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<script type="text/javascript" src="/corehtml/pmc/jatsreader/ptpmc_3.22/js/jr.boots.min.js"> </script><title>Toward Improved Therapy for Classic Galactosemia - 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="Toward Improved Therapy for Classic Galactosemia">
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<meta name="citation_publisher" content="National Center for Biotechnology Information (US)">
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<meta name="citation_date" content="2011/03/03">
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<meta name="citation_author" content="Matthew B Boxer">
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<meta name="citation_author" content="Min Shen">
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<meta name="citation_author" content="Cordelle Tanega">
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<meta name="citation_author" content="Manshu Tang">
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<meta name="citation_author" content="Kent Lai">
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<meta name="citation_author" content="Douglas S Auld">
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<meta name="citation_pmid" content="21735606">
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<meta name="DC.Title" content="Toward Improved Therapy for Classic Galactosemia">
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<meta name="DC.Type" content="Text">
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<meta name="DC.Publisher" content="National Center for Biotechnology Information (US)">
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<meta name="DC.Contributor" content="Matthew B Boxer">
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<meta name="DC.Contributor" content="Min Shen">
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<meta name="DC.Contributor" content="Cordelle Tanega">
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<meta name="DC.Contributor" content="Manshu Tang">
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<meta name="DC.Contributor" content="Kent Lai">
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<meta name="DC.Contributor" content="Douglas S Auld">
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<meta name="DC.Date" content="2011/03/03">
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<meta name="description" content="Classic Galactosemia is a rare genetic metabolic disorder (1/60,000 births) that is characterized by decreased production of galactose-1-phosphate uridyltransferase (GALT), an enzyme responsible for the conversion of galactose-1-phosphate (gal-1-p) to glucose-1-phosphate. The resulting elevated intracellular concentrations of gal-1-p are believed to be the major pathogenic mechanism in Classic Galactosemia that leads to a myriad of secondary symptoms and, if untreated, death of the patient. Galactokinase (GALK) is an upstream enzyme in the Leloir pathway that is responsible for conversion of galactose to gal-1-p. Therefore, it was hypothesized that the identification of a small-molecule inhibitor of GALK would act to decrease levels of gal-1-p and allow for a novel entry into therapies for this disorder. In collaboration with Professor Kent Lai of the University of Utah, a quantitative high-throughput screening (qHTS) assay has been performed at the NIH Chemical Genomics Center (NCGC) and has identified a potent and selective inhibitor of GALK bearing a 1,4-dihydropyrimidine core. Several rounds of medicinal chemistry were performed and generated a small molecule capable of inhibiting GALK with an IC50 of 1.0μM. The probe ML152 (CID-664331; SID-87550830) is selective for GALK against CDP-ME, and thus represents an advancement over previously reported inhibitors. The probe was the best analog for inhibition of GALK in a purified enzyme assay. Its mechanism of action, as determined via substrate competition and kinetic assays, are consistent with it being ATP competitive.">
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<meta name="og:title" content="Toward Improved Therapy for Classic Galactosemia">
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<meta name="og:type" content="book">
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<meta name="og:description" content="Classic Galactosemia is a rare genetic metabolic disorder (1/60,000 births) that is characterized by decreased production of galactose-1-phosphate uridyltransferase (GALT), an enzyme responsible for the conversion of galactose-1-phosphate (gal-1-p) to glucose-1-phosphate. The resulting elevated intracellular concentrations of gal-1-p are believed to be the major pathogenic mechanism in Classic Galactosemia that leads to a myriad of secondary symptoms and, if untreated, death of the patient. Galactokinase (GALK) is an upstream enzyme in the Leloir pathway that is responsible for conversion of galactose to gal-1-p. Therefore, it was hypothesized that the identification of a small-molecule inhibitor of GALK would act to decrease levels of gal-1-p and allow for a novel entry into therapies for this disorder. In collaboration with Professor Kent Lai of the University of Utah, a quantitative high-throughput screening (qHTS) assay has been performed at the NIH Chemical Genomics Center (NCGC) and has identified a potent and selective inhibitor of GALK bearing a 1,4-dihydropyrimidine core. Several rounds of medicinal chemistry were performed and generated a small molecule capable of inhibiting GALK with an IC50 of 1.0μM. The probe ML152 (CID-664331; SID-87550830) is selective for GALK against CDP-ME, and thus represents an advancement over previously reported inhibitors. The probe was the best analog for inhibition of GALK in a purified enzyme assay. Its mechanism of action, as determined via substrate competition and kinetic assays, are consistent with it being ATP competitive.">
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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="_NBK56237_"><span class="title" itemprop="name">Toward Improved Therapy for Classic Galactosemia</span></h1><p class="contribs">Boxer MB, Shen M, Tanega C, et al.</p><p class="fm-aai"><a href="#_NBK56237_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>Classic Galactosemia is a rare genetic metabolic disorder (1/60,000 births) that is characterized by decreased production of galactose-1-phosphate uridyltransferase (GALT), an enzyme responsible for the conversion of galactose-1-phosphate (gal-1-p) to glucose-1-phosphate. The resulting elevated intracellular concentrations of gal-1-p are believed to be the major pathogenic mechanism in Classic Galactosemia that leads to a myriad of secondary symptoms and, if untreated, death of the patient. Galactokinase (GALK) is an upstream enzyme in the Leloir pathway that is responsible for conversion of galactose to gal-1-p. Therefore, it was hypothesized that the identification of a small-molecule inhibitor of GALK would act to decrease levels of gal-1-p and allow for a novel entry into therapies for this disorder. In collaboration with Professor Kent Lai of the University of Utah, a quantitative high-throughput screening (qHTS) assay has been performed at the NIH Chemical Genomics Center (NCGC) and has identified a potent and selective inhibitor of GALK bearing a 1,4-dihydropyrimidine core. Several rounds of medicinal chemistry were performed and generated a small molecule capable of inhibiting GALK with an IC<sub>50</sub> of 1.0μM. The probe <a href="/pcsubstance/?term=ML152[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML152</a> (CID-664331; <a href="https://pubchem.ncbi.nlm.nih.gov/substance/87550830" ref="pagearea=abstract&targetsite=entrez&targetcat=link&targettype=pubchem">SID-87550830</a>) is selective for GALK against CDP-ME, and thus represents an advancement over previously reported inhibitors. The probe was the best analog for inhibition of GALK in a purified enzyme assay. Its mechanism of action, as determined via substrate competition and kinetic assays, are consistent with it being ATP competitive.</p></div><div class="h2"></div><p><b>Assigned Assay Grant #:</b> R03 MH085689</p><p><b>Screening Center Name & PI:</b> NIH Chemical Genomics Center & Dr. Christopher P. Austin</p><p><b>Chemistry Center Name & PI:</b> NIH Chemical Genomics Center & Dr. Christopher P. Austin</p><p><b>Assay Submitter & Institution:</b> Kent Lai, University of Utah School of Medicine</p><p><b>PubChem Summary Bioassay Identifier (AID):</b>
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<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2114" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2114</a></p><div id="ml152.s1"><h2 id="_ml152_s1_">Probe Structure & Characteristics</h2><div id="ml152.fu1" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56237/bin/ml152fu1.jpg" alt="ML152." /></div><h3><span class="title"><a href="/pcsubstance/?term=ML152[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML152</a></span></h3></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml152tu1"><a href="/books/NBK56237/table/ml152.tu1/?report=objectonly" target="object" title="Table" class="img_link icnblk_img figpopup" rid-figpopup="figml152tu1" rid-ob="figobml152tu1"><img class="small-thumb" src="/books/NBK56237/table/ml152.tu1/?report=thumb" src-large="/books/NBK56237/table/ml152.tu1/?report=previmg" alt="Image " /></a><div class="icnblk_cntnt"><h4 id="ml152.tu1"><a href="/books/NBK56237/table/ml152.tu1/?report=objectonly" target="object" rid-ob="figobml152tu1">Table</a></h4></div></div></div><div id="ml152.s2"><h2 id="_ml152_s2_">Recommendations for scientific use of the probe</h2><p>Classic Galactosemia is a potentially lethal disease caused by deficient galactose-1-phosphate uridyltransferase (GALT) that results in the buildup of galactose-1-phosphate (gal-1-P) in the blood. Galactokinase (GALK) is the enzyme responsible for converting galactose into gal-1-p. A pharmacological inhibitor of GALK is therefore sought for a potential therapy for galactosemia by reducing levels of gal-1-P. The probe developed herein inhibits GALK with a potency of 1.0μM in a luminescence based biochemical assay and should be useful for studying the role of GALK modulation in the progression of Classic Galactosemia.