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<script type="text/javascript" src="/corehtml/pmc/jatsreader/ptpmc_3.22/js/jr.boots.min.js"> </script><title>Modulators of Lipid Storage - 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="Modulators of Lipid Storage">
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<meta name="citation_publisher" content="National Center for Biotechnology Information (US)">
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<meta name="citation_date" content="2013/05/03">
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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="Yaqin Zhang">
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<meta name="citation_author" content="Li Liu">
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<meta name="citation_author" content="Douglas S. Auld">
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<meta name="citation_author" content="Mathias Beller">
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<meta name="citation_pmid" content="23905198">
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<meta name="DC.Title" content="Modulators of Lipid Storage">
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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="Yaqin Zhang">
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<meta name="DC.Contributor" content="Li Liu">
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<meta name="DC.Contributor" content="Douglas S. Auld">
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<meta name="DC.Contributor" content="Mathias Beller">
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<meta name="DC.Date" content="2013/05/03">
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<meta name="description" content="The primary goal of the project was the identification of chemical probes decreasing the storage lipid content of cells. For this purpose, a cell-based assay using embryonic Drosophila melanogaster cells called “S3” was developed. Cells were incubated with the free fatty acid oleate to induce storage lipid deposition. Lipids are stored in specialized organelles, the so-called lipid droplets, which were stained with the lipophilic dye BODIPY493/503 (Invitrogen/Molecular Probes). We identified three unique chemotypes showing potent lipid droplet reduction phenotypes in the S3 cell based assay. The three probes identified, ML206, ML219 and ML220, had EC50s of 8, 2 and 705 nM, respectively. While ML206 showed little translation into mammalian cell lines, both ML219 and ML220 showed moderate translation into acute myeloid leukemia (AML)12 cells.">
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<meta name="og:title" content="Modulators of Lipid Storage">
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<meta name="og:description" content="The primary goal of the project was the identification of chemical probes decreasing the storage lipid content of cells. For this purpose, a cell-based assay using embryonic Drosophila melanogaster cells called “S3” was developed. Cells were incubated with the free fatty acid oleate to induce storage lipid deposition. Lipids are stored in specialized organelles, the so-called lipid droplets, which were stained with the lipophilic dye BODIPY493/503 (Invitrogen/Molecular Probes). We identified three unique chemotypes showing potent lipid droplet reduction phenotypes in the S3 cell based assay. The three probes identified, ML206, ML219 and ML220, had EC50s of 8, 2 and 705 nM, respectively. While ML206 showed little translation into mammalian cell lines, both ML219 and ML220 showed moderate translation into acute myeloid leukemia (AML)12 cells.">
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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="_NBK153217_"><span class="title" itemprop="name">Modulators of Lipid Storage</span></h1><p class="contribs">Boxer MB, Shen M, Zhang Y, et al.</p><p class="fm-aai"><a href="#_NBK153217_pubdet_">Publication Details</a></p></div></div><div class="jig-ncbiinpagenav body-content whole_rhythm" data-jigconfig="allHeadingLevels: ['h2'],smoothScroll: false" itemprop="text"><div id="_abs_rndgid_" itemprop="description"><p>The primary goal of the project was the identification of chemical probes decreasing the storage lipid content of cells. For this purpose, a cell-based assay using embryonic <i>Drosophila melanogaster</i> cells called “S3” was developed. Cells were incubated with the free fatty acid oleate to induce storage lipid deposition. Lipids are stored in specialized organelles, the so-called lipid droplets, which were stained with the lipophilic dye BODIPY493/503 (Invitrogen/Molecular Probes). We identified three unique chemotypes showing potent lipid droplet reduction phenotypes in the S3 cell based assay. The three probes identified, <a href="/pcsubstance/?term=ML206[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML206</a>, <a href="/pcsubstance/?term=ML219[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML219</a> and <a href="/pcsubstance/?term=ML220[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML220</a>, had EC<sub>50</sub>s of 8, 2 and 705 nM, respectively. While <a href="/pcsubstance/?term=ML206[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML206</a> showed little translation into mammalian cell lines, both <a href="/pcsubstance/?term=ML219[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML219</a> and <a href="/pcsubstance/?term=ML220[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML220</a> showed moderate translation into acute myeloid leukemia (AML)12 cells.</p></div><div class="h2"></div><p><b>Assigned Assay Grant #:</b> R03MH085686</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>: Dr. Mathias Beller, Max-Planck-Institut für Biophysikalische Chemie</p><p><b>PubChem Summary Bioassay Identifier (AID):</b>
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<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1623" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1623</a></p><div id="ml220.s1"><h2 id="_ml220_s1_">Probe Structure & Characteristics</h2><div id="ml220.fu1a" class="figure bk_fig"><div class="graphic"><img src="/books/NBK153217/bin/ml220fu1a.jpg" alt="ML206." /></div><h3><span class="title">ML206</span></h3></div><div id="ml220.fu1b" class="figure bk_fig"><div class="graphic"><img src="/books/NBK153217/bin/ml220fu1b.jpg" alt="ML219." /></div><h3><span class="title">ML219</span></h3></div><div id="ml220.fu1c" class="figure bk_fig"><div class="graphic"><img src="/books/NBK153217/bin/ml220fu1c.jpg" alt="ML220." /></div><h3><span class="title">ML220</span></h3></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml220tu1"><a href="/books/NBK153217/table/ml220.tu1/?report=objectonly" target="object" title="Table" class="img_link icnblk_img figpopup" rid-figpopup="figml220tu1" rid-ob="figobml220tu1"><img class="small-thumb" src="/books/NBK153217/table/ml220.tu1/?report=thumb" src-large="/books/NBK153217/table/ml220.tu1/?report=previmg" alt="Image " /></a><div class="icnblk_cntnt"><h4 id="ml220.tu1"><a href="/books/NBK153217/table/ml220.tu1/?report=objectonly" target="object" rid-ob="figobml220tu1">Table</a></h4></div></div></div><div id="ml220.s2"><h2 id="_ml220_s2_">Recommendations for Scientific Use of the Probe</h2><p>Lipid droplets (LDs) are the universal lipid storage organelles. Lipids remobilized from LDs are used both for energy production and anabolic reactions, such as membrane biosynthesis. Nearly all mammalian components of adipocyte lipolysis are conserved in the fruit fly, <i>Drosophila melanogaster,</i> and the LDs of both species are coated with a similar set of proteins.<sup><a class="bibr" href="#ml220.r1" rid="ml220.r1">1</a>–<a class="bibr" href="#ml220.r9" rid="ml220.r9">9</a></sup> These facts, combined with the rich genetic toolbox available for <i>Drosophila melanogaster</i>, positions the fruit fly as a prime <i>in vivo</i> model to study the mechanisms and pathways involved in lipid droplet biogenesis, regulation and breakdown. In this <i>Drosophila melanogaster</i> cell-based screen, the probes <a href="/pcsubstance/?term=ML206[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML206</a>, <a href="/pcsubstance/?term=ML219[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML219</a> and <a href="/pcsubstance/?term=ML220[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML220</a> show a potent LD reduction phenotype, with EC<sub>50</sub>s of 8 nM, 2 nM, and 705 nM, respectively. When looking at translation of this phenotype into mammalian cell lines, <a href="/pcsubstance/?term=ML206[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML206</a> showed no significant activity in HepG2-, HeLa- or AML12-cells with <50% efficacy when compared to Triacsin C. Moderate translation into mammalian cells was seen for <a href="/pcsubstance/?term=ML219[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML219</a>, which gave an EC<sub>50</sub> of 11 nM with 46% efficacy with AML12-cells. The third probe, <a href="/pcsubstance/?term=ML220[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML220</a>, also showed moderate translation in the AML12 cell line, reducing LD’s with an EC<sub>50</sub> of 1.25 μM with >57% efficacy. With all three compounds showing extremely potent LD phenotypes in S3-cell based assays and modest translation into AML12-cells for <a href="/pcsubstance/?term=ML219[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML219</a> and <a href="/pcsubstance/?term=ML220[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML220</a>, the utility of these compounds lies predominantly in <i>Drosophila melanogaster</i> studies, but they do show promise for use in mammalian systems. The target and mechanism of action for these probes in <i>Drosophila melanogaster</i> cells are not currently known, so studies using affinity chromatography, photoaffinity labeling and RNAi screening will hopefully elucidate their targets and provide insight into underlying mechanisms of LD biogenesis, maintenance and/or breakdown in mammalian systems.</p></div><div id="ml220.s3"><h2 id="_ml220_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 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). 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 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="ml220.s4"><h3>2.1. Assays</h3><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml220t1"><a href="/books/NBK153217/table/ml220.t1/?report=objectonly" target="object" title="Table 1" class="img_link icnblk_img figpopup" rid-figpopup="figml220t1" rid-ob="figobml220t1"><img class="small-thumb" src="/books/NBK153217/table/ml220.t1/?report=thumb" src-large="/books/NBK153217/table/ml220.t1/?report=previmg" alt="Table 1. Screens Deposited in PubChem." /></a><div class="icnblk_cntnt"><h4 id="ml220.t1"><a href="/books/NBK153217/table/ml220.t1/?report=objectonly" target="object" rid-ob="figobml220t1">Table 1</a></h4><p class="float-caption no_bottom_margin">Screens Deposited in PubChem. </p></div></div></div><div id="ml220.s5"><h3>Primary qHTS assay for lipid storage modulators [AID:2685]</h3><p><b>Assay details and protocol.