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<script type="text/javascript" src="/corehtml/pmc/jatsreader/ptpmc_3.22/js/jr.boots.min.js"> </script><title>A high-throughput screen for pre-mRNA splicing modulators - Probe Reports from the NIH Molecular Libraries Program - NCBI Bookshelf</title>
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<meta name="citation_inbook_title" content="Probe Reports from the NIH Molecular Libraries Program [Internet]">
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<meta name="citation_title" content="A high-throughput screen for pre-mRNA splicing modulators">
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<meta name="citation_publisher" content="National Center for Biotechnology Information (US)">
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<meta name="citation_date" content="2010/09/02">
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<meta name="citation_author" content="Douglas Auld">
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<meta name="citation_author" content="Min Shen">
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<meta name="citation_author" content="Craig Thomas">
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<meta name="citation_pmid" content="21433360">
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<meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK47344/">
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<meta name="DC.Title" content="A high-throughput screen for pre-mRNA splicing modulators">
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<meta name="DC.Publisher" content="National Center for Biotechnology Information (US)">
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<meta name="DC.Contributor" content="Douglas Auld">
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<meta name="DC.Contributor" content="Min Shen">
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<meta name="DC.Contributor" content="Craig Thomas">
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<meta name="DC.Date" content="2010/09/02">
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<meta name="description" content="The premature aging disease Hutchinson-Gilford Progeria Syndrome (HGPS) is caused by a point mutation in the lamin A/C (LMNA) gene, leading to the activation of an exonic cryptic splice site. This study proposed to use this aberrant splicing event as a model system to identify small molecule modulators of alternative pre-mRNA splicing to: (1) identify specific modulators of LMNA pre-mRNA splicing as lead compounds in the search for drugs with therapeutic potential in HGPS; and (2) identify general pre-mRNA splicing inhibitors, as useful experimental tools in the study of pre-mRNA splicing mechanisms. Successful execution of this screen will represent the first unbiased identification of modulators of pre-mRNA splicing, and will provide proof of principle for the application of HTS approaches to the field of pre-mRNA splicing. Among the more interesting chemotypes uncovered by this screen was a series of substituted quinazolines. It was posited that these agents might alter pre-mRNA splicing via inhibition of a class of kinases known as the cdc2-like kinases (Clk). The Clk family is known to alter the actions of the spliceosome by phosphorylating the serine-arginine rich (SR) proteins that are critical elements of spliceosome assembly. These agents were found to be inhibitors of Clk1 and Clk4 and optimization efforts yielded agents with low-nanomolar potency and high selectivity for this class of kinases. The results of these efforts form the basis for the naming of ML106 (CID-3232621) and ML105 (CID-3234998) as probes of Clk4.">
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<meta name="og:title" content="A high-throughput screen for pre-mRNA splicing modulators">
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<meta name="og:description" content="The premature aging disease Hutchinson-Gilford Progeria Syndrome (HGPS) is caused by a point mutation in the lamin A/C (LMNA) gene, leading to the activation of an exonic cryptic splice site. This study proposed to use this aberrant splicing event as a model system to identify small molecule modulators of alternative pre-mRNA splicing to: (1) identify specific modulators of LMNA pre-mRNA splicing as lead compounds in the search for drugs with therapeutic potential in HGPS; and (2) identify general pre-mRNA splicing inhibitors, as useful experimental tools in the study of pre-mRNA splicing mechanisms. Successful execution of this screen will represent the first unbiased identification of modulators of pre-mRNA splicing, and will provide proof of principle for the application of HTS approaches to the field of pre-mRNA splicing. Among the more interesting chemotypes uncovered by this screen was a series of substituted quinazolines. It was posited that these agents might alter pre-mRNA splicing via inhibition of a class of kinases known as the cdc2-like kinases (Clk). The Clk family is known to alter the actions of the spliceosome by phosphorylating the serine-arginine rich (SR) proteins that are critical elements of spliceosome assembly. These agents were found to be inhibitors of Clk1 and Clk4 and optimization efforts yielded agents with low-nanomolar potency and high selectivity for this class of kinases. The results of these efforts form the basis for the naming of ML106 (CID-3232621) and ML105 (CID-3234998) as probes of Clk4.">
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id="jr-fip-info-p"><a id="jr-fip-prev" class="wsprkl btn" title="Jump to previuos match">◀</a><button id="jr-fip-matches">no matches yet</button><a id="jr-fip-next" class="wsprkl btn" title="Jump to next match">▶</a></nav></nav></div><div id="jr-epub-interstitial" class="hidden"></div><div id="jr-content"><article data-type="main"><div class="main-content lit-style" itemscope="itemscope" itemtype="http://schema.org/CreativeWork"><div class="meta-content fm-sec"><div class="fm-sec"><h1 id="_NBK47344_"><span class="title" itemprop="name">A high-throughput screen for pre-mRNA splicing modulators</span></h1><p class="contribs">Auld D, Shen M, Thomas C.</p><p class="fm-aai"><a href="#_NBK47344_pubdet_">Publication Details</a></p></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 premature aging disease Hutchinson-Gilford Progeria Syndrome (HGPS) is caused by a point mutation in the lamin A/C (LMNA) gene, leading to the activation of an exonic cryptic splice site. This study proposed to use this aberrant splicing event as a model system to identify small molecule modulators of alternative pre-mRNA splicing to: (1) identify specific modulators of LMNA pre-mRNA splicing as lead compounds in the search for drugs with therapeutic potential in HGPS; and (2) identify general pre-mRNA splicing inhibitors, as useful experimental tools in the study of pre-mRNA splicing mechanisms. Successful execution of this screen will represent the first unbiased identification of modulators of pre-mRNA splicing, and will provide proof of principle for the application of HTS approaches to the field of pre-mRNA splicing. Among the more interesting chemotypes uncovered by this screen was a series of substituted quinazolines. It was posited that these agents might alter pre-mRNA splicing via inhibition of a class of kinases known as the cdc2-like kinases (Clk). The Clk family is known to alter the actions of the spliceosome by phosphorylating the serine-arginine rich (SR) proteins that are critical elements of spliceosome assembly. These agents were found to be inhibitors of Clk1 and Clk4 and optimization efforts yielded agents with low-nanomolar potency and high selectivity for this class of kinases. The results of these efforts form the basis for the naming of ML106 (CID-3232621) and ML105 (CID-3234998) as probes of Clk4.</p></div><div class="h2"></div><p><b>Assigned Assay Grant #:</b> 1 R03 MH084827-01</p><p><b>Screening Center Name & PI:</b> NIH Chemical Genomics Center, Christopher Austin</p><p><b>Chemistry Center Name & PI:</b> NIH Chemical Genomics Center, Christopher Austin</p><p><b>Assay Submitter & Institution:</b> Dr. Tom Misteli, NCI, NIH</p><p><b>PubChem Summary Bioassay Identifier (AID):</b>
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<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1997" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1997</a></p><div id="ml106.s1"><h2 id="_ml106_s1_">Probe Structure & Characteristics: <a href="/pcsubstance/?term=ML106[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML106</a></h2><div id="ml106.fu1" class="figure"><div class="graphic"><img src="/books/NBK47344/bin/ml106fu1.jpg" alt="Image ml106fu1" /></div></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml106tu1"><a href="/books/NBK47344/table/ml106.tu1/?report=objectonly" target="object" title="Table" class="img_link icnblk_img figpopup" rid-figpopup="figml106tu1" rid-ob="figobml106tu1"><img class="small-thumb" src="/books/NBK47344/table/ml106.tu1/?report=thumb" src-large="/books/NBK47344/table/ml106.tu1/?report=previmg" alt="Image " /></a><div class="icnblk_cntnt"><h4 id="ml106.tu1"><a href="/books/NBK47344/table/ml106.tu1/?report=objectonly" target="object" rid-ob="figobml106tu1">Table</a></h4></div></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml106tu2"><a href="/books/NBK47344/table/ml106.tu2/?report=objectonly" target="object" title="Table" class="img_link icnblk_img figpopup" rid-figpopup="figml106tu2" rid-ob="figobml106tu2"><img class="small-thumb" src="/books/NBK47344/table/ml106.tu2/?report=thumb" src-large="/books/NBK47344/table/ml106.tu2/?report=previmg" alt="Image " /></a><div class="icnblk_cntnt"><h4 id="ml106.tu2"><a href="/books/NBK47344/table/ml106.tu2/?report=objectonly" target="object" rid-ob="figobml106tu2">Table</a></h4></div></div><p>Note: Throughout this probe report, MLSMR and NCGC identification numbers are used to identify compounds. A complete listing of PubChem CIDs can be found on page 21 (<a class="figpopup" href="/books/NBK47344/table/ml106.t1/?report=objectonly" target="object" rid-figpopup="figml106t1" rid-ob="figobml106t1">Table 2</a>).</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml106t1"><a href="/books/NBK47344/table/ml106.t1/?report=objectonly" target="object" title="Table 2" class="img_link icnblk_img figpopup" rid-figpopup="figml106t1" rid-ob="figobml106t1"><img class="small-thumb" src="/books/NBK47344/table/ml106.t1/?report=thumb" src-large="/books/NBK47344/table/ml106.t1/?report=previmg" alt="Table 2. Compound identification numbers." /></a><div class="icnblk_cntnt"><h4 id="ml106.t1"><a href="/books/NBK47344/table/ml106.t1/?report=objectonly" target="object" rid-ob="figobml106t1">Table 2</a></h4><p class="float-caption no_bottom_margin">Compound identification numbers. </p></div></div></div><div id="ml106.s3"><h2 id="_ml106_s3_">Recommendations for the scientific use of this probe</h2><p>The probe is a member of a series of inhibitors for Clk4 (CDC-like kinase 4). This compound can be used to study the implications of Clk4 in gene splicing events and other cellular events.</p></div><div id="ml106.s4"><h2 id="_ml106_s4_">Probe Structure & Characteristics: <a href="/pcsubstance/?term=ML105[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML105</a></h2><div id="ml106.fu2" class="figure"><div class="graphic"><img src="/books/NBK47344/bin/ml106fu2.jpg" alt="Image ml106fu2" /></div></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml106tu3"><a href="/books/NBK47344/table/ml106.tu3/?report=objectonly" target="object" title="Table" class="img_link icnblk_img figpopup" rid-figpopup="figml106tu3" rid-ob="figobml106tu3"><img class="small-thumb" src="/books/NBK47344/table/ml106.tu3/?report=thumb" src-large="/books/NBK47344/table/ml106.tu3/?report=previmg" alt="Image " /></a><div class="icnblk_cntnt"><h4 id="ml106.tu3"><a href="/books/NBK47344/table/ml106.tu3/?report=objectonly" target="object" rid-ob="figobml106tu3">Table</a></h4></div></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml106tu4"><a href="/books/NBK47344/table/ml106.tu4/?report=objectonly" target="object" title="Table" class="img_link icnblk_img figpopup" rid-figpopup="figml106tu4" rid-ob="figobml106tu4"><img class="small-thumb" src="/books/NBK47344/table/ml106.tu4/?report=thumb" src-large="/books/NBK47344/table/ml106.tu4/?report=previmg" alt="Image " /></a><div class="icnblk_cntnt"><h4 id="ml106.tu4"><a href="/books/NBK47344/table/ml106.tu4/?report=objectonly" target="object" rid-ob="figobml106tu4">Table</a></h4></div></div></div><div id="ml106.s6"><h2 id="_ml106_s6_">Recommendations for the scientific use of this probe</h2><p>The probe is a member of a series of inhibitors for Clk4 (CDC-like kinase 4). This compound can