</p></div><div id="ml152.s3"><h2 id="_ml152_s3_">1. Introduction</h2><p>Classic Galactosemia is a potentially lethal disorder with a high mortality rate when left untreated. Typically, removal of lactose and galactose from the diet of individuals with deficient GALT function is sufficient for the prevention of lethality. While morbidity rates for those affected with Classic Galactosemia have decreased, the lack of an effective therapy has been shown to cause developmental delay, neurological disorders, and premature ovarian failure<a class="bibr" href="#ml152.r1" rid="ml152.r1">1</a>. Although the exact pathogenic mechanism of Classic Galactosemia is not known, elevated galactose-1-phosphate (gal-1-p) levels have been denoted as a probable cause. The high levels of gal-1-p are believed to arise from deficient GALT, which is the second enzyme in the Leloir pathway that converts gal-1-p to uridine diphosphogalactose (UDP-gal) and uridine diphosphoglucose (UDP-glu) to glucose-1-phosphate (glu-1-p). Upstream from this enzyme liess GALK, which converts galactose to gal-1-p. This, and the fact that GALK-deficient patients have much milder and benign phenotypes, position GALK as a key target for reduction of gal-1-p levels and a potential therapeutic target for Classic Galactosemia<a class="bibr" href="#ml152.r2" rid="ml152.r2 ml152.r3 ml152.r4">2–4</a>. The current probe (CID-664331) represents the first small molecule GALK inhibitor with good potency against the purified GALK enzyme and high selectivity against CMK kinase (a related kinase in the GHMP kinase family).</p></div><div id="ml152.s4"><h2 id="_ml152_s4_">2. Materials and Methods</h2><p>All air and/or moisture sensitive reactions were performed under positive pressure of nitrogen with oven-dried glassware. Anhydrous solvents such as dichloromethane, <i>N, N</i>-dimethylforamide (DMF), acetonitrile, methanol and triethylamine were obtained by purchasing from Sigma-Aldrich. Preparative purification was performed on a Waters semi-preparative HPLC. The column used was a Phenomenex Luna C18 (5 micron, 30 × 75 mm) at a flow rate of 45 ml/min. The mobile phase consisted of acetonitrile and water (each containing 0.1% trifluoroacetic acid). A gradient of 10% to 50% acetonitrile over 8 minutes was used during the purification. Fraction collection was triggered by UV detection (220 nM). Analytical analysis was performed on an Agilent LC/MS (Agilent Technologies, Santa Clara, CA).</p><p>Method 1: A 7 minute gradient of 4% to 100% Acetonitrile (containing 0.025% trifluoroacetic acid) in water (containing 0.05% trifluoroacetic acid) was used with an 8 minute run time at a flow rate of 1 mL/min. A Phenomenex Luna C18 column (3 micron, 3 × 75 mm) was used at a temperature of 50°C.</p><p>Method 2: A 3 minute gradient of 4% to 100% Acetonitrile (containing 0.025% trifluoroacetic acid) in water (containing 0.05% trifluoroacetic acid) was used with a 4.5 minute run time at a flow rate of 1 mL/min. A Phenomenex Gemini Phenyl column (3 micron, 3 × 100 mm) was used at a temperature of 50 °C.</p><p>Purity determination was performed using an Agilent Diode Array Detector on both Method 1 and Method 2. Mass determination was performed using an Agilent 6130 mass spectrometer with electrospray ionization in the positive mode. <sup>1</sup>H NMR spectra were recorded on Varian 400 MHz spectrometers. Chemical Shifts are reported in ppm with tetramethylsilane (TMS) as internal standard (0 ppm) for CDCl<sub>3</sub> solutions or undeuterated solvent (DMSO-h6 at 2.49 ppm) for DMSO-d6 solutions. All of the analogs for assay have purity greater than 95% based on both analytical methods. High resolution mass spectrometry was recorded on an Agilent 6210 Time-of-Flight LC/MS system. Confirmation of molecular formula was accomplished using electrospray ionization in the positive mode with the Agilent Masshunter software (version B.02).</p><div id="ml152.s5"><h3>2.1. Assays</h3><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml152t1"><a href="/books/NBK56237/table/ml152.t1/?report=objectonly" target="object" title="Table 1" class="img_link icnblk_img figpopup" rid-figpopup="figml152t1" rid-ob="figobml152t1"><img class="small-thumb" src="/books/NBK56237/table/ml152.t1/?report=thumb" src-large="/books/NBK56237/table/ml152.t1/?report=previmg" alt="Table 1. Screens Deposited in PubChem." /></a><div class="icnblk_cntnt"><h4 id="ml152.t1"><a href="/books/NBK56237/table/ml152.t1/?report=objectonly" target="object" rid-ob="figobml152t1">Table 1</a></h4><p class="float-caption no_bottom_margin">Screens Deposited in PubChem. </p></div></div><div id="ml152.s6"><h4>Primary qHTS assay for inhibitors of GALK [<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1868" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1868</a>]</h4><p><b>Assay details and protocol:</b> The primary assay monitored ATP depletion using Promega’s KinaseGlo™ technology, where ATP levels are measured through luminescence generated from firefly luciferase, a bioluminescent ATP-dependent enzyme<a class="bibr" href="#ml152.r5" rid="ml152.r5">5</a>, <a class="bibr" href="#ml152.r6" rid="ml152.r6">6</a>. ATP was held at 35μM, near its reported K<sub>M</sub> value, and the K<sub>M</sub> for galactose was determined under the 1536-well assay conditions to be 50–100μM (<a class="figpopup" href="/books/NBK56237/figure/ml152.f1/?report=objectonly" target="object" rid-figpopup="figml152f1" rid-ob="figobml152f1">Figure 1A</a>). As well, we confirmed the IC<sub>50</sub> for a commercially available CD45 inhibitor (N-(9,10-dioxo-9,10-dihydrophenanthren-2-yl)pivalamide), previously found by Dr. Lai to inhibit GALK (<a class="figpopup" href="/books/NBK56237/figure/ml152.f1/?report=objectonly" target="object" rid-figpopup="figml152f1" rid-ob="figobml152f1">Figure 1B</a>)<a class="bibr" href="#ml152.r1" rid="ml152.r1">1</a>. This was used as the positive control for the assay. The assay used 5 nM GalK and a 1 hr incubation time, which gave sufficient signal:background and stability for the HTS. The percent conversion of ATP under these conditions was estimated to be approximately 50% using an ATP standard curve.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml152f1" co-legend-rid="figlgndml152f1"><a href="/books/NBK56237/figure/ml152.f1/?report=objectonly" target="object" title="Figure 1" class="img_link icnblk_img figpopup" rid-figpopup="figml152f1" rid-ob="figobml152f1"><img class="small-thumb" src="/books/NBK56237/bin/ml152f1.gif" src-large="/books/NBK56237/bin/ml152f1.jpg" alt="Figure 1. Galactose KM determination and sensitivity to a control inhibitor in the 1536-well GalK assay." /></a><div class="icnblk_cntnt" id="figlgndml152f1"><h4 id="ml152.f1"><a href="/books/NBK56237/figure/ml152.f1/?report=objectonly" target="object" rid-ob="figobml152f1">Figure 1</a></h4><p class="float-caption no_bottom_margin">Galactose K<sub>M</sub> determination and sensitivity to a control inhibitor in the 1536-well GalK assay. <i>A</i>. Galactose variation in the 1536-well assay using ATP depletion. Rates (from determining the remaining ATP at time seven points starting at 5 min to 55 min; <a href="/books/NBK56237/figure/ml152.f1/?report=objectonly" target="object" rid-ob="figobml152f1">(more...)</a></p></div></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml152t2"><a href="/books/NBK56237/table/ml152.t2/?report=objectonly" target="object" title="Table 2" class="img_link icnblk_img figpopup" rid-figpopup="figml152t2" rid-ob="figobml152t2"><img class="small-thumb" src="/books/NBK56237/table/ml152.t2/?report=thumb" src-large="/books/NBK56237/table/ml152.t2/?report=previmg" alt="Table 2. Stepwise Protocol Used for the 1536-Well Assay." /></a><div class="icnblk_cntnt"><h4 id="ml152.t2"><a href="/books/NBK56237/table/ml152.t2/?report=objectonly" target="object" rid-ob="figobml152t2">Table 2</a></h4><p class="float-caption no_bottom_margin">Stepwise Protocol Used for the 1536-Well Assay. </p></div></div></div><div id="ml152.s7"><h4>Confirmatory assay. [<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2499" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-2499</a>]</h4><p>As in the primary screen, the confirmatory assay monitored ATP depletion using Promega’s KinaseGlo™ technology, where ATP levels are measured through luminescence generated from firefly luciferase, a bioluminescent ATP-dependent enzyme<a class="bibr" href="#ml152.r5" rid="ml152.r5">5</a>, <a class="bibr" href="#ml152.r6" rid="ml152.r6">6</a>. ATP was held at 35μM, near its reported K<sub>M</sub> value, and the K<sub>M</sub> for galactose was determined under the 1536-well assay conditions to be 50 – 100μM. In this assay, NCGC00187642 (CID-664331; <a href="https://pubchem.ncbi.nlm.nih.gov/substance/87550830" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-87550830</a>) was found to be a 1μM inhibitor of GALK (<a class="figpopup" href="/books/NBK56237/figure/ml152.f2/?report=objectonly" target="object" rid-figpopup="figml152f2" rid-ob="figobml152f2">Figure 2</a>).</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml152f2" co-legend-rid="figlgndml152f2"><a href="/books/NBK56237/figure/ml152.f2/?report=objectonly" target="object" title="Figure 2" class="img_link icnblk_img figpopup" rid-figpopup="figml152f2" rid-ob="figobml152f2"><img class="small-thumb" src="/books/NBK56237/bin/ml152f2.gif" src-large="/books/NBK56237/bin/ml152f2.jpg" alt="Figure 2. Dose response curve for NCGC00187642 (CID-664331; SID-87550830) in confirmatory assay." /></a><div class="icnblk_cntnt" id="figlgndml152f2"><h4 id="ml152.f2"><a href="/books/NBK56237/figure/ml152.f2/?report=objectonly" target="object" rid-ob="figobml152f2">Figure 2</a></h4><p class="float-caption no_bottom_margin">Dose response curve for NCGC00187642 (CID-664331; SID-87550830) in confirmatory assay. </p></div></div></div><div id="ml152.s8"><h4>Secondary assays</h4><p>The selectivity for these analogs against CDP-ME, another member of the GHMP kinase family, was performed using a bioluminescent Kinase Glo™ assay that detects ATP depletion after kinase reaction (PubChem ID 2506). NCGC00187642 (CID-664331; <a href="https://pubchem.ncbi.nlm.nih.gov/substance/87550830" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-87550830</a>); all analogs showed no activity in this assay (up to 57μM), highlighting the selectivity of this chemotype. All analogs were also tested for cytotoxicity using Promega CellTiter Glo on HEK293 cells that were treated with compounds and analyzed after 48 hours. All compounds shown in <a class="figpopup" href="/books/NBK56237/table/ml152.t5/?report=objectonly" target="object" rid-figpopup="figml152t5" rid-ob="figobml152t5">table 5</a> showed no cytotoxic effect (PubChem ID 2547). In addition, the ability of these compounds to undergo redox recycling, which may lead to false positive results, was also evaluated using an endpoint colorimetric assay; this assay detects the presence of H<sub>2</sub>O<sub>2</sub> in the kinase reaction buffer (PubChem ID 2502). The lead compound and analogs are inactive in the redox recycling, further confirming that these compounds have genuine GALK target activity.