</b> The qHTS was performed with embryonic <i>Drosophila</i> S3 cells (Bloomington Drosophila Stock Center [DGRC]), that showed excellent oleic acid feeding characteristics and good performance during automated liquid handling in 1,536-well format (<a class="figpopup" href="/books/NBK153217/table/ml220.t2/?report=objectonly" target="object" rid-figpopup="figml220t2" rid-ob="figobml220t2">Table 2</a>). We dispensed 4 μl of cells at 1.25 × 10<sup>6</sup> cells/ml into LoBase Aurora COC 1,536-well plates (black walled, clear bottom) with a bottle-valve solenoid-based dispenser (Kalypsys) to obtain 5,000 cells/well. A total of 23 nl of compound solution of different concentrations was transferred to the assay plates using a Kalypsys pin tool equipped with a 1,536-pin array containing 10-nl slotted pins (FP1S10, 0.457-mm diameter, 50.8 mm long; V&P Scientific). One microliter of oleic acid (400 μM) was added, and the plate was lidded with stainless steel rubber gasket-lined lids containing pinholes. After 18–24 h incubation at 24 °C and 95% humidity, 4 μl of BODIPY 493/503 (Molecular Probes) was added to the wells to stain lipid droplets, and the Cell Tracker Red CMTPX dye (Molecular Probes) was added to enumerate cell number. Fluorescence was detected by excitation of the fluorophores with a 488 nm laser on an Acumen Explorer (TTP Lab Tech). The total intensity in channel 1 (500–530 nm) reflected lipid droplet accumulation. Cells were detected using channel 3 (575–640 nm) with 5 μm width and 100 μm depth filters. The ratio of the total intensity in PMT channel 1 over total intensity of channel 3 was also calculated. Percent activity was computed relative to an internal control (100% inhibited lipid droplet deposition due to the presence of 20 μM Triacsin C), which was added to 32 wells/plate. Example data for the assay is shown in <a class="figpopup" href="/books/NBK153217/figure/ml220.f1/?report=objectonly" target="object" rid-figpopup="figml220f1" rid-ob="figobml220f1">Figure 1</a>.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml220t2"><a href="/books/NBK153217/table/ml220.t2/?report=objectonly" target="object" title="Table 2" class="img_link icnblk_img figpopup" rid-figpopup="figml220t2" rid-ob="figobml220t2"><img class="small-thumb" src="/books/NBK153217/table/ml220.t2/?report=thumb" src-large="/books/NBK153217/table/ml220.t2/?report=previmg" alt="Table 2. Stepwise protocol used for the S3 cell 1536-well assay." /></a><div class="icnblk_cntnt"><h4 id="ml220.t2"><a href="/books/NBK153217/table/ml220.t2/?report=objectonly" target="object" rid-ob="figobml220t2">Table 2</a></h4><p class="float-caption no_bottom_margin">Stepwise protocol used for the S3 cell 1536-well assay. </p></div></div><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml220f1" co-legend-rid="figlgndml220f1"><a href="/books/NBK153217/figure/ml220.f1/?report=objectonly" target="object" title="Figure 1" class="img_link icnblk_img figpopup" rid-figpopup="figml220f1" rid-ob="figobml220f1"><img class="small-thumb" src="/books/NBK153217/bin/ml220f1.gif" src-large="/books/NBK153217/bin/ml220f1.jpg" alt="Figure 1. Primary lipid droplet storage assay." /></a><div class="icnblk_cntnt" id="figlgndml220f1"><h4 id="ml220.f1"><a href="/books/NBK153217/figure/ml220.f1/?report=objectonly" target="object" rid-ob="figobml220f1">Figure 1</a></h4><p class="float-caption no_bottom_margin">Primary lipid droplet storage assay. The primary assay used the Acumen Explorer and was a two-color assay using the lipid-specific dye (BODIPY493/503; PMT) and a cell mask stain (CellTracker Red; PMT: 575–640 nm) to determine lipid content and <a href="/books/NBK153217/figure/ml220.f1/?report=objectonly" target="object" rid-ob="figobml220f1">(more...)</a></p></div></div></div><div id="ml220.s6"><h3>Confirmatory assay. [AID 1569, AID 488913 and AID 504432]</h3><p><b>Secondary assays</b>. Cytotoxicity was tested using CellTiter-Glo to measure ATP levels using a final compound concentration of 46.2 μM to 0.3 nM and a 24 hr time point [<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/488908" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 488908</a> and <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504433" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504433</a>]. To test for the possibility that compounds found to be active in the lipid S3 assay remained active in mammalian cell lines, we employed HeLa [<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/488900" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 488900</a>], HepG2 [<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/492960" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 492960</a>] and AML12 cells [<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504434" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504434</a>] with an analogous assay protocol (<a class="figpopup" href="/books/NBK153217/table/ml220.t3/?report=objectonly" target="object" rid-figpopup="figml220t3" rid-ob="figobml220t3">Table 3</a>).</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml220t3"><a href="/books/NBK153217/table/ml220.t3/?report=objectonly" target="object" title="Table 3" class="img_link icnblk_img figpopup" rid-figpopup="figml220t3" rid-ob="figobml220t3"><img class="small-thumb" src="/books/NBK153217/table/ml220.t3/?report=thumb" src-large="/books/NBK153217/table/ml220.t3/?report=previmg" alt="Table 3. Stepwise Protocol Used for the Hela, HepG2 or AML12 cell 1536-Well Assay." /></a><div class="icnblk_cntnt"><h4 id="ml220.t3"><a href="/books/NBK153217/table/ml220.t3/?report=objectonly" target="object" rid-ob="figobml220t3">Table 3</a></h4><p class="float-caption no_bottom_margin">Stepwise Protocol Used for the Hela, HepG2 or AML12 cell 1536-Well Assay. </p></div></div></div><div id="ml220.s7"><h3>2.2. Probe Chemical Characterization</h3><div id="ml220.s8"><h4>ML206</h4><p><sup>1</sup>H NMR (400 MHz, DMSO-<i>d</i><sub>6</sub>); 8.60 (m. 2H), 8.08 (m. 1H), 8.00 (m, 1H), 7.51–7.73 (m, 3H), 7.25 (m, 1H), 5.14 (m, 1H), 2.38–2.56 (m, 2H), 1.70–2.01 (m, 4H). Method 1, retention time, 5.336 min; Method 2, retention time 3.267 min; HRMS:° <i>m/z</i> °(M<sup>+</sup>) = 354.1298 (Calculated for C<sub>19</sub>H<sub>16</sub>F<sub>2</sub>N<sub>4</sub>O = 354.1292). [α]<sub>D</sub> +82 (c=0.22, MeOH). Solubility (PBS, pH 7.4, 23 °C) = 20.4 μg/mL. Stability profile over 48 hrs (PBS, pH 7.4, 23°C) is show in <a class="figpopup" href="/books/NBK153217/figure/ml220.f2/?report=objectonly" target="object" rid-figpopup="figml220f2" rid-ob="figobml220f2">Figure 2</a>. Calculated properties for <a href="/pcsubstance/?term=ML206[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML206</a> are shown in <a class="figpopup" href="/books/NBK153217/table/ml220.t4/?report=objectonly" target="object" rid-figpopup="figml220t4" rid-ob="figobml220t4">Table 4</a>.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml220f2" co-legend-rid="figlgndml220f2"><a href="/books/NBK153217/figure/ml220.f2/?report=objectonly" target="object" title="Figure 2" class="img_link icnblk_img figpopup" rid-figpopup="figml220f2" rid-ob="figobml220f2"><img class="small-thumb" src="/books/NBK153217/bin/ml220f2.gif" src-large="/books/NBK153217/bin/ml220f2.jpg" alt="Figure 2. Stability of ML206 in PBS buffer (pH 7.4, 23°C) plotted as AUC vs. time for a 48 hr period using LC/MS Method 1 described in Section 2." /></a><div class="icnblk_cntnt" id="figlgndml220f2"><h4 id="ml220.f2"><a href="/books/NBK153217/figure/ml220.f2/?report=objectonly" target="object" rid-ob="figobml220f2">Figure 2</a></h4><p class="float-caption no_bottom_margin">Stability of ML206 in PBS buffer (pH 7.4, 23°C) plotted as AUC vs. time for a 48 hr period using LC/MS Method 1 described in Section 2. No instability was observed, and >90% of compound remained at 48 hours. </p></div></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml220t4"><a href="/books/NBK153217/table/ml220.t4/?report=objectonly" target="object" title="Table 4" class="img_link icnblk_img figpopup" rid-figpopup="figml220t4" rid-ob="figobml220t4"><img class="small-thumb" src="/books/NBK153217/table/ml220.t4/?report=thumb" src-large="/books/NBK153217/table/ml220.t4/?report=previmg" alt="Table 4. Probe properties." /></a><div class="icnblk_cntnt"><h4 id="ml220.t4"><a href="/books/NBK153217/table/ml220.t4/?report=objectonly" target="object" rid-ob="figobml220t4">Table 4</a></h4><p class="float-caption no_bottom_margin">Probe properties. </p></div></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml220t5"><a href="/books/NBK153217/table/ml220.t5/?report=objectonly" target="object" title="Table 5" class="img_link icnblk_img figpopup" rid-figpopup="figml220t5" rid-ob="figobml220t5"><img class="small-thumb" src="/books/NBK153217/table/ml220.t5/?report=thumb" src-large="/books/NBK153217/table/ml220.t5/?report=previmg" alt="Table 5. Probe and analogs submitted to the MLSMR." /></a><div class="icnblk_cntnt"><h4 id="ml220.t5"><a href="/books/NBK153217/table/ml220.t5/?report=objectonly" target="object" rid-ob="figobml220t5">Table 5</a></h4><p class="float-caption no_bottom_margin">Probe and analogs submitted to the MLSMR. </p></div></div></div><div id="ml220.s10"><h4>ML219</h4><p><sup>1</sup>H NMR (400 MHz, DMSO-<i>d</i><sub>6</sub>) δ ppm 8.29 (t, <i>J</i>=5.67 Hz, 1 H), 7.39 (d, <i>J</i>=5.48 Hz, 1 H), 7.08 – 7.15 (m, 2 H), 7.04 (s, 1 H), 6.96 – 7.02 (m, 2 H), 6.87 (d, <i>J</i>=7.43 Hz, 1 H), 6.75 – 6.83 (m, 2 H), 6.66 (dd, <i>J</i>=8.22, 1.96 Hz, 1 H), 5.71 (s, 2 H), 3.93 (qd, <i>J</i>=6.98, 4.70 Hz, 4 H), 3.34 – 3.43 (m, 2 H), 2.70 (t, <i>J</i>=7.24 Hz, 2 H), 2.19 (s, 3 H), 1.25 (dt, <i>J</i>=10.27, 6.90 Hz, 6 H). Method 1, retention time, 6.811 min; Method 2, retention time 3.787 min; HRMS:° <i>m/z</i> °(M+H<sup>+</sup>) = 462.1985 (Calculated for C<sub>27</sub>H<sub>30</sub>N<sub>2</sub>O<sub>3</sub>S = 462.1977). Solubility (PBS, pH 7.4, 23 °C) = 5.9 μg/mL. Stability profile over 48 hrs (PBS, pH 7.4, 23 °C) is shown in <a class="figpopup" href="/books/NBK153217/figure/ml220.f3/?report=objectonly" target="object" rid-figpopup="figml220f3" rid-ob="figobml220f3">Figure 3</a>.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml220f3" co-legend-rid="figlgndml220f3"><a href="/books/NBK153217/figure/ml220.f3/?report=objectonly" target="object" title="Figure 3" class="img_link icnblk_img figpopup" rid-figpopup="figml220f3" rid-ob="figobml220f3"><img class="small-thumb" src="/books/NBK153217/bin/ml220f3.gif" src-large="/books/NBK153217/bin/ml220f3.jpg" alt="Figure 3. Stability of ML219 in PBS buffer (pH 7.4, 23°C) plotted as AUC vs. time for a 48 hr period, using LC/MS Method 1 described in Section 2." /></a><div class="icnblk_cntnt" id="figlgndml220f3"><h4 id="ml220.f3"><a href="/books/NBK153217/figure/ml220.f3/?report=objectonly" target="object" rid-ob="figobml220f3">Figure 3</a></h4><p class="float-caption no_bottom_margin">Stability of ML219 in PBS buffer (pH 7.4, 23°C) plotted as AUC vs. time for a 48 hr period, using LC/MS Method 1 described in Section 2. No instability was observed, and >90% of compound remained at 48 hours. </p></div></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml220t6"><a href="/books/NBK153217/table/ml220.t6/?report=objectonly" target="object" title="Table 6" class="img_link icnblk_img figpopup" rid-figpopup="figml220t6" rid-ob="figobml220t6"><img class="small-thumb" src="/books/NBK153217/table/ml220.t6/?report=thumb" src-large="/books/NBK153217/table/ml220.t6/?report=previmg" alt="Table 6. Probe and analogs submitted to the MLSMR." /></a><div class="icnblk_cntnt"><h4 id="ml220.t6"><a href="/books/NBK153217/table/ml220.t6/?report=objectonly" target="object" rid-ob="figobml220t6">Table 6</a></h4><p class="float-caption no_bottom_margin">Probe and analogs submitted to the MLSMR. </p></div></div></div><div id="ml220.s11"><h4>ML220</h4><p><sup>1</sup>H NMR (400 MHz, DMSO-<i>d</i><sub>6</sub>) δ ppm 8.03 (dd,
