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<script type="text/javascript" src="/corehtml/pmc/jatsreader/ptpmc_3.22/js/jr.boots.min.js"> </script><title>Discovery of ML355, a Potent and Selective Inhibitor of Human 12-Lipoxygenase - 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="Discovery of ML355, a Potent and Selective Inhibitor of Human 12-Lipoxygenase">
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<meta name="citation_publisher" content="National Center for Biotechnology Information (US)">
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<meta name="citation_date" content="2014/09/18">
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<meta name="citation_author" content="Diane Luci">
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<meta name="citation_author" content="J. Brian Jameson, II">
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<meta name="citation_author" content="Adam Yasgar">
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<meta name="citation_author" content="Giovanni Diaz">
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<meta name="citation_author" content="Netra Joshi">
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<meta name="citation_author" content="Auric Kantz">
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<meta name="citation_author" content="Kate Markham">
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<meta name="citation_author" content="Steve Perry">
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<meta name="citation_author" content="Norine Kuhn">
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<meta name="citation_author" content="Jennifer Yeung">
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<meta name="citation_author" content="Lena Schultz">
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<meta name="citation_author" content="Michael Holinstat">
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<meta name="citation_author" content="Jerry Nadler">
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<meta name="citation_author" content="David A. Taylor-Fishwick">
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<meta name="citation_author" content="Ajit Jadhav">
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<meta name="citation_author" content="Anton Simeonov">
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<meta name="citation_author" content="Theodore R. Holman">
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<meta name="citation_author" content="David J. Maloney">
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<meta name="citation_pmid" content="25506969">
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<meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK259188/">
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<meta name="DC.Title" content="Discovery of ML355, a Potent and Selective Inhibitor of Human 12-Lipoxygenase">
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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="Diane Luci">
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<meta name="DC.Contributor" content="J. Brian Jameson, II">
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<meta name="DC.Contributor" content="Adam Yasgar">
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<meta name="DC.Contributor" content="Giovanni Diaz">
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<meta name="DC.Contributor" content="Netra Joshi">
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<meta name="DC.Contributor" content="Auric Kantz">
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<meta name="DC.Contributor" content="Kate Markham">
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<meta name="DC.Contributor" content="Steve Perry">
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<meta name="DC.Contributor" content="Norine Kuhn">
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<meta name="DC.Contributor" content="Jennifer Yeung">
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<meta name="DC.Contributor" content="Lena Schultz">
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<meta name="DC.Contributor" content="Michael Holinstat">
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<meta name="DC.Contributor" content="Jerry Nadler">
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<meta name="DC.Contributor" content="David A. Taylor-Fishwick">
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<meta name="DC.Contributor" content="Ajit Jadhav">
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<meta name="DC.Contributor" content="Anton Simeonov">
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<meta name="DC.Contributor" content="Theodore R. Holman">
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<meta name="DC.Contributor" content="David J. Maloney">
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<meta name="DC.Date" content="2014/09/18">
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<meta name="description" content="Human lipoxygenases (LOXs) are enzymes involved in catalyzing the oxidation of polyunsaturated fatty acids to provide the corresponding bioactive hydroxyeicosatetraenoic acid (HETE) metabolites as the end product [1-3]. These eicosanoid signaling molecules are involved in a number of physiologic responses such as platelet aggregation, inflammation and cell proliferation [4-6]. As a result, modulation of these responses through the inhibition of the lipoxygenase enzymes is of great interest. Our group has particular interest in platelet-type 12-(S)-LOX (12-LOX) because of its demonstrated role in skin diseases, diabetes, platelet hemostasis, thrombosis and cancer [7-11]. However, despite the potential of 12-LOX as a therapeutic target, few potent and selective inhibitors have been reported. The lack of high quality 12-LOX inhibitors prompted us to initiate a high-throughput screening campaign as part of the MLPCN program which ultimately led to the discovery of ML127. While potent and selective, ML127 demonstrated limited tolerance for structural modifications, which hampered continued medicinal chemistry efforts thus a continued discovery efforts to develop additional novel inhibitors of 12-LOX is needed. Herein, we report the identification and medicinal chemistry optimization of an unrelated, second chemotype, ML355, which displays nM potency against 12-LOX and excellent selectivity over related lipoxygenases and cyclooxygenases. ML355 has favorable absorption, distribution, metabolism, and excretion (ADME) properties, inhibits PAR-4 induced aggregation and calcium mobilization in human platelets, and reduces 12-HETE in mouse/human beta cells suggesting its potential utility in animal models for antiplatelet therapy and diabetes.">
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<meta name="og:title" content="Discovery of ML355, a Potent and Selective Inhibitor of Human 12-Lipoxygenase">
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<meta name="og:type" content="book">
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<meta name="og:description" content="Human lipoxygenases (LOXs) are enzymes involved in catalyzing the oxidation of polyunsaturated fatty acids to provide the corresponding bioactive hydroxyeicosatetraenoic acid (HETE) metabolites as the end product [1-3]. These eicosanoid signaling molecules are involved in a number of physiologic responses such as platelet aggregation, inflammation and cell proliferation [4-6]. As a result, modulation of these responses through the inhibition of the lipoxygenase enzymes is of great interest. Our group has particular interest in platelet-type 12-(S)-LOX (12-LOX) because of its demonstrated role in skin diseases, diabetes, platelet hemostasis, thrombosis and cancer [7-11]. However, despite the potential of 12-LOX as a therapeutic target, few potent and selective inhibitors have been reported. The lack of high quality 12-LOX inhibitors prompted us to initiate a high-throughput screening campaign as part of the MLPCN program which ultimately led to the discovery of ML127. While potent and selective, ML127 demonstrated limited tolerance for structural modifications, which hampered continued medicinal chemistry efforts thus a continued discovery efforts to develop additional novel inhibitors of 12-LOX is needed. Herein, we report the identification and medicinal chemistry optimization of an unrelated, second chemotype, ML355, which displays nM potency against 12-LOX and excellent selectivity over related lipoxygenases and cyclooxygenases. ML355 has favorable absorption, distribution, metabolism, and excretion (ADME) properties, inhibits PAR-4 induced aggregation and calcium mobilization in human platelets, and reduces 12-HETE in mouse/human beta cells suggesting its potential utility in animal models for antiplatelet therapy and diabetes.">