</p></div></div><div id="ml152.s9"><h3>2.2. Probe Chemical Characterization</h3><div id="ml152.f6" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56237/bin/ml152f6.jpg" alt="Scheme 1. Synthesis of CID-664331." /></div><h3><span class="label">Scheme 1</span><span class="title">Synthesis of CID-664331</span></h3></div><p><sup>1</sup>H NMR (400 MHz, DMSO-<i>d</i><sub>6</sub>); 10.37 (br. s. 1H), 9.98 (br. S. 1H), 7.40 (m, 2H), 7.16 (m, 2H), 2.52 (m, 2H), 2.39 (m, 2H), 2.26 (m, 2H), 1.83 (m, 6H), 1.62 (m, 2H). Method 1, retention time, 5.671 min; Method 2, retention time 3.704 min; HRMS: <i>m/z</i> (M+H<sup>+</sup>) = 336.1592 (Calculated for C<sub>19</sub>H<sub>20</sub>N<sub>4</sub>O<sub>2</sub> = 336.1586). Solubility (PBS, pH 7.4, 23°C) = 2.3 μg/ml. Stability profile over 48 hrs (PBS, pH 7.4, 23°C) is shown below in <a class="figpopup" href="/books/NBK56237/figure/ml152.f3/?report=objectonly" target="object" rid-figpopup="figml152f3" rid-ob="figobml152f3">Figure 3</a>.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml152f3" co-legend-rid="figlgndml152f3"><a href="/books/NBK56237/figure/ml152.f3/?report=objectonly" target="object" title="Figure 3" class="img_link icnblk_img figpopup" rid-figpopup="figml152f3" rid-ob="figobml152f3"><img class="small-thumb" src="/books/NBK56237/bin/ml152f3.gif" src-large="/books/NBK56237/bin/ml152f3.jpg" alt="Figure 3. PBS Buffer Stability of NCGC00187642/CID664331." /></a><div class="icnblk_cntnt" id="figlgndml152f3"><h4 id="ml152.f3"><a href="/books/NBK56237/figure/ml152.f3/?report=objectonly" target="object" rid-ob="figobml152f3">Figure 3</a></h4><p class="float-caption no_bottom_margin">PBS Buffer Stability of NCGC00187642/CID664331. Stability of NCGC00187642/CID664331 in PBS buffer (pH 7.4, 23°C) plotted as AUC vs. time for a 48 hr period using LCMS Method 1 described in Section 2. No instability was observed, and 100% of compound <a href="/books/NBK56237/figure/ml152.f3/?report=objectonly" target="object" rid-ob="figobml152f3">(more...)</a></p></div></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml152tu2"><a href="/books/NBK56237/table/ml152.tu2/?report=objectonly" target="object" title="Table" class="img_link icnblk_img figpopup" rid-figpopup="figml152tu2" rid-ob="figobml152tu2"><img class="small-thumb" src="/books/NBK56237/table/ml152.tu2/?report=thumb" src-large="/books/NBK56237/table/ml152.tu2/?report=previmg" alt="MLS Numbers for Probe and Analogs." /></a><div class="icnblk_cntnt"><h4 id="ml152.tu2"><a href="/books/NBK56237/table/ml152.tu2/?report=objectonly" target="object" rid-ob="figobml152tu2">Table</a></h4><p class="float-caption no_bottom_margin">MLS Numbers for Probe and Analogs. </p></div></div></div><div id="ml152.s11"><h3>2.3. Probe Preparation</h3><p>Cyclohexane-1,3-dione (0.191g, 1.703mmol, 1.5equiv.) and 1-(benzo[d]oxazol-2-yl)guanidine (.2g, 1.135mmol, 1.0equiv.) were added to a 2 – 5 ml Biotage microwave vial with a stir bar, and the powders were mixed well. Cyclopentanone (0.102ml, 1.135mmol, 1.5equiv.) was added, then the microwave vial was capped and quickly dropped into an oil bath at 120°C with vigorous stirring. The vial was stirred at 120°C for 6 hours, then removed from the oil bath; the reaction flask was carefully vented using a needle, then the cap was removed and ~6 ml of DMSO was added to the hot flask. Once all of the compound has dissolved, purification was done by directly injecting to a Waters® reverse phase purification system to give NCGC00187642/CID-664331 as a TFA salt (0.072g, 14%).</p></div></div><div id="ml152.s12"><h2 id="_ml152_s12_">3. Results</h2><p>Please check subsections for a detailed description of the results.</p><div id="ml152.s13"><h3>3.1. Summary of Screening Results</h3><p><b>Primary assay summary:</b> The primary qHTS showed excellent performance, and the statistics of the screen are shown below.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml152t3"><a href="/books/NBK56237/table/ml152.t3/?report=objectonly" target="object" title="Table 3" class="img_link icnblk_img figpopup" rid-figpopup="figml152t3" rid-ob="figobml152t3"><img class="small-thumb" src="/books/NBK56237/table/ml152.t3/?report=thumb" src-large="/books/NBK56237/table/ml152.t3/?report=previmg" alt="Table 3. Primary Assay Statistics." /></a><div class="icnblk_cntnt"><h4 id="ml152.t3"><a href="/books/NBK56237/table/ml152.t3/?report=objectonly" target="object" rid-ob="figobml152t3">Table 3</a></h4><p class="float-caption no_bottom_margin">Primary Assay Statistics. </p></div></div><p><b>Identification of lead:</b> A very low active rate was observed, and the results of the qHTS are shown in <a class="figpopup" href="/books/NBK56237/table/ml152.t4/?report=objectonly" target="object" rid-figpopup="figml152t4" rid-ob="figobml152t4">Table 4</a>. A total of 149 compounds were found to show certain inhibition. These were re-confirmed in the primary assay. Several counter-screens for assay related artifacts were then applied. These included an assay for inhibitors of KinaseGlo<a class="bibr" href="#ml152.r5" rid="ml152.r5">5</a>, redox-activity<a class="bibr" href="#ml152.r8" rid="ml152.r8">8</a> and selectivity against the related GHMP kinase, CDP-Me kinase (CMK) from bacteria. The latter used KinaseGlo again for detection. This identified NCGC00187642 (CID-664331; <a href="https://pubchem.ncbi.nlm.nih.gov/substance/87550830" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-87550830</a>) as a selective inhibitor of GALK.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml152t4"><a href="/books/NBK56237/table/ml152.t4/?report=objectonly" target="object" title="Table 4" class="img_link icnblk_img figpopup" rid-figpopup="figml152t4" rid-ob="figobml152t4"><img class="small-thumb" src="/books/NBK56237/table/ml152.t4/?report=thumb" src-large="/books/NBK56237/table/ml152.t4/?report=previmg" alt="Table 4. Concentration-Response Curve Class Distribution from Primary Screen." /></a><div class="icnblk_cntnt"><h4 id="ml152.t4"><a href="/books/NBK56237/table/ml152.t4/?report=objectonly" target="object" rid-ob="figobml152t4">Table 4</a></h4><p class="float-caption no_bottom_margin">Concentration-Response Curve Class Distribution from Primary Screen. </p></div></div></div><div id="ml152.s14"><h3>3.2. Dose Response Curve for Probe</h3><div id="ml152.f4" class="figure bk_fig"><div class="graphic"><img src="/books/NBK56237/bin/ml152f4.jpg" alt="Figure 4. Potency and selectivity of NCGC00187642 (CID-664331; SID-87550830) against CMK for the lead compound." /></div><h3><span class="label">Figure 4</span><span class="title">Potency and selectivity of NCGC00187642 (CID-664331; <a href="https://pubchem.ncbi.nlm.nih.gov/substance/87550830" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-87550830</a>) against CMK for the lead compound</span></h3><div class="caption"><p>The lead compound was observed to be active against GALK with an IC<sub>50</sub> of 1μM but inactive against CDP-Me kinase. Results showed no redox or cytotoxic activity of the compound (cytotoxicity was determined after 48 hours). All assays were performed at least in duplicate on at least two separate days.</p></div></div></div><div id="ml152.s15"><h3>3.3. Scaffold/Moiety Chemical Liabilities</h3><p>No obvious liabilities predicted.</p></div><div id="ml152.s16"><h3>3.4. SAR Table</h3><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml152t5"><a href="/books/NBK56237/table/ml152.t5/?report=objectonly" target="object" title="Table 5" class="img_link icnblk_img figpopup" rid-figpopup="figml152t5" rid-ob="figobml152t5"><img class="small-thumb" src="/books/NBK56237/table/ml152.t5/?report=thumb" src-large="/books/NBK56237/table/ml152.t5/?report=previmg" alt="Table 5. SAR of select analogs in GALK purified enzyme assay." /></a><div class="icnblk_cntnt"><h4 id="ml152.t5"><a href="/books/NBK56237/table/ml152.t5/?report=objectonly" target="object" rid-ob="figobml152t5">Table 5</a></h4><p class="float-caption no_bottom_margin">SAR of select analogs in GALK purified enzyme assay. </p></div></div></div><div id="ml152.s17"><h3>3.5. Cellular Activity</h3><p>Not available for this probe.</p></div><div id="ml152.s18"><h3>3.6. Profiling Assays</h3><p>Not available for this probe.</p></div></div><div id="ml152.s19"><h2 id="_ml152_s19_">4. Discussion</h2><p>SAR was seen with potencies ranging from 1μM to > 50μM (<a class="figpopup" href="/books/NBK56237/table/ml152.t5/?report=objectonly" target="object" rid-figpopup="figml152t5" rid-ob="figobml152t5">Table 5</a>). Entries 1 – 4 show the effect of varying the ketone starting material, giving variations of the spiro group, with the 5-membered ring giving the best results (entry 3). The dimethyl, 4- and 6-membered rings resulted in a ~10-fold loss of potency (compare entries 1, 2 and 4). The dione was also varied, giving a 5-membered compound as well as 5-phenyl and 5,5-dimethyl analogs that showed loss in potency (entries 5 – 7). Adding hydrophobic groups to the benzoxazole guanidine showed moderate to large loss of potency (6- to >50-fold). Changing the benzoxazole to a N-methylbenzimidazole was accompanied by a >50-fold loss in potency as well. From these results, it was determined that NCGC00187642 (CID-664331; <a href="https://pubchem.ncbi.nlm.nih.gov/substance/87550830" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-87550830</a>) was the best analog for the inhibition of GALK in the purified enzyme assay.</p><div id="ml152.s20"><h3>4.1. Comparison to existing art and how the new probe is an improvement</h3><p>This probe is an advancement over previously reported inhibitors, which were all non-selective electrophilic Michael-acceptors, and/or redox active compounds, which likely engender a promiscuous activity profile. The probe described here is selective for GALK against CDP-ME (a related kinase in the GHMP family), as well as other kinase assays run to date at the NIH Chemical Genomics Center. NCGC00187642 (CID-664331; <a href="https://pubchem.ncbi.nlm.nih.gov/substance/87550830" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-87550830</a>) did not show activity in 42 unique assays, including both biochemical and cell-based assays.</p></div><div id="ml152.s21"><h3>4.2. Mechanism of Action Studies</h3><p>The mechanism of action of NCGC00187642 (CID-664331; <a href="https://pubchem.ncbi.nlm.nih.gov/substance/87550830" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-87550830</a>) was determined via substrate competition and kinetic assays. Results are consistent with the lead compound being ATP competitive (<a class="figpopup" href="/books/NBK56237/figure/ml152.f5/?report=objectonly" target="object" rid-figpopup="figml152f5" rid-ob="figobml152f5">Figure 5</a>).