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<i>J</i>=8.41, 1.56 Hz, 1 H), 7.94 (s, 1 H), 7.42 (s, 1 H), 7.35 (dd,
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<i>J</i>=8.02, 1.37 Hz, 1 H), 7.18 (ddd, <i>J</i>=8.41, 7.24, 1.57 Hz, 1 H), 6.85 – 6.96 (m, 1 H), 4.28 (dt, <i>J</i>=13.16, 3.30 Hz, 2 H), 3.96 (q, <i>J</i>=7.17 Hz, 2 H), 2.76 – 2.99 (m, 2 H), 2.43 – 2.55 (m, 1 H), 1.73 (dd, <i>J</i>=13.11, 2.93 Hz, 2 H), 1.31 – 1.45 (m, 8 H), 1.06 (t, <i>J</i>=7.14 Hz, 3 H). Method 1, retention time, 4.995 min; Method 2, retention time 3.281 min; HRMS:° <i>m/z</i> °(M<sup>+</sup>) = 395.1623 (Calculated for C<sub>19</sub>H<sub>26</sub>ClN<sub>3</sub>O<sub>4</sub> = 395.1612). Solubility (PBS, pH 7.4, 23 °C) = >75.0 μg/mL. Stability profile over 48 hrs (PBS, pH 7.4, 23 °C) is shown in <a class="figpopup" href="/books/NBK153217/figure/ml220.f4/?report=objectonly" target="object" rid-figpopup="figml220f4" rid-ob="figobml220f4">Figure 4</a>.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml220f4" co-legend-rid="figlgndml220f4"><a href="/books/NBK153217/figure/ml220.f4/?report=objectonly" target="object" title="Figure 4" class="img_link icnblk_img figpopup" rid-figpopup="figml220f4" rid-ob="figobml220f4"><img class="small-thumb" src="/books/NBK153217/bin/ml220f4.gif" src-large="/books/NBK153217/bin/ml220f4.jpg" alt="Figure 4. Stability of ML220 in PBS buffer (pH 7.4, 23°C) plotted as AUC vs. time for a 48 hr period, using LC/MS Method 1 described in Section 2." /></a><div class="icnblk_cntnt" id="figlgndml220f4"><h4 id="ml220.f4"><a href="/books/NBK153217/figure/ml220.f4/?report=objectonly" target="object" rid-ob="figobml220f4">Figure 4</a></h4><p class="float-caption no_bottom_margin">Stability of ML220 in PBS buffer (pH 7.4, 23°C) plotted as AUC vs. time for a 48 hr period, using LC/MS Method 1 described in Section 2. No instability was observed, and >90% of compound remained at 48 hours. </p></div></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml220t7"><a href="/books/NBK153217/table/ml220.t7/?report=objectonly" target="object" title="Table 7" class="img_link icnblk_img figpopup" rid-figpopup="figml220t7" rid-ob="figobml220t7"><img class="small-thumb" src="/books/NBK153217/table/ml220.t7/?report=thumb" src-large="/books/NBK153217/table/ml220.t7/?report=previmg" alt="Table 7. Probe and analogs submitted to the MLSMR." /></a><div class="icnblk_cntnt"><h4 id="ml220.t7"><a href="/books/NBK153217/table/ml220.t7/?report=objectonly" target="object" rid-ob="figobml220t7">Table 7</a></h4><p class="float-caption no_bottom_margin">Probe and analogs submitted to the MLSMR. </p></div></div></div></div><div id="ml220.s12"><h3>2.3. Probe Preparation</h3><div id="ml220.s13"><h4>Synthesis of ML206</h4><p><a class="figpopup" href="/books/NBK153217/figure/ml220.f8/?report=objectonly" target="object" rid-figpopup="figml220f8" rid-ob="figobml220f8">Scheme 1</a>, Steps 1-3 were adapted directly from Guo <i>et al</i>.<sup><a class="bibr" href="#ml220.r14" rid="ml220.r14">14</a></sup></p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml220f8" co-legend-rid="figlgndml220f8"><a href="/books/NBK153217/figure/ml220.f8/?report=objectonly" target="object" title="Scheme 1" class="img_link icnblk_img figpopup" rid-figpopup="figml220f8" rid-ob="figobml220f8"><img class="small-thumb" src="/books/NBK153217/bin/ml220f8.gif" src-large="/books/NBK153217/bin/ml220f8.jpg" alt="Scheme 1. Synthesis of enantiomerically pure ML206." /></a><div class="icnblk_cntnt" id="figlgndml220f8"><h4 id="ml220.f8"><a href="/books/NBK153217/figure/ml220.f8/?report=objectonly" target="object" rid-ob="figobml220f8">Scheme 1</a></h4><p class="float-caption no_bottom_margin">Synthesis of enantiomerically pure ML206. </p></div></div><p><a class="figpopup" href="/books/NBK153217/figure/ml220.f8/?report=objectonly" target="object" rid-figpopup="figml220f8" rid-ob="figobml220f8">Scheme 1</a>, Step 4. 1-(2,4-difluorophenyl)-4,5,6,7-tetrahydro-1H-indazol-4-amine (0.1 g, 0.401 mmol) and picolinic acid (0.049 g, 0.401 mmol) were dissolved in DMF (2 ml) and DIPEA (0.091 ml, 0.522 mmol) was added, followed by HATU (0.183 g, 0.481 mmol); the reaction was stirred at RT for 2 hrs, then purified by directly injecting to a Waters® reverse phase purification system.</p><p><a class="figpopup" href="/books/NBK153217/figure/ml220.f8/?report=objectonly" target="object" rid-figpopup="figml220f8" rid-ob="figobml220f8">Scheme 1</a>, Step 5. Chiral chromotography was performed on a Chiralpac IB column (2 cm × 25 cm, 20 micron) using EtOH/Hexane (60/40, v/v) as the eluant at a flow rate of 4.0 mL/min, with a 30 min run time to give enantiomerically pure <a href="/pcsubstance/?term=ML206[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML206</a>.</p></div><div id="ml220.s14"><h4>Synthesis for determination of absolute configuration of ML206</h4><p><u>Scheme 4, Step 6.</u> NaOH (0.762 ml, 0.762 mmol), then (Boc)<sub>2</sub>O (0.195 ml, 0.838 mmol) were added to the solution of racemic 1-(2,4-difluorophenyl)-4,5,6,7-tetrahydro-1H-indazol-4-amine (0.19 g, 0.762 mmol) in DCM (8 ml). The mixture was stirred at RT for 3hrs. The organic layer was washed with H2O twice, dried over Na2SO4, and concentrated. The crude product was submitted for chiral separation.</p><p><u>Scheme 4, Step 7.</u> Chiral chromatography was performed on a Chiralpac OJ column (20 × 50 cm, 20 micron) with MeOH as the eluant, a flow rate of 40 mL/min and a 40 min run time.</p><p><a class="figpopup" href="/books/NBK153217/figure/ml220.f10/?report=objectonly" target="object" rid-figpopup="figml220f10" rid-ob="figobml220f10">Scheme 3</a>, Step 8. TFA (0.2 ml, 2.60 mmol) was added to the solution of the optically pure tert-butyl 1-(2,4-difluorophenyl)-4,5,6,7-tetrahydro-1H-indazol-4-ylcarbamate (17 mg, 0.049 mmol) in DCM (1 ml). The mixture was stirred at RT overnight. The solvent was then removed <i>in vacuo</i> and placed on a high vacuum pump (~1 torr) overnight. The crude product was used in the next reactions without purification.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml220f10" co-legend-rid="figlgndml220f10"><a href="/books/NBK153217/figure/ml220.f10/?report=objectonly" target="object" title="Scheme 3" class="img_link icnblk_img figpopup" rid-figpopup="figml220f10" rid-ob="figobml220f10"><img class="small-thumb" src="/books/NBK153217/bin/ml220f10.gif" src-large="/books/NBK153217/bin/ml220f10.jpg" alt="Scheme 3. Synthesis of ML220." /></a><div class="icnblk_cntnt" id="figlgndml220f10"><h4 id="ml220.f10"><a href="/books/NBK153217/figure/ml220.f10/?report=objectonly" target="object" rid-ob="figobml220f10">Scheme 3</a></h4><p class="float-caption no_bottom_margin">Synthesis of ML220. </p></div></div><p><a class="figpopup" href="/books/NBK153217/figure/ml220.f10/?report=objectonly" target="object" rid-figpopup="figml220f10" rid-ob="figobml220f10">Scheme 3</a>, Step 9-11.</p><p>The enantiomerically pure amine was coupled with either picolinic acid, <i>R</i>-Mosher acid or <i>S</i>-Mosher acid using the amide coupling described for step 4 <a class="figpopup" href="/books/NBK153217/figure/ml220.f8/?report=objectonly" target="object" rid-figpopup="figml220f8" rid-ob="figobml220f8">scheme 1</a>. All products were purified using a Waters® reverse phase purification system.</p></div><div id="ml220.s15"><h4>Synthesis of ML219</h4><p><a class="figpopup" href="/books/NBK153217/figure/ml220.f9/?report=objectonly" target="object" rid-figpopup="figml220f9" rid-ob="figobml220f9">Scheme 2</a>, Step 1. HATU (0.751 g, 1.974 mmol) and DIPEA (0.94 ml, 5.38 mmol) were added to a solution of 4H-thieno[3,2-b]pyrrole-5-carboxylic acid (0.3 g, 1.794 mmol) in DMF (25 ml). The solution was stirred at room temperature for 30 min, and then 2-(3,4-diethoxyphenyl)ethanamine (0.413 g, 1.974 mmol) was added. The reaction was stirred at room temperature overnight. Water (40 ml) was added to the mixture. The solid was filtered and washed with water, and dried. The crude product was used in the next reaction without further purification.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml220f9" co-legend-rid="figlgndml220f9"><a href="/books/NBK153217/figure/ml220.f9/?report=objectonly" target="object" title="Scheme 2" class="img_link icnblk_img figpopup" rid-figpopup="figml220f9" rid-ob="figobml220f9"><img class="small-thumb" src="/books/NBK153217/bin/ml220f9.gif" src-large="/books/NBK153217/bin/ml220f9.jpg" alt="Scheme 2. Synthesis of ML219." /></a><div class="icnblk_cntnt" id="figlgndml220f9"><h4 id="ml220.f9"><a href="/books/NBK153217/figure/ml220.f9/?report=objectonly" target="object" rid-ob="figobml220f9">Scheme 2</a></h4><p class="float-caption no_bottom_margin">Synthesis of ML219. </p></div></div><p><a class="figpopup" href="/books/NBK153217/figure/ml220.f9/?report=objectonly" target="object" rid-figpopup="figml220f9" rid-ob="figobml220f9">Scheme 2</a>, Step 2. Sodium hydride (3.68 mg of 60 wt% dispersion in mineral oil, 0.092 mmol) was added to the solution of N-(3,4-diethoxyphenethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide (30 mg, 0.084 mmol) in DMF (1.5 ml). The mixture was stirred at room temperature for 15 min. 1-(bromomethyl)-3-methylbenzene (0.014 ml, 0.1 mmol) was added to this mixture. The reaction mixture was stirred at room temperature overnight. The DMF solution was purified by directly injecting to a Waters® reverse phase purification system to give <a href="/pcsubstance/?term=ML219[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML219</a> as a TFA salt.