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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="_NBK259188_"><span class="title" itemprop="name">Discovery of ML355, a Potent and Selective Inhibitor of Human 12-Lipoxygenase</span></h1><p class="contribs">Luci D, Jameson JB II, Yasgar A, et al.</p><p class="fm-aai"><a href="#_NBK259188_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>Human lipoxygenases (LOXs) are enzymes involved in catalyzing the oxidation of polyunsaturated fatty acids to provide the corresponding bioactive hydroxyeicosatetraenoic acid (HETE) metabolites as the end product [<a class="bibr" href="#ml355.r1" rid="ml355.r1">1</a>-<a class="bibr" href="#ml355.r3" rid="ml355.r3">3</a>]. These eicosanoid signaling molecules are involved in a number of physiologic responses such as platelet aggregation, inflammation and cell proliferation [<a class="bibr" href="#ml355.r4" rid="ml355.r4">4</a>-<a class="bibr" href="#ml355.r6" rid="ml355.r6">6</a>]. As a result, modulation of these responses through the inhibition of the lipoxygenase enzymes is of great interest. Our group has particular interest in platelet-type 12-(<i>S</i>)-LOX (12-LOX) because of its demonstrated role in skin diseases, diabetes, platelet hemostasis, thrombosis and cancer [<a class="bibr" href="#ml355.r7" rid="ml355.r7">7</a>-<a class="bibr" href="#ml355.r11" rid="ml355.r11">11</a>]. However, despite the potential of 12-LOX as a therapeutic target, few potent and selective inhibitors have been reported. The lack of high quality 12-LOX inhibitors prompted us to initiate a high-throughput screening campaign as part of the MLPCN program which ultimately led to the discovery of <a href="/pcsubstance/?term=ML127[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML127</a>. While potent and selective, <a href="/pcsubstance/?term=ML127[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML127</a> demonstrated limited tolerance for structural modifications, which hampered continued medicinal chemistry efforts thus a continued discovery efforts to develop additional novel inhibitors of 12-LOX is needed. Herein, we report the identification and medicinal chemistry optimization of an unrelated, second chemotype, <a href="/pcsubstance/?term=ML355[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML355</a>, which displays nM potency against 12-LOX and excellent selectivity over related lipoxygenases and cyclooxygenases. <a href="/pcsubstance/?term=ML355[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML355</a> has favorable absorption, distribution, metabolism, and excretion (ADME) properties, inhibits PAR-4 induced aggregation and calcium mobilization in human platelets, and reduces 12-HETE in mouse/human beta cells suggesting its potential utility in animal models for antiplatelet therapy and diabetes.</p></div><div class="h2"></div><p><b>Assigned Assay Grant #:</b> <a href="/nuccore/1365464663" class="bk_tag" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=nuccore">MH081283</a></p><p><b>Screening Center Name & PI:</b> NIH Chemical Genomics Center, Christopher P. Austin</p><p><b>Chemistry Center Name & PI:</b> NIH Chemical Genomics Center, Christopher P. Austin</p><p><b>Assay Submitter & Institution:</b> Theodore R. Holman, University of California, Santa Cruz.</p><p>PubChem Summary Bioassay Identifier (AID): <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2164" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2164</a></p><div id="ml355.s1"><h2 id="_ml355_s1_">Probe Structure & Characteristics</h2><div id="ml355.f1" class="figure"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Image%20ml355f1&p=BOOKS&id=259188_ml355f1.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img src="/books/NBK259188/bin/ml355f1.jpg" alt="Image ml355f1" class="tileshop" title="Click on image to zoom" /></a></div></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml355t1"><a href="/books/NBK259188/table/ml355.t1/?report=objectonly" target="object" title="Table" class="img_link icnblk_img figpopup" rid-figpopup="figml355t1" rid-ob="figobml355t1"><img class="small-thumb" src="/books/NBK259188/table/ml355.t1/?report=thumb" src-large="/books/NBK259188/table/ml355.t1/?report=previmg" alt="Image " /></a><div class="icnblk_cntnt"><h4 id="ml355.t1"><a href="/books/NBK259188/table/ml355.t1/?report=objectonly" target="object" rid-ob="figobml355t1">Table</a></h4></div></div></div><div id="ml355.s2"><h2 id="_ml355_s2_">1. Recommendations for Scientific Use of the Probe</h2><p>12-LOX has been implicated in the pathophysiology of a variety of diseases including arterial thrombosis and diabetes (T1D and T2D). Thus, targeted inhibition of 12-LOX has been proposed as a therapeutic strategy to mitigate the effects of these diseases by ultimately reducing the production of the bioactive metabolite 12-HETE. The <a href="/pcsubstance/?term=ML355[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML355</a> probe displays potent and selective inhibition of 12-LOX <i>in vitro</i> and demonstrated good activity in cell-based assays. <a href="/pcsubstance/?term=ML355[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML355</a> has been shown to decrease calcium mobilization and PAR-4 induced platelet aggregation in patient derived human platelets and to significantly inhibit AA/IONO-induced 12-HETE in mouse BTC3 cells and human islets. Hence the <a href="/pcsubstance/?term=ML355[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML355</a> probe can be used by researchers to interrogate the role of 12-LOX in both diabetes and anti-platelet <i>in vivo</i> models via pharmacological inhibition. Moreover, <a href="/pcsubstance/?term=ML355[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML355</a> has shown favorable ADME properties enabling the scientific community to explore its potential therapeutic applications for other diseases where the 12-LOX role is crucial.</p></div><div id="ml355.s3"><h2 id="_ml355_s3_">2. Materials and Methods</h2><p><b>General Methods for Chemistry:</b> All air or moisture sensitive reactions were performed under positive pressure of nitrogen with oven-dried glassware. Anhydrous solvents such as dichloromethane, <i>N,N</i>-dimethylformamide (DMF), acetonitrile, methanol and triethylamine were purchased from Sigma-Aldrich. Preparative purification was performed on a Waters semi-preparative HPLC system. The column used was a Phenomenex Luna C18 (5 micron, 30 × 75 mm) at a flow rate of 45 mL/min. The mobile phase consisted of acetonitrile and water (each containing 0.1% trifluoroacetic acid). A gradient of 10% to 50% acetonitrile over 8 minutes was used during the purification. Fraction collection was triggered by UV detection (220 nm). Analytical analysis was performed on an Agilent LC/MS (Agilent Technologies, Santa Clara, CA). Method 1: A 7 minute gradient of 4% to 100% Acetonitrile (containing 0.025% trifluoroacetic acid) in water (containing 0.05% trifluoroacetic acid) was used with an 8 minute run time at a flow rate of 1 mL/min. A Phenomenex Luna C18 column (3 micron, 3 × 75 mm) was used at a temperature of 50 °C. Method 2: A 3 minute gradient of 4% to 100% Acetonitrile (containing 0.025% trifluoroacetic acid) in water (containing 0.05% trifluoroacetic acid) was used with a 4.5 minute run time at a flow rate of 1 mL/min. A Phenomenex Gemini Phenyl column (3 micron, 3 × 100 mm) was used at a temperature of 50 °C. Purity determination was performed using an Agilent Diode Array Detector for both Method 1 and Method 2. Mass determination was performed using an Agilent 6130 mass spectrometer with electrospray ionization in the positive mode. <sup>1</sup>H NMR spectra were recorded on Varian 400 MHz spectrometers. Chemical shifts are reported in ppm with undeuterated solvent (DMSO-d<sub>6</sub> at 2.49 ppm) as internal standard for DMSO-<i>d</i><sub>6</sub> solutions. All of the analogs tested in the biological assays have purity greater than 95%, based on both analytical methods. High resolution mass spectrometry was recorded on Agilent 6210 Time-of-Flight LC/MS system. Confirmation of molecular formula was accomplished using electrospray ionization in the positive mode with the Agilent Masshunter software (version B.02).</p><p><b>Biological Reagents:</b> All commercial fatty acids (Sigma-Aldrich Chemical Company) were re-purified using a Higgins HAIsil Semi-Preparative (5 µm, 250 × 10mm) C-18 column. Solution A was 99.9% MeOH and 0.1% acetic acid; solution B was 99.9% H<sub>2</sub>O and 0.1% acetic acid. An isocratic elution of 85% A:15% B was used to purify all fatty acids, which were stored at −80 ºC for a maximum of 6 months.</p><p><b>Human Platelets:</b> Human platelets were obtained from healthy volunteers within the Thomas Jefferson University community and the Philadelphia area. These studies were approved by the Thomas Jefferson University Institutional Review Board, and informed consent was obtained from all donors before blood draw. Blood was centrifuged at 200 <i>g</i> for 13 min at room temperature. Platelet-rich plasma was transferred into a conical tube containing a 10% acid citrate dextrose solution (39 mM citric acid, 75 mM sodium citrate, and 135 mM glucose, pH 7.4) and centrifuged at 2000 <i>g</i> for 15 min at room temperature. Platelets were resuspended in Tyrode's buffer (12 mM NaHCO<sub>3</sub>, 127 mM NaCl, 5 mM KCl, 0.5 mM NaH<sub>2</sub>PO<sub>4</sub>, 1 mM MgCl<sub>2</sub>, 5 mM glucose, and 10 mM HEPES), and the final platelet concentration was adjusted to 3 × 10<sup>8</sup> platelets/mL after counting with a ZI Coulter particle counter (Beckman Coulter, Fullerton, CA). Reported results are the data obtained using platelets from at least three different subjects. Agonists and inhibitors were used at concentrations indicated in the figures and figure legends.