</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml152f5" co-legend-rid="figlgndml152f5"><a href="/books/NBK56237/figure/ml152.f5/?report=objectonly" target="object" title="Figure 5" class="img_link icnblk_img figpopup" rid-figpopup="figml152f5" rid-ob="figobml152f5"><img class="small-thumb" src="/books/NBK56237/bin/ml152f5.gif" src-large="/books/NBK56237/bin/ml152f5.jpg" alt="Figure 5. Substrate competition with NCGC00187642 (CID-664331; SID-87550830)." /></a><div class="icnblk_cntnt" id="figlgndml152f5"><h4 id="ml152.f5"><a href="/books/NBK56237/figure/ml152.f5/?report=objectonly" target="object" rid-ob="figobml152f5">Figure 5</a></h4><p class="float-caption no_bottom_margin">Substrate competition with NCGC00187642 (CID-664331; SID-87550830). <i>A</i> Kinase reaction run at two different ATP concentrations, while keeping the galactose concentration at K<sub>M</sub>, showed increase in potency of the compound as exhibited by a left shift of <a href="/books/NBK56237/figure/ml152.f5/?report=objectonly" target="object" rid-ob="figobml152f5">(more...)</a></p></div></div></div><div id="ml152.s22"><h3>4.3. Planned Future Studies</h3><p>Medicinal chemistry efforts will be coupled with <i>in vitro</i> assays to develop a soluble, cell-permeable and metabolically stable compound to study the inhibition of GALK in a cell-based assay. Initial libraries will be synthesized and tested in purified enzyme assays (GALK and CDP-ME kinase) to assess potency and selectivity. Compounds with potencies ≤ 1μM that are >100 fold selective over all GHMP kinases would be carried on for subsequent <i>in vitro</i> ADME assays (PAMPA, solubility, Caco-2, hepatocyte stability). These assays will be necessary to further refine the profile of these chemotypes to possess the appropriate properties for cell-based assays. After analyzing the results from the <i>in vitro</i> ADME assays, subsequent rounds of SAR and library expansion may be necessary in a cyclical fashion.</p><p>After optimized probes are developed, we plan to test these in GALT-deficient primary patient fibroblasts for gal-1-p reduction (Lai Lab). We will also perform toxico-metabolomics studies of the selected inhibitors. Briefly, normal and patient cells will be incubated with the selected inhibitors for 16 hours or longer. Metabolomic profiles for a minimum of 58 intermediates of TCA cycle, amino acid biosynthesis, fatty acid oxidation, etc. will be elucidated by the Metabolomics Core Facility at the University of Utah on a fee-for-service basis.</p></div></div><div id="ml152.s23"><h2 id="_ml152_s23_">Probe properties</h2><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml152tu3"><a href="/books/NBK56237/table/ml152.tu3/?report=objectonly" target="object" title="Table" class="img_link icnblk_img figpopup" rid-figpopup="figml152tu3" rid-ob="figobml152tu3"><img class="small-thumb" src="/books/NBK56237/table/ml152.tu3/?report=thumb" src-large="/books/NBK56237/table/ml152.tu3/?report=previmg" alt="Properties computed from Structure." /></a><div class="icnblk_cntnt"><h4 id="ml152.tu3"><a href="/books/NBK56237/table/ml152.tu3/?report=objectonly" target="object" rid-ob="figobml152tu3">Table</a></h4><p class="float-caption no_bottom_margin">Properties computed from Structure. </p></div></div></div><div id="ml152.s24"><h2 id="_ml152_s24_">5. References</h2><dl class="temp-labeled-list"><dl class="bkr_refwrap"><dt>1.</dt><dd><div class="bk_ref" id="ml152.r1">Wierenga KJ, Lai K, Buchwald P, Tang M. High-throughput screening for human galactokinase inhibitors. <span><span class="ref-journal">J Biomol Screen. </span>2008;<span class="ref-vol">13</span>:415–423.</span> [<a href="/pmc/articles/PMC2705177/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2705177</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/18490662" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18490662</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>2.</dt><dd><div class="bk_ref" id="ml152.r2">Gitzelmann R. Galactose-1-phosphate in the pathophysiology of galactosemia. <span><span class="ref-journal">Eur J Pediatr. </span>1995;<span class="ref-vol">154</span>:S45–9.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/7671964" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 7671964</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>3.</dt><dd><div class="bk_ref" id="ml152.r3">Lai K, Langley SD, Khwaja FW, Schmitt EW, Elsas LJ. GALT deficiency causes UDP-hexose deficit in human galactosemic cells. <span><span class="ref-journal">Glycobiology. </span>2003;<span class="ref-vol">13</span>:285–94.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/12626383" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 12626383</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>4.</dt><dd><div class="bk_ref" id="ml152.r4">Lai K, Willis AC, Elsas LJ. The biochemical role of glutamine 188 in human galactose-1- phosphate uridyltransferase. <span><span class="ref-journal">J Biol Chem. </span>1999;<span class="ref-vol">274</span>:6559–66.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/10037750" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 10037750</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>5.</dt><dd><div class="bk_ref" id="ml152.r5">Auld DS, et al. 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 class="bkr_refwrap"><dt>6.</dt><dd><div class="bk_ref" id="ml152.r6">Inglese J, et al. Quantitative high-throughput screening: A titration-based approach that efficiently identifies biological activities in large chemical libraries. <span><span class="ref-journal">Proc Natl Acad Sci U S A. </span>2006;<span class="ref-vol">103</span>:11473–11478.</span> [<a href="/pmc/articles/PMC1518803/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC1518803</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/16864780" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 16864780</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>7.</dt><dd><div class="bk_ref" id="ml152.r7">Shukla SJ, et al. Identification of pregnane X receptor ligands using time-resolved fluorescence resonance energy transfer and quantitative high-throughput screening. <span><span class="ref-journal">Assay Drug Dev Technol. </span>2009;<span class="ref-vol">7</span>:143–169.</span> [<a href="/pmc/articles/PMC3116688/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3116688</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/19505231" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19505231</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>8.</dt><dd><div class="bk_ref" id="ml152.r8">Soares KM, et al. Profiling the NIH Small Molecule Repository for Compounds That Generate H(2)O(2) by Redox Cycling in Reducing Environments. <span><span class="ref-journal">Assay Drug Dev Technol. </span>2010;<span class="ref-vol">8</span>(2):152–74.</span> [<a href="/pmc/articles/PMC3098569/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3098569</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/20070233" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 20070233</span></a>]</div></dd></dl></dl></div><div id="bk_toc_contnr"></div></div></div><div class="fm-sec"><h2 id="_NBK56237_pubdet_">Publication Details</h2><h3>Author Information and Affiliations</h3><p class="contrib-group"><h4>Authors</h4><span itemprop="author">Matthew B Boxer</span>,<sup>a</sup> <span itemprop="author">Min Shen</span>,<sup>a</sup> <span itemprop="author">Cordelle Tanega</span>,<sup>a</sup> <span itemprop="author">Manshu Tang</span>,<sup>b</sup> <span itemprop="author">Kent Lai</span>,<sup>b</sup> and <span itemprop="author">Douglas S Auld</span><sup>a</sup>.</p><h4>Affiliations</h4><div class="affiliation"><sup>a</sup>
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NIH Chemical Genomics Center, National Human Genome Research Institute, National Institutes of Health, 9800 Medical Center Drive, MSC 3370 Bethesda, Maryland 20850</div><div class="affiliation"><sup>b</sup>
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Division of Medical Genetics, Department of Pediatrics, University of Utah School of Medicine, 50 N Mario Capecchi Drive, Salt Lake City, UT 84132</div><h3>Publication History</h3><p class="small">Received: <span itemprop="datePublished">March 18, 2010</span>; Last Update: <span itemprop="dateModified">March 3, 2011</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>Boxer MB, Shen M, Tanega C, et al. Toward Improved Therapy for Classic Galactosemia. 2010 Mar 18 [Updated 2011 Mar 3]. 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/ml154/?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/ml151/?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="figobml152fu1"><div id="ml152.fu1" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK56237/bin/ml152fu1.jpg" alt="ML152." /></div><h3><span class="title"><a href="/pcsubstance/?term=ML152[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML152</a></span></h3></div></article><article data-type="table-wrap" id="figobml152tu1"><div id="ml152.tu1" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK56237/table/ml152.tu1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml152.tu1_lrgtbl__"><table><thead><tr><th id="hd_h_ml152.tu1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">CID/ML#</th><th id="hd_h_ml152.tu1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Target Name</th><th id="hd_h_ml152.tu1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">IC<sub>50</sub>/EC<sub>50</sub> (nM) [SID, AID]</th><th id="hd_h_ml152.tu1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Anti-target Name(s)</th><th id="hd_h_ml152.tu1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">IC<sub>50</sub>/EC<sub>50</sub> (μM) [SID, AID]</th><th id="hd_h_ml152.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Fold Selective</th><th id="hd_h_ml152.tu1_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Secondary Assay(s) Name: IC<sub>50</sub>/EC<sub>50</sub> (nM) [SID, AID]</th></tr></thead><tbody><tr><td headers="hd_h_ml152.tu1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">664331/<a href="/pcsubstance/?term=ML152[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML152</a></td><td headers="hd_h_ml152.tu1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">GALK</td><td headers="hd_h_ml152.tu1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">1.0μM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/87550830" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87550830</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2499" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2499</a>]</td><td headers="hd_h_ml152.tu1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">CDP-Me</td><td headers="hd_h_ml152.tu1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">>10μM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/87550830" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87550830</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2506" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2506</a>]</td><td headers="hd_h_ml152.