</p></div><div id="ml220.s16"><h4>Synthesis of ML220</h4><p><a class="figpopup" href="/books/NBK153217/figure/ml220.f10/?report=objectonly" target="object" rid-figpopup="figml220f10" rid-ob="figobml220f10">Scheme 3</a>, Step 1. A solution of NaOH (0.028 g, 0.698 mmol) in Water (1 ml) was added to a suspension of 1-aminocyclohexanecarboxylic acid (0.1 g, 0.698 mmol) in 1,4-dioxane (3 ml) at RT. The mixture was stirred at RT for 20 min (clear solution). A solution of 2,4-dichloro-1-isocyanatobenzene (0.131 g, 0.698 mmol) in dioxane (2 ml) was added dropwise to the reaction mixture at RT. The resulting solution was stirred at RT for 1 hr, and then evaporated to dryness under reduced pressure to give the title compound.</p><p><a class="figpopup" href="/books/NBK153217/figure/ml220.f10/?report=objectonly" target="object" rid-figpopup="figml220f10" rid-ob="figobml220f10">Scheme 3</a>, Step 2. HATU (81 mg, 0.214 mmol), then DIPEA (0.102 ml, 0.584 mmol) were added to a solution of 2-(3-(2-chlorophenyl)ureido)-2-methylpropanoic acid (50 mg, 0.195 mmol) in DMF (1.5 ml). The solution was stirred at RT for 30 min, and then ethyl piperidine-4-carboxylate (0.033 ml, 0.214 mmol) was added. The reaction was stirred at RT for 3 hrs. The DMF solution was purified by directly injecting to a Waters® reverse phase purification system to give <a href="/pcsubstance/?term=ML220[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML220</a> as a TFA salt.</p></div></div></div><div id="ml220.s17"><h2 id="_ml220_s17_">3. Results</h2><div id="ml220.s18"><h3>3.1. Dose Response Curves for Probe</h3><div id="ml220.f5" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%205.%20Potency%20confirmation.&p=BOOKS&id=153217_ml220f5.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/NBK153217/bin/ml220f5.jpg" alt="Figure 5. Potency confirmation." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 5</span><span class="title">Potency confirmation</span></h3><div class="caption"><p>Confirmatory data for the probe compounds in S3, Hela, HepG2 and AML12 cells, along with
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cytotoxicity determined in S3 cells. The lipid reduction % Activity plotted is from the
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lipid data. <b>A.</b> The racemic compound CID16192366. <b>B.</b> The enantiomerically
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pure compound where the absolute configuration was confirmed as (<i>R)</i> for <a href="/pcsubstance/?term=ML206[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML206</a>. <b>C.</b> The enantiomerically pure compound where the absolute configuration was confirmed as (<i>S</i>) for CID42288155. <b>D</b>. The probe compound <a href="/pcsubstance/?term=ML219[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML219</a> which also showed weak activity in AML12 cells. E. The probe compound <a href="/pcsubstance/?term=ML220[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML220</a> which also showed weak activity in AML12 cells.</p></div></div><p>The enantiomerically pure compounds have also been transferred to the assay provider’s lab, and Dr. Beller has confirmed activity in S3 cells using the same lipid-specific BODIPY dye, but with wide-field microscope-based analysis (<a class="figpopup" href="/books/NBK153217/figure/ml220.f6/?report=objectonly" target="object" rid-figpopup="figml220f6" rid-ob="figobml220f6">Figure 6</a>). These results support high and low potency for the (<i>R</i>) and (<i>S</i>) enantiomers respectively, as well as low cytotoxicity in S3 cells.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml220f6" co-legend-rid="figlgndml220f6"><a href="/books/NBK153217/figure/ml220.f6/?report=objectonly" target="object" title="Figure 6" class="img_link icnblk_img figpopup" rid-figpopup="figml220f6" rid-ob="figobml220f6"><img class="small-thumb" src="/books/NBK153217/bin/ml220f6.gif" src-large="/books/NBK153217/bin/ml220f6.jpg" alt="Figure 6. BODIPY and Hoechst staining of S3 cells treated with compounds." /></a><div class="icnblk_cntnt" id="figlgndml220f6"><h4 id="ml220.f6"><a href="/books/NBK153217/figure/ml220.f6/?report=objectonly" target="object" rid-ob="figobml220f6">Figure 6</a></h4><p class="float-caption no_bottom_margin">BODIPY and Hoechst staining of S3 cells treated with compounds. Data was collected at the Max Planck Institute for Biophysical Chemistry, Department of Molecular Developmental Biology. </p></div></div></div><div id="ml220.s19"><h3>3.2. Cellular Activity</h3><p>Primary and all follow-up assays were performed in <i>Drosophila melanogaster</i> cells (S3), HeLa, HepG2 and AML12 cells, with activity seen in varying levels in 3 of these cell lines (S3, HepG2 and AML12). Therefore, the compounds are deemed to have sufficient cell permeability/activity. No significant cytotoxicity (as compared to EC<sub>50</sub> values) was seen for these probes (and the majority of their analogs) in any of the 4 cell lines. <a href="/pcsubstance/?term=ML206[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML206</a>, <a href="/pcsubstance/?term=ML219[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML219</a> and <a href="/pcsubstance/?term=ML220[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML220</a> showed >1000-fold cytotoxicity windows in S3-cells, with only minimal cytotoxicity at 46 μM (<a class="figpopup" href="/books/NBK153217/figure/ml220.f6/?report=objectonly" target="object" rid-figpopup="figml220f6" rid-ob="figobml220f6">Figure 6</a>).</p></div><div id="ml220.s20"><h3>3.3. Profiling Assays</h3><p>Profiling assays have not been done with these probes, as they show minimal activity in mammalian cell-lines, and thus such profiles are not deemed valuable at this point.</p></div></div><div id="ml220.s21"><h2 id="_ml220_s21_">4. Discussion</h2><div id="ml220.s22"><h3>4.1. Comparison to Existing Art and How the New Probe is an Improvement</h3><p>We have previously reported that inhibition of COPI function by Exo1 (<a href="/pcsubstance/?term=ML084[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML084</a>) results in lipid overstorage, but we had not discovered inhibitors of LD accumulation at that time.<sup><a class="bibr" href="#ml220.r16" rid="ml220.r16">16</a></sup> Few substances are available as reagents for <i>in vitro</i> lipid storage research (i.e. Triacsin C<sup><a class="bibr" href="#ml220.r15" rid="ml220.r15">15</a></sup>, Exo1 (<a href="/pcsubstance/?term=ML084[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML084</a>)<sup><a class="bibr" href="#ml220.r16" rid="ml220.r16">16</a>,<a class="bibr" href="#ml220.r17" rid="ml220.r17">17</a></sup>, Isoproterenol and other β-adrenergic agonists<sup><a class="bibr" href="#ml220.r18" rid="ml220.r18">18</a></sup> and natural products, such as Magnolol<sup><a class="bibr" href="#ml220.r19" rid="ml220.r19">19</a></sup>). While these compounds are useful, the biogenesis, maintenance, breakdown, signaling pathways and mechanisms associated with LDs are not fully understood, and the discovery of novel and potent LD storage modulators is still highly sought. Particularly, the identification of compounds targeting new proteins is desirable to help shed light on specific aspects of these pathways. The previous probes discovered (<a href="/pcsubstance/?term=ML206[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML206</a> and <a href="/pcsubstance/?term=ML207[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML207</a> (CID 46916320)) are extremely potent lipid storage inhibitors in <i>Drosophila melanogaster</i>, and the new additions (<a href="/pcsubstance/?term=ML219[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML219</a> and <a href="/pcsubstance/?term=ML220[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML220</a>) show similar potent phenotypes in the S3-cell line with improved translation into the mammalian cell line. This renders these new probes as considerable improvements over the existing art. Currently, there is an ongoing effort to discover the target(s) of all three probes using photoaffinity labeling, affinity chromatography and RNAi screening experiments.</p><div id="ml220.f7" class="figure bk_fig"><div class="graphic"><img src="/books/NBK153217/bin/ml220f7.jpg" alt="Figure 7. Previous ML probes that modulate lipid storage in S3 cells." /></div><h3><span class="label">Figure 7</span><span class="title">Previous ML probes that modulate lipid storage in S3 cells</span></h3></div></div></div><div id="ml220.s23"><h2 id="_ml220_s23_">5. References</h2><dl class="temp-labeled-list"><dl class="bkr_refwrap"><dt>1.</dt><dd><div class="bk_ref" id="ml220.r1">Bohni R, Riesgo-Escovar J, Oldham S, Brogiolo W, Stocker H, Andruss BF, Beckingham K, Hafen E. Autonomous control of cell and organ size by CHICO, a Drosophila homolog of vertebrate IRS1-4. <span><span class="ref-journal">Cell. </span>1999;<span class="ref-vol">97</span>:865–75.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/10399915" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 10399915</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>2.</dt><dd><div class="bk_ref" id="ml220.r2">Gronke S, Beller M, Fellert S, Ramakrishnan H, Jackle H, Kuhnlein RP. Control of fat storage by a Drosophila PAT domain protein. <span><span class="ref-journal">Curr Biol. </span>2003;<span class="ref-vol">13</span>:603–6.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/12676093" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 12676093</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>3.</dt><dd><div class="bk_ref" id="ml220.r3">Gronke S, Mildner A, Fellert S, Tennagels N, Petry S, Muller G, Jackle H, Kuhnlein RP. Brummer lipase is an evolutionary conserved fat storage regulator in Drosophila. <span><span class="ref-journal">Cell Metab. </span>2005;<span class="ref-vol">1</span>:323–30.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/16054079" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 16054079</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>4.</dt><dd><div class="bk_ref" id="ml220.r4">Gronke S, Muller G, Hirsch J, Fellert S, Andreou A, Haase T, Jackle H, Kuhnlein RP. Dual lipolytic control of body fat storage and mobilization in Drosophila. <span><span class="ref-journal">PLoS Biol. </span>2007;<span class="ref-vol">5</span>:e137.</span> [<a href="/pmc/articles/PMC1865564/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC1865564</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/17488184" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17488184</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>5.</dt><dd><div class="bk_ref" id="ml220.r5">Teixeira L, Rabouille C, Rorth P, Ephrussi A, Vanzo NF. Drosophila Perilipin/ADRP homologue Lsd2 regulates lipid metabolism. <span><span class="ref-journal">Mech Dev. </span>2003;<span class="ref-vol">120</span>:1071–81.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/14550535" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 14550535</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>6.</dt><dd><div class="bk_ref" id="ml220.r6">Bartz R, Zehmer JK, Zhu M, Chen Y, Serrero G, Zhao Y, Liu P. Dynamic activity of lipid droplets: protein phosphorylation and GTP-mediated protein translocation. <span><span class="ref-journal">J Proteome Res. </span>2007;<span class="ref-vol">6</span>:3256–65.