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be used to study the implications of Clk4 in gene splicing events and other cellular
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events.</p></div><div id="ml106.s7"><h2 id="_ml106_s7_">The Role of Clk4 in alternate splicing</h2><div id="ml106.s8"><h3>Specific Aim</h3><p>The premature aging disease Hutchinson-Gilford Progeria Syndrome (HGPS) is caused by a point mutation in the lamin A/C (LMNA) gene, leading to the activation of an exonic cryptic splice site. We will use this aberrant splicing event as a model system to identify small molecule modulators of alternative pre-mRNA splicing.</p><p>In this effort, we have aimed to 1) identify specific modulators of LMNA pre-mRNA splicing; these compounds are leads in the search for drugs with therapeutic potential in HGPS and 2) identify general pre-mRNA splicing inhibitors; these compounds will be useful as experimental tools in the study of pre-mRNA splicing mechanisms.</p><p>To this end, we will screen compounds in Molecular Libraries Small Molecule Repository (MLSMR) available at the NIH Chemical Genomics Center. Active compounds will be subjected to a variety of secondary assays, and will be directly tested for their potency in reversing HGPS cellular phenotypes in patient cells. Successful execution of this screen will represent the first unbiased identification of modulators of pre-mRNA splicing, and will provide proof of principle for the application of HTS approaches to the field of pre-mRNA splicing.</p><p>Currently, oligonucleotides have been used that are chemically modified so as to evade the RNAse H-dependent degradation pathway, which gives rise to the traditional antisense response. Commonly used chemical modifications include 2-O-methyl, 2′-O-methoxyethyl, phosphoramidate, PNAs (protein nucleic acids) or morpholino chemistry, and these chemical modifications have been optimized for extended persistence in cells ensuring prolonged activity. For example, Morpholino oligonucleotides have a half-life of several weeks in cultured cells. We have a morpholino-based oligonucleotide capable of correcting LMNA splicing. However, the limiting factor in oligonucleotide-based approaches with regards to their use in therapeutic applications is their delivery.</p><p>Small molecules that target the regulatory machinery of a cell in a manner that alters splicing events (either upregulating all splicing events or altering the ratio of aberrant splicing events to correct splicing events) would be novel molecular tools to study HGPS, and other diseases caused by aberrant splicing events.</p><p>The cdc2-like kinases (Clk) are made up of 4 isoforms (Clk1, Clk2, Clk3 and clk4). The Clk kinases are capable of both autophosphorylation and the phosphorylation of exogenous substrates. Importantly, the Clk family has been shown to be important constructs for the phosphorylation of serine and arginine rich (SR) proteins that are key components of the splicesome.<a class="bibr" href="#ml106.r1" rid="ml106.r1">1</a> Based upon these data (and other reports) the Clk kinases potentially play an important role in RNA splicing.</p><p>The primary phenotypic pre-mRNA splicing assay revealed a small molecule quinazoline (a chemotype related to known privileged structures for kinase inhibition). The lead compound was profiled for activity versus the Clk class of kinases and found to be active versus Clk4 (approx. 1 μM) (<a class="figpopup" href="/books/NBK47344/figure/ml106.f1/?report=objectonly" target="object" rid-figpopup="figml106f1" rid-ob="figobml106f1">figure 1</a>). From this point, the optimization of this chemotype revolved around achieving better potency and establishing selectivity for the Clk class of kinases. The results of these efforts form the basis for the naming of NCGC00010037 and NCGC00012420 as probes of Clk4.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml106f1" co-legend-rid="figlgndml106f1"><a href="/books/NBK47344/figure/ml106.f1/?report=objectonly" target="object" title="Figure 1" class="img_link icnblk_img figpopup" rid-figpopup="figml106f1" rid-ob="figobml106f1"><img class="small-thumb" src="/books/NBK47344/bin/ml106f1.gif" src-large="/books/NBK47344/bin/ml106f1.jpg" alt="Figure 1. The lead quinazoline structure from the primary screen and the data from the primary screen that denotes the ratiometric output favoring corrected splicing in the LaminA GFP/RFP phenotype assay." /></a><div class="icnblk_cntnt" id="figlgndml106f1"><h4 id="ml106.f1"><a href="/books/NBK47344/figure/ml106.f1/?report=objectonly" target="object" rid-ob="figobml106f1">Figure 1</a></h4><p class="float-caption no_bottom_margin">The lead quinazoline structure from the primary screen and the data from the primary screen that denotes the ratiometric output favoring corrected splicing in the LaminA GFP/RFP phenotype assay. </p></div></div></div></div><div id="ml106.s10"><h2 id="_ml106_s10_">Assay Implementation and Screening</h2><div id="ml106.s11"><h3>PubChem Bioassay Name</h3><p><i>Clk4 inhibition screen</i></p></div><div id="ml106.s12"><h3>Screens deposited to PubChem</h3><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml106tu5"><a href="/books/NBK47344/table/ml106.tu5/?report=objectonly" target="object" title="Table" class="img_link icnblk_img figpopup" rid-figpopup="figml106tu5" rid-ob="figobml106tu5"><img class="small-thumb" src="/books/NBK47344/table/ml106.tu5/?report=thumb" src-large="/books/NBK47344/table/ml106.tu5/?report=previmg" alt="Image " /></a><div class="icnblk_cntnt"><h4 id="ml106.tu5"><a href="/books/NBK47344/table/ml106.tu5/?report=objectonly" target="object" rid-ob="figobml106tu5">Table</a></h4></div></div></div><div id="ml106.s13"><h3>Primary Assay Description</h3><div id="ml106.s14"><h4>Overview</h4><p>Accurate determination of enzymological parameters requires the measurement of the initial rate of reaction. In principle, this can be determined by following either the formation of product or depletion of substrate, provided that < 5% of the substrate is converted. However, for high-throughput screening (HTS) where a signal:background ratio of ≥2-fold is desirable, measurement of product formation will yield the most robust assay signal. In some cases, such as the cleavage of profluorescent peptide substrate by a protease to form a fluorescent product, the HTS assay can be performed with the %conversion as low as ~10%. However, HTS assays for other classes of enzymes such as protein kinases have been performed using either product formation or substrate depletion, oftentimes at much higher conversion levels.</p><p>The application of bioluminescence to ATPases assay has relied on a substrate depletion format. In these assays, the ATP dependence of firefly luciferase is used to measure the remaining ATP concentration, where the luminescence signal is inversely proportional to kinase activity.<a class="bibr" href="#ml106.r3" rid="ml106.r3 ml106.r4 ml106.r5">2–4</a> To provide a signal:background of approximately 2-fold, the substrate must be depleted by at least 50%. While operating enzyme assays under these high conversion conditions is not at all optimal for classical enzymological studies, this is acceptable for HTS, as shifts in potency are typically less than 2-fold with %conversion <80%.<a class="bibr" href="#ml106.r6" rid="ml106.r6">5</a> Given that HTS assays typically show a variability in potency determinations ~2–3-fold, shifts due to high conversions in the range of 50–80% will not be easily discernable from the assay noise even if the assay is performed at lower conversions. Therefore, ATP-depletion has become a popular choice to perform generic HTS assays for ATPases, particularly protein kinases.</p><p>We used two bioluminescent assays for the Clk4 assay (<a class="figpopup" href="/books/NBK47344/figure/ml106.f2/?report=objectonly" target="object" rid-figpopup="figml106f2" rid-ob="figobml106f2">Figure 2</a>). Measurement of ATP depletion employed the Kinase-Glo™ assay system, where a firefly luciferase detection reagent containing D-luciferin and buffer components are added to detect the remaining ATP, following the Clk4 kinase assay (<a class="figpopup" href="/books/NBK47344/figure/ml106.f2/?report=objectonly" target="object" rid-figpopup="figml106f2" rid-ob="figobml106f2">Figure 2A</a>). The second system, ADP-Glo<sup>®</sup> measures kinase activity by quantifying the amount of ADP formed after kinase reaction. Bioluminescent detection of ADP levels is achieved through the addition two different detection reagents (<a class="figpopup" href="/books/NBK47344/figure/ml106.f2/?report=objectonly" target="object" rid-figpopup="figml106f2" rid-ob="figobml106f2">Figure 2B</a>). First, a reagent that stops the protein kinase reaction and depletes the remaining ATP is added. Then a second reagent is added to stop ATP degradation. In addition, the second reagent also contains an enzyme, such as pyruvate kinase, that efficiently converts the ADP to ATP and the same firefly luciferase/D-luciferin components present in Kinase-Glo, which generate the luminescent signal proportional to the ADP concentration produced. Therefore, the two assay formats show opposite luminescent signal changes in response to protein kinase inhibitors.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml106f2" co-legend-rid="figlgndml106f2"><a href="/books/NBK47344/figure/ml106.f2/?report=objectonly" target="object" title="Figure 2" class="img_link icnblk_img figpopup" rid-figpopup="figml106f2" rid-ob="figobml106f2"><img class="small-thumb" src="/books/NBK47344/bin/ml106f2.gif" src-large="/books/NBK47344/bin/ml106f2.jpg" alt="Figure 2. Bioluminescent assays used for Clk4 qHTS." /></a><div class="icnblk_cntnt" id="figlgndml106f2"><h4 id="ml106.f2"><a href="/books/NBK47344/figure/ml106.f2/?report=objectonly" target="object" rid-ob="figobml106f2">Figure 2</a></h4><p class="float-caption no_bottom_margin">Bioluminescent assays used for Clk4 qHTS. <i>A</i>. Bioluminescent measurement of ATP depletion using Kinase-Glo. <i>B</i>. Bioluminescent measurement of ADP formation using ADP-Glo. </p></div></div></div><div id="ml106.s15"><h4>Assay protocols</h4><p>The optimized 1536-well protocols are given in Table 1.