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">> 10-fold</td><td headers="hd_h_ml152.tu1_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Redox, cytotox<br /><br /><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87550830" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-87550830</a><br /><br /><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2502" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-2502</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2547" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2547</a></td></tr></tbody></table></div></div></article><article data-type="table-wrap" id="figobml152t1"><div id="ml152.t1" class="table"><h3><span class="label">Table 1</span><span class="title">Screens Deposited in PubChem</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK56237/table/ml152.t1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml152.t1_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml152.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">PubChem AID</th><th id="hd_h_ml152.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Type</th><th id="hd_h_ml152.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Target</th><th id="hd_h_ml152.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Conc. Range</th><th id="hd_h_ml152.t1_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_ml152.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1868" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1868</a></td><td headers="hd_h_ml152.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Primary qHTS</td><td headers="hd_h_ml152.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">GALK</td><td headers="hd_h_ml152.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">57.5 μM – 3.7 nM</td><td headers="hd_h_ml152.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">274,749</td></tr><tr><td headers="hd_h_ml152.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2499" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2499</a></td><td headers="hd_h_ml152.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Confirmatory</td><td headers="hd_h_ml152.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">GALK</td><td headers="hd_h_ml152.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">57.5 μM – 0.3 nM</td><td headers="hd_h_ml152.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">43</td></tr><tr><td headers="hd_h_ml152.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2506" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2506</a></td><td headers="hd_h_ml152.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Anti-target</td><td headers="hd_h_ml152.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">CDP-ME (CMK)</td><td headers="hd_h_ml152.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">57.5 μM – 0.3 nM</td><td headers="hd_h_ml152.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">43</td></tr><tr><td headers="hd_h_ml152.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2502" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2502</a></td><td headers="hd_h_ml152.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Anti-target</td><td headers="hd_h_ml152.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Redox activity</td><td headers="hd_h_ml152.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">57.5 μM – 0.3 nM</td><td headers="hd_h_ml152.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">43</td></tr><tr><td headers="hd_h_ml152.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2547" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2547</a></td><td headers="hd_h_ml152.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Anti-target</td><td headers="hd_h_ml152.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Cytotoxicity</td><td headers="hd_h_ml152.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">57.5 μM – 0.3 nM</td><td headers="hd_h_ml152.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">174</td></tr><tr><td headers="hd_h_ml152.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2114" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2114</a></td><td headers="hd_h_ml152.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Summary</td><td headers="hd_h_ml152.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">GALK</td><td headers="hd_h_ml152.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">NA</td><td headers="hd_h_ml152.t1_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="figobml152f1"><div id="ml152.f1" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK56237/bin/ml152f1.jpg" alt="Figure 1. Galactose KM determination and sensitivity to a control inhibitor in the 1536-well GalK assay." /></div><h3><span class="label">Figure 1</span><span class="title">Galactose K<sub>M</sub> determination and sensitivity to a control inhibitor in the 1536-well GalK assay</span></h3><div class="caption"><p><b>A</b>. Galactose variation in the 1536-well assay using ATP depletion. Rates (from determining the remaining ATP at time seven points starting at 5 min to 55 min; rates shown are 0.05 μM/min, 0.35 μM/min, 0.82 μM/min, 1.51 μM/min, 1.58 μM/min, 1.49 μM/min). <b>B</b> IC<sub>50</sub> determination of the positive control, a CD45 inhibitor. IC<sub>50</sub> = 3μM.</p></div></div></article><article data-type="table-wrap" id="figobml152t2"><div id="ml152.t2" class="table"><h3><span class="label">Table 2</span><span class="title">Stepwise Protocol Used for the 1536-Well Assay</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK56237/table/ml152.t2/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml152.t2_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml152.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;">Step</th><th id="hd_h_ml152.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;">Value</th><th id="hd_h_ml152.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;">Description</th></tr></thead><tbody><tr><td headers="hd_h_ml152.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">1a</td><td headers="hd_h_ml152.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">3μl</td><td headers="hd_h_ml152.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">35μM ATP buffer (2–48)</td></tr><tr><td headers="hd_h_ml152.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">1b</td><td headers="hd_h_ml152.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">4μl</td><td headers="hd_h_ml152.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Buffer only to column 1</td></tr><tr><td headers="hd_h_ml152.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">2</td><td headers="hd_h_ml152.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">23nl</td><td headers="hd_h_ml152.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">ATP titration (Col 1), CD45 inhibitor, DMSO</td></tr><tr><td headers="hd_h_ml152.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">3</td><td headers="hd_h_ml152.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">23nl</td><td headers="hd_h_ml152.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">40μM to 0.24nM</td></tr><tr><td headers="hd_h_ml152.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">4a</td><td headers="hd_h_ml152.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">1μl</td><td headers="hd_h_ml152.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Col 4, Buffer (no GalK)</td></tr><tr><td headers="hd_h_ml152.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">4b</td><td headers="hd_h_ml152.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">1μl</td><td headers="hd_h_ml152.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Col 2&3, 5–48 GalK (5 nM), 100 μM galactose</td></tr><tr><td headers="hd_h_ml152.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">5</td><td headers="hd_h_ml152.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">1 hr</td><td headers="hd_h_ml152.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Room temperature incubation</td></tr><tr><td headers="hd_h_ml152.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">6</td><td headers="hd_h_ml152.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">4μl</td><td headers="hd_h_ml152.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">KinaseGlo-Plus detection</td></tr><tr><td headers="hd_h_ml152.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">7</td><td headers="hd_h_ml152.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">10 min</td><td headers="hd_h_ml152.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Room temperature incubation</td></tr><tr><td headers="hd_h_ml152.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">8</td><td headers="hd_h_ml152.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Read</td><td headers="hd_h_ml152.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">ViewLux</td></tr><tr><th headers="hd_h_ml152.t2_1_1_1_1 hd_h_ml152.t2_1_1_1_2 hd_h_ml152.t2_1_1_1_3" id="hd_b_ml152.t2_1_1_11_1" colspan="3" rowspan="1" style="text-align:left;vertical-align:bottom;">Step Notes</th></tr><tr><td headers="hd_h_ml152.t2_1_1_1_1 hd_b_ml152.t2_1_1_11_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">1</td><td headers="hd_h_ml152.t2_1_1_1_2 hd_b_ml152.t2_1_1_11_1 hd_h_ml152.t2_1_1_1_3" colspan="2" rowspan="1" style="text-align:left;vertical-align:top;">White solid Kalypsys plates 1a: (HEPES pH 8.0, 5mM MgCl2, 60mM NaCl, 1 mM DTT, 0.01% BSA, 35μM ATP) 1b: no ATP buffer</td></tr><tr><td headers="hd_h_ml152.t2_1_1_1_1 hd_b_ml152.t2_1_1_11_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">2</td><td headers="hd_h_ml152.t2_1_1_1_2 hd_b_ml152.t2_1_1_11_1 hd_h_ml152.t2_1_1_1_3" colspan="2" rowspan="1" style="text-align:left;vertical-align:top;">Pintool addition of compound. Controls (column 2–4); and ATP 35μM titration 1:2 dilution (column 1)</td></tr><tr><td headers="hd_h_ml152.t2_1_1_1_1 hd_b_ml152.t2_1_1_11_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">3</td><td headers="hd_h_ml152.t2_1_1_1_2 hd_b_ml152.t2_1_1_11_1 hd_h_ml152.t2_1_1_1_3" colspan="2" rowspan="1" style="text-align:left;vertical-align:top;">Pintool transfer (DMSO; iPA, MeOH, 3-sec dry wash cycle)</td></tr><tr><td headers="hd_h_ml152.t2_1_1_1_1 hd_b_ml152.t2_1_1_11_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">4</td><td headers="hd_h_ml152.t2_1_1_1_2 hd_b_ml152.t2_1_1_11_1 hd_h_ml152.t2_1_1_1_3" colspan="2" rowspan="1" style="text-align:left;vertical-align:top;">4a: buffer, 4b: buffer + 5 nM GalK & 100μM galactose (kept on ice)</td></tr><tr><td headers="hd_h_ml152.t2_1_1_1_1 hd_b_ml152.t2_1_1_11_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">5</td><td