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/17608402" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17608402</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>7.</dt><dd><div class="bk_ref" id="ml220.r7">Beller M, Riedel D, Jansch L, Dieterich G, Wehland J, Jackle H, Kuhnlein RP. Characterization of the Drosophila lipid droplet subproteome. <span><span class="ref-journal">Mol Cell Proteomics. </span>2006;<span class="ref-vol">5</span>:1082–94.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/16543254" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 16543254</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>8.</dt><dd><div class="bk_ref" id="ml220.r8">Cermelli S, Guo Y, Gross SP, Welte MA. The lipid-droplet proteome reveals that droplets are a protein-storage depot. <span><span class="ref-journal">Curr Biol. </span>2006;<span class="ref-vol">16</span>:1783–95.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/16979555" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 16979555</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>9.</dt><dd><div class="bk_ref" id="ml220.r9">Liu P, Ying Y, Zhao Y, Mundy DI, Zhu M, Anderson RG. Chinese hamster ovary K2 cell lipid droplets appear to be metabolic organelles involved in membrane traffic. <span><span class="ref-journal">J Biol Chem. </span>2004;<span class="ref-vol">279</span>:3787–92.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/14597625" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 14597625</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>10.</dt><dd><div class="bk_ref" id="ml220.r10">Brasaemle DL. <span><span class="ref-journal">J Lipid Res. </span>2007;<span class="ref-vol">48</span>:2547–2559.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/17878492" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17878492</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>11.</dt><dd><div class="bk_ref" id="ml220.r11">Gieselmann V. <span><span class="ref-journal">Biochimica et Biophysica Acta. </span>1995;<span class="ref-vol">1270</span>:103–136.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/7727535" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 7727535</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>12.</dt><dd><div class="bk_ref" id="ml220.r12">Winchester B, Vellodi A, Young E. <span><span class="ref-journal">Biochem Soc Trans. </span>2000;<span class="ref-vol">28</span>:150–154.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/10816117" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 10816117</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>13.</dt><dd><div class="bk_ref" id="ml220.r13">Chien S, Reiter LT, Bier E, Gribskov M. <span><span class="ref-journal">Nucleic Acids Res. </span>2002;<span class="ref-vol">30</span>:149–151.</span> [<a href="/pmc/articles/PMC99119/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC99119</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/11752278" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 11752278</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>14.</dt><dd><div class="bk_ref" id="ml220.r14">Guo S, Song Y, Huang Q, Yuan H, Wan B, Wang Y, He R, Beconi MG, Franzblau SG, Kozikowski AP. <span><span class="ref-journal">J Med Chem. </span>2010;<span class="ref-vol">53</span>:649–659.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/20000470" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 20000470</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>15.</dt><dd><div class="bk_ref" id="ml220.r15">Igal RA, Wang P, Coleman RA. Triacsin C blocks de novo synthesis of glycerolipids and cholesterol esters but not recycling of fatty acid into phospholipid: evidence for functionally separate pools of acyl-CoA. <span><span class="ref-journal">Biochem J. </span>1997;<span class="ref-vol">324</span>(Pt 2):529–34.</span> [<a href="/pmc/articles/PMC1218462/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC1218462</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/9182714" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 9182714</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>16.</dt><dd><div class="bk_ref" id="ml220.r16">Beller M, Sztalryd C, Southall N, Bell M, Jackle H, Auld DS, Oliver B. COPI complex is a regulator of lipid homeostasis. <span><span class="ref-journal">PLoS Biol. </span>2008;<span class="ref-vol">6</span>:2530–2549.</span> [<a href="/pmc/articles/PMC2586367/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2586367</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/19067489" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19067489</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>17.</dt><dd><div class="bk_ref" id="ml220.r17">Feng Y, Yu S, Lasell TK, Jadhav AP, Macia E, Chardin P, Melancon P, Roth M, Mitchison T, Kirchhausen T. Exo1: a new chemical inhibitor of the exocytic pathway. <span><span class="ref-journal">Proc Natl Acad Sci U S A. </span>2003;<span class="ref-vol">100</span>:6469–74.</span> [<a href="/pmc/articles/PMC164470/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC164470</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/12738886" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 12738886</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>18.</dt><dd><div class="bk_ref" id="ml220.r18">Wright EE, Simpson ER. Inhibition of the lipolytic action of beta-adrenergic agonists in human adipocytes by alpha-adrenergic agonists. <span><span class="ref-journal">J Lipid Res. </span>1981;<span class="ref-vol">22</span>:1265–70.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/6119348" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 6119348</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>19.</dt><dd><div class="bk_ref" id="ml220.r19">Chen JS, Chen YL, Greenberg AS, Chen YJ, Wang SM. Magnolol stimulates lipolysis in lipid-laden RAW 264.7 macrophages. <span><span class="ref-journal">J Cell Biochem. </span>2005;<span class="ref-vol">94</span>:1028–37.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/15597343" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 15597343</span></a>]</div></dd></dl></dl></div><div id="bk_toc_contnr"></div></div></div><div class="fm-sec"><h2 id="_NBK153217_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><sup>,*</sup> <span itemprop="author">Min Shen</span>,<sup>A</sup> <span itemprop="author">Yaqin Zhang</span>,<sup>A</sup> <span itemprop="author">Li Liu</span>,<sup>A</sup> <span itemprop="author">Douglas S. Auld</span>,<sup>A</sup> and <span itemprop="author">Mathias Beller</span><sup>B</sup>.</p><h4>Affiliations</h4><div class="affiliation"><sup>A</sup>
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NIH Chemical Genomics Center, NIH Center for Translational Therapeutics, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD</div><div class="affiliation"><sup>B</sup>
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Max-Planck-Institut für biophysikalische Chemie, Abteilung für Molekulare Entwicklungsbiologie, Göttingen, Germany</div><div class="affiliation">
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<sup>*</sup> To whom correspondence should be addressed: NIH Chemical Genomics Center, NIH Center for Translational Therapeutics, National Human Genome Research Institute, National Institutes of Health, 9800 Medical Center Drive, Building B, Bethesda, MD 20892-3370. Phone: 301-217-4681. Fax: 301-217-5736. Email:
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<a href="mailto:dev@null" data-email="vog.hin.liam@mrexob" class="oemail">vog.hin.liam@mrexob</a></div><h3>Publication History</h3><p class="small">Received: <span itemprop="datePublished">December 15, 2010</span>; Last Update: <span itemprop="dateModified">May 3, 2013</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, Zhang Y, et al. Modulators of Lipid Storage. 2010 Dec 15 [Updated 2013 May 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/ml221/?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/ml218/?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="figobml220fu1a"><div id="ml220.fu1a" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK153217/bin/ml220fu1a.jpg" alt="ML206." /></div><h3><span class="title">ML206</span></h3></div></article><article data-type="fig" id="figobml220fu1b"><div id="ml220.fu1b" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK153217/bin/ml220fu1b.jpg" alt="ML219." /></div><h3><span class="title">ML219</span></h3></div></article><article data-type="fig" id="figobml220fu1c"><div id="ml220.fu1c" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK153217/bin/ml220fu1c.jpg" alt="ML220." /></div><h3><span class="title">ML220</span></h3></div></article><article data-type="table-wrap" id="figobml220tu1"><div id="ml220.tu1" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK153217/table/ml220.tu1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml220.tu1_lrgtbl__"><table><thead><tr><th id="hd_h_ml220.tu1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">CID/ML#</th><th id="hd_h_ml220.tu1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Target Name</th><th id="hd_h_ml220.tu1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">IC50/EC50 (nM) [SID, AID]</th><th id="hd_h_ml220.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_ml220.tu1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">IC50/EC50 (μM) [SID, AID]</th><th id="hd_h_ml220.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Fold Selective</th><th id="hd_h_ml220.tu1_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Secondary Assay(s)<br />Name: IC50/EC50 (nM) [SID, AID]</th></tr></thead><tbody><tr><td headers="hd_h_ml220.tu1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">CID 42288156/<a href="/pcsubstance/?term=ML206[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML206</a></td><td headers="hd_h_ml220.tu1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Cellular phenotype for lipid droplet storage</td><td headers="hd_h_ml220.tu1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">8 nM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/99381576" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 99381576</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/488913" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 488913</a>]</td><td headers="hd_h_ml220.tu1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Cytotoxicity</td><td headers="hd_h_ml220.tu1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">8.9 μM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/99381576" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 99381576</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/488908" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 488908</a>]</td><td headers="hd_h_ml220.