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml106tu6"><a href="/books/NBK47344/table/ml106.tu6/?report=objectonly" target="object" title="Table" class="img_link icnblk_img figpopup" rid-figpopup="figml106tu6" rid-ob="figobml106tu6"><img class="small-thumb" src="/books/NBK47344/table/ml106.tu6/?report=thumb" src-large="/books/NBK47344/table/ml106.tu6/?report=previmg" alt="Image " /></a><div class="icnblk_cntnt"><h4 id="ml106.tu6"><a href="/books/NBK47344/table/ml106.tu6/?report=objectonly" target="object" rid-ob="figobml106tu6">Table</a></h4></div></div><p><a class="figpopup" href="/books/NBK47344/figure/ml106.f3/?report=objectonly" target="object" rid-figpopup="figml106f3" rid-ob="figobml106f3">Figure 3. Performance of the two 1,536-well Clk4 kinase assays</a></p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml106f3" co-legend-rid="figlgndml106f3"><a href="/books/NBK47344/figure/ml106.f3/?report=objectonly" target="object" title="Figure 3" class="img_link icnblk_img figpopup" rid-figpopup="figml106f3" rid-ob="figobml106f3"><img class="small-thumb" src="/books/NBK47344/bin/ml106f3.gif" src-large="/books/NBK47344/bin/ml106f3.jpg" alt="Figure 3. Performance of the two 1,536-well Clk4 kinase assays." /></a><div class="icnblk_cntnt" id="figlgndml106f3"><h4 id="ml106.f3"><a href="/books/NBK47344/figure/ml106.f3/?report=objectonly" target="object" rid-ob="figobml106f3">Figure 3</a></h4><p class="float-caption no_bottom_margin">Performance of the two 1,536-well Clk4 kinase assays. Kinase-Glo (A) and ADP-Glo (B) qHTS experiments. Light grey represents the compounds that fit to active CRC classes, and dark grey is data classified as inactive. Data for one of the ADP-Glo qHTS experiments <a href="/books/NBK47344/figure/ml106.f3/?report=objectonly" target="object" rid-ob="figobml106f3">(more...)</a></p></div></div><p><a class="figpopup" href="/books/NBK47344/figure/ml106.f4/?report=objectonly" target="object" rid-figpopup="figml106f4" rid-ob="figobml106f4">Figure 4. ADP-Glo and Kinase-Glo show excellent agreement in potency values</a></p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml106f4" co-legend-rid="figlgndml106f4"><a href="/books/NBK47344/figure/ml106.f4/?report=objectonly" target="object" title="Figure 4" class="img_link icnblk_img figpopup" rid-figpopup="figml106f4" rid-ob="figobml106f4"><img class="small-thumb" src="/books/NBK47344/bin/ml106f4.gif" src-large="/books/NBK47344/bin/ml106f4.jpg" alt="Figure 4. ADP-Glo and Kinase-Glo show excellent agreement in potency values." /></a><div class="icnblk_cntnt" id="figlgndml106f4"><h4 id="ml106.f4"><a href="/books/NBK47344/figure/ml106.f4/?report=objectonly" target="object" rid-ob="figobml106f4">Figure 4</a></h4><p class="float-caption no_bottom_margin">ADP-Glo and Kinase-Glo show excellent agreement in potency values. Comparison of potency values for run 1 (A) and run 2 (B) of the ADP-Glo qHTS against the Kinase-Glo qHTS. Black solid circles represent potencies derived from high quality CRCs and open <a href="/books/NBK47344/figure/ml106.f4/?report=objectonly" target="object" rid-ob="figobml106f4">(more...)</a></p></div></div><p>Both Kinase-Glo and ADP-Glo employ an optimized variant of firefly luciferase from <i>Photuris pennsylvanica</i> (Ultra-Glo™); we have previously determined the SAR of firefly luciferase inhibitors and determined that formulations of Ultra-Glo are much more resistant to inhibition than formulations containing <i>Photinus Pyralis</i> firefly luciferase.<a class="bibr" href="#ml106.r7" rid="ml106.r7">6</a>,<a class="bibr" href="#ml106.r7" rid="ml106.r7">7</a> Consistent with the robust nature of the Ultra-Glo formulations and the SAR of firefly luciferase inhibitors, we did not observe any interference with Kinase-Glo in the presence of ATP alone in any of the Clk4 kinase inhibitors identified here.</p></div><div id="ml106.s16"><h4>Identification of CID-3232621 & CID-3234998</h4><p>We used the 1,536-well protocols to screen a combinatorial library focused against protein
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kinases (<a class="figpopup" href="/books/NBK47344/figure/ml106.f5/?report=objectonly" target="object" rid-figpopup="figml106f5" rid-ob="figobml106f5">Figure 5</a>). This library contains substituted
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4-methyl-6-phenylquinazoline (<b>1</b>), 4-methyl-2-phenylquinazoline (<b>2</b>),
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phenylpyrimidines (<b>3–5</b>), and pteridinone (<b>6</b>) scaffolds known to
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target protein kinases. The 1,352 member library contains several hundred analogs around each
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scaffold that provide dense structure-activity relationships (SAR) around active scaffolds. We
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clustered the activity from this library using the high quality CRCs (1a, 1b, and 2a) to define
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active scaffolds (see <a class="figpopup" href="/books/NBK47344/figure/ml106.f8/?report=objectonly" target="object" rid-figpopup="figml106f8" rid-ob="figobml106f8">Scheme 1</a> for CRC class descriptions).</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml106f5" co-legend-rid="figlgndml106f5"><a href="/books/NBK47344/figure/ml106.f5/?report=objectonly" target="object" title="Figure 5" class="img_link icnblk_img figpopup" rid-figpopup="figml106f5" rid-ob="figobml106f5"><img class="small-thumb" src="/books/NBK47344/bin/ml106f5.gif" src-large="/books/NBK47344/bin/ml106f5.jpg" alt="Figure 5. Summary of activity from the qHTS." /></a><div class="icnblk_cntnt" id="figlgndml106f5"><h4 id="ml106.f5"><a href="/books/NBK47344/figure/ml106.f5/?report=objectonly" target="object" rid-ob="figobml106f5">Figure 5</a></h4><p class="float-caption no_bottom_margin">Summary of activity from the qHTS. <i>A)</i> Percentage of high quality actives (CRC classes 1a, 1b and 2a), inconclusive (all other active CRC classes) and inactive compounds (CRC class 4) obtained from the Kinase-Glo and duplicate runs of the ADP-Glo assay. <a href="/books/NBK47344/figure/ml106.f5/?report=objectonly" target="object" rid-ob="figobml106f5">(more...)</a></p></div></div><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml106f8" co-legend-rid="figlgndml106f8"><a href="/books/NBK47344/figure/ml106.f8/?report=objectonly" target="object" title="Scheme 1" class="img_link icnblk_img figpopup" rid-figpopup="figml106f8" rid-ob="figobml106f8"><img class="small-thumb" src="/books/NBK47344/bin/ml106f8.gif" src-large="/books/NBK47344/bin/ml106f8.jpg" alt="Scheme 1. Example qHTS data and classification scheme for assignment of resulting curve-fit data into classes." /></a><div class="icnblk_cntnt" id="figlgndml106f8"><h4 id="ml106.f8"><a href="/books/NBK47344/figure/ml106.f8/?report=objectonly" target="object" rid-ob="figobml106f8">Scheme 1</a></h4><p class="float-caption no_bottom_margin">Example qHTS data and classification scheme for assignment of resulting curve-fit data into classes. <i>Top</i>, qHTS curve-fit data from AID-361 binned into curve classifications 1-4 based classification criteria. <i>Below</i>, Examples of curves fitting the following <a href="/books/NBK47344/figure/ml106.f8/?report=objectonly" target="object" rid-ob="figobml106f8">(more...)</a></p></div></div></div></div></div><div id="ml106.s17"><h2 id="_ml106_s17_">Probe Characterization: CID-3232621/<a href="/pcsubstance/?term=ML106[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML106</a></h2><p><a class="figpopup" href="/books/NBK47344/figure/ml106.f6/?report=objectonly" target="object" rid-figpopup="figml106f6" rid-ob="figobml106f6">Figure 6. Confirmation data for CID-322621</a></p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml106f6" co-legend-rid="figlgndml106f6"><a href="/books/NBK47344/figure/ml106.f6/?report=objectonly" target="object" title="Figure 6" class="img_link icnblk_img figpopup" rid-figpopup="figml106f6" rid-ob="figobml106f6"><img class="small-thumb" src="/books/NBK47344/bin/ml106f6.gif" src-large="/books/NBK47344/bin/ml106f6.jpg" alt="Figure 6. Confirmation data for CID-322621." /></a><div class="icnblk_cntnt" id="figlgndml106f6"><h4 id="ml106.f6"><a href="/books/NBK47344/figure/ml106.f6/?report=objectonly" target="object" rid-ob="figobml106f6">Figure 6</a></h4><p class="float-caption no_bottom_margin">Confirmation data for CID-322621. Normalized data for the ADP-Glo (black; IC<sub>50</sub> = 75 nM), Kinase-Glo (cyan; IC<sub>50</sub> = 63 nM), and Bell-Brooks Transcreener (Green; IC<sub>50</sub> = 50 nM) assays against Clk4 are shown. Also shown is the fluorescent interference data <a href="/books/NBK47344/figure/ml106.f6/?report=objectonly" target="object" rid-ob="figobml106f6">(more...)</a></p></div></div><div id="ml106.s18"><h3>Mode of action</h3><p>The probe is a member of a series of highly specific inhibitors of the Clk family of kinases (CDC
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like kinases) with registered activity versus Clk4 at 30 nM. Profiles of this agent suggest that this agent has low nM activity versus the other 3 members of this family (Clk1, Clk2 and Clk3) and Drk1A. The only other reported Clk4 inhibitor is TG003 (reported IC<sub>50</sub> of 15 nM; IC<sub>50</sub> value of 44 nM in our study)1 was found to be far more promiscuous versus the same panel (see dendrograms below). Although it has not been shown, it is likely that CID-3232621 is an ATP competitive kinase inhibitor (based upon structural similarity to other well-characterized ATP competitive kinase inhibitors).</p><div id="ml106.fu3" class="figure"><div class="graphic"><img src="/books/NBK47344/bin/ml106fu3.jpg" alt="Image ml106fu3" /></div></div></div><div id="ml106.s19"><h3>Synthesis of analogs</h3><div id="ml106.s20"><h4>General procedure for the synthesis of analogues</h4><div id="ml106.fu4" class="figure"><div class="graphic"><img src="/books/NBK47344/bin/ml106fu4.jpg" alt="Image ml106fu4" /></div></div><div id="ml106.s21"><h5>Procedure for the preparation of 6-bromo-N-(thiophen-2-ylmethyl)quinazolin-4-amine (1)</h5><p>To a solution of 6-bromo-4-chloroquinazoline (.3 g, 1.232 mmol) in DMF were added thiophen-2-ylmethanamine (0.139 g, 1.232 mmol) and N-ethyl-N-isopropylpropan-2-amine (0.214 mL, 1.232 mmol). The reaction mixture was stirred at rt for 2h. Upon completion, the reaction mixture was diluted with EtOAc (100 mL) and washed with 10% KHSO<sub>4</sub> (25 mL) and three times with 3N LiCl (3 x 30 mL). The organic layer was concentrated <i>in vacuo</i>, and the residue was purified directly on silica column. Gradient elution with ethyl acetate (15→75%) in hexanes provided the title compound as a colorless solid: yield (0.39 g, 1.218 mmol, 99 %).</p><div id="ml106.fu5" class="figure"><div class="graphic"><img src="/books/NBK47344/bin/ml106fu5.jpg" alt="Image ml106fu5" /></div></div></div><div id="ml106.s22"><h5>Procedure for the preparation of 6-(benzo[d][1,3]dioxol-5-yl)-N-(thiophen-2-ylmethyl)quinazolin-4-amine</h5><p>To a solution of 6-bromo-N-(thiophen-2-ylmethyl)quinazolin-4-amine (.1 g, 0.312 mmol) in DMF (3 mL) were added benzo[d][1,3]dioxol-5-ylboronic acid (0.078 g, 0.468 mmol), sodium carbonate (0.066 g, 0.625 mmol), and tetrakis (0.036 g, 0.031 mmol). The reaction mixture was heated in the microwave at 150 °C for 1h. Upon completion, the reaction mixture was diluted with EtOAc (75 mL) and washed with NaHCO<sub>3</sub> (50 mL), three times with 3N LiCl (3 x 30 mL), and brine (30 mL). The organic layer was collected, filtered through a pad of Celite, and concentrated <i>in vacuo</i>. The residue was purified directly on silica column. Gradient elution with ethyl acetate (5→65%) in hexanes provided the title compound as a colorless solid: yield (0.065 g, 0.180 mmol, 58 %). <sup>1</sup>H NMR (DMSO-<i>d</i><sub>6</sub>) δ 4.97 (d, <i>J</i> = 5.53 Hz, 2H), 6.09 (s, 2H), 6.92 – 7.01 (m, 1H), 7.02 – 7.18 (m, 2H), 7.29 – 7.41 (m, 2H), 7.44 (s, 1H), 7.73 (d, <i>J</i> = 8.56 Hz, 1H), 8.08 (d, <i>J</i> = 8.46 Hz, 1H), 8.51 (d, <i>J</i> = 4.70 Hz, 2H), 8.99 (d, <i>J =</i> 4.89 Hz, 1H); <sup>13</sup>C NMR (DMSO-<i>d</i><sub>6</sub>) δ 54.88, 101.28, 107.14, 108.70, 115.04, 119.52, 120.54, 125.11, 125.79, 126.54, 127.99, 131.04, 133.21, 137.02, 142.18, 147.14, 148.11, 148.26, 154.75, 159.05. MS (ESI) <i>m</i>/<i>z</i> 362.1 (M+H)<sup>+</sup> (C<sub>20</sub>H<sub>16</sub>N<sub>3</sub>O<sub>2</sub>S requires 362.1). LC (ESE) retention time: 3 min gradient elution with acetonitrile (4–100%) in water = 3.050; 7 min gradient elution with acetonitrile (4–100%) in water = 4.531 min.