headers="hd_h_ml152.t2_1_1_1_2 hd_b_ml152.t2_1_1_11_1 hd_h_ml152.t2_1_1_1_3" colspan="2" rowspan="1" style="text-align:left;vertical-align:top;">Room temperature incubation</td></tr><tr><td headers="hd_h_ml152.t2_1_1_1_1 hd_b_ml152.t2_1_1_11_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">6</td><td headers="hd_h_ml152.t2_1_1_1_2 hd_b_ml152.t2_1_1_11_1 hd_h_ml152.t2_1_1_1_3" colspan="2" rowspan="1" style="text-align:left;vertical-align:top;">Kinase-Glo Plus at room temperature</td></tr><tr><td headers="hd_h_ml152.t2_1_1_1_1 hd_b_ml152.t2_1_1_11_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">7</td><td headers="hd_h_ml152.t2_1_1_1_2 hd_b_ml152.t2_1_1_11_1 hd_h_ml152.t2_1_1_1_3" colspan="2" rowspan="1" style="text-align:left;vertical-align:top;">Room temperature incubation</td></tr><tr><td headers="hd_h_ml152.t2_1_1_1_1 hd_b_ml152.t2_1_1_11_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">8</td><td headers="hd_h_ml152.t2_1_1_1_2 hd_b_ml152.t2_1_1_11_1 hd_h_ml152.t2_1_1_1_3" colspan="2" rowspan="1" style="text-align:left;vertical-align:top;">ViewLux, 1 sec exposure 2x bin</td></tr></tbody></table></div></div></article><article data-type="fig" id="figobml152f2"><div id="ml152.f2" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK56237/bin/ml152f2.jpg" alt="Figure 2. Dose response curve for NCGC00187642 (CID-664331; SID-87550830) in confirmatory assay." /></div><h3><span class="label">Figure 2</span><span class="title">Dose response curve for NCGC00187642 (CID-664331; <a href="https://pubchem.ncbi.nlm.nih.gov/substance/87550830" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-87550830</a>) in confirmatory assay</span></h3></div></article><article data-type="fig" id="figobml152f6"><div id="ml152.f6" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK56237/bin/ml152f6.jpg" alt="Scheme 1. Synthesis of CID-664331." /></div><h3><span class="label">Scheme 1</span><span class="title">Synthesis of CID-664331</span></h3></div></article><article data-type="fig" id="figobml152f3"><div id="ml152.f3" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK56237/bin/ml152f3.jpg" alt="Figure 3. PBS Buffer Stability of NCGC00187642/CID664331." /></div><h3><span class="label">Figure 3</span><span class="title">PBS Buffer Stability of NCGC00187642/CID664331</span></h3><div class="caption"><p>Stability of NCGC00187642/CID664331 in PBS buffer (pH 7.4, 23°C) plotted as AUC vs. time for a 48 hr period using LCMS Method 1 described in <a href="#ml152.s4">Section 2</a>. No instability was observed, and 100% of compound remained at 48 hours.</p></div></div></article><article data-type="table-wrap" id="figobml152tu2"><div id="ml152.tu2" class="table"><h3><span class="title">MLS Numbers for Probe and Analogs</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK56237/table/ml152.tu2/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml152.tu2_lrgtbl__"><table class="no_top_margin"><tbody><tr><td rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Probe</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">NCGC00187642</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">MLS000039538</td></tr><tr><td rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Analog</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">NCGC00187643</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">MLS003178546</td></tr><tr><td rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Analog</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">NCGC00188578</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">MLS003178547</td></tr><tr><td rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Analog</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">NCGC00188580</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">MLS003178548</td></tr><tr><td rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Analog</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">NCGC00188579</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">MLS003178549</td></tr><tr><td rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Analog</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">NCGC00188583</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">MLS003178550</td></tr></tbody></table></div></div></article><article data-type="table-wrap" id="figobml152t3"><div id="ml152.t3" class="table"><h3><span class="label">Table 3</span><span class="title">Primary Assay Statistics</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK56237/table/ml152.t3/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml152.t3_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml152.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><i>Parameter</i></th><th id="hd_h_ml152.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><i>qHTS</i></th></tr></thead><tbody><tr><td headers="hd_h_ml152.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">System</td><td headers="hd_h_ml152.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Kalypsys/Viewlux detection</td></tr><tr><td headers="hd_h_ml152.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Plates Screened</td><td headers="hd_h_ml152.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">1192 (4 batches)</td></tr><tr><td headers="hd_h_ml152.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Plates Failed QC</td><td headers="hd_h_ml152.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">30 (4 titration series)</td></tr><tr><td headers="hd_h_ml152.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Compound (total # tested)</td><td headers="hd_h_ml152.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">277,329</td></tr><tr><td headers="hd_h_ml152.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Concentration-Response Titrations</td><td headers="hd_h_ml152.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">277,329</td></tr><tr><td headers="hd_h_ml152.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Sample wells</td><td headers="hd_h_ml152.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">1,561,627</td></tr><tr><td headers="hd_h_ml152.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Number of data points</td><td headers="hd_h_ml152.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">1,561,627</td></tr><tr><td headers="hd_h_ml152.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Z′</td><td headers="hd_h_ml152.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0.48 ± 0.16</td></tr><tr><td headers="hd_h_ml152.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Signal/Background</td><td headers="hd_h_ml152.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">4.5 ± 1.6</td></tr><tr><td headers="hd_h_ml152.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">CV</td><td headers="hd_h_ml152.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">17 ± 9</td></tr><tr><td headers="hd_h_ml152.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">MSR of CD45 control</td><td headers="hd_h_ml152.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">2</td></tr></tbody></table></div></div></article><article data-type="table-wrap" id="figobml152t4"><div id="ml152.t4" class="table"><h3><span class="label">Table 4</span><span class="title">Concentration-Response Curve Class Distribution from Primary Screen<a class="bibr" href="#ml152.r6" rid="ml152.r6">6</a>,<a class="bibr" href="#ml152.r7" rid="ml152.r7">7</a></span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK56237/table/ml152.t4/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml152.t4_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml152.t4_1_1_1_1" rowspan="2" colspan="1" headers="hd_h_ml152.t4_1_1_1_1" style="text-align:left;vertical-align:middle;">Activity</th><th id="hd_h_ml152.t4_1_1_1_2" rowspan="2" colspan="1" headers="hd_h_ml152.t4_1_1_1_2" style="text-align:left;vertical-align:middle;">Distribution</th><th id="hd_h_ml152.t4_1_1_1_3" colspan="7" rowspan="1" style="text-align:left;vertical-align:top;">Concentration-Response Curve Class</th></tr><tr><th headers="hd_h_ml152.t4_1_1_1_3" id="hd_h_ml152.t4_1_1_2_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">1.1</th><th headers="hd_h_ml152.t4_1_1_1_3" id="hd_h_ml152.t4_1_1_2_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">1.2</th><th headers="hd_h_ml152.t4_1_1_1_3" id="hd_h_ml152.t4_1_1_2_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">2.1</th><th headers="hd_h_ml152.t4_1_1_1_3" id="hd_h_ml152.t4_1_1_2_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">2.2</th><th headers="hd_h_ml152.t4_1_1_1_3" id="hd_h_ml152.t4_1_1_2_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">3</th><th headers="hd_h_ml152.t4_1_1_1_3" id="hd_h_ml152.t4_1_1_2_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">4</th><th headers="hd_h_ml152.t4_1_1_1_3" id="hd_h_ml152.t4_1_1_2_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">5</th></tr></thead><tbody><tr><td headers="hd_h_ml152.t4_1_1_1_1" rowspan="2" colspan="1" style="text-align:left;vertical-align:middle;">Activation</td><td headers="hd_h_ml152.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Cmpd No</td><td headers="hd_h_ml152.t4_1_1_1_3 hd_h_ml152.t4_1_1_2_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0</td><td headers="hd_h_ml152.t4_1_1_1_3 hd_h_ml152.t4_1_1_2_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0</td><td headers="hd_h_ml152.t4_1_1_1_3 hd_h_ml152.t4_1_1_2_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0</td><td headers="hd_h_ml152.t4_1_1_1_3 hd_h_ml152.t4_1_1_2_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">13</td><td headers="hd_h_ml152.t4_1_1_1_3 hd_h_ml152.t4_1_1_2_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0</td><td headers="hd_h_ml152.t4_1_1_1_3 hd_h_ml152.t4_1_1_2_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"></td><td headers="hd_h_ml152.t4_1_1_1_3 hd_h_ml152.t4_1_1_2_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"></td></tr><tr><td headers="hd_h_ml152.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">% library</td><td headers="hd_h_ml152.t4_1_1_1_3 hd_h_ml152.t4_1_1_2_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0%</td><td headers="hd_h_ml152.t4_1_1_1_3 hd_h_ml152.t4_1_1_2_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0%</td><td headers="hd_h_ml152.t4_1_1_1_3 hd_h_ml152.t4_1_1_2_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0%</td><td headers="hd_h_ml152.t4_1_1_1_3 hd_h_ml152.t4_1_1_2_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0%</td><td headers="hd_h_ml152.t4_1_1_1_3 hd_h_ml152.t4_1_1_2_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0.00%</td><td headers="hd_h_ml152.t4_1_1_1_3 hd_h_ml152.t4_1_1_2_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">277167</td><td headers="hd_h_ml152.t4_1_1_1_3 hd_h_ml152.t4_1_1_2_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0</td></tr><tr><td headers="hd_h_ml152.t4_1_1_1_1" rowspan="2" colspan="1" style="text-align:left;vertical-align:middle;">Inhibition</td><td headers="hd_h_ml152.