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">>1000-fold</td><td headers="hd_h_ml220.tu1_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">HeLa cell lipid droplet storage;<br />HepG2 cell lipid droplet storage;<br />AML12 cell lipid droplet storage;<br />SID: <a href="https://pubchem.ncbi.nlm.nih.gov/substance/99381576" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99381576</a><br />AID: <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/488900" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">488900</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/492960" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">492960</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504434" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">504434</a></td></tr><tr><td headers="hd_h_ml220.tu1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">CID 46947845/<a href="/pcsubstance/?term=ML219[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML219</a></td><td headers="hd_h_ml220.tu1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Cellular phenotype for lipid droplet storage</td><td headers="hd_h_ml220.tu1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">2 nM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/99460848" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 99460848</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504432" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504432</a>]</td><td headers="hd_h_ml220.tu1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Cytotoxicity</td><td headers="hd_h_ml220.tu1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">11.2 μM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/99460848" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 99460848</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504433" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504433</a>]</td><td headers="hd_h_ml220.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">>1000-fold</td><td headers="hd_h_ml220.tu1_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">HepG2 cell lipid droplet storage;<br />AML12 cell lipid droplet storage;<br />SID: <a href="https://pubchem.ncbi.nlm.nih.gov/substance/99460848" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99460848</a><br />AID: <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/492960" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">492960</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504434" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">504434</a></td></tr><tr><td headers="hd_h_ml220.tu1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">CID 46947860/<a href="/pcsubstance/?term=ML220[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML220</a></td><td headers="hd_h_ml220.tu1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Cellular phenotype for lipid droplet storage</td><td headers="hd_h_ml220.tu1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">705 nM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/99460839" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 99460839</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504432" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504432</a>]</td><td headers="hd_h_ml220.tu1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Cytotoxicity</td><td headers="hd_h_ml220.tu1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">inactive [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/99460839" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 99460839</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504433" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504433</a>]</td><td headers="hd_h_ml220.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">>1000-fold</td><td headers="hd_h_ml220.tu1_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">HepG2 cell lipid droplet storage;<br />AML12 cell lipid droplet storage;<br />SID: <a href="https://pubchem.ncbi.nlm.nih.gov/substance/99460839" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99460839</a><br />AID: <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/492960" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">492960</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504434" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">504434</a></td></tr></tbody></table></div></div></article><article data-type="table-wrap" id="figobml220t1"><div id="ml220.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/NBK153217/table/ml220.t1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml220.t1_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml220.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">AID</th><th id="hd_h_ml220.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Type</th><th id="hd_h_ml220.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Target</th><th id="hd_h_ml220.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Conc. Range</th><th id="hd_h_ml220.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_ml220.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/2685" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2685</a></td><td headers="hd_h_ml220.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Primary qHTS</td><td headers="hd_h_ml220.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Lipid Storage in S3 cell</td><td headers="hd_h_ml220.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">46.2 μM – 3 nM</td><td headers="hd_h_ml220.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">321194</td></tr><tr><td headers="hd_h_ml220.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/1569" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1569</a></td><td headers="hd_h_ml220.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Confirmatory</td><td headers="hd_h_ml220.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Lipid Storage in S3 cell</td><td headers="hd_h_ml220.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">46.2 μM – 0.3 nM</td><td headers="hd_h_ml220.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">749</td></tr><tr><td headers="hd_h_ml220.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/488913" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">488913</a></td><td headers="hd_h_ml220.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Confirmatory (for probe SAR)</td><td headers="hd_h_ml220.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Lipid Storage in S3 cell</td><td headers="hd_h_ml220.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">46.2 μM – 0.3 nM</td><td headers="hd_h_ml220.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">153</td></tr><tr><td headers="hd_h_ml220.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/504432" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">504432</a></td><td headers="hd_h_ml220.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Confirmatory (for probe SAR)</td><td headers="hd_h_ml220.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Lipid Storage in S3 cell</td><td headers="hd_h_ml220.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">46.2 μM – 0.3 nM</td><td headers="hd_h_ml220.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">37</td></tr><tr><td headers="hd_h_ml220.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/488908" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">488908</a></td><td headers="hd_h_ml220.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Anti-target (for probe SAR)</td><td headers="hd_h_ml220.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Cytotoxicity</td><td headers="hd_h_ml220.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">46.2 μM – 0.3 nM</td><td headers="hd_h_ml220.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">151</td></tr><tr><td headers="hd_h_ml220.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/504433" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">504433</a></td><td headers="hd_h_ml220.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Anti-target (for probe SAR)</td><td headers="hd_h_ml220.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Cytotoxicity</td><td headers="hd_h_ml220.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">46.2 μM – 0.3 nM</td><td headers="hd_h_ml220.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">37</td></tr><tr><td headers="hd_h_ml220.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/488900" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">488900</a></td><td headers="hd_h_ml220.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Secondary (for probe SAR)</td><td headers="hd_h_ml220.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Lipid Storage in Hela cell</td><td headers="hd_h_ml220.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">46.2 μM – 0.3 nM</td><td headers="hd_h_ml220.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">56</td></tr><tr><td headers="hd_h_ml220.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/492960" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">492960</a></td><td headers="hd_h_ml220.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Secondary (for probe SAR)</td><td headers="hd_h_ml220.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Lipid Storage in HepG2 cell</td><td headers="hd_h_ml220.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">46.2 μM – 0.3 nM</td><td headers="hd_h_ml220.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">112</td></tr><tr><td headers="hd_h_ml220.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/504434" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">504434</a></td><td headers="hd_h_ml220.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Secondary (for probe SAR)</td><td headers="hd_h_ml220.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Lipid Storage in AML12 cell</td><td headers="hd_h_ml220.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">46.2 μM – 0.3 nM</td><td headers="hd_h_ml220.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">132</td></tr><tr><td headers="hd_h_ml220.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/1623" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1623</a></td><td headers="hd_h_ml220.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Summary</td><td headers="hd_h_ml220.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Lipid Storage</td><td headers="hd_h_ml220.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">NA</td><td headers="hd_h_ml220.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="table-wrap" id="figobml220t2"><div id="ml220.t2" class="table"><h3><span class="label">Table 2</span><span class="title">Stepwise protocol used for the S3 cell 1536-well assay</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK153217/table/ml220.t2/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml220.t2_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml220.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Step</th><th id="hd_h_ml220.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Value</th><th id="hd_h_ml220.