</p></div></div></div><div id="ml106.s23"><h3>SAR for CID-3232621</h3><p>The NCGC compound library has numerous quinazoline-based small molecules, and the qHTS screening format allows for the identification of nascent SAR from the preliminary screening of Clk4. From this effort, a great deal of information was provided regarding the SAR pathways of utility and the SAR pathways of futility. A visual description of this data is shown below. From this data, we planned and executed a matrix library at the 4-amine substitution and the 3-aryl substitution. A total of 63 compounds were synthesized and examined. This focused library gave very tight SAR and was found to be very active in terms of Clk4 inhibition. The details are shown below, with 47 compounds of the 63 registering a 1.1 class curve and 21 compounds possessed an IC<sub>50</sub> value below 1 μM (3 compounds were found to be sub-100 nM). CID-3232621 was the most active with an IC<sub>50</sub> of 63 nM and a max response of ~100% inhibition.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml106tu7"><a href="/books/NBK47344/table/ml106.tu7/?report=objectonly" target="object" title="Table" class="img_link icnblk_img figpopup" rid-figpopup="figml106tu7" rid-ob="figobml106tu7"><img class="small-thumb" src="/books/NBK47344/table/ml106.tu7/?report=thumb" src-large="/books/NBK47344/table/ml106.tu7/?report=previmg" alt="Image " /></a><div class="icnblk_cntnt"><h4 id="ml106.tu7"><a href="/books/NBK47344/table/ml106.tu7/?report=objectonly" target="object" rid-ob="figobml106tu7">Table</a></h4></div></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml106tu8"><a href="/books/NBK47344/table/ml106.tu8/?report=objectonly" target="object" title="Table" class="img_link icnblk_img figpopup" rid-figpopup="figml106tu8" rid-ob="figobml106tu8"><img class="small-thumb" src="/books/NBK47344/table/ml106.tu8/?report=thumb" src-large="/books/NBK47344/table/ml106.tu8/?report=previmg" alt="Image " /></a><div class="icnblk_cntnt"><h4 id="ml106.tu8"><a href="/books/NBK47344/table/ml106.tu8/?report=objectonly" target="object" rid-ob="figobml106tu8">Table</a></h4></div></div><p><a class="figpopup" href="/books/NBK47344/figure/ml106.fu6/?report=objectonly" target="object" rid-figpopup="figml106fu6" rid-ob="figobml106fu6">SAR from Primary Clk4 Screen.</a></p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml106fu6" co-legend-rid="figlgndml106fu6"><a href="/books/NBK47344/figure/ml106.fu6/?report=objectonly" target="object" title="Figure" class="img_link icnblk_img figpopup" rid-figpopup="figml106fu6" rid-ob="figobml106fu6"><img class="small-thumb" src="/books/NBK47344/bin/ml106fu6.gif" src-large="/books/NBK47344/bin/ml106fu6.jpg" alt="SAR from Primary Clk4 Screen." /></a><div class="icnblk_cntnt" id="figlgndml106fu6"><h4 id="ml106.fu6"><a href="/books/NBK47344/figure/ml106.fu6/?report=objectonly" target="object" rid-ob="figobml106fu6">Figure</a></h4><p class="float-caption no_bottom_margin">SAR from Primary Clk4 Screen. </p></div></div><p><a class="figpopup" href="/books/NBK47344/figure/ml106.fu7/?report=objectonly" target="object" rid-figpopup="figml106fu7" rid-ob="figobml106fu7">SAR from focused synthetic library.</a></p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml106fu7" co-legend-rid="figlgndml106fu7"><a href="/books/NBK47344/figure/ml106.fu7/?report=objectonly" target="object" title="Figure" class="img_link icnblk_img figpopup" rid-figpopup="figml106fu7" rid-ob="figobml106fu7"><img class="small-thumb" src="/books/NBK47344/bin/ml106fu7.gif" src-large="/books/NBK47344/bin/ml106fu7.jpg" alt="SAR from focused synthetic library." /></a><div class="icnblk_cntnt" id="figlgndml106fu7"><h4 id="ml106.fu7"><a href="/books/NBK47344/figure/ml106.fu7/?report=objectonly" target="object" rid-ob="figobml106fu7">Figure</a></h4><p class="float-caption no_bottom_margin">SAR from focused synthetic library. </p></div></div><p>This probe compound has been made available through the MLSMR (MLS002276464).</p><p><i>Canonical SMILES C12=NC=NC(NCC3=CSC=C3)=C1C=C(C4=CC(OCO5)=C5C=C4)C=C2</i></p></div><div id="ml106.s24"><h3>Description of secondary assays used in probe characterization</h3><p>A bioluminescence assay for ATP depletion was used (Kinase-Glo, Promega) to confirm activity in the bioluminescent ADP formation assay (ADP-Glo, Promega). These assays show opposite luminescent responses to kinase inhibition. As well, we used the fluorescent polarization assay from Bell Brooks Labs (Transcreener) wherein the ADP formed in the kinase reaction is measured using an antibody to a fluorescently-labeled ADP.</p><p>For specific selectivity the <b>Ambit Panel</b> was determined. Kinase profiles were performed by Ambit Biosciences (San Diego, CA, USA: <a href="http://www.ambitbio.com/" ref="pagearea=body&targetsite=external&targetcat=link&targettype=uri">http://www.ambitbio.com/</a>) utilizing KINOME<i>scan</i>™. Activity was recorded via a competition binding assay of selected kinases that are fused to a proprietary tag. Measurements of the amount of kinase bound to an immobilized, active-site directed ligand in the presence and absence of the test compound provide a % of DMSO control for binding of ligand. Activities between 0 and 10 were selected for Kd determinations. Dendrogram representations were generated by an in-house visualization tool designated PhyloChem. Dendrogram clustering and apexes are based on the human phylogenetic kinase data available at <a href="http://kinase.com/human/kinome" ref="pagearea=body&targetsite=external&targetcat=link&targettype=uri">http://kinase.com/human/kinome</a>. The results of this profile are discussed on page 12.</p></div><div id="ml106.s25"><h3>Compound preparation</h3><p>Compound is prepared in DMSO at 10 mM stock concentration.</p></div><div id="ml106.s26"><h3>Known probe properties</h3><div id="ml106.s27"><h4>1. Probe</h4><div id="ml106.s28"><h5>a. Chemical name of probe compound</h5><p>6-(1,3-benzodioxol-5-yl)-N-(thiophen-2-ylmethyl)quinazolin-4-amine (<a href="/pcsubstance/?term=ML106[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML106</a>)</p></div><div id="ml106.s29"><h5>b. Probe chemical structure including stereochemistry</h5><div id="ml106.fu8" class="figure"><div class="graphic"><img src="/books/NBK47344/bin/ml106fu8.jpg" alt="Image ml106fu8" /></div></div></div><div id="ml106.s30"><h5>c. Structural Verification Information of probe SID</h5><p>See <a href="#ml106.s17">Probe Characterization</a>.</p></div><div id="ml106.s31"><h5>d. PubChem CID (corresponding to the SID)</h5><p><a href="https://pubchem.ncbi.nlm.nih.gov/substance/3232621" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-3232621</a></p></div><div id="ml106.s32"><h5>e. Availability from a vendor</h5><p>Aliquots of CID-3232621 are available from the NCGC upon request. Additionally, compound has been provided through the MLSMR (MLS-003115039 and MLS-002276464).</p></div><div id="ml106.s33"><h5>f. Mode of action for biological activity of probe</h5><p>Although it has not been shown, it is likely that CID-3232621 is an ATP competitive kinase inhibitor (based upon structural similarity to other well characterized ATP competitive kinase inhibitors).</p></div><div id="ml106.s34"><h5>g. Detailed synthetic pathway for making probe</h5><p>See <a href="#ml106.s19">Scheme</a>.</p></div><div id="ml106.s35"><h5>h. Summary of probe properties (solubility, absorbance/fluorescence, reactivity, toxicity, etc.)</h5><p>Compound is soluble at 10 mM in DMSO. The compound is not fluorescent with blue excitation wavelengths (~340 nm). Solubility in buffer has not been determined.</p></div><div id="ml106.s36"><h5>i. Summary of known probe properties: CID-3232621</h5><p>See <a href="#ml106.s1">above</a>.</p></div></div></div></div><div id="ml106.s37"><h2 id="_ml106_s37_">Probe Characterization: CID-3234998/<a href="/pcsubstance/?term=ML105[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML105</a></h2><p><a class="figpopup" href="/books/NBK47344/figure/ml106.f7/?report=objectonly" target="object" rid-figpopup="figml106f7" rid-ob="figobml106f7">Figure 7. Confirmation data for CID: 3234998</a></p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml106f7" co-legend-rid="figlgndml106f7"><a href="/books/NBK47344/figure/ml106.f7/?report=objectonly" target="object" title="Figure 7" class="img_link icnblk_img figpopup" rid-figpopup="figml106f7" rid-ob="figobml106f7"><img class="small-thumb" src="/books/NBK47344/bin/ml106f7.gif" src-large="/books/NBK47344/bin/ml106f7.jpg" alt="Figure 7. Confirmation data for CID: 3234998." /></a><div class="icnblk_cntnt" id="figlgndml106f7"><h4 id="ml106.f7"><a href="/books/NBK47344/figure/ml106.f7/?report=objectonly" target="object" rid-ob="figobml106f7">Figure 7</a></h4><p class="float-caption no_bottom_margin">Confirmation data for CID: 3234998. Normalized data for the ADP-Glo (blue; IC<sub>50</sub> = 100 nM), Kinase-Glo (cyan; IC<sub>50</sub> = 70 nM), and Bell-Brooks Transcreener (black; IC<sub>50</sub> = 50 nM) assays against Clk4 are shown. Also shown is the fluorescent interference data <a href="/books/NBK47344/figure/ml106.f7/?report=objectonly" target="object" rid-ob="figobml106f7">(more...)</a></p></div></div><div id="ml106.s38"><h3>Mode of action</h3><p>The probe is a member of a series of highly specific inhibitors of the Clk family of kinases (CDC like kinases) with registered activity versus Clk4 at 30 nM. Profiles of this agent suggest that this agent has low nM activity versus the other 3 members of this family (Clk1, Clk2 and Clk3) and Drk1A. The only other reported Clk4 inhibitor is TG003 (IC50 value of 44 nM in our study) was found to be far more promiscuous versus the same panel (see dendrograms below). Although it has not been shown, it is likely that NCGC00010037 is an ATP competitive kinase inhibitor (based upon structural similarity to other well characterized ATP competitive kinase inhibitors).</p></div><div id="ml106.s39"><h3>Synthesis of analogs</h3><p><i>The synthesis of CID-3234998 and selected analogues has not yet been undertaken.</i></p></div><div id="ml106.s40"><h3>SAR for CID-3234998</h3><p>The NCGC compound library has numerous pyrimidine-based small molecules, and the qHTS screening format allows for the identification of nascent SAR from the preliminary screening of Clk4. From this effort, a great deal of information was provided regarding the SAR pathways of utility and the SAR pathways of futility. A visual description of this data is shown below.</p><p><a class="figpopup" href="/books/NBK47344/figure/ml106.fu9/?report=objectonly" target="object" rid-figpopup="figml106fu9" rid-ob="figobml106fu9">SAR from Primary Clk4 Screen.