</p><p><b>Over expression and Purification of 12-Human Lipoxygenase, 5-Human Lipoxygenase, 12/15-Mouse Lipoxygenase and the 15-Human Lipoxygenases:</b> Human platelet 12-lipoxygenase (12-LOX), human reticulocyte 15-lipoxygenase-1 (15-LOX-1), and human epithelial 15-lipoxygenase-2 (15-LOX-2), were expressed as N-terminally, His<sub>6</sub>-tagged proteins and purified to greater than 90% purity, as evaluated by SDS-PAGE analysis. Human 5-lipoxygenase was expressed as a non-tagged protein and used as a crude ammonium sulfate protein fraction, as published previously. 12/15-mouse lipoxygenase (12/15-mLOX) was expressed as a non-tagged protein and purified on Bio-Rad Uno Q1 with NaCl as the eluent. Iron content of 12-LOX was determined with a Finnigan inductively coupled plasma mass spectrometer (ICP-MS), using cobalt-EDTA as an internal standard. Iron concentrations were compared to standardized iron solutions and used to normalize enzyme concentrations.</p><p><b>High-throughput Screen Materials:</b> Dimethyl sulfoxide (DMSO) ACS grade was from Fisher, while ferrous ammonium sulfate, Xylenol Orange (XO), sulfuric acid, and Triton X-100 were obtained from Sigma-Aldrich.</p><div id="ml355.s4"><h3>2.1. Assays</h3><p><b>12-Lipoxygenase qHTS Assay (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1452" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1452</a>).</b> All screening operations were performed on a fully integrated robotic system (Kalypsys Inc, San Diego, CA). Three μL of 12-LOX solution (80 nM final concentration) was dispensed into 1536-well Greiner black clear-bottom assay plates. Compounds and controls (23 nL) were transferred via Kalypsys pintool equipped with 1536-pin array. The plates were incubated for 15 min at room temperature, and then a 1 μL aliquot of arachidonic acid substrate solution (50 µM final concentration) was added to start the reaction. The reaction was stopped after 6.5 min by the addition of 4 µL FeXO solution (final concentrations of 200 µM Xylenol Orange (XO) and 300 µM of ferrous ammonium sulfate in 50 mM sulfuric acid). The plates were incubated at room temperature for 30 minutes. The absorbances at 405 and 573 nm were recorded using ViewLux high throughput CCD imager (Perkin-Elmer, Waltham, MA).</p><p><b>Human Islet (12-HETE Inhibition) Assay.</b> To validate the functional (phenotypic) inhibition of 12-LOX activity by <a href="/pcsubstance/?term=ML355[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML355</a>, a cell based assay was developed to quantitate the 12-HETE expression, a downstream target of the 12-LOX protein. Human donor islets obtained from integrated islet distribution program were incubated overnight in CMRL media containing 10% Fetal Bovine Serum, 1U penicillin 1µg streptomycin (pen/strep). Islets were equilibrated in serum free media for 1 hour prior to pretreatment with 10µM <a href="/pcsubstance/?term=ML355[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML355</a> for 30 mins. For 12-HETE induction, islets were treated with 100 µM arachidonic acid and 5 µM A23187 for 4 hours at 37 ºC. Islets were harvested, centrifuged at 1000 RPM for 5 minutes with cleared supernatant and islet pellet being stored at -80 ºC. For extraction of the supernatants, samples were acidified to pH 3 with 1N hydrochloric acid (HCl) for 30 minutes and spun at 1000 RPM for five minutes. Samples were added to a prepared column prewashed with ethanol (EtOH), water and hexane. The samples were eluted with ethyl acetate and dried under nitrogen gas before reconstitution in 500 mL of 12-HETE ELISA sample buffer. Cell pellets were extracted using CHCl<sub>3</sub> / MeOH and dried under nitrogen gas before reconstitution in 250 µL of ELISA sample buffer. 12-HETE levels in samples were determined using the Enzo Life Science 12-HETE ELISA kit.</p><p><b>Mouse Beta cells (12-HETE Inhibition) Assay.</b> Cells were gown to 90% confluency in 24 well plates in DMEM, pre-treated with <a href="/pcsubstance/?term=ML355[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML355</a> and stimulated with arachidonic acid and A23187. After four hours, the media was removed and spun at 1000 RPM for 5 minutes. The cleared supernatant was stored at -80 ºC prior to analysis. For analysis, supernatants were extracted on SepPak c18 SPE and dried under nitrogen gas before reconstitution in 12-HETE ELISA buffer. Levels of 12-HETE was evaluated using the Enzo Life Science 12-HETE ELISA using the manufacturer's recommended protocol.</p></div><div id="ml355.s5"><h3>2.2. Probe Chemical Characterization</h3><div id="ml355.f2" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=*Purity%20%3E98%25%20as%20determined%20by%20LC%2FMS%20and%201H%20NMR%20analyses&p=BOOKS&id=259188_ml355f2.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img src="/books/NBK259188/bin/ml355f2.jpg" alt="*Purity >98% as determined by LC/MS and 1H NMR analyses" class="tileshop" title="Click on image to zoom" /></a></div><div class="caption"><p>*Purity >98% as determined by LC/MS and <sup>1</sup>H NMR analyses.</p></div></div><p><b><i>N-(</i></b><b>benzo</b><b><i>[d]</i></b><b>thiazol-2-yl)-4-((2-hydroxy-3-methoxybenzyl)amino) benzenesulfonamide (CID-70701426);</b> ( LC-MS room temperature = 2.26 (Method 2) M+1 = 442.0; <sup>1</sup>H NMR (400 MHz, DMSO-<i>d</i><sub>6</sub>) δ 12.86 (s, 1H), 8.73 (d, <i>J</i> = 0.5 Hz, 1H), 7.75 (ddd, <i>J</i> = 7.9, 1.2, 0.6 Hz, 1H), 7.54 – 7.46 (m, 2H), 7.40 – 7.31 (m, 1H), 7.28 – 7.16 (m, 2H), 6.93 – 6.79 (m, 2H), 6.78 – 6.55 (m, 4H), 4.23 (d, <i>J</i> = 5.8 Hz, 2H) and 3.78 (s, 3H); <sup>13</sup>C NMR (DMSO-<i>d</i><sub>6</sub>) δ ppm 152.4, 147.7, 144.3, 128.2, 125.7, 122.9, 120.4, 119.0, 111.4, 110.9, 56.2 and 40.6; HRMS (ESI) <i>m/z</i> (M+H)<sup>+</sup> calcd. For C<sub>21</sub>H<sub>19</sub>N<sub>3</sub>O<sub>4</sub>S<sub>2</sub>, 441.0817; found 441.0819.</p><div id="ml355.f3" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%201.%20Stability%20of%20ML355%20measured%20as%20percent%20composition%20of%20probe%20molecule%20in%20aqueous%20solution%20(contains%2020%20%25%20acetonitrile)%20at%20r.t.%20over%20the%20indicated%20time%20period%20in%20(A)%20pH%202%20buffer%20%2C%20(B)%20PBS%20pH%207.4%20buffer%2C%20(C)%201M%20HEPES%20pH%207.3%20Lipoxygenase%20UV-Vis%20assay%20buffer%2C%20(D)%20and%20in%20the%20presence%20of%205%20mM%20glutathione%20(reduced%20form).&p=BOOKS&id=259188_ml355f3.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img src="/books/NBK259188/bin/ml355f3.jpg" alt="Figure 1. Stability of ML355 measured as percent composition of probe molecule in aqueous solution (contains 20 % acetonitrile) at r.t. over the indicated time period in (A) pH 2 buffer , (B) PBS pH 7.4 buffer, (C) 1M HEPES pH 7.3 Lipoxygenase UV-Vis assay buffer, (D) and in the presence of 5 mM glutathione (reduced form)." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 1</span><span class="title">Stability of ML355 measured as percent composition of probe molecule in aqueous solution (contains 20 % acetonitrile) at r.t. over the indicated time period in (A) pH 2 buffer , (B) PBS pH 7.4 buffer, (C) 1M HEPES pH 7.3 Lipoxygenase UV-Vis assay buffer, (D) and in the presence of 5 mM glutathione (reduced form)</span></h3></div><div id="ml355.f4" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%202.%20Structures%20of%20the%20five%20analogs%20that%20have%20been%20submitted%20to%20the%20MLSMR%20with%20their%20corresponding%20Compound%20IDs%20and%20MLS%20IDs%20listed%20in%20Table%202.&p=BOOKS&id=259188_ml355f4.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img src="/books/NBK259188/bin/ml355f4.jpg" alt="Figure 2. Structures of the five analogs that have been submitted to the MLSMR with their corresponding Compound IDs and MLS IDs listed in Table 2." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 2</span><span class="title">Structures of the five analogs that have been submitted to the MLSMR with their corresponding Compound IDs and MLS IDs listed in <a class="figpopup" href="/books/NBK259188/table/ml355.t2/?report=objectonly" target="object" rid-figpopup="figml355t2" rid-ob="figobml355t2">Table 2</a></span></h3></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml355t3"><a href="/books/NBK259188/table/ml355.t3/?report=objectonly" target="object" title="Table 1" class="img_link icnblk_img figpopup" rid-figpopup="figml355t3" rid-ob="figobml355t3"><img class="small-thumb" src="/books/NBK259188/table/ml355.t3/?report=thumb" src-large="/books/NBK259188/table/ml355.t3/?report=previmg" alt="Table 1. List of probe ML355 and related analogs that have been submitted to the MLSMR." /></a><div class="icnblk_cntnt"><h4 id="ml355.t3"><a href="/books/NBK259188/table/ml355.t3/?report=objectonly" target="object" rid-ob="figobml355t3">Table 1</a></h4><p class="float-caption no_bottom_margin">List of probe ML355 and related analogs that have been submitted to the MLSMR. </p></div></div></div><div id="ml355.s6"><h3>2.3. Probe Preparation</h3><p>Preparation of <b><i>N-(</i></b><b>benzo</b><b><i>[d]</i></b><b>thiazol-2-yl)-4-((2-hydroxy-3-methoxybenzyl)amino) benzenesulfonamide (<a href="/pcsubstance/?term=ML355[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML355</a>)</b> is a two-step process described below and illustrated in <a class="figpopup" href="/books/NBK259188/figure/ml355.f5/?report=objectonly" target="object" rid-figpopup="figml355f5" rid-ob="figobml355f5">Scheme 1</a>.