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Cmpd No</td><td headers="hd_h_ml152.t4_1_1_1_3 hd_h_ml152.t4_1_1_2_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">3</td><td headers="hd_h_ml152.t4_1_1_1_3 hd_h_ml152.t4_1_1_2_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">12</td><td headers="hd_h_ml152.t4_1_1_1_3 hd_h_ml152.t4_1_1_2_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">1</td><td headers="hd_h_ml152.t4_1_1_1_3 hd_h_ml152.t4_1_1_2_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">18</td><td headers="hd_h_ml152.t4_1_1_1_3 hd_h_ml152.t4_1_1_2_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">115</td><td headers="hd_h_ml152.t4_1_1_1_3 hd_h_ml152.t4_1_1_2_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">99.94%</td><td headers="hd_h_ml152.t4_1_1_1_3 hd_h_ml152.t4_1_1_2_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0.00%</td></tr><tr><td headers="hd_h_ml152.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">% library</td><td headers="hd_h_ml152.t4_1_1_1_3 hd_h_ml152.t4_1_1_2_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0 %</td><td headers="hd_h_ml152.t4_1_1_1_3 hd_h_ml152.t4_1_1_2_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0 %</td><td headers="hd_h_ml152.t4_1_1_1_3 hd_h_ml152.t4_1_1_2_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0%</td><td headers="hd_h_ml152.t4_1_1_1_3 hd_h_ml152.t4_1_1_2_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0.01%</td><td headers="hd_h_ml152.t4_1_1_1_3 hd_h_ml152.t4_1_1_2_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0.04%</td><td headers="hd_h_ml152.t4_1_1_1_3 hd_h_ml152.t4_1_1_2_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"></td><td headers="hd_h_ml152.t4_1_1_1_3 hd_h_ml152.t4_1_1_2_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"></td></tr></tbody></table></div></div></article><article data-type="fig" id="figobml152f4"><div id="ml152.f4" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK56237/bin/ml152f4.jpg" alt="Figure 4. Potency and selectivity of NCGC00187642 (CID-664331; SID-87550830) against CMK for the lead compound." /></div><h3><span class="label">Figure 4</span><span class="title">Potency and selectivity of NCGC00187642 (CID-664331; <a href="https://pubchem.ncbi.nlm.nih.gov/substance/87550830" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-87550830</a>) against CMK for the lead compound</span></h3><div class="caption"><p>The lead compound was observed to be active against GALK with an IC<sub>50</sub> of 1μM but inactive against CDP-Me kinase. Results showed no redox or cytotoxic activity of the compound (cytotoxicity was determined after 48 hours). All assays were performed at least in duplicate on at least two separate days.</p></div></div></article><article data-type="table-wrap" id="figobml152t5"><div id="ml152.t5" class="table"><h3><span class="label">Table 5</span><span class="title">SAR of select analogs in GALK purified enzyme assay</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK56237/table/ml152.t5/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml152.t5_lrgtbl__"><table class="no_margin"><thead><tr><th id="hd_h_ml152.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Entry</th><th id="hd_h_ml152.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Structure</th><th id="hd_h_ml152.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Compound ID<sup>a</sup></th><th id="hd_h_ml152.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">IC<sub>50</sub> (μM)<sup>b</sup></th><th id="hd_h_ml152.t5_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">%Max Resp.<sup>c</sup></th></tr></thead><tbody><tr><td headers="hd_h_ml152.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">1</td><td headers="hd_h_ml152.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">
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<div class="graphic"><img src="/books/NBK56237/bin/ml152fu2.jpg" alt="Image ml152fu2.jpg" /></div></td><td headers="hd_h_ml152.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00187643-01<br />(CID-44607593)<br /><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87357364" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-87357364</a>)</td><td headers="hd_h_ml152.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">16.8</td><td headers="hd_h_ml152.t5_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">−74%</td></tr><tr><td headers="hd_h_ml152.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">2</td><td headers="hd_h_ml152.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">
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<div class="graphic"><img src="/books/NBK56237/bin/ml152fu3.jpg" alt="Image ml152fu3.jpg" /></div></td><td headers="hd_h_ml152.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00187641-01<br />(CID-44607596)<br />(<a href="https://pubchem.ncbi.nlm.nih.gov/substance/87357362" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-87357362</a>)</td><td headers="hd_h_ml152.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">16.8</td><td headers="hd_h_ml152.t5_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">−74%</td></tr><tr><td headers="hd_h_ml152.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">3</td><td headers="hd_h_ml152.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">
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<div class="graphic"><img src="/books/NBK56237/bin/ml152fu4.jpg" alt="Image ml152fu4.jpg" /></div></td><td headers="hd_h_ml152.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00187642-02<br />(CID-664331)<br />(<a href="https://pubchem.ncbi.nlm.nih.gov/substance/87550830" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-87550830</a>)</td><td headers="hd_h_ml152.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">1.0</td><td headers="hd_h_ml152.t5_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">−86%</td></tr><tr><td headers="hd_h_ml152.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">4</td><td headers="hd_h_ml152.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">
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<div class="graphic"><img src="/books/NBK56237/bin/ml152fu5.jpg" alt="Image ml152fu5.jpg" /></div></td><td headers="hd_h_ml152.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00186052-01<br />CID-1294862)<br />(<a href="https://pubchem.ncbi.nlm.nih.gov/substance/85256913" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-85256913</a>)</td><td headers="hd_h_ml152.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">11.9</td><td headers="hd_h_ml152.t5_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">−99%</td></tr><tr><td headers="hd_h_ml152.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">5</td><td headers="hd_h_ml152.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">
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<div class="graphic"><img src="/books/NBK56237/bin/ml152fu6.jpg" alt="Image ml152fu6.jpg" /></div></td><td headers="hd_h_ml152.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00188034-01<br />(CID-1286615)<br />(<a href="https://pubchem.ncbi.nlm.nih.gov/substance/87357368" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-87357368</a>)</td><td headers="hd_h_ml152.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">6.0</td><td headers="hd_h_ml152.t5_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">−82%</td></tr><tr><td headers="hd_h_ml152.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">6</td><td headers="hd_h_ml152.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">
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<div class="graphic"><img src="/books/NBK56237/bin/ml152fu7.jpg" alt="Image ml152fu7.jpg" /></div></td><td headers="hd_h_ml152.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00188035-01<br />(CID-44607600)<br />(<a href="https://pubchem.ncbi.nlm.nih.gov/substance/87357369" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-87357369</a>)</td><td headers="hd_h_ml152.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">14.9</td><td headers="hd_h_ml152.t5_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">−36%</td></tr><tr><td headers="hd_h_ml152.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">7</td><td headers="hd_h_ml152.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">
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<div class="graphic"><img src="/books/NBK56237/bin/ml152fu8.jpg" alt="Image ml152fu8.jpg" /></div></td><td headers="hd_h_ml152.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00188036-01<br />(CID-44607594)<br />(<a href="https://pubchem.ncbi.nlm.nih.gov/substance/87357370" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-87357370</a>)</td><td headers="hd_h_ml152.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">13.3</td><td headers="hd_h_ml152.t5_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">−75%</td></tr><tr><td headers="hd_h_ml152.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">8</td><td headers="hd_h_ml152.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">
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<div class="graphic"><img src="/books/NBK56237/bin/ml152fu9.jpg" alt="Image ml152fu9.jpg" /></div></td><td headers="hd_h_ml152.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00188578-01<br />(CID-44623889)<br />(<a href="https://pubchem.ncbi.nlm.nih.gov/substance/87550843" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-87550843</a>)</td><td headers="hd_h_ml152.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">6.0</td><td headers="hd_h_ml152.t5_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">−81%</td></tr><tr><td headers="hd_h_ml152.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">9</td><td headers="hd_h_ml152.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">
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<div class="graphic"><img src="/books/NBK56237/bin/ml152fu10.jpg" alt="Image ml152fu10.jpg" /></div></td><td headers="hd_h_ml152.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00188580-01<br />(CID-44623882)<br />(<a href="https://pubchem.ncbi.nlm.nih.gov/substance/87550845" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-87550845</a>)</td><td headers="hd_h_ml152.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">21</td><td headers="hd_h_ml152.t5_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">−58%</td></tr><tr><td headers="hd_h_ml152.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">10</td><td headers="hd_h_ml152.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">