</a></p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml106fu9" co-legend-rid="figlgndml106fu9"><a href="/books/NBK47344/figure/ml106.fu9/?report=objectonly" target="object" title="Figure" class="img_link icnblk_img figpopup" rid-figpopup="figml106fu9" rid-ob="figobml106fu9"><img class="small-thumb" src="/books/NBK47344/bin/ml106fu9.gif" src-large="/books/NBK47344/bin/ml106fu9.jpg" alt="SAR from Primary Clk4 Screen." /></a><div class="icnblk_cntnt" id="figlgndml106fu9"><h4 id="ml106.fu9"><a href="/books/NBK47344/figure/ml106.fu9/?report=objectonly" target="object" rid-ob="figobml106fu9">Figure</a></h4><p class="float-caption no_bottom_margin">SAR from Primary Clk4 Screen. </p></div></div><p><i>Canonical SMILES C1(CNC2=CC=NC(C3=CC(OCO4)=C4C=C3)=N2)=CC=CN=C1</i></p></div><div id="ml106.s41"><h3>Description of secondary assays used in probe characterization</h3><p>A bioluminescence assay for ATP depletion was used (Kinase-Glo, Promega) to confirm activity in the bioluminescent ADP formation assay (ADP-Glo, Promega). These assays show opposite luminescent responses to kinase inhibition. As well, we used the fluorescent polarization assay from Bell Brooks Labs (Transcreener) wherein the ADP formed in the kinase reaction is measured using an antibody to a fluorescently-labeled ADP.</p></div><div id="ml106.s42"><h3>Compound preparation</h3><p>Compound is prepared in DMSO at 10 mM stock concentration.</p></div><div id="ml106.s43"><h3>Known probe properties</h3><div id="ml106.s44"><h4>2. Probe</h4><div id="ml106.s45"><h5>a. Chemical name</h5><p>2-(benzo[d][1,3]dioxol-5-yl)-N-(pyridin-3-ylmethyl)pyrimidin-4-amine (<a href="/pcsubstance/?term=ML105[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML105</a>)</p></div><div id="ml106.s46"><h5>b. Probe chemical structure</h5><div id="ml106.fu10" class="figure"><div class="graphic"><img src="/books/NBK47344/bin/ml106fu10.jpg" alt="Image ml106fu10" /></div></div></div><div id="ml106.s47"><h5>c. Structural Verification Information of probe SID</h5><p><a href="https://pubchem.ncbi.nlm.nih.gov/substance/4239891" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-4239891</a></p></div><div id="ml106.s48"><h5>d. PubChem CID (corresponding to the SID)</h5><p>CID-3234998</p></div><div id="ml106.s49"><h5>e. Availability from a vendor</h5><p>Aliquots of NCGC-00012420 are available from the NCGC upon request. Compound has been also made available through the MLSMR (MLS-003115040)</p></div><div id="ml106.s50"><h5>f. Mode of action for biological activity of probe</h5><p>Although it has not been shown, it is likely that CID-3234998 (NCGC-00012420) is an ATP competitive kinase inhibitor (based upon structural similarity to other well characterized ATP competitive kinase inhibitors).</p></div><div id="ml106.s51"><h5>g. Detailed synthetic pathway for making probe</h5><p>NA</p></div><div id="ml106.s52"><h5>h. Summary of probe properties (solubility, absorbance/fluorescence, reactivity, toxicity, etc.)</h5><p>Compound is soluble at 10 mM in DMSO. The compound is not fluorescent with blue excitation wavelengths (~340 nm). Solubility in buffer has not been determined.</p></div><div id="ml106.s53"><h5>i. Summary of known probe properties</h5><p>See <a href="#ml106.s4">above</a>.</p></div></div></div></div><div id="ml106.s54"><h2 id="_ml106_s54_">Bibliography</h2><dl class="temp-labeled-list"><dl class="bkr_refwrap"><dt>1.</dt><dd><div class="bk_ref" id="ml106.r1">Muraki M, Ohkawara B, Hosoya T, Onogi H, Koizumi J, Koizumi T, Sumi K, Yomoda J, Murray MV, Kimura H, Furuichi K, Shibuya H, Krainer AR, Suzuki M, Hagiwara M. Manipulation of alternative splicing by a newly developed inhibitor of Clks. <span><span class="ref-journal">J Biol Chem. </span>2004;<span class="ref-vol">279</span>:24246–54.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/15010457" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 15010457</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>2.</dt><dd><div class="bk_ref" id="ml106.r2">Fan F, Wood KV. Bioluminescent assays for high-throughput screening. <span><span class="ref-journal">Assay Drug Dev Technol. </span>2007;<span class="ref-vol">5</span>:127–36.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/17355205" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17355205</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>2.</dt><dd><div class="bk_ref" id="ml106.r3">Schroter T, Minond D, Weiser A, Dao C, Habel J, Spicer T, Chase P, Baillargeon P, Scampavia L, Schurer S, Chung C, Mader C, Southern M, Tsinoremas N, LoGrasso P, Hodder P. Comparison of miniaturized time-resolved fluorescence resonance energy transfer and enzyme-coupled luciferase high-throughput screening assays to discover inhibitors of Rho-kinase II (ROCK-II). <span><span class="ref-journal">J Biomol Screen. </span>2008;<span class="ref-vol">13</span>:17–28.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/18227223" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18227223</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>3.</dt><dd><div class="bk_ref" id="ml106.r4">Singh P, Harden BJ, Lillywhite BJ, Broad PM. Identification of kinase inhibitors by an ATP depletion method. <span><span class="ref-journal">Assay Drug Dev Technol. </span>2004;<span class="ref-vol">2</span>:161–9.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/15165512" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 15165512</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>4.</dt><dd><div class="bk_ref" id="ml106.r5">Wu G, Yuan Y, Hodge CN. Determining appropriate substrate conversion for enzymatic assays in high-throughput screening. <span><span class="ref-journal">J Biomol Screen. </span>2003;<span class="ref-vol">8</span>:694–700.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/14711395" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 14711395</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>5.</dt><dd><div class="bk_ref" id="ml106.r6">Auld DS, Southall NT, Jadhav A, Johnson RL, Diller DJ, Simeonov A, Austin CP, Inglese J. Characterization of chemical libraries for luciferase inhibitory activity. <span><span class="ref-journal">J Med Chem. </span>2008;<span class="ref-vol">51</span>:2372–86.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/18363348" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18363348</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>6.</dt><dd><div class="bk_ref" id="ml106.r7">Auld DS, Zhang YQ, Southall NT, Rai G, Landsman M, Maclure J, Langevin D, Thomas CJ, Austin CP, Inglese J. A Basis for Reduced Chemical Library Inhibition of Firefly Luciferase Obtained from Directed Evolution. <span><span class="ref-journal">J Med Chem. </span>2009;<span class="ref-vol">52</span>:1450–1458.</span> [<a href="/pmc/articles/PMC3430137/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3430137</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/19215089" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19215089</span></a>]</div></dd></dl></dl></div><div id="bk_toc_contnr"></div></div></div><div class="fm-sec"><h2 id="_NBK47344_pubdet_">Publication Details</h2><h3>Author Information and Affiliations</h3><p class="contrib-group"><h4>Authors</h4><span itemprop="author">Douglas Auld</span>, <span itemprop="author">Min Shen</span>, and <span itemprop="author">Craig Thomas</span>.</p><h4>Affiliations</h4><div class="affiliation"><sup>1</sup> NIH Chemical Genomics Center</div><h3>Publication History</h3><p class="small">Received: <span itemprop="datePublished">May 18, 2009</span>; Last Update: <span itemprop="dateModified">September 2, 2010</span>.</p><h3>Copyright</h3><div><div class="half_rhythm"><a href="/books/about/copyright/">Copyright Notice</a></div></div><h3>Publisher</h3><p>National Center for Biotechnology Information (US), Bethesda (MD)</p><h3>NLM Citation</h3><p>Auld D, Shen M, Thomas C. A high-throughput screen for pre-mRNA splicing modulators. 2009 May 18 [Updated 2010 Sep 2]. 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></p></div><div class="small-screen-prev"><a href="/books/n/mlprobe/y1/?report=reader"><svg xmlns="http://www.w3.org/2000/svg" viewBox="0 0 100 100" preserveAspectRatio="none"><path d="M75,30 c-80,60 -80,0 0,60 c-30,-60 -30,0 0,-60"></path><text x="20" y="28" textLength="60" style="font-size:25px">Prev</text></svg></a></div><div class="small-screen-next"><a href="/books/n/mlprobe/ml104/?report=reader"><svg xmlns="http://www.w3.org/2000/svg" viewBox="0 0 100 100" preserveAspectRatio="none"><path d="M25,30c80,60 80,0 0,60 c30,-60 30,0 0,-60"></path><text x="20" y="28" textLength="60" style="font-size:25px">Next</text></svg></a></div></article><article data-type="fig" id="figobml106fu1"><div id="ml106.fu1" class="figure"><div class="graphic"><img data-src="/books/NBK47344/bin/ml106fu1.jpg" alt="Image ml106fu1" /></div></div></article><article data-type="table-wrap" id="figobml106tu1"><div id="ml106.tu1" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK47344/table/ml106.tu1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml106.tu1_lrgtbl__"><table><tbody><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">PubChem CID</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CID-3232621</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Internal ID</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC-00010037</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Molecular Weight</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">361.417</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Molecular Formula</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">C20H15N3O2S</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">XLogP</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">4.5</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">H-Bond Donor</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">1</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">H-Bond Acceptor</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">5</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Rotatable Bond Count</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">4</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Exact Mass</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">361.0885</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Topological Polar Surface Area</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">84.5</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Heavy Atom Count</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">26</td></tr></tbody></table></div></div></article><article data-type="table-wrap" id="figobml106tu2"><div id="ml106.tu2" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK47344/table/ml106.tu2/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml106.tu2_lrgtbl__"><table><thead><tr><th id="hd_h_ml106.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">CID/ML</th><th id="hd_h_ml106.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Target Name</th><th id="hd_h_ml106.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">EC50 (μM) [SID, AID]</th><th id="hd_h_ml106.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Anti-target Name(s)</th><th id="hd_h_ml106.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Selectivity Score</th><th id="hd_h_ml106.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Secondary Assay(s) Name:</th></tr></thead><tbody><tr><td headers="hd_h_ml106.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">CID-3232621/<a href="/pcsubstance/?term=ML106[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML106</a></td><td headers="hd_h_ml106.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Clk4</td><td headers="hd_h_ml106.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0.06 ± 0.01 [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/85239684" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-85239684</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1970" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1970</a>]</td><td headers="hd_h_ml106.