-ob="figobml246t3">Table 3</a> summarizes preliminary results obtained in an invasion assay with <a href="/pcsubstance/?term=ML246[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML246</a>. An examination of the number of cells that have invaded through the 3D collagen channel reflects their migratory aptitude. Gratifyingly, the novel PNC-reducer probe <a href="/pcsubstance/?term=ML246[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML246</a> showed dose dependent inhibition of migration with IC<sub>50</sub>s 3–4 μM. The numbers of cells in the output port after the assay duration were also counted as a measurement of proliferation. Here too, the probe showed dose-dependent inhibition activity, suggesting that the anti-invasive effect of the compound is concomitant with an anti-proliferative effect. With this data in hand, we measured the cytotoxicity of the probe (via the ATP viability assay, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/588740" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 588740</a>) to be 9,960 nM. The ATP data and the pattern of activity demonstrated in the NCI-60 panel (<i>vide infra</i>, <a class="figpopup" href="/books/NBK143533/figure/ml246.f9/?report=objectonly" target="object" rid-figpopup="figml246f9" rid-ob="figobml246f9">Figure 9</a>) lead us to conclude that the cell proliferation modulation, especially at the concentrations causing PNC reduction, is mostly likely due to growth inhibition and not cytotoxicity.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml246t3"><a href="/books/NBK143533/table/ml246.t3/?report=objectonly" target="object" title="Table 3" class="img_link icnblk_img figpopup" rid-figpopup="figml246t3" rid-ob="figobml246t3"><img class="small-thumb" src="/books/NBK143533/table/ml246.t3/?report=thumb" src-large="/books/NBK143533/table/ml246.t3/?report=previmg" alt="Table 3. BellBrook Labs Proliferation and Migration Data for Key Compounds." /></a><div class="icnblk_cntnt"><h4 id="ml246.t3"><a href="/books/NBK143533/table/ml246.t3/?report=objectonly" target="object" rid-ob="figobml246t3">Table 3</a></h4><p class="float-caption no_bottom_margin">BellBrook Labs Proliferation and Migration Data for Key Compounds. </p></div></div><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml246f9" co-legend-rid="figlgndml246f9"><a href="/books/NBK143533/figure/ml246.f9/?report=objectonly" target="object" title="Figure 9" class="img_link icnblk_img figpopup" rid-figpopup="figml246f9" rid-ob="figobml246f9"><img class="small-thumb" src="/books/NBK143533/bin/ml246f9.gif" src-large="/books/NBK143533/bin/ml246f9.jpg" alt="Figure 9. NCI-60 panel: Dose response growth inhibition curves for ML246 in 60 cancer cell lines." /></a><div class="icnblk_cntnt" id="figlgndml246f9"><h4 id="ml246.f9"><a href="/books/NBK143533/figure/ml246.f9/?report=objectonly" target="object" rid-ob="figobml246f9">Figure 9</a></h4><p class="float-caption no_bottom_margin">NCI-60 panel: Dose response growth inhibition curves for ML246 in 60 cancer cell lines. The curves are grouped according to the type of cancer. Concentrations able to produce a percentage of growth below zero indicate loss of cell density and can be correlated <a href="/books/NBK143533/figure/ml246.f9/?report=objectonly" target="object" rid-ob="figobml246f9">(more...)</a></p></div></div><p>We also wanted to test the ability of the chemical series to affect anchorage independent growth
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in a soft agar assay, a stringent method to detect malignant transformation of cells <i>in
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vitro</i>. To that end, <a class="figpopup" href="/books/NBK143533/figure/ml246.f8/?report=objectonly" target="object" rid-figpopup="figml246f8" rid-ob="figobml246f8">Figure 8</a> depicts our initial
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assessment of <a href="/pcsubstance/?term=ML246[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML246</a> in such an assay with PC3M cells. As shown in panel 8A,
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<a href="/pcsubstance/?term=ML246[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML246</a>