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<div class="graphic"><img src="/books/NBK56237/bin/ml152fu11.jpg" alt="Image ml152fu11.jpg" /></div></td><td headers="hd_h_ml152.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00188577-01<br />(CID-44623881)<br />(<a href="https://pubchem.ncbi.nlm.nih.gov/substance/87550842" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-87550842</a>)</td><td headers="hd_h_ml152.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">37</td><td headers="hd_h_ml152.t5_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">−38%</td></tr><tr><td headers="hd_h_ml152.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">11</td><td headers="hd_h_ml152.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">
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<div class="graphic"><img src="/books/NBK56237/bin/ml152fu12.jpg" alt="Image ml152fu12.jpg" /></div></td><td headers="hd_h_ml152.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00188572-01<br />(CID-44623883)<br />(<a href="https://pubchem.ncbi.nlm.nih.gov/substance/87550837" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-87550837</a>)</td><td headers="hd_h_ml152.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">6.7</td><td headers="hd_h_ml152.t5_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">−73%</td></tr><tr><td headers="hd_h_ml152.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">12</td><td headers="hd_h_ml152.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">
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<div class="graphic"><img src="/books/NBK56237/bin/ml152fu13.jpg" alt="Image ml152fu13.jpg" /></div></td><td headers="hd_h_ml152.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00188579-01<br />(CID-44623888)<br />(<a href="https://pubchem.ncbi.nlm.nih.gov/substance/87550844" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-87550844</a>)</td><td headers="hd_h_ml152.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">7.5</td><td headers="hd_h_ml152.t5_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">−73%</td></tr><tr><td headers="hd_h_ml152.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">13</td><td headers="hd_h_ml152.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">
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<div class="graphic"><img src="/books/NBK56237/bin/ml152fu14.jpg" alt="Image ml152fu14.jpg" /></div></td><td headers="hd_h_ml152.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00188581-01<br />(CID-44623886)<br />(<a href="https://pubchem.ncbi.nlm.nih.gov/substance/87550846" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-87550846</a>)</td><td headers="hd_h_ml152.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">>57</td><td headers="hd_h_ml152.t5_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NA</td></tr><tr><td headers="hd_h_ml152.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">14</td><td headers="hd_h_ml152.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">
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<div class="graphic"><img src="/books/NBK56237/bin/ml152fu15.jpg" alt="Image ml152fu15.jpg" /></div></td><td headers="hd_h_ml152.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00188583-01<br />(CID-44623884)<br />(<a href="https://pubchem.ncbi.nlm.nih.gov/substance/87550848" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-87550848</a>)</td><td headers="hd_h_ml152.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">>57</td><td headers="hd_h_ml152.t5_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NA</td></tr></tbody></table></div><div class="tblwrap-foot"><div><dl class="temp-labeled-list small"><dl class="bkr_refwrap"><dt>a</dt><dd><div id="ml152.tfn1"><p class="no_margin">All compounds were synthesized</p></div></dd></dl><dl class="bkr_refwrap"><dt>b</dt><dd><div id="ml152.tfn2"><p class="no_margin">IC<sub>50</sub> values were determined utilizing the luminescen GALK-luminescent ATP-depletion assay.</p></div></dd></dl><dl class="bkr_refwrap"><dt>c</dt><dd><div id="ml152.tfn3"><p class="no_margin">Max Resp. represents the % inhibition at 57 μM compound.</p></div></dd></dl></dl></div></div></div></article><article data-type="fig" id="figobml152f5"><div id="ml152.f5" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK56237/bin/ml152f5.jpg" alt="Figure 5. Substrate competition with NCGC00187642 (CID-664331; SID-87550830)." /></div><h3><span class="label">Figure 5</span><span class="title">Substrate competition with NCGC00187642 (CID-664331; <a href="https://pubchem.ncbi.nlm.nih.gov/substance/87550830" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-87550830</a>)</span></h3><div class="caption"><p><b>A</b> Kinase reaction run at two different ATP concentrations, while keeping the galactose concentration at K<sub>M</sub>, showed increase in potency of the compound as exhibited by a left shift of the dose-response curve of the ATP competition assay. <b>B</b> No significant change in the IC<sub>50</sub>/potency of the lead was observed in the presence of three different galactose concentrations run at K<sub>M</sub> concentration of ATP. <b>C</b> Dose response kinetics study of the lead compound. K<sub>M</sub> of ATP increased as concentration of the inhibitor increased but the K<sub>M</sub> for galactose remained the same; hence further showing a potential ATP competition.</p></div></div></article><article data-type="table-wrap" id="figobml152tu3"><div id="ml152.tu3" class="table"><h3><span class="title">Properties computed from Structure</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK56237/table/ml152.tu3/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml152.tu3_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml152.tu3_1_1_1_1" rowspan="2" colspan="1" headers="hd_h_ml152.tu3_1_1_1_1" style="text-align:center;vertical-align:middle;">Calculated Property</th><th id="hd_h_ml152.tu3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Probe Identity</th></tr><tr><th headers="hd_h_ml152.tu3_1_1_1_2" id="hd_h_ml152.tu3_1_1_2_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;">CID-664331 (MLS000039538)</th></tr></thead><tbody><tr><td headers="hd_h_ml152.tu3_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Molecular Weight [g/mol]</td><td headers="hd_h_ml152.tu3_1_1_1_2 hd_h_ml152.tu3_1_1_2_1" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">336.3877</td></tr><tr><td headers="hd_h_ml152.tu3_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Molecular Formula</td><td headers="hd_h_ml152.tu3_1_1_1_2 hd_h_ml152.tu3_1_1_2_1" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">C19H20N4O2</td></tr><tr><td headers="hd_h_ml152.tu3_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">XLogP3-AA</td><td headers="hd_h_ml152.tu3_1_1_1_2 hd_h_ml152.tu3_1_1_2_1" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">2.1</td></tr><tr><td headers="hd_h_ml152.tu3_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">H-Bond Donor</td><td headers="hd_h_ml152.tu3_1_1_1_2 hd_h_ml152.tu3_1_1_2_1" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">2</td></tr><tr><td headers="hd_h_ml152.tu3_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">H-Bond Acceptor</td><td headers="hd_h_ml152.tu3_1_1_1_2 hd_h_ml152.tu3_1_1_2_1" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">6</td></tr><tr><td headers="hd_h_ml152.tu3_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Rotatable Bond Count</td><td headers="hd_h_ml152.tu3_1_1_1_2 hd_h_ml152.tu3_1_1_2_1" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">2</td></tr><tr><td headers="hd_h_ml152.tu3_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Tautomer Count</td><td headers="hd_h_ml152.tu3_1_1_1_2 hd_h_ml152.tu3_1_1_2_1" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">34</td></tr><tr><td headers="hd_h_ml152.tu3_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Exact Mass</td><td headers="hd_h_ml152.tu3_1_1_1_2 hd_h_ml152.tu3_1_1_2_1" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">336.158626</td></tr><tr><td headers="hd_h_ml152.tu3_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">MonoIsotopic Mass</td><td headers="hd_h_ml152.tu3_1_1_1_2 hd_h_ml152.tu3_1_1_2_1" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">336.158626</td></tr><tr><td headers="hd_h_ml152.tu3_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Topological Polar Surface Area</td><td headers="hd_h_ml152.tu3_1_1_1_2 hd_h_ml152.tu3_1_1_2_1" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">79.5</td></tr><tr><td headers="hd_h_ml152.tu3_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Heavy Atom Count</td><td headers="hd_h_ml152.tu3_1_1_1_2 hd_h_ml152.tu3_1_1_2_1" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">25</td></tr><tr><td headers="hd_h_ml152.tu3_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Formal Charge</td><td headers="hd_h_ml152.tu3_1_1_1_2 hd_h_ml152.tu3_1_1_2_1" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">0</td></tr><tr><td headers="hd_h_ml152.tu3_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Isotope Atom Count</td><td headers="hd_h_ml152.tu3_1_1_1_2 hd_h_ml152.tu3_1_1_2_1" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">0</td></tr><tr><td headers="hd_h_ml152.tu3_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Defined Atom StereoCenter Count</td><td headers="hd_h_ml152.tu3_1_1_1_2 hd_h_ml152.tu3_1_1_2_1" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">0</td></tr><tr><td headers="hd_h_ml152.tu3_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Undefined Atom StereoCenter Count</td><td headers="hd_h_ml152.tu3_1_1_1_2 hd_h_ml152.tu3_1_1_2_1" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">0</td></tr><tr><td headers="hd_h_ml152.tu3_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Defined Bond StereoCenter Count</td><td headers="hd_h_ml152.tu3_1_1_1_2 hd_h_ml152.tu3_1_1_2_1" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">0</td></tr><tr><td headers="hd_h_ml152.tu3_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Undefined Bond StereoCenter Count</td><td headers="hd_h_ml152.tu3_1_1_1_2 hd_h_ml152.tu3_1_1_2_1" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">0</td></tr><tr><td headers="hd_h_ml152.tu3_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Covalently-Bonded Unit Count</td><td headers="hd_h_ml152.tu3_1_1_1_2 hd_h_ml152.tu3_1_1_2_1" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">1</td></tr><tr><td headers="hd_h_ml152.tu3_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Complexity</td><td headers="hd_h_ml152.tu3_1_1_1_2 hd_h_ml152.tu3_1_1_2_1" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">635</td></tr></tbody></table></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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