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">> 400 kinases</td><td headers="hd_h_ml106.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0.014</td><td headers="hd_h_ml106.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">% competition kinase panel</td></tr></tbody></table></div></div></article><article data-type="table-wrap" id="figobml106t1"><div id="ml106.t1" class="table"><h3><span class="label">Table 2</span><span class="title">Compound identification numbers</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK47344/table/ml106.t1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml106.t1_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml106.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Compound CID</th><th id="hd_h_ml106.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">NCGC ID</th></tr></thead><tbody><tr><td headers="hd_h_ml106.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CID-3232621</td><td headers="hd_h_ml106.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">NCGC-00010037</td></tr><tr><td headers="hd_h_ml106.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CID-3234998</td><td headers="hd_h_ml106.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">NCGC-00012420</td></tr></tbody></table></div></div></article><article data-type="fig" id="figobml106fu2"><div id="ml106.fu2" class="figure"><div class="graphic"><img data-src="/books/NBK47344/bin/ml106fu2.jpg" alt="Image ml106fu2" /></div></div></article><article data-type="table-wrap" id="figobml106tu3"><div id="ml106.tu3" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK47344/table/ml106.tu3/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml106.tu3_lrgtbl__"><table><tbody><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">PubChem CID</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CID-3234998</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Internal ID</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC-00012420</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Molecular Weight</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">306.3</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Molecular Formula</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">C17H14N4O2</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">XLogP</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">2.4</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">H-Bond Donor</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">1</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">H-Bond Acceptor</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">6</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Rotatable Bond Count</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">4</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Exact Mass</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">306.1117</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Topological Polar Surface Area</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">69.2</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Heavy Atom Count</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">23</td></tr></tbody></table></div></div></article><article data-type="table-wrap" id="figobml106tu4"><div id="ml106.tu4" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK47344/table/ml106.tu4/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml106.tu4_lrgtbl__"><table><thead><tr><th id="hd_h_ml106.tu4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">CID/ML</th><th id="hd_h_ml106.tu4_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Target Name</th><th id="hd_h_ml106.tu4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">EC50 (μM) [SID, AID]</th><th id="hd_h_ml106.tu4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Anti-target Name(s)</th><th id="hd_h_ml106.tu4_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Selectivity Score</th><th id="hd_h_ml106.tu4_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Secondary Assay(s) Name:</th></tr></thead><tbody><tr><td headers="hd_h_ml106.tu4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">CID-3234998/<a href="/pcsubstance/?term=ML105[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML105</a></td><td headers="hd_h_ml106.tu4_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Clk4</td><td headers="hd_h_ml106.tu4_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0.06 ± 0.01 [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/4239891" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-4239891</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1770" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1770</a>]</td><td headers="hd_h_ml106.tu4_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">> 400 kinases</td><td headers="hd_h_ml106.tu4_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">ND</td><td headers="hd_h_ml106.tu4_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">% competition kinase panel</td></tr></tbody></table></div></div></article><article data-type="fig" id="figobml106f1"><div id="ml106.f1" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK47344/bin/ml106f1.jpg" alt="Figure 1. The lead quinazoline structure from the primary screen and the data from the primary screen that denotes the ratiometric output favoring corrected splicing in the LaminA GFP/RFP phenotype assay." /></div><h3><span class="label">Figure 1</span><span class="title">The lead quinazoline structure from the primary screen and the data from the primary screen that denotes the ratiometric output favoring corrected splicing in the LaminA GFP/RFP phenotype assay</span></h3></div></article><article data-type="table-wrap" id="figobml106tu5"><div id="ml106.tu5" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK47344/table/ml106.tu5/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml106.tu5_lrgtbl__"><table><thead><tr><th id="hd_h_ml106.tu5_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">PubChem AID</th><th id="hd_h_ml106.tu5_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Type</th><th id="hd_h_ml106.tu5_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Target</th><th id="hd_h_ml106.tu5_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Conc. Range</th><th id="hd_h_ml106.tu5_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Samples Tested</th></tr></thead><tbody><tr><td headers="hd_h_ml106.tu5_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1770" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1770</a></td><td headers="hd_h_ml106.tu5_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Primary qHTS Kinase-Glo</td><td headers="hd_h_ml106.tu5_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Clk4 inhibitor</td><td headers="hd_h_ml106.tu5_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">66.7 μM – 4.3 nM</td><td headers="hd_h_ml106.tu5_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">1352</td></tr><tr><td headers="hd_h_ml106.tu5_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1771" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1771</a></td><td headers="hd_h_ml106.tu5_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Primary qHTS ADP-Glo</td><td headers="hd_h_ml106.tu5_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Clk4 inhibitor</td><td headers="hd_h_ml106.tu5_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">66.7 μM – 4.3 nM</td><td headers="hd_h_ml106.tu5_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">1352</td></tr><tr><td headers="hd_h_ml106.tu5_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1795" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1795</a></td><td headers="hd_h_ml106.tu5_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Primary qHTS ADP-FP</td><td headers="hd_h_ml106.tu5_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Clk4 inhibitor</td><td headers="hd_h_ml106.tu5_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">66.7 μM – 4.3 nM</td><td headers="hd_h_ml106.tu5_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">1352</td></tr><tr><td headers="hd_h_ml106.tu5_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1969" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1969</a></td><td headers="hd_h_ml106.tu5_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Confirmatory-ADP-Glo</td><td headers="hd_h_ml106.tu5_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Clk4 inhibitor</td><td headers="hd_h_ml106.tu5_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">66.7 μM – 4.3 nM</td><td headers="hd_h_ml106.tu5_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">63</td></tr><tr><td headers="hd_h_ml106.tu5_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1970" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1970</a></td><td headers="hd_h_ml106.tu5_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Confirmatory Kinase-Glo</td><td headers="hd_h_ml106.tu5_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Clk4 inhibitor</td><td headers="hd_h_ml106.tu5_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">66.7 µM – 0.8 nM</td><td headers="hd_h_ml106.tu5_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">166</td></tr><tr><td headers="hd_h_ml106.tu5_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1983" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1983</a></td><td headers="hd_h_ml106.tu5_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Confirmatory-ADP-FP</td><td headers="hd_h_ml106.tu5_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Clk4 inhibitor</td><td headers="hd_h_ml106.tu5_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">66.7 μM – 4.3 nM</td><td headers="hd_h_ml106.tu5_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">63</td></tr><tr><td headers="hd_h_ml106.tu5_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2705" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2705</a></td><td headers="hd_h_ml106.tu5_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Selectivity</td><td headers="hd_h_ml106.tu5_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Dyrk1A</td><td headers="hd_h_ml106.tu5_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">76.9 μM – 0.04 nM</td><td headers="hd_h_ml106.tu5_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">98</td></tr><tr><td headers="hd_h_ml106.tu5_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2710" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2710</a></td><td headers="hd_h_ml106.tu5_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Anti-target</td><td headers="hd_h_ml106.tu5_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Reaction Biology Panel (Clk1–4, Dyrk1A, Dyrk1B)</td><td headers="hd_h_ml106.tu5_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">10 μM – 0.5 nM</td><td headers="hd_h_ml106.tu5_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">20</td></tr><tr><td headers="hd_h_ml106.tu5_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1997" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1997</a></td><td headers="hd_h_ml106.tu5_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Summary</td><td headers="hd_h_ml106.tu5_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Clk4 inhibitor</td><td headers="hd_h_ml106.tu5_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">NA</td><td headers="hd_h_ml106.tu5_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">NA</td></tr></tbody></table></div></div></article><article data-type="fig" id="figobml106f2"><div id="ml106.f2" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK47344/bin/ml106f2.jpg" alt="Figure 2. Bioluminescent assays used for Clk4 qHTS." /></div><h3><span class="label">Figure 2</span><span class="title">Bioluminescent assays used for Clk4 qHTS</span></h3><div class="caption"><p><b>A</b>. Bioluminescent measurement of ATP depletion using Kinase-Glo. <b>B</b>. Bioluminescent measurement of ADP formation using ADP-Glo.