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml355f5" co-legend-rid="figlgndml355f5"><a href="/books/NBK259188/figure/ml355.f5/?report=objectonly" target="object" title="Scheme 1" class="img_link icnblk_img figpopup" rid-figpopup="figml355f5" rid-ob="figobml355f5"><img class="small-thumb" src="/books/NBK259188/bin/ml355f5.gif" src-large="/books/NBK259188/bin/ml355f5.jpg" alt="Scheme 1. Synthetic route to ML355." /></a><div class="icnblk_cntnt" id="figlgndml355f5"><h4 id="ml355.f5"><a href="/books/NBK259188/figure/ml355.f5/?report=objectonly" target="object" rid-ob="figobml355f5">Scheme 1</a></h4><p class="float-caption no_bottom_margin">Synthetic route to ML355. </p></div></div><ol class="upper-alpha"><li class="half_rhythm"><div>4-aminobenzenesulfonamide, 2-hydroxy-3-methoxybenzaldehyde in ethanol (EtOH) was heated to reflux for 4 hr. During this time an orange precipitate form and the reaction mixture were cooled to room temperature before sodium borohydride (0.330 g, 8.71 mmol) was added. After addition of the sodium borohydride the reaction mixture turned to a clear solution and was allowed to stir at room temperature for 30 min. Slowly 1 M hydrochloric acid (HCl) in diethyl ether (Et<sub>2</sub>O) was added yielding a colorless precipitate which was filtered and washed with ethanol followed by a wash with ether to give a colorless solid product 4-((2-hydroxy-3-methoxybenzyl)amino)benzenesulfonamide with a yield of 92%.</div></li><li class="half_rhythm"><div>4-((2-hydroxy-3-methoxybenzyl)amino)benzenesulfonamide, 2-bromobenzo[d]thiazole, potassium carbonate (K<sub>2</sub>CO<sub>3</sub>), N,N'-dimethylethylenediamine and copper (I) iodide were added to a 15 mL round bottom flask in DMF and the reaction mixture was heated for 18 hr at 90 ºC. The reaction mixture was cooled to room temperature, filtered through a pad of celite, and rinsed the ethyl acetate (EtOAc). The filtrate was transferred to a separation funnel, washed with water (3X), and saturated ammonium chloride solution (2X). The organic layer was concentrated to 2 mL of solvent and passed through a thiol cartridge, dried over sodium sulfate (Na<sub>2</sub>SO<sub>4</sub>), filtered and concentrated to a tan solid. The crude solid was purified on the Isco reverse phase 30 g gold<sup>®</sup> column to provide a 30% yield of <i>N-(</i>benzo<i>[d]</i>thiazol-2-yl)-4-((2-hydroxy-3-methoxybenzyl)amino)benzenesulfonamide (<a href="/pcsubstance/?term=ML355[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML355</a>) as an off-white solid.</div></li></ol></div></div><div id="ml355.s7"><h2 id="_ml355_s7_">3. Results</h2><div id="ml355.s8"><h3>3.1. Dose Response Curves for Probe</h3><div id="ml355.f6" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%203.%20Dose%20response%20curves%20for%20probe%20ML355%20showing%20inhibition%20in%20the%20UV-Vis%20assay.&p=BOOKS&id=259188_ml355f6.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img src="/books/NBK259188/bin/ml355f6.jpg" alt="Figure 3. Dose response curves for probe ML355 showing inhibition in the UV-Vis assay." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 3</span><span class="title">Dose response curves for probe ML355 showing inhibition in the UV-Vis assay</span></h3></div></div><div id="ml355.s9"><h3>3.2. Cellular Activity</h3><div id="ml355.f7" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%204.%20Dose%20dependent%20inhibition%20of%20stimulated%2012-HETE%20by%20ML355.&p=BOOKS&id=259188_ml355f7.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img src="/books/NBK259188/bin/ml355f7.jpg" alt="Figure 4. Dose dependent inhibition of stimulated 12-HETE by ML355." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 4</span><span class="title">Dose dependent inhibition of stimulated 12-HETE by ML355</span></h3><div class="caption"><p>Mouse beta cells (BTC3) were treated with arachidonic acid and calcium ionophore (AA/IONO) alone or in the presence of <a href="/pcsubstance/?term=ML355[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML355</a>. Graphed are the levels of 12-HETE expressed as a percentage of that detected in cells stimulated with AA/IONO alone. 12-HETE was measured by ELISA. Result showed inhibition of 12-HETE expression by the <a href="/pcsubstance/?term=ML355[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML355</a> probe.</p></div></div><div id="ml355.f8" class="figure bk_fig"><div class="graphic"><img src="/books/NBK259188/bin/ml355f8.jpg" alt="Figure 5. Histogram of 12-HETE expression (measured by ELISA) in human primary donor islets stimulated with Arachidonic acid and calcium ionophore (AA/IONO) alone or in the presence of 10 µM of ML355." /></div><h3><span class="label">Figure 5</span><span class="title">Histogram of 12-HETE expression (measured by ELISA) in human primary donor islets stimulated with Arachidonic acid and calcium ionophore (AA/IONO) alone or in the presence of 10 µM of ML355</span></h3><div class="caption"><p>Result showed inhibition of the of the 12-HETE expression on stimulated human islets upon treatment of <a href="/pcsubstance/?term=ML355[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML355</a>.</p></div></div></div><div id="ml355.s10"><h3>3.3. Profiling Assays</h3><p>Selective profiling of <a href="/pcsubstance/?term=ML355[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML355</a> and selected analogs showed inactivity of <a href="/pcsubstance/?term=ML355[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML355</a> against 15-LOX-1, 15-LOX-2 and 5-LOX but good 12-LOX with 0.29 µM (<a class="figpopup" href="/books/NBK259188/table/ml355.t2/?report=objectonly" target="object" rid-figpopup="figml355t2" rid-ob="figobml355t2">Table 2</a>). Moreover, <a href="/pcsubstance/?term=ML355[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML355</a> demonstrated excellent microsomal stability with both rat (T<sub>1/2</sub> >30 minutes) and mouse (T<sub>1/2</sub> >300 minutes) and was found to be stable to mouse plasma over a 2 hour period (100% remaining). <a href="/pcsubstance/?term=ML355[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML355</a> showed no degradation over various aqueous buffers (pH 2-9) and was stable to 5 mM glutathione suggesting excellent stability. One remaining liability is the aqueous solubility which is <5 µM, however improved solubility is observed in the assay buffer (qualitative analysis). <a href="/pcsubstance/?term=ML355[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML355</a> showed moderate permeability in the Caco-2 assay (1.5 × 10<sup>−6</sup> cm/s) and does not appear to be a substrate for Pgp given the efflux ratio of <2. <i>In vivo</i> PK studies where <a href="/pcsubstance/?term=ML355[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML355</a> was administered as a solution via IV (3mpk) and PO (30mpk) demonstrated that <a href="/pcsubstance/?term=ML355[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML355</a> is orally bioavailable (%F = 20) with good half-life (T<sub>1/2</sub> = 2.9 hours). At 30 mpk dosing, <a href="/pcsubstance/?term=ML355[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML355</a> achieves a C<sub>max</sub> of over 135 times the <i>in vitro</i> IC<sub>50</sub> and remains over IC<sub>50</sub> value for over 12 hours. The compound has low clearance (3.4 mL/min/kg) and good overall exposure (AUC<sub>inf</sub>) of 38 µM. Although, the volume of distribution (V<sub>D</sub>) observed was low (0.55 L/kg), the rest of the PK profiling results suggested a reasonable distribution between tissue and blood. These profiling results provided informative data necessary to develop an appropriate dosing regimen for future <i>in vivo</i> studies.