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Description</th></tr></thead><tbody><tr><td headers="hd_h_ml220.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><b>1</b></td><td headers="hd_h_ml220.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">4μL</td><td headers="hd_h_ml220.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">S3 cells (2,000/well) 5% FBS Schneider’s medium</td></tr><tr><td headers="hd_h_ml220.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><b>2</b></td><td headers="hd_h_ml220.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Time</td><td headers="hd_h_ml220.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">24°C incubation O/N</td></tr><tr><td headers="hd_h_ml220.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><b>3</b></td><td headers="hd_h_ml220.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">23 nL</td><td headers="hd_h_ml220.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Controls: SB 408124-lipid over storage; Triacsin C – lipid under storage 20 μM each</td></tr><tr><td headers="hd_h_ml220.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><b>4</b></td><td headers="hd_h_ml220.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">23 nL</td><td headers="hd_h_ml220.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">46 μM to 3 nM compound</td></tr><tr><td headers="hd_h_ml220.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><b>5</b></td><td headers="hd_h_ml220.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">1 μL</td><td headers="hd_h_ml220.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Oleic acid 400 μM, Schneider’s medium</td></tr><tr><td headers="hd_h_ml220.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><b>6</b></td><td headers="hd_h_ml220.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Time</td><td headers="hd_h_ml220.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">24°C incubation O/N</td></tr><tr><td headers="hd_h_ml220.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><b>7</b></td><td headers="hd_h_ml220.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">4 μL</td><td headers="hd_h_ml220.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Dyes (Bodipy/Cell Tracker Red)</td></tr><tr><td headers="hd_h_ml220.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><b>8</b></td><td headers="hd_h_ml220.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Time</td><td headers="hd_h_ml220.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">2 hrs at r.t.</td></tr><tr><td headers="hd_h_ml220.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><b>9</b></td><td headers="hd_h_ml220.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Detect</td><td headers="hd_h_ml220.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Acumen (488 nm ex; 500–530 em; 575–640 em)</td></tr></tbody></table></div></div></article><article data-type="fig" id="figobml220f1"><div id="ml220.f1" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%201.%20Primary%20lipid%20droplet%20storage%20assay.&p=BOOKS&id=153217_ml220f1.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/NBK153217/bin/ml220f1.jpg" alt="Figure 1. Primary lipid droplet storage assay." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 1</span><span class="title">Primary lipid droplet storage assay</span></h3><div class="caption"><p>The primary assay used the Acumen Explorer and was a two-color assay using the lipid-specific dye (BODIPY493/503; PMT) and a cell mask stain (CellTracker Red; PMT: 575–640 nm) to determine lipid content and cell numbers, respectively. Shown are the fluorescent peak intensity histograms and .tif images from the Acumen (top left) for S3 cells fed or not fed with oleic acid (OA). Top right shows the ratio (lipid/cell) for OA treatment; 400 μM OA was used in the assay. Lower left shows the example replicate CRCs for the acyl CoA inhibitor (Triacsin C). All data is from the 1536-well assay protocol.</p></div></div></article><article data-type="table-wrap" id="figobml220t3"><div id="ml220.t3" class="table"><h3><span class="label">Table 3</span><span class="title">Stepwise Protocol Used for the Hela, HepG2 or AML12 cell 1536-Well Assay</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK153217/table/ml220.t3/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml220.t3_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml220.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Step</th><th id="hd_h_ml220.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Value</th><th id="hd_h_ml220.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Description</th></tr></thead><tbody><tr><td headers="hd_h_ml220.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><b>1</b></td><td headers="hd_h_ml220.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">4 μL</td><td headers="hd_h_ml220.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">HeLa, HepG2 or AML12 cells (700/well) 2% FBS OPTI-MEM-phenol red free</td></tr><tr><td headers="hd_h_ml220.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><b>2</b></td><td headers="hd_h_ml220.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Time</td><td headers="hd_h_ml220.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">37°C incubation O/N</td></tr><tr><td headers="hd_h_ml220.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><b>3</b></td><td headers="hd_h_ml220.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">23 nL</td><td headers="hd_h_ml220.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Controls: SB 408124-lipid over storage; Triacsin C – lipid under storage; 20 μM each</td></tr><tr><td headers="hd_h_ml220.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><b>4</b></td><td headers="hd_h_ml220.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">23 nL</td><td headers="hd_h_ml220.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">46 μM to 3 nM compound</td></tr><tr><td headers="hd_h_ml220.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><b>5</b></td><td headers="hd_h_ml220.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">1 μL</td><td headers="hd_h_ml220.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Oleic acid 400 μM in medium</td></tr><tr><td headers="hd_h_ml220.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><b>6</b></td><td headers="hd_h_ml220.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Time</td><td headers="hd_h_ml220.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">37 °C incubation O/N</td></tr><tr><td headers="hd_h_ml220.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><b>7</b></td><td headers="hd_h_ml220.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">4 μL</td><td headers="hd_h_ml220.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Dyes (Bodipy/Cell Tracker Red)</td></tr><tr><td headers="hd_h_ml220.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><b>8</b></td><td headers="hd_h_ml220.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Time</td><td headers="hd_h_ml220.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">37 °C incubation 2hrs</td></tr><tr><td headers="hd_h_ml220.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><b>9</b></td><td headers="hd_h_ml220.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Time</td><td headers="hd_h_ml220.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">30 min at r.t.</td></tr><tr><td headers="hd_h_ml220.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><b>10</b></td><td headers="hd_h_ml220.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Detect</td><td headers="hd_h_ml220.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Acumen (488 nm ex; 500–530 em; 575–640 em)</td></tr></tbody></table></div></div></article><article data-type="fig" id="figobml220f2"><div id="ml220.f2" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%202.%20Stability%20of%20ML206%20in%20PBS%20buffer%20(pH%207.4%2C%2023%B0C)%20plotted%20as%20AUC%20vs.%20time%20for%20a%2048%20hr%20period%20using%20LC%2FMS%20Method%201%20described%20in%20Section%202.&p=BOOKS&id=153217_ml220f2.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/NBK153217/bin/ml220f2.jpg" alt="Figure 2. Stability of ML206 in PBS buffer (pH 7.4, 23°C) plotted as AUC vs. time for a 48 hr period using LC/MS Method 1 described in Section 2." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 2</span><span class="title">Stability of ML206 in PBS buffer (pH 7.4, 23°C) plotted as AUC vs. time for a 48 hr period using LC/MS Method 1 described in <a href="#ml220.s3">Section 2</a></span></h3><div class="caption"><p>No instability was observed, and >90% of compound remained at 48 hours.</p></div></div></article><article data-type="table-wrap" id="figobml220t4"><div id="ml220.t4" class="table"><h3><span class="label">Table 4</span><span class="title">Probe properties</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK153217/table/ml220.t4/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml220.t4_lrgtbl__"><table class="no_top_margin"><tbody><tr><th id="hd_b_ml220.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;"></th><th id="hd_b_ml220.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;"><a href="/pcsubstance/?term=ML206[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML206</a></th><th id="hd_b_ml220.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;"><a href="/pcsubstance/?term=ML219[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML219</a></th><th id="hd_b_ml220.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;"><a href="/pcsubstance/?term=ML220[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML220</a></th></tr><tr><th id="hd_b_ml220.t4_1_1_2_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Molecular Weight [g/mol]</th><td headers="hd_b_ml220.t4_1_1_2_1 hd_b_ml220.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">354.353346</td><td headers="hd_b_ml220.t4_1_1_2_1 hd_b_ml220.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">462.6037</td><td headers="hd_b_ml220.t4_1_1_2_1 hd_b_ml220.