</p></div></div></article><article data-type="table-wrap" id="figobml106tu6"><div id="ml106.tu6" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK47344/table/ml106.tu6/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml106.tu6_lrgtbl__"><table><thead><tr><th id="hd_h_ml106.tu6_1_1_1_1" colspan="4" rowspan="1" style="text-align:center;vertical-align:top;">Kinase-Glo</th><th id="hd_h_ml106.tu6_1_1_1_2" colspan="3" rowspan="1" style="text-align:center;vertical-align:top;">ADP-Glo</th></tr><tr><th headers="hd_h_ml106.tu6_1_1_1_1 hd_h_ml106.tu6_1_1_1_2" id="hd_h_ml106.tu6_1_1_2_1" colspan="7" rowspan="1" style="text-align:left;vertical-align:top;"><span class="hr"></span></th></tr><tr><th headers="hd_h_ml106.tu6_1_1_1_1 hd_h_ml106.tu6_1_1_2_1" id="hd_h_ml106.tu6_1_1_3_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Step</th><th headers="hd_h_ml106.tu6_1_1_1_1 hd_h_ml106.tu6_1_1_2_1" id="hd_h_ml106.tu6_1_1_3_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Parameter</th><th headers="hd_h_ml106.tu6_1_1_1_1 hd_h_ml106.tu6_1_1_2_1" id="hd_h_ml106.tu6_1_1_3_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Value</th><th headers="hd_h_ml106.tu6_1_1_1_1 hd_h_ml106.tu6_1_1_2_1" id="hd_h_ml106.tu6_1_1_3_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Description</th><th headers="hd_h_ml106.tu6_1_1_1_2 hd_h_ml106.tu6_1_1_2_1" id="hd_h_ml106.tu6_1_1_3_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Parameter</th><th headers="hd_h_ml106.tu6_1_1_1_2 hd_h_ml106.tu6_1_1_2_1" id="hd_h_ml106.tu6_1_1_3_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Value</th><th headers="hd_h_ml106.tu6_1_1_1_2 hd_h_ml106.tu6_1_1_2_1" id="hd_h_ml106.tu6_1_1_3_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Description</th></tr></thead><tbody><tr><td headers="hd_h_ml106.tu6_1_1_1_1 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><b>1</b></td><td headers="hd_h_ml106.tu6_1_1_1_1 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Reagent</td><td headers="hd_h_ml106.tu6_1_1_1_1 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">2 μL</td><td headers="hd_h_ml106.tu6_1_1_1_1 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">ATP/peptide</td><td headers="hd_h_ml106.tu6_1_1_1_2 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Reagent</td><td headers="hd_h_ml106.tu6_1_1_1_2 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">2 μL</td><td headers="hd_h_ml106.tu6_1_1_1_2 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">ATP/peptide</td></tr><tr><td headers="hd_h_ml106.tu6_1_1_1_1 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><b>2</b></td><td headers="hd_h_ml106.tu6_1_1_1_1 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Library</td><td headers="hd_h_ml106.tu6_1_1_1_1 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">23 nL</td><td headers="hd_h_ml106.tu6_1_1_1_1 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0.5 nM- 46 μM</td><td headers="hd_h_ml106.tu6_1_1_1_2 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Library</td><td headers="hd_h_ml106.tu6_1_1_1_2 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">23 nL</td><td headers="hd_h_ml106.tu6_1_1_1_2 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0.6 nM- 55.2 μM</td></tr><tr><td headers="hd_h_ml106.tu6_1_1_1_1 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><b>3</b></td><td headers="hd_h_ml106.tu6_1_1_1_1 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Controls</td><td headers="hd_h_ml106.tu6_1_1_1_1 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">23 nL</td><td headers="hd_h_ml106.tu6_1_1_1_1 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">TG003</td><td headers="hd_h_ml106.tu6_1_1_1_2 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Controls</td><td headers="hd_h_ml106.tu6_1_1_1_2 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">23 nL</td><td headers="hd_h_ml106.tu6_1_1_1_2 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">TG003</td></tr><tr><td headers="hd_h_ml106.tu6_1_1_1_1 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><b>4</b></td><td headers="hd_h_ml106.tu6_1_1_1_1 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Reagent</td><td headers="hd_h_ml106.tu6_1_1_1_1 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">1 μL</td><td headers="hd_h_ml106.tu6_1_1_1_1 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Clk4</td><td headers="hd_h_ml106.tu6_1_1_1_2 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Reagent</td><td headers="hd_h_ml106.tu6_1_1_1_2 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0.5 μL</td><td headers="hd_h_ml106.tu6_1_1_1_2 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Clk4</td></tr><tr><td headers="hd_h_ml106.tu6_1_1_1_1 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><b>5</b></td><td headers="hd_h_ml106.tu6_1_1_1_1 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Time</td><td headers="hd_h_ml106.tu6_1_1_1_1 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">4.5 hrs</td><td headers="hd_h_ml106.tu6_1_1_1_1 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">r.t. incubation</td><td headers="hd_h_ml106.tu6_1_1_1_2 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Time</td><td headers="hd_h_ml106.tu6_1_1_1_2 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">1 hr</td><td headers="hd_h_ml106.tu6_1_1_1_2 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">r.t. incubation</td></tr><tr><td headers="hd_h_ml106.tu6_1_1_1_1 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><b>6</b></td><td headers="hd_h_ml106.tu6_1_1_1_1 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Reagent</td><td headers="hd_h_ml106.tu6_1_1_1_1 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">3 μL</td><td headers="hd_h_ml106.tu6_1_1_1_1 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Kinase-Glo</td><td headers="hd_h_ml106.tu6_1_1_1_2 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Reagent</td><td headers="hd_h_ml106.tu6_1_1_1_2 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">2.5 μL</td><td headers="hd_h_ml106.tu6_1_1_1_2 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Deplete ATP</td></tr><tr><td headers="hd_h_ml106.tu6_1_1_1_1 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><b>7</b></td><td headers="hd_h_ml106.tu6_1_1_1_1 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Read</td><td headers="hd_h_ml106.tu6_1_1_1_1 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">2 sec</td><td headers="hd_h_ml106.tu6_1_1_1_1 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">ViewLux</td><td headers="hd_h_ml106.tu6_1_1_1_2 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Time</td><td headers="hd_h_ml106.tu6_1_1_1_2 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">45 min</td><td headers="hd_h_ml106.tu6_1_1_1_2 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">r.t. incubation</td></tr><tr><td headers="hd_h_ml106.tu6_1_1_1_1 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><b>8</b></td><td headers="hd_h_ml106.tu6_1_1_1_1 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"></td><td headers="hd_h_ml106.tu6_1_1_1_1 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"></td><td headers="hd_h_ml106.tu6_1_1_1_1 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"></td><td headers="hd_h_ml106.tu6_1_1_1_2 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Reagent</td><td headers="hd_h_ml106.tu6_1_1_1_2 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">5 μL</td><td headers="hd_h_ml106.tu6_1_1_1_2 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">ADP→ATP/Luc</td></tr><tr><td headers="hd_h_ml106.tu6_1_1_1_1 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><b>9</b></td><td headers="hd_h_ml106.tu6_1_1_1_1 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"></td><td headers="hd_h_ml106.tu6_1_1_1_1 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"></td><td headers="hd_h_ml106.tu6_1_1_1_1 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"></td><td headers="hd_h_ml106.tu6_1_1_1_2 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Time</td><td headers="hd_h_ml106.tu6_1_1_1_2 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">30 min</td><td headers="hd_h_ml106.tu6_1_1_1_2 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">r.t. incubation</td></tr><tr><td headers="hd_h_ml106.tu6_1_1_1_1 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><b>10</b></td><td headers="hd_h_ml106.tu6_1_1_1_1 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"></td><td headers="hd_h_ml106.tu6_1_1_1_1 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"></td><td headers="hd_h_ml106.tu6_1_1_1_1 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"></td><td headers="hd_h_ml106.tu6_1_1_1_2 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Read</td><td headers="hd_h_ml106.tu6_1_1_1_2 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">2 sec</td><td headers="hd_h_ml106.tu6_1_1_1_2 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">ViewLux</td></tr><tr><td headers="hd_h_ml106.tu6_1_1_1_1 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_1 hd_h_ml106.tu6_1_1_3_2 hd_h_ml106.tu6_1_1_3_3 hd_h_ml106.tu6_1_1_3_4 hd_h_ml106.tu6_1_1_1_2 hd_h_ml106.tu6_1_1_3_5 hd_h_ml106.tu6_1_1_3_6 hd_h_ml106.tu6_1_1_3_7" colspan="7" rowspan="1" style="text-align:left;vertical-align:top;"><span class="hr"></span></td></tr><tr><td headers="hd_h_ml106.tu6_1_1_1_1 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Step</td><td headers="hd_h_ml106.tu6_1_1_1_1 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_2 hd_h_ml106.tu6_1_1_3_3 hd_h_ml106.tu6_1_1_3_4" colspan="3" rowspan="1" style="text-align:left;vertical-align:top;">Notes</td><td headers="hd_h_ml106.tu6_1_1_1_2 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_5 hd_h_ml106.tu6_1_1_3_6 hd_h_ml106.tu6_1_1_3_7" colspan="3" rowspan="1" style="text-align:left;vertical-align:top;">Notes</td></tr><tr><td headers="hd_h_ml106.tu6_1_1_1_1 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_1 hd_h_ml106.tu6_1_1_3_2 hd_h_ml106.tu6_1_1_3_3 hd_h_ml106.tu6_1_1_3_4 hd_h_ml106.tu6_1_1_1_2 hd_h_ml106.tu6_1_1_3_5 hd_h_ml106.tu6_1_1_3_6 hd_h_ml106.tu6_1_1_3_7" colspan="7" rowspan="1" style="text-align:left;vertical-align:top;"><span class="hr"></span></td></tr><tr><td headers="hd_h_ml106.tu6_1_1_1_1 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><b>1</b></td><td headers="hd_h_ml106.tu6_1_1_1_1 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_2 hd_h_ml106.tu6_1_1_3_3 hd_h_ml106.tu6_1_1_3_4" colspan="3" rowspan="1" style="text-align:left;vertical-align:top;">100 μM RS peptide 1 μM ATP (final) concentration in buffer: FRD dispense</td><td headers="hd_h_ml106.tu6_1_1_1_2 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_5 hd_h_ml106.tu6_1_1_3_6 hd_h_ml106.tu6_1_1_3_7" colspan="3" rowspan="1" style="text-align:left;vertical-align:top;">100 μM RS peptide 1 μM ATP (final) concentration in buffer: FRD dispense</td></tr><tr><td headers="hd_h_ml106.tu6_1_1_1_1 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><b>2</b></td><td headers="hd_h_ml106.tu6_1_1_1_1 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_2 hd_h_ml106.tu6_1_1_3_3 hd_h_ml106.tu6_1_1_3_4" colspan="3" rowspan="1" style="text-align:left;vertical-align:top;">Pin-tool transfer compound library for a (final) range of 46 μM to 0.5 nM</td><td headers="hd_h_ml106.tu6_1_1_1_2 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_5 