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demonstrates a dose dependent reduction in the number of colonies after 14 days at very low concentrations (3.8, 18.6 nM), with no impact on cell viability. Thus, the probe exhibits potent inhibition of anchorage independent growth in PC3M cells. <a class="figpopup" href="/books/NBK143533/figure/ml246.f8/?report=objectonly" target="object" rid-figpopup="figml246f8" rid-ob="figobml246f8">Figure 8B</a> shows two images of representative colonies after treatment at the relevant concentrations; a clear reduction if the number of PC-3M cell colonies can be seen especially at 18.6 nM.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml246f8" co-legend-rid="figlgndml246f8"><a href="/books/NBK143533/figure/ml246.f8/?report=objectonly" target="object" title="Figure 8" class="img_link icnblk_img figpopup" rid-figpopup="figml246f8" rid-ob="figobml246f8"><img class="small-thumb" src="/books/NBK143533/bin/ml246f8.gif" src-large="/books/NBK143533/bin/ml246f8.jpg" alt="Figure 8. A: Histogram representing number and size (μm) of PC3M soft agar colonies after 14 days treatment with ML246 at 3." /></a><div class="icnblk_cntnt" id="figlgndml246f8"><h4 id="ml246.f8"><a href="/books/NBK143533/figure/ml246.f8/?report=objectonly" target="object" rid-ob="figobml246f8">Figure 8</a></h4><p class="float-caption no_bottom_margin"><i>A</i>: Histogram representing number and size (μm) of PC3M soft agar colonies after 14 days treatment with ML246 at 3.8 and 18.6 nM. <i>B:</i> Representative images of colonies at these concentrations. </p></div></div></div><div id="ml246.s16"><h3>3.3. Profiling Assays</h3><p><a href="/pcsubstance/?term=ML246[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML246</a>
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is a novel chemical entity and has no previously disclosed interactions with biological targets. The
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probe has been screened against 44 GPCR and CNS relevant targets in the PDSP comprehensive binding panel (<a class="figpopup" href="/books/NBK143533/table/ml246.t4/?report=objectonly" target="object" rid-figpopup="figml246t4" rid-ob="figobml246t4">Table 4</a>). With the exception of the sigma 2 receptor, <a href="/pcsubstance/?term=ML246[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML246</a> was mostly inactive against all these targets. Notably, this includes dopaminergic activity, as the original hit (<b>8</b>) was found to be an allosteric D1 agonist in PubChem <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504660" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504660</a>.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml246t4"><a href="/books/NBK143533/table/ml246.t4/?report=objectonly" target="object" title="Table 4" class="img_link icnblk_img figpopup" rid-figpopup="figml246t4" rid-ob="figobml246t4"><img class="small-thumb" src="/books/NBK143533/table/ml246.t4/?report=thumb" src-large="/books/NBK143533/table/ml246.t4/?report=previmg" alt="Table 4. Profiling of the probe, ML246, in the PDSP comprehensive panel." /></a><div class="icnblk_cntnt"><h4 id="ml246.t4"><a href="/books/NBK143533/table/ml246.t4/?report=objectonly" target="object" rid-ob="figobml246t4">Table 4</a></h4><p class="float-caption no_bottom_margin">Profiling of the probe, ML246, in the PDSP comprehensive panel. </p></div></div><p><a href="/pcsubstance/?term=ML246[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML246</a> has good stability in PBS buffer. It also has good stability in mouse plasma and is not rapidly metabolized by mouse liver microsomes in the presence of NADPH (<a class="figpopup" href="/books/NBK143533/table/ml246.t5/?report=objectonly" target="object" rid-figpopup="figml246t5" rid-ob="figobml246t5">Table 5</a>). At a 50 mpk high dose, the probe provided significant <i>in vivo</i> exposure in mice, with a C<sub>max</sub> of 20.3 μM @ 30 min and a C<sub>last</sub> of 1.3 μM @ 48 hrs. Some mortality was observed (3/18), but this is expected, as the exposure is well beyond the expected therapeutic levels. Subsequent experiments with 25 mpk intraperitoneal (IP) dosing provided very good exposure without any sign of toxicity (<a class="figpopup" href="/books/NBK143533/table/ml246.t6/?report=objectonly" target="object" rid-figpopup="figml246t6" rid-ob="figobml246t6">Table 6</a>).</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml246t5"><a href="/books/NBK143533/table/ml246.t5/?report=objectonly" target="object" title="Table 5" class="img_link icnblk_img figpopup" rid-figpopup="figml246t5" rid-ob="figobml246t5"><img class="small-thumb" src="/books/NBK143533/table/ml246.t5/?report=thumb" src-large="/books/NBK143533/table/ml246.t5/?report=previmg" alt="Table 5. In vitro ADME data for ML246." /></a><div class="icnblk_cntnt"><h4 id="ml246.t5"><a href="/books/NBK143533/table/ml246.t5/?report=objectonly" target="object" rid-ob="figobml246t5">Table 5</a></h4><p class="float-caption no_bottom_margin"><i>In vitro</i> ADME data for ML246. </p></div></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml246t6"><a href="/books/NBK143533/table/ml246.t6/?report=objectonly" target="object" title="Table 6" class="img_link icnblk_img figpopup" rid-figpopup="figml246t6" rid-ob="figobml246t6"><img class="small-thumb" src="/books/NBK143533/table/ml246.t6/?report=thumb" src-large="/books/NBK143533/table/ml246.t6/?report=previmg" alt="Table 6. Comparison of Mouse Pharmacokinetics Parameters of Probe after IP Dosing of 50 or 25 mpk in Male C57BL/6 Mice." /></a><div class="icnblk_cntnt"><h4 id="ml246.t6"><a href="/books/NBK143533/table/ml246.t6/?report=objectonly" target="object" rid-ob="figobml246t6">Table 6</a></h4><p class="float-caption no_bottom_margin">Comparison of Mouse Pharmacokinetics Parameters of Probe after IP Dosing of 50 or 25 mpk in