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml355t2"><a href="/books/NBK259188/table/ml355.t2/?report=objectonly" target="object" title="Table 2" class="img_link icnblk_img figpopup" rid-figpopup="figml355t2" rid-ob="figobml355t2"><img class="small-thumb" src="/books/NBK259188/table/ml355.t2/?report=thumb" src-large="/books/NBK259188/table/ml355.t2/?report=previmg" alt="Table 2. Selectivity profiling of ML355 and other top compounds." /></a><div class="icnblk_cntnt"><h4 id="ml355.t2"><a href="/books/NBK259188/table/ml355.t2/?report=objectonly" target="object" rid-ob="figobml355t2">Table 2</a></h4><p class="float-caption no_bottom_margin">Selectivity profiling of ML355 and other top compounds. NT = not tested while NI = No inhibition. </p></div></div></div></div><div id="ml355.s11"><h2 id="_ml355_s11_">4. Discussion</h2><div id="ml355.s12"><h3>4.1. Comparison to Existing Art and How the New Probe is an Improvement</h3><p>Previously reported inhibitors of 12-LOX (<a class="figpopup" href="/books/NBK259188/figure/ml355.f9/?report=objectonly" target="object" rid-figpopup="figml355f9" rid-ob="figobml355f9">Figure 6</a>) such as baicalein [<a class="bibr" href="#ml355.r12" rid="ml355.r12">12</a>] and nor-dihydroguairetic acid (NDGA) [<a class="bibr" href="#ml355.r13" rid="ml355.r13">13</a>], “bromo-phenols” or “pyrazole derivatives” all possess several liabilities. These compounds are not only less potent, selective, but are also not easily amendable to further optimization. Our previously described 12-LOX inhibitor (<a href="/pcsubstance/?term=ML127[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML127</a>) demonstrates potent inhibition (<500 nM) and excellent selectivity but is not very tolerant of structural modifications [<a class="bibr" href="#ml355.r14" rid="ml355.r14">14</a>]. Few regions of the molecule are amendable for optimization limiting available avenues for medicinal chemistry efforts. In addition, while the 8-HQ scaffold of <a href="/pcsubstance/?term=ML127[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML127</a> has not yet emerged as a liability for the series, the known promiscuous metal chelation for related compounds of that type suggested one should proceed with caution. In comparison, the new <a href="/pcsubstance/?term=ML355[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML355</a> probe series demonstrated potent (290 nM) activity towards 12-LOX and excellent selectivity against related enzymes 15-LOX-1, 5-LOX, 15-LOX-2, and COX ½ (<a class="figpopup" href="/books/NBK259188/table/ml355.t4/?report=objectonly" target="object" rid-figpopup="figml355t4" rid-ob="figobml355t4">Table 3</a>). Additionally, this chemotype is structurally distinct from all previously reported inhibitors (including <a href="/pcsubstance/?term=ML127[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML127</a>) and it possesses a very drug-like scaffold. This series is readily amendable to structural modifications and displays clear and tractable SAR. Most importantly, <a href="/pcsubstance/?term=ML355[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML355</a> exhibits a favorable <i>in vitro</i> ADME and <i>in vivo</i> PK profile with activity in disease relevant cell-based systems like diabetes through12-HETE reduction in β-cells.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml355f9" co-legend-rid="figlgndml355f9"><a href="/books/NBK259188/figure/ml355.f9/?report=objectonly" target="object" title="Figure 6" class="img_link icnblk_img figpopup" rid-figpopup="figml355f9" rid-ob="figobml355f9"><img class="small-thumb" src="/books/NBK259188/bin/ml355f9.gif" src-large="/books/NBK259188/bin/ml355f9.jpg" alt="Figure 6. Prior Art Inhibitors of 12-Lipxygenase." /></a><div class="icnblk_cntnt" id="figlgndml355f9"><h4 id="ml355.f9"><a href="/books/NBK259188/figure/ml355.f9/?report=objectonly" target="object" rid-ob="figobml355f9">Figure 6</a></h4><p class="float-caption no_bottom_margin">Prior Art Inhibitors of 12-Lipxygenase. </p></div></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml355t4"><a href="/books/NBK259188/table/ml355.t4/?report=objectonly" target="object" title="Table 3" class="img_link icnblk_img figpopup" rid-figpopup="figml355t4" rid-ob="figobml355t4"><img class="small-thumb" src="/books/NBK259188/table/ml355.t4/?report=thumb" src-large="/books/NBK259188/table/ml355.t4/?report=previmg" alt="Table 3. Comparison of ML355 to previously identified 12-LOX inhibitors." /></a><div class="icnblk_cntnt"><h4 id="ml355.t4"><a href="/books/NBK259188/table/ml355.t4/?report=objectonly" target="object" rid-ob="figobml355t4">Table 3</a></h4><p class="float-caption no_bottom_margin">Comparison of ML355 to previously identified 12-LOX inhibitors. ND = Not Determined </p></div></div></div></div><div id="ml355.s13"><h2 id="_ml355_s13_">5. References</h2><dl class="temp-labeled-list"><dl class="bkr_refwrap"><dt>1.</dt><dd><div class="bk_ref" id="ml355.r1">Yamamato S, Suzuki H, Ueda N. Arachidonate 12-lipoxygenases (Review). <span><span class="ref-journal">Prog Lipid Res. </span>1997;<span class="ref-vol">36</span>(1):23–41.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/9373619" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 9373619</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>2.</dt><dd><div class="bk_ref" id="ml355.r2">Solomon EI, Zhou J, Neese F, Pavel EG. New insights from spectroscopy into the structure/function relationships of lipoxygenases. <span><span class="ref-journal">Chem. Biol. </span>1997;<span class="ref-vol">4</span>:795–808.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/9384534" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 9384534</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>3.</dt><dd><div class="bk_ref" id="ml355.r3">Brash AR. Lipoxygenases: Occurrence, Functions, Catalysis and Acquisition of Substrate. <span><span class="ref-journal">J. Biol. Chem. </span>1999;<span class="ref-vol">274</span>:23679–23682.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/10446122" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 10446122</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>4.</dt><dd><div class="bk_ref" id="ml355.r4">Kuhn H, O'Donnell VB. Inflammation and immune regulation by 12/15-lipoxygenases. <span><span class="ref-journal">Prog Lipid Res. </span>2006;<span class="ref-vol">45</span>(4):334–56.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/16678271" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 16678271</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>5.</dt><dd><div class="bk_ref" id="ml355.r5">Nie D, Tang K, Diglio C, Honn K. Eicosanoid regulation of angiogenesis: role of endothelial arachidonate 12-lipoxygenase. <span><span class="ref-journal">Blood. </span>2000;<span class="ref-vol">95</span>:2304–2311.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/10733500" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 10733500</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>6.</dt><dd><div class="bk_ref" id="ml355.r6">Yeung J, Apopa PL, Vesci J, Kenyon V, Rai G, Jadhav A, Simeonov A, Holman TR, Maloney DJ, Boutaud O, Holinstat M. Protein Kinase C Regulation of 12-Lipoxygenase-Mediated Human Platelet Activation. <span><span class="ref-journal">Mol. Pharmacol. </span>2012;<span class="ref-vol">81</span>(3):420–430.</span> [<a href="/pmc/articles/PMC3286293/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3286293</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/22155783" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 22155783</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>7.</dt><dd><div class="bk_ref" id="ml355.r7">Catalano A, Procopio A. New aspects on the role of lipoxygenases in cancer progression. <span><span class="ref-journal">Histol Histopathol. </span>2005;<span class="ref-vol">20</span>:969–975.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/15944947" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 15944947</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>8.</dt><dd><div class="bk_ref" id="ml355.r8">Thomas CP, Morgan LT, Maskrey BH, Murphy RC, Kuhn H, Hazen SL, Goodall AH, Hamali HA, Collins PW, O'Donnell VD. Phospholipid-esterified eicosanoids are generated in agonist-activated human platelets and enhance tissue factor-dependant thrombin generation. <span><span class="ref-journal">J. Biol. Chem. </span>2010;<span class="ref-vol">285</span>:6891–6903.</span> [<a href="/pmc/articles/PMC2844139/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2844139</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/20061396" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 20061396</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>9.</dt><dd><div class="bk_ref" id="ml355.r9">Chen M, Yang ZD, Smith KM, Carter JD, Nadler JL. Activation of 12-Lipoxygenase in pro-inflammatory cytokine-mediated β-cell toxicity. <span><span class="ref-journal">Diabetologia. </span>2005;<span class="ref-vol">48</span>:486–495.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/15729574" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 15729574</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>10.</dt><dd><div class="bk_ref" id="ml355.r10">Timár J, Silletti S, Bazaz R, Raz A, Honn KV. Regulation of melanoma-cell motility by the lipoxygenase metabolite 12-(S)-HETE. <span><span class="ref-journal">Int. J. Cancer. </span>1993;<span class="ref-vol">55</span>:1003–1010.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/8253518" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 8253518</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>11.</dt><dd><div class="bk_ref" id="ml355.r11">Ma K, Nunemaker CS, Wu R, Chakrabarti SK, Taylor-Fishwick DA, Nadler JL. 12-Lipoxygenase Products Reduce Insulin Secretion and β-cell Viability in Human Islets. <span><span class="ref-journal">J. Clin. Endocrinol. Metab. </span>2010;<span class="ref-vol">95</span>:887–893.