hd_h_ml106.tu6_1_1_3_6 hd_h_ml106.tu6_1_1_3_7" colspan="3" rowspan="1" style="text-align:left;vertical-align:top;">Pin-tool transfer compound library for a (final) range of 55.2 μM to 0.6 nM</td></tr><tr><td headers="hd_h_ml106.tu6_1_1_1_1 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><b>3</b></td><td headers="hd_h_ml106.tu6_1_1_1_1 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_2 hd_h_ml106.tu6_1_1_3_3 hd_h_ml106.tu6_1_1_3_4" colspan="3" rowspan="1" style="text-align:left;vertical-align:top;"></td><td headers="hd_h_ml106.tu6_1_1_1_2 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_5 hd_h_ml106.tu6_1_1_3_6 hd_h_ml106.tu6_1_1_3_7" colspan="3" rowspan="1" style="text-align:left;vertical-align:top;"></td></tr><tr><td headers="hd_h_ml106.tu6_1_1_1_1 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><b>4</b></td><td headers="hd_h_ml106.tu6_1_1_1_1 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_2 hd_h_ml106.tu6_1_1_3_3 hd_h_ml106.tu6_1_1_3_4" colspan="3" rowspan="1" style="text-align:left;vertical-align:top;">Clk4 at 25 nM final, FRD dispense</td><td headers="hd_h_ml106.tu6_1_1_1_2 hd_h_ml106.tu6_1_1_2_1 hd_h_ml106.tu6_1_1_3_5 hd_h_ml106.tu6_1_1_3_6 hd_h_ml106.tu6_1_1_3_7" colspan="3" rowspan="1" style="text-align:left;vertical-align:top;">Clk4 at 25 nM final, FRD dispense</td></tr></tbody></table></div></div></article><article data-type="fig" id="figobml106f3"><div id="ml106.f3" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK47344/bin/ml106f3.jpg" alt="Figure 3. Performance of the two 1,536-well Clk4 kinase assays." /></div><h3><span class="label">Figure 3</span><span class="title">Performance of the two 1,536-well Clk4 kinase assays</span></h3><div class="caption"><p>Kinase-Glo (A) and ADP-Glo (B) qHTS experiments. Light grey represents the compounds that fit to active CRC classes, and dark grey is data classified as inactive. Data for one of the ADP-Glo qHTS experiments is shown. C). Data and curve-fits for the highest confidence CRCs classes obtained in the Kinase-Glo qHTS. D). Data and curve-fits for the highest confidence CRCs classes obtained in the ADP-Glo qHTS, both replicates are shown.</p></div></div></article><article data-type="fig" id="figobml106f4"><div id="ml106.f4" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK47344/bin/ml106f4.jpg" alt="Figure 4. ADP-Glo and Kinase-Glo show excellent agreement in potency values." /></div><h3><span class="label">Figure 4</span><span class="title">ADP-Glo and Kinase-Glo show excellent agreement in potency values</span></h3><div class="caption"><p>Comparison of potency values for run 1 (A) and run 2 (B) of the ADP-Glo qHTS against the Kinase-Glo qHTS. Black solid circles represent potencies derived from high quality CRCs and open squares represent potencies derived from lower confidence CRCs.</p></div></div></article><article data-type="fig" id="figobml106f5"><div id="ml106.f5" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK47344/bin/ml106f5.jpg" alt="Figure 5. Summary of activity from the qHTS." /></div><h3><span class="label">Figure 5</span><span class="title">Summary of activity from the qHTS</span></h3><div class="caption"><p><b>A)</b> Percentage of high quality actives (CRC classes 1a, 1b and 2a), inconclusive (all other active CRC classes) and inactive compounds (CRC class 4) obtained from the Kinase-Glo and duplicate runs of the ADP-Glo assay. <b>B</b>) Scaffolds in the 1,352 member kinase-focused library. C). Distribution of activity per in clusters for each scaffold (1-6) obtained from the qHTS. <b>D</b>) Potency distribution in clusters for each scaffold (1-6) obtained from the qHTS.</p></div></div></article><article data-type="fig" id="figobml106f8"><div id="ml106.f8" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK47344/bin/ml106f8.jpg" alt="Scheme 1. Example qHTS data and classification scheme for assignment of resulting curve-fit data into classes." /></div><h3><span class="label">Scheme 1</span><span class="title">Example qHTS data and classification scheme for assignment of resulting curve-fit data into classes</span></h3><div class="caption"><p><b>Top</b>, qHTS curve-fit data from <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/361" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-361</a> binned into curve classifications 1-4 based classification criteria. <b>Below</b>, Examples of curves fitting the following classification criteria: Class 1 curves display two asymptotes, an inflection point, and r2 ≥ 0.9; subclasses 1a (blue) vs. 1b (orange) are differentiated by full (>80%) vs. partial (≤ 80%) response. Class 2 curves display a single left-hand asymptote and inflection point; subclasses 2a (blue) and 2b (orange) are differentiated by a max response and r2, >80% and >0.9 or <80% and <0.9, respectively. Class 3 curves have a single left-hand asymptote, no inflection point, and a response >3SD the mean activity of the sample field. Class 4 defines those samples showing no activity across the concentration range. Specifically, for the identification of the Probe series 1, the filtering shown in <a class="figpopup" href="/books/NBK47344/figure/ml106.f3/?report=objectonly" target="object" rid-figpopup="figml106f3" rid-ob="figobml106f3">Figure 3</a> below was used.</p></div></div></article><article data-type="fig" id="figobml106f6"><div id="ml106.f6" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK47344/bin/ml106f6.jpg" alt="Figure 6. Confirmation data for CID-322621." /></div><h3><span class="label">Figure 6</span><span class="title">Confirmation data for CID-322621</span></h3><div class="caption"><p>Normalized data for the ADP-Glo (black; IC<sub>50</sub> = 75 nM), Kinase-Glo (cyan; IC<sub>50</sub> = 63 nM), and Bell-Brooks Transcreener (Green; IC<sub>50</sub> = 50 nM) assays against Clk4 are shown. Also shown is the fluorescent interference data for the compound against the Transcreener label (pink) and interference with Kinase-Glo (using 1 μM ATP and Ultra-Glo luciferase alone; orange).</p></div></div></article><article data-type="fig" id="figobml106fu3"><div id="ml106.fu3" class="figure"><div class="graphic"><img data-src="/books/NBK47344/bin/ml106fu3.jpg" alt="Image ml106fu3" /></div></div></article><article data-type="fig" id="figobml106fu4"><div id="ml106.fu4" class="figure"><div class="graphic"><img data-src="/books/NBK47344/bin/ml106fu4.jpg" alt="Image ml106fu4" /></div></div></article><article data-type="fig" id="figobml106fu5"><div id="ml106.fu5" class="figure"><div class="graphic"><img data-src="/books/NBK47344/bin/ml106fu5.jpg" alt="Image ml106fu5" /></div></div></article><article data-type="table-wrap" id="figobml106tu7"><div id="ml106.tu7" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK47344/table/ml106.tu7/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml106.tu7_lrgtbl__"><table><thead><tr><th id="hd_h_ml106.tu7_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Curve Class</th><th id="hd_h_ml106.tu7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"># of Compounds</th></tr></thead><tbody><tr><td headers="hd_h_ml106.tu7_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">−1.1</td><td headers="hd_h_ml106.tu7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">47</td></tr><tr><td headers="hd_h_ml106.tu7_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">−1.2</td><td headers="hd_h_ml106.tu7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">11</td></tr><tr><td headers="hd_h_ml106.tu7_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">−2.1</td><td headers="hd_h_ml106.tu7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">2</td></tr><tr><td headers="hd_h_ml106.tu7_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">−2.2</td><td headers="hd_h_ml106.tu7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">1</td></tr><tr><td headers="hd_h_ml106.tu7_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">−2.4</td><td headers="hd_h_ml106.tu7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">1</td></tr><tr><td headers="hd_h_ml106.tu7_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">4</td><td headers="hd_h_ml106.tu7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">1</td></tr></tbody></table></div></div></article><article data-type="table-wrap" id="figobml106tu8"><div id="ml106.tu8" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK47344/table/ml106.tu8/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml106.tu8_lrgtbl__"><table><thead><tr><th id="hd_h_ml106.tu8_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">AC50 Range</th><th id="hd_h_ml106.tu8_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"># of Compounds</th></tr></thead><tbody><tr><td headers="hd_h_ml106.tu8_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><=100nM</td><td headers="hd_h_ml106.tu8_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">3</td></tr><tr><td headers="hd_h_ml106.tu8_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.1–lµM</td><td headers="hd_h_ml106.tu8_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">18</td></tr><tr><td headers="hd_h_ml106.tu8_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">1–10µM</td><td headers="hd_h_ml106.tu8_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">23</td></tr><tr><td headers="hd_h_ml106.tu8_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">>10µM</td><td headers="hd_h_ml106.tu8_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">19</td></tr></tbody></table></div></div></article><article data-type="fig" id="figobml106fu6"><div id="ml106.fu6" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK47344/bin/ml106fu6.jpg" alt="SAR from Primary Clk4 Screen." /></div><h3><span class="title">SAR from Primary Clk4 Screen</span></h3></div></article><article data-type="fig" id="figobml106fu7"><div id="ml106.fu7" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK47344/bin/ml106fu7.jpg" alt="SAR from focused synthetic library." /></div><h3><span class="title">SAR from focused synthetic library</span></h3></div></article><article data-type="fig" id="figobml106fu8"><div id="ml106.fu8" class="figure"><div class="graphic"><img data-src="/books/NBK47344/bin/ml106fu8.jpg" alt="Image ml106fu8" /></div></div></article><article data-type="fig" id="figobml106f7"><div id="ml106.f7" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK47344/bin/ml106f7.jpg" alt="Figure 7. Confirmation data for CID: 3234998." /></div><h3><span class="label">Figure 7</span><span class="title">Confirmation data for CID: 3234998</span></h3><div class="caption"><p>Normalized data for the ADP-Glo (blue; IC<sub>50</sub> = 100 nM), Kinase-Glo (cyan; IC<sub>50</sub> = 70 nM), and Bell-Brooks Transcreener (black; IC<sub>50</sub> = 50 nM) assays against Clk4 are shown. Also shown is the fluorescent interference data for the compound against the Transcreener label (grey) and interference with Kinase-Glo (using 1 μM ATP and Ultra-Glo luciferase alone; green).</p></div></div></article><article data-type="fig" id="figobml106fu9"><div id="ml106.fu9" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK47344/bin/ml106fu9.jpg" alt="SAR from Primary Clk4 Screen." /></div><h3><span class="title">SAR from Primary Clk4 Screen</span></h3></div></article><article data-type="fig" id="figobml106fu10"><div id="ml106.fu10" class="figure"><div class="graphic"><img data-src="/books/NBK47344/bin/ml106fu10.jpg" alt="Image ml106fu10" /></div></div></article></div><div id="jr-scripts"><script src="/corehtml/pmc/jatsreader/ptpmc_3.22/js/libs.min.js"> </script><script src="/corehtml/pmc/jatsreader/ptpmc_3.22/js/jr.min.js"> </script></div></div>
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