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Male C57BL/6 Mice. </p></div></div><p>The NCI-60 panel is a collection of human cancerous cell lines used by the National Cancer Institute as a profiling tool for the development and screening of novel anticancer drugs.[<a class="bk_pop" href="#ml246.r5">5</a>] This panel allows the evaluation of compounds in 60 cell lines of various origins and compares the pattern of activity with other known anticancer agents to impute a possible mechanism of action. Compounds are tested at a single concentration (10 μM) first, and if a growth was below 40% in at least eight cell lines, they are sent for dose-response testing. The dose response curves for <a href="/pcsubstance/?term=ML246[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML246</a> are displayed in <a class="figpopup" href="/books/NBK143533/figure/ml246.f9/?report=objectonly" target="object" rid-figpopup="figml246f9" rid-ob="figobml246f9">Figure 9</a>.</p><p>In general, we observed potent dose dependent growth inhibition ≥1 μM concentration across all cell lines. At the highest concentration (100 μM), we observed cell death (as represented by curves below 0% percentage growth) in all cell lines. Thus, the chemical series appears to exhibit growth inhibitory effects at concentrations that cause reduction of the PNC. Fortunately, cytotoxicity is observed only at higher concentrations, and it should be possible to decouple this effect from PNC disruption with proper dosage.</p></div></div><div id="ml246.s17"><h2 id="_ml246_s17_">4. Discussion</h2><div id="ml246.s18"><h3>4.1. Comparison to Existing Art and How the New Probe is an Improvement</h3><p>Existing PNC reducers are chemotherapy drugs that induce PNC disruption by known cytotoxic mechanisms, like topoisomerase or polymerase III inhibition; or DNA intercalation. Drugs used against metastatic cancers also operate through such cytotoxic mechanisms. Hence, we chose to develop PNC reducers that did not show activity in assays which measured cytotoxicity via these mechanisms. Extensive SAR explorations yielded a probe molecule that is able to reduce the PNC population in PC3M cells at submicromolar concentrations with cytotoxicity observed at ~10μM (25-fold selectivity). The probe shows ~10-fold selectivity between growth inhibition and cytotoxicity in the NCI-60 panel. Thus the chemical series represents first-in-class molecules that reduce the prevalence of the PNC and do not operate via known cytotoxic mechanisms.</p></div></div><div id="ml246.s19"><h2 id="_ml246_s19_">5. References</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="ml246.r1">Kamath RV, Thor AD, Wang C, Edgerton SM, Slusarczyk A, Leary DJ, Wang J, Wiley EL, Jovanovic B, Wu Q, Nayar R, Kovarik P, Shi F, Huang S. <span><span class="ref-journal">Cancer Res. </span>2005;<span class="ref-vol">65</span>(1):246–53.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/15665301" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 15665301</span></a>]</div></dd><dt>2.</dt><dd><div class="bk_ref" id="ml246.r2">Slusarczyk A, Kamath R, Wang C, Anchel D, Pollock C, Lewandowska MA, Fitzpatrick T, Bazett-Jones DP, Huang S. <span><span class="ref-journal">Cold Spring Harb Symp Quant Biol. </span>2010;<span class="ref-vol">75</span>:599–605.</span> [<a href="/pmc/articles/PMC4374480/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC4374480</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/21289045" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 21289045</span></a>]</div></dd><dt>3.</dt><dd><div class="bk_ref" id="ml246.r3">Kamath RV, Leary DJ, Huang S. <span><span class="ref-journal">Mol Biol Cell. </span>2001;<span class="ref-vol">12</span>:3808–20.</span> [<a href="/pmc/articles/PMC60757/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC60757</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/11739782" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 11739782</span></a>]</div></dd><dt>4.</dt><dd><div class="bk_ref" id="ml246.r4"><a href="http://www.bellbrooklabs.com/PDFs/Poster_3Dinvasion_LabAuto_Jan2011.pdf" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">http://www<wbr style="display:inline-block"></wbr>.bellbrooklabs<wbr style="display:inline-block"></wbr>.com/PDFs/Poster<wbr style="display:inline-block"></wbr>_3Dinvasion_LabAuto_Jan2011.pdf</a></div></dd><dt>5.