</span> [<a href="/pmc/articles/PMC2840856/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2840856</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/20089617" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 20089617</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>12.</dt><dd><div class="bk_ref" id="ml355.r12">Svensson H, Grenegard M, Ollinger K, Lindstrom E. Inhibition of 12-lipoxygenase reduces platelet activation and prevents their mitogenic function. <span><span class="ref-journal">Platelets. </span>2013.</span> [Epub ahead of print] [<a href="https://pubmed.ncbi.nlm.nih.gov/23534390" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 23534390</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>13.</dt><dd><div class="bk_ref" id="ml355.r13">Whitman S, Gezginci M, Timmermann BN, Holman TR. Structure-activity relationship studies of nordihydroguaiaretic acid inhibitors toward soybean, 12-human, and 15-human lipoxygenase. <span><span class="ref-journal">J. Med. Chem. </span>2002;<span class="ref-vol">45</span>:2659–2661.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/12036375" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 12036375</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>14.</dt><dd><div class="bk_ref" id="ml355.r14">Kenyon V, Rai G, Jadhav A, Schultz L, Armstrong M, Jameson BJ, Perry S, Joshi N, Bougie JM, Leister W, Taylor-Fishwick DA, Nadler JL, Holinstat M, Simeonov A, Maloney DJ, Holman TR. Discovery of Potent and Selective Inhibitors of Human Platelet-Type 12-Lipoxygenase. <span><span class="ref-journal">J. Med. Chem. </span>2011;<span class="ref-vol">54</span>:5485–5497.</span> [<a href="/pmc/articles/PMC3150642/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3150642</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/21739938" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 21739938</span></a>]</div></dd></dl></dl></div><div id="bk_toc_contnr"></div></div></div><div class="fm-sec"><h2 id="_NBK259188_pubdet_">Publication Details</h2><h3>Author Information and Affiliations</h3><p class="contrib-group"><h4>Authors</h4><span itemprop="author">Diane Luci</span>,<sup>1</sup> <span itemprop="author">J. Brian Jameson, II</span>,<sup>2</sup> <span itemprop="author">Adam Yasgar</span>,<sup>1</sup> <span itemprop="author">Giovanni Diaz</span>,<sup>2</sup> <span itemprop="author">Netra Joshi</span>,<sup>2</sup> <span itemprop="author">Auric Kantz</span>,<sup>2</sup> <span itemprop="author">Kate Markham</span>,<sup>2</sup> <span itemprop="author">Steve Perry</span>,<sup>2</sup> <span itemprop="author">Norine Kuhn</span>,<sup>3</sup> <span itemprop="author">Jennifer Yeung</span>,<sup>4</sup> <span itemprop="author">Lena Schultz</span>,<sup>1</sup> <span itemprop="author">Michael Holinstat</span>,<sup>4</sup> <span itemprop="author">Jerry Nadler</span>,<sup>3</sup> <span itemprop="author">David A. Taylor-Fishwick</span>,<sup>3</sup> <span itemprop="author">Ajit Jadhav</span>,<sup>1</sup> <span itemprop="author">Anton Simeonov</span>,<sup>1</sup> <span itemprop="author">Theodore R. Holman</span>,<sup>2</sup> and <span itemprop="author">David J. Maloney</span><sup>1</sup><sup>,*</sup>.</p><h4>Affiliations</h4><div class="affiliation"><sup>1</sup>
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National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD</div><div class="affiliation"><sup>2</sup>
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Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA</div><div class="affiliation"><sup>3</sup>
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Strelitz Diabetes Center, Eastern Virginia Medical School, Norfolk, VA, United States</div><div class="affiliation"><sup>4</sup>
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Department of Medicine, Cardeza Foundation for Hematologic Research, Thomas Jefferson University, Philadelphia, PA</div><div class="affiliation">
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<sup>*</sup> To whom correspondence should be addressed:
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<span class="before-email-separator"></span><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="vog.hin.liam@dyenolam" class="oemail">vog.hin.liam@dyenolam</a></div><h3>Publication History</h3><p class="small">Received: <span itemprop="datePublished">April 12, 2013</span>; Last Update: <span itemprop="dateModified">September 18, 2014</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>Luci D, Jameson JB II, Yasgar A, et al. Discovery of ML355, a Potent and Selective Inhibitor of Human 12-Lipoxygenase. 2013 Apr 12 [Updated 2014 Sep 18]. In: Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-. <span class="bk_cite_avail"></span></p></div><div class="small-screen-prev"><a href="/books/n/mlprobe/ml356/?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/ml354/?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="figobml355f1"><div id="ml355.f1" class="figure"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Image%20ml355f1&p=BOOKS&id=259188_ml355f1.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img data-src="/books/NBK259188/bin/ml355f1.jpg" alt="Image ml355f1" class="tileshop" title="Click on image to zoom" /></a></div></div></article><article data-type="table-wrap" id="figobml355t1"><div id="ml355.t1" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK259188/table/ml355.t1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml355.t1_lrgtbl__"><table><thead><tr><th id="hd_h_ml355.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CID/ML#</th><th id="hd_h_ml355.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Target Name</th><th id="hd_h_ml355.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">IC50/EC50 (nM) [SID, AID]</th><th id="hd_h_ml355.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Anti-target Name(s)</th><th id="hd_h_ml355.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">IC50/EC50 (μM) [SID, AID]</th><th id="hd_h_ml355.t1_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Fold Selective</th><th id="hd_h_ml355.t1_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Secondary Assay(s) Name: IC50/EC50 (nM) [SID, AID]</th></tr></thead><tbody><tr><td headers="hd_h_ml355.t1_1_1_1_1" rowspan="4" colspan="1" style="text-align:left;vertical-align:top;"><b>CID 70701426/<a href="/pcsubstance/?term=ML355[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML355</a></b></td><td headers="hd_h_ml355.t1_1_1_1_2" rowspan="4" colspan="1" style="text-align:left;vertical-align:top;">12-LOX</td><td headers="hd_h_ml355.t1_1_1_1_3" rowspan="4" colspan="1" style="text-align:left;vertical-align:top;">290 nM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/160844040" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 160844040</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/493216" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 493216</a>]</td><td headers="hd_h_ml355.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">15-LOX-1</td><td headers="hd_h_ml355.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">>100 [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/160844040" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 160844040</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/493219" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 493219</a>]</td><td headers="hd_h_ml355.t1_1_1_1_6" rowspan="4" colspan="1" style="text-align:left;vertical-align:top;"><10% inhibition at 15 μM</td><td headers="hd_h_ml355.t1_1_1_1_7" rowspan="4" colspan="1" style="text-align:left;vertical-align:top;">COX-1/2 [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/160844040" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 160844040</a>]</td></tr><tr><td headers="hd_h_ml355.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">15-LOX-2</td><td headers="hd_h_ml355.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">>100 [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/160844040" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 160844040</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/493220" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 493220</a>]</td></tr><tr><td headers="hd_h_ml355.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">5-LOX</td><td headers="hd_h_ml355.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">>100 [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/160844040" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 160844040</a>]</td></tr><tr><td headers="hd_h_ml355.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">COX-1/2</td><td headers="hd_h_ml355.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><10% inhibition at 15 µM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/1160844040" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 160844040</a>]</td></tr></tbody></table></div></div></article><article data-type="fig" id="figobml355f2"><div id="ml355.f2" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=*Purity%20%3E98%25%20as%20determined%20by%20LC%2FMS%20and%201H%20NMR%20analyses&p=BOOKS&id=259188_ml355f2.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img data-src="/books/NBK259188/bin/ml355f2.jpg" alt="*Purity >98% as determined by LC/MS and 1H NMR analyses" class="tileshop" title="Click on image to zoom" /></a></div><div class="caption"><p>*Purity >98% as determined by LC/MS and <sup>1</sup>H NMR analyses.</p></div></div></article><article data-type="fig" id="figobml355f3"><div id="ml355.f3" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%201.%20Stability%20of%20ML355%20measured%20as%20percent%20composition%20of%20probe%20molecule%20in%20aqueous%20solution%20(contains%2020%20%25%20acetonitrile)%20at%20r.t.%20over%20the%20indicated%20time%20period%20in%20(A)%20pH%202%20buffer%20%2C%20(B)%20PBS%20pH%207.4%20buffer%2C%20(C)%201M%20HEPES%20pH%207.3%20Lipoxygenase%20UV-Vis%20assay%20buffer%2C%20(D)%20and%20in%20the%20presence%20of%205%20mM%20glutathione%20(reduced%20form).&p=BOOKS&id=259188_ml355f3.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img data-src="/books/NBK259188/bin/ml355f3.jpg" alt="Figure 1. Stability of ML355 measured as percent composition of probe molecule in aqueous solution (contains 20 % acetonitrile) at r.t. over the indicated time period in (A) pH 2 buffer , (B) PBS pH 7.4 buffer, (C) 1M HEPES pH 7.3 Lipoxygenase UV-Vis assay buffer, (D) and in the presence of 5 mM glutathione (reduced form)." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 1</span><span class="title">Stability of ML355 measured as percent composition of probe molecule in aqueous solution (contains 20 % acetonitrile) at r.t. over the indicated time period in (A) pH 2 buffer , (B) PBS pH 7.4 buffer, (C) 1M HEPES pH 7.3 Lipoxygenase UV-Vis assay buffer, (D) and in the presence of 5 mM glutathione (reduced form)</span></h3></div></article><article data-type="fig" id="figobml355f4"><div id="ml355.f4" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%202.%20Structures%20of%20the%20five%20analogs%20that%20have%20been%20submitted%20to%20the%20MLSMR%20with%20their%20corresponding%20Compound%20IDs%20and%20MLS%20IDs%20listed%20in%20Table%202.&p=BOOKS&id=259188_ml355f4.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img data-src="/books/NBK259188/bin/ml355f4.jpg" alt="Figure 2. Structures of the five analogs that have been submitted to the MLSMR with their corresponding Compound IDs and MLS IDs listed in Table 2." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 2</span><span class="title">Structures of the five analogs that have been submitted to the MLSMR with their corresponding Compound IDs and MLS IDs listed in <a class="figpopup" href="/books/NBK259188/table/ml355.t2/?report=objectonly" target="object" rid-figpopup="figml355t2" rid-ob="figobml355t2">Table 2</a></span></h3></div></article><article data-type="table-wrap" id="figobml355t2"><div id="ml355.t2" class="table"><h3><span class="label">Table 2</span><span class="title">Selectivity profiling of ML355 and other top compounds</span></h3><div class="caption"><p>NT = not tested while NI = No inhibition.</p></div><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK259188/table/ml355.t2/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml355.t2_lrgtbl__"><table><thead><tr><th id="hd_h_ml355.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Analog</th><th id="hd_h_ml355.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">12-LOX<i><sup>a</sup></i></th><th id="hd_h_ml355.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">15-LOX-1</th><th id="hd_h_ml355.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">15-LOX-2</th><th id="hd_h_ml355.t2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">5-LOX</th><th id="hd_h_ml355.t2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">COX-1/2</th><th id="hd_h_ml355.t2_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Redox Activity</th></tr></thead><tbody><tr><td headers="hd_h_ml355.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="/pcsubstance/?term=ML355[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML355</a></td><td headers="hd_h_ml355.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>0.29</b></td><td headers="hd_h_ml355.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>>100</b></td><td headers="hd_h_ml355.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>>100</b></td><td headers="hd_h_ml355.t2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>> 50</b></td><td headers="hd_h_ml355.t2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>NI</b></td><td headers="hd_h_ml355.t2_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>NI</b></td></tr><tr><td headers="hd_h_ml355.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">30</td><td headers="hd_h_ml355.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">1.2</td><td headers="hd_h_ml355.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">>70</td><td headers="hd_h_ml355.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">>100</td><td headers="hd_h_ml355.t2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">>15</td><td headers="hd_h_ml355.t2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NI</td><td headers="hd_h_ml355.t2_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NT</td></tr><tr><td headers="hd_h_ml355.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">31</td><td headers="hd_h_ml355.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.38</td><td headers="hd_h_ml355.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">>100</td><td headers="hd_h_ml355.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">>100</td><td headers="hd_h_ml355.t2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">>35</td><td headers="hd_h_ml355.t2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NI</td><td headers="hd_h_ml355.t2_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NT</td></tr><tr><td headers="hd_h_ml355.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">33</td><td headers="hd_h_ml355.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.21</td><td headers="hd_h_ml355.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.7</td><td headers="hd_h_ml355.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">> 40</td><td headers="hd_h_ml355.t2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">>50</td><td headers="hd_h_ml355.t2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NI</td><td headers="hd_h_ml355.t2_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NI</td></tr></tbody></table></div></div></article><article data-type="table-wrap" id="figobml355t3"><div id="ml355.t3" class="table"><h3><span class="label">Table 1</span><span class="title">List of probe ML355 and related analogs that have been submitted to the MLSMR</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK259188/table/ml355.t3/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml355.t3_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml355.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Internal ID</th><th id="hd_h_ml355.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS ID</th><th id="hd_h_ml355.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">SID</th><th id="hd_h_ml355.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CID</th><th id="hd_h_ml355.t3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">ML #</th><th id="hd_h_ml355.t3_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Type</th><th id="hd_h_ml355.t3_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Source</th></tr></thead><tbody><tr><td headers="hd_h_ml355.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>NCGC00263773</b></td><td headers="hd_h_ml355.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS004813829</td><td headers="hd_h_ml355.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/160844040" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">160844040</a></td><td headers="hd_h_ml355.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">70701426</td><td headers="hd_h_ml355.t3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="/pcsubstance/?term=ML355[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML355</a></td><td headers="hd_h_ml355.t3_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Probe</td><td headers="hd_h_ml355.t3_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC</td></tr><tr><td headers="hd_h_ml355.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>NCGC00319027</b></td><td headers="hd_h_ml355.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS004813830</td><td headers="hd_h_ml355.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/160844073" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">160844073</a></td><td headers="hd_h_ml355.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">70701393</td><td headers="hd_h_ml355.t3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml355.t3_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Analog</td><td headers="hd_h_ml355.t3_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC</td></tr><tr><td headers="hd_h_ml355.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>NCGC00264087</b></td><td headers="hd_h_ml355.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS004813831</td><td headers="hd_h_ml355.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/160844041" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">160844041</a></td><td headers="hd_h_ml355.