</dt><dd><div class="bk_ref" id="ml246.r5">Roth HJ, Eger K. <span><span class="ref-journal">Arch. Pharmaz. </span>1975;<span class="ref-vol">308</span>:179–185.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/1130955" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 1130955</span></a>]</div></dd><dt>6.</dt><dd><div class="bk_ref" id="ml246.r6">Mohamed MS, Rashad AE, Zaki MEA, Fatahala SS. <span><span class="ref-journal">Acta Pharm. </span>2005;<span class="ref-vol">55</span>:237–249.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/16375835" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 16375835</span></a>]</div></dd><dt>7.</dt><dd><div class="bk_ref" id="ml246.r7"> [Last accessed 10/25/2011]. <a href="http://dtp.nci.nih.gov/branches/btb/ivclsp.html" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">http://dtp<wbr style="display:inline-block"></wbr>.nci.nih.gov<wbr style="display:inline-block"></wbr>/branches/btb/ivclsp.html</a>.</div></dd></dl></div><div id="bk_toc_contnr"></div></div></div>
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<div class="post-content"><div><div class="half_rhythm"><a href="/books/about/copyright/">Copyright Notice</a></div><div class="small"><span class="label">Bookshelf ID: NBK143533</span><span class="label">PMID: <a href="https://pubmed.ncbi.nlm.nih.gov/23762948" title="PubMed record of this page" ref="pagearea=meta&targetsite=entrez&targetcat=link&targettype=pubmed">23762948</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/ml247/" title="Previous page in this title">< Prev</a><a class="active page_link next" href="/books/n/mlprobe/ml245/" title="Next page in this title">Next ></a></div></div></div></div>
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<div xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance"></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Views</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="PDF_download" id="Shutter"></a></div><div class="portlet_content"><ul xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="simple-list"><li><a href="/books/NBK143533/?report=reader">PubReader</a></li><li><a href="/books/NBK143533/?report=printable">Print View</a></li><li><a data-jig="ncbidialog" href="#_ncbi_dlg_citbx_NBK143533" data-jigconfig="width:400,modal:true">Cite this Page</a><div id="_ncbi_dlg_citbx_NBK143533" style="display:none" title="Cite this Page"><div class="bk_tt">Frankowski K, Patnaik S, Schoenen F, et al. Discovery and Development of Small Molecules That Reduce PNC Prevalence. 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="#ml246.s1" ref="log$=inpage&link_id=inpage">Probe Structure & Characteristics</a></li><li><a href="#ml246.s2" ref="log$=inpage&link_id=inpage">Recommendations for Scientific Use of the Probe</a></li><li><a href="#ml246.s3" ref="log$=inpage&link_id=inpage">Materials and Methods</a></li><li><a href="#ml246.s13" ref="log$=inpage&link_id=inpage">Results</a></li><li><a href="#ml246.s17" ref="log$=inpage&link_id=inpage">Discussion</a></li><li><a href="#ml246.s19" ref="log$=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=3036108" ref="log$=recordlinks">PMC</a><div class="brieflinkpop offscreen_noflow">PubMed Central citations</div></li><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pcassay&DbFrom=books&Cmd=Link&LinkName=books_pcassay_probe&IdsFromResult=3036108" ref="log$=recordlinks">PubChem BioAssay for Chemical Probe</a><div class="brieflinkpop offscreen_noflow">PubChem BioAssay records reporting screening data for the development of the chemical probe(s) described in this book chapter</div></li><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pcsubstance&DbFrom=books&Cmd=Link&LinkName=books_pcsubstance&IdsFromResult=3036108" ref="log$=recordlinks">PubChem Substance</a><div class="brieflinkpop offscreen_noflow">Related PubChem Substances</div></li><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pubmed&DbFrom=books&Cmd=Link&LinkName=books_pubmed_refs&IdsFromResult=3036108" ref="log$=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/35696646" ref="ordinalpos=1&linkpos=1&log$=relatedarticles&logdbfrom=pubmed">Discovery and Optimization of Pyrrolopyrimidine Derivatives as Selective Disruptors of the Perinucleolar Compartment, a Marker of Tumor Progression toward Metastasis.</a><span class="source">[J Med Chem. 2022]</span><div class="brieflinkpop offscreen_noflow">Discovery and Optimization of Pyrrolopyrimidine Derivatives as Selective Disruptors of the Perinucleolar Compartment, a Marker of Tumor Progression toward Metastasis.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Frankowski KJ, Patnaik S, Wang C, Southall N, Dutta D, De S, Li D, Dextras C, Lin YH, Bryant-Connah M, 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. 2022 Jun 23; 65(12):8303-8331. 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