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">70701387</td><td headers="hd_h_ml355.t3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml355.t3_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Analog</td><td headers="hd_h_ml355.t3_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC</td></tr><tr><td headers="hd_h_ml355.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>NCGC00319012</b></td><td headers="hd_h_ml355.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS004813832</td><td headers="hd_h_ml355.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/160844065" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">160844065</a></td><td headers="hd_h_ml355.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">70701378</td><td headers="hd_h_ml355.t3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml355.t3_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Analog</td><td headers="hd_h_ml355.t3_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC</td></tr><tr><td headers="hd_h_ml355.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>NCGC00343716</b></td><td headers="hd_h_ml355.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS004813833</td><td headers="hd_h_ml355.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/160844087" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">160844087</a></td><td headers="hd_h_ml355.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">70701397</td><td headers="hd_h_ml355.t3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml355.t3_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Analog</td><td headers="hd_h_ml355.t3_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC</td></tr><tr><td headers="hd_h_ml355.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>NCGC00319030</b></td><td headers="hd_h_ml355.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS004813834</td><td headers="hd_h_ml355.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/160844076" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">160844076</a></td><td headers="hd_h_ml355.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">70701412</td><td headers="hd_h_ml355.t3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td><td headers="hd_h_ml355.t3_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Analog</td><td headers="hd_h_ml355.t3_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC</td></tr></tbody></table></div></div></article><article data-type="fig" id="figobml355f5"><div id="ml355.f5" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Scheme%201.%20Synthetic%20route%20to%20ML355.&p=BOOKS&id=259188_ml355f5.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img data-src="/books/NBK259188/bin/ml355f5.jpg" alt="Scheme 1. Synthetic route to ML355." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Scheme 1</span><span class="title">Synthetic route to ML355</span></h3></div></article><article data-type="fig" id="figobml355f6"><div id="ml355.f6" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%203.%20Dose%20response%20curves%20for%20probe%20ML355%20showing%20inhibition%20in%20the%20UV-Vis%20assay.&p=BOOKS&id=259188_ml355f6.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img data-src="/books/NBK259188/bin/ml355f6.jpg" alt="Figure 3. Dose response curves for probe ML355 showing inhibition in the UV-Vis assay." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 3</span><span class="title">Dose response curves for probe ML355 showing inhibition in the UV-Vis assay</span></h3></div></article><article data-type="fig" id="figobml355f7"><div id="ml355.f7" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%204.%20Dose%20dependent%20inhibition%20of%20stimulated%2012-HETE%20by%20ML355.&p=BOOKS&id=259188_ml355f7.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img data-src="/books/NBK259188/bin/ml355f7.jpg" alt="Figure 4. Dose dependent inhibition of stimulated 12-HETE by ML355." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 4</span><span class="title">Dose dependent inhibition of stimulated 12-HETE by ML355</span></h3><div class="caption"><p>Mouse beta cells (BTC3) were treated with arachidonic acid and calcium ionophore (AA/IONO) alone or in the presence of <a href="/pcsubstance/?term=ML355[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML355</a>. Graphed are the levels of 12-HETE expressed as a percentage of that detected in cells stimulated with AA/IONO alone. 12-HETE was measured by ELISA. Result showed inhibition of 12-HETE expression by the <a href="/pcsubstance/?term=ML355[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML355</a> probe.</p></div></div></article><article data-type="fig" id="figobml355f8"><div id="ml355.f8" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK259188/bin/ml355f8.jpg" alt="Figure 5. Histogram of 12-HETE expression (measured by ELISA) in human primary donor islets stimulated with Arachidonic acid and calcium ionophore (AA/IONO) alone or in the presence of 10 µM of ML355." /></div><h3><span class="label">Figure 5</span><span class="title">Histogram of 12-HETE expression (measured by ELISA) in human primary donor islets stimulated with Arachidonic acid and calcium ionophore (AA/IONO) alone or in the presence of 10 µM of ML355</span></h3><div class="caption"><p>Result showed inhibition of the of the 12-HETE expression on stimulated human islets upon treatment of <a href="/pcsubstance/?term=ML355[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML355</a>.</p></div></div></article><article data-type="fig" id="figobml355f9"><div id="ml355.f9" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%206.%20Prior%20Art%20Inhibitors%20of%2012-Lipxygenase.&p=BOOKS&id=259188_ml355f9.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img data-src="/books/NBK259188/bin/ml355f9.jpg" alt="Figure 6. Prior Art Inhibitors of 12-Lipxygenase." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 6</span><span class="title">Prior Art Inhibitors of 12-Lipxygenase</span></h3></div></article><article data-type="table-wrap" id="figobml355t4"><div id="ml355.t4" class="table"><h3><span class="label">Table 3</span><span class="title">Comparison of ML355 to previously identified 12-LOX inhibitors</span></h3><div class="caption"><p>ND = Not Determined</p></div><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK259188/table/ml355.t4/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml355.t4_lrgtbl__"><table><thead><tr><th id="hd_h_ml355.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Compound</th><th id="hd_h_ml355.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">12-LOX<br /><br />(IC<sub>50</sub>)</th><th id="hd_h_ml355.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Selective</th><th id="hd_h_ml355.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Cell-Active</th></tr></thead><tbody><tr><td headers="hd_h_ml355.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="/pcsubstance/?term=ML355[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML355</a></td><td headers="hd_h_ml355.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">290 nM</td><td headers="hd_h_ml355.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Yes</td><td headers="hd_h_ml355.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Yes</td></tr><tr><td headers="hd_h_ml355.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b>NDGA</b></td><td headers="hd_h_ml355.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">5100 nM</td><td headers="hd_h_ml355.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">No</td><td headers="hd_h_ml355.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">ND</td></tr><tr><td headers="hd_h_ml355.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b>Baicalein</b></td><td headers="hd_h_ml355.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">600 nM</td><td headers="hd_h_ml355.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">No</td><td headers="hd_h_ml355.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Yes</td></tr><tr><td headers="hd_h_ml355.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="/pcsubstance/?term=ML127[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML127</a></td><td headers="hd_h_ml355.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">430 nM</td><td headers="hd_h_ml355.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Yes</td><td headers="hd_h_ml355.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Yes</td></tr><tr><td headers="hd_h_ml355.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b>“bromo-phenols”</b></td><td headers="hd_h_ml355.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">700-7000 nM</td><td headers="hd_h_ml355.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">No</td><td headers="hd_h_ml355.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">ND</td></tr><tr><td headers="hd_h_ml355.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">“<b>pyrazole derivatives”</b></td><td headers="hd_h_ml355.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">9200 nM</td><td headers="hd_h_ml355.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">No</td><td headers="hd_h_ml355.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Yes</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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