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">395.88044</td></tr><tr><th id="hd_b_ml220.t4_1_1_3_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Molecular Formula</th><td headers="hd_b_ml220.t4_1_1_3_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">C19H16F2N4O</td><td headers="hd_b_ml220.t4_1_1_3_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">C27H30N2O3S</td><td headers="hd_b_ml220.t4_1_1_3_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">C19H26ClN3O4</td></tr><tr><th id="hd_b_ml220.t4_1_1_4_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">XLogP3-AA</th><td headers="hd_b_ml220.t4_1_1_4_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">2.9</td><td headers="hd_b_ml220.t4_1_1_4_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">5.83</td><td headers="hd_b_ml220.t4_1_1_4_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">3.04</td></tr><tr><th id="hd_b_ml220.t4_1_1_5_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">H-Bond Donor</th><td headers="hd_b_ml220.t4_1_1_5_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">1</td><td headers="hd_b_ml220.t4_1_1_5_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">1</td><td headers="hd_b_ml220.t4_1_1_5_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">2</td></tr><tr><th id="hd_b_ml220.t4_1_1_6_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">H-Bond Acceptor</th><td headers="hd_b_ml220.t4_1_1_6_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">5</td><td headers="hd_b_ml220.t4_1_1_6_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">3</td><td headers="hd_b_ml220.t4_1_1_6_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">4</td></tr><tr><th id="hd_b_ml220.t4_1_1_7_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Rotatable Bond Count</th><td headers="hd_b_ml220.t4_1_1_7_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">3</td><td headers="hd_b_ml220.t4_1_1_7_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">10</td><td headers="hd_b_ml220.t4_1_1_7_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">6</td></tr><tr><th id="hd_b_ml220.t4_1_1_8_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Tautomer Count</th><td headers="hd_b_ml220.t4_1_1_8_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">2</td><td headers="hd_b_ml220.t4_1_1_8_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">2</td><td headers="hd_b_ml220.t4_1_1_8_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">3</td></tr><tr><th id="hd_b_ml220.t4_1_1_9_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Exact Mass</th><td headers="hd_b_ml220.t4_1_1_9_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">354.129218</td><td headers="hd_b_ml220.t4_1_1_9_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">462.197714</td><td headers="hd_b_ml220.t4_1_1_9_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">395.161184</td></tr><tr><th id="hd_b_ml220.t4_1_1_10_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">MonoIsotopic Mass</th><td headers="hd_b_ml220.t4_1_1_10_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">354.129218</td><td headers="hd_b_ml220.t4_1_1_10_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">462.197714</td><td headers="hd_b_ml220.t4_1_1_10_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">395.161184</td></tr><tr><th id="hd_b_ml220.t4_1_1_11_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Topological Polar Surface Area</th><td headers="hd_b_ml220.t4_1_1_11_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">59.8</td><td headers="hd_b_ml220.t4_1_1_11_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">80.7</td><td headers="hd_b_ml220.t4_1_1_11_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">87.7</td></tr><tr><th id="hd_b_ml220.t4_1_1_12_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Heavy Atom Count</th><td headers="hd_b_ml220.t4_1_1_12_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">26</td><td headers="hd_b_ml220.t4_1_1_12_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">33</td><td headers="hd_b_ml220.t4_1_1_12_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">27</td></tr><tr><th id="hd_b_ml220.t4_1_1_13_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Formal Charge</th><td headers="hd_b_ml220.t4_1_1_13_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0</td><td headers="hd_b_ml220.t4_1_1_13_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0</td><td headers="hd_b_ml220.t4_1_1_13_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0</td></tr><tr><th id="hd_b_ml220.t4_1_1_14_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Complexity</th><td headers="hd_b_ml220.t4_1_1_14_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">508</td><td headers="hd_b_ml220.t4_1_1_14_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">620</td><td headers="hd_b_ml220.t4_1_1_14_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">547</td></tr><tr><th id="hd_b_ml220.t4_1_1_15_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Isotope Atom Count</th><td headers="hd_b_ml220.t4_1_1_15_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0</td><td headers="hd_b_ml220.t4_1_1_15_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0</td><td headers="hd_b_ml220.t4_1_1_15_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0</td></tr><tr><th id="hd_b_ml220.t4_1_1_16_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Defined Atom StereoCenter Count</th><td headers="hd_b_ml220.t4_1_1_16_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">1</td><td headers="hd_b_ml220.t4_1_1_16_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0</td><td headers="hd_b_ml220.t4_1_1_16_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0</td></tr><tr><th id="hd_b_ml220.t4_1_1_17_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Undefined Atom StereoCenter Count</th><td headers="hd_b_ml220.t4_1_1_17_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0</td><td headers="hd_b_ml220.t4_1_1_17_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0</td><td headers="hd_b_ml220.t4_1_1_17_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0</td></tr><tr><th id="hd_b_ml220.t4_1_1_18_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Defined Bond StereoCenter Count</th><td headers="hd_b_ml220.t4_1_1_18_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0</td><td headers="hd_b_ml220.t4_1_1_18_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0</td><td headers="hd_b_ml220.t4_1_1_18_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0</td></tr><tr><th id="hd_b_ml220.t4_1_1_19_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Undefined Bond StereoCenter Count</th><td headers="hd_b_ml220.t4_1_1_19_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0</td><td headers="hd_b_ml220.t4_1_1_19_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0</td><td headers="hd_b_ml220.t4_1_1_19_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0</td></tr><tr><th id="hd_b_ml220.t4_1_1_20_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Covalently-Bonded Unit Count</th><td headers="hd_b_ml220.t4_1_1_20_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">1</td><td headers="hd_b_ml220.t4_1_1_20_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">1</td><td headers="hd_b_ml220.t4_1_1_20_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">1</td></tr></tbody></table></div></div></article><article data-type="table-wrap" id="figobml220t5"><div id="ml220.t5" class="table"><h3><span class="label">Table 5</span><span class="title">Probe and analogs submitted to the MLSMR</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK153217/table/ml220.t5/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml220.t5_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml220.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">NCGC ID</th><th id="hd_h_ml220.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Probe/Analog</th><th id="hd_h_ml220.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">MLS ID</th><th id="hd_h_ml220.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">SID</th></tr></thead><tbody><tr><td headers="hd_h_ml220.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00189555-02</td><td headers="hd_h_ml220.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Probe (<a href="/pcsubstance/?term=ML206[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML206</a>)</td><td headers="hd_h_ml220.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS003221389</td><td headers="hd_h_ml220.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99381576" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99381576</a></td></tr><tr><td headers="hd_h_ml220.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00238536-01</td><td headers="hd_h_ml220.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Analog</td><td headers="hd_h_ml220.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS003221390</td><td headers="hd_h_ml220.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99381578" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99381578</a></td></tr><tr><td headers="hd_h_ml220.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00189556-01</td><td headers="hd_h_ml220.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Analog</td><td headers="hd_h_ml220.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS003221391</td><td headers="hd_h_ml220.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99381577" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99381577</a></td></tr><tr><td headers="hd_h_ml220.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00238548-02</td><td headers="hd_h_ml220.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Analog</td><td headers="hd_h_ml220.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS003221392</td><td headers="hd_h_ml220.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/110167680" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">110167680</a></td></tr><tr><td headers="hd_h_ml220.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00238543-02</td><td headers="hd_h_ml220.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Analog</td><td headers="hd_h_ml220.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS003221393</td><td headers="hd_h_ml220.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/143472015" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">143472015</a></td></tr><tr><td headers="hd_h_ml220.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00238537-02</td><td headers="hd_h_ml220.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Analog</td><td headers="hd_h_ml220.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS003221394</td><td headers="hd_h_ml220.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/110167679" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">110167679</a></td></tr></tbody></table></div></div></article><article data-type="fig" id="figobml220f3"><div id="ml220.f3" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%203.%20Stability%20of%20ML219%20in%20PBS%20buffer%20(pH%207.4%2C%2023%B0C)%20plotted%20as%20AUC%20vs.%20time%20for%20a%2048%20hr%20period%2C%20using%20LC%2FMS%20Method%201%20described%20in%20Section%202.&p=BOOKS&id=153217_ml220f3.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/NBK153217/bin/ml220f3.jpg" alt="Figure 3. Stability of ML219 in PBS buffer (pH 7.4, 23°C) plotted as AUC vs. time for a 48 hr period, using LC/MS Method 1 described in Section 2." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 3</span><span class="title">Stability of ML219 in PBS buffer (pH 7.4, 23°C) plotted as AUC vs. time for a 48 hr period, using LC/MS Method 1 described in <a href="#ml220.s3">Section 2</a></span></h3><div class="caption"><p>No instability was observed, and >90% of compound remained at 48 hours.</p></div></div></article><article data-type="table-wrap" id="figobml220t6"><div id="ml220.t6" class="table"><h3><span class="label">Table 6</span><span class="title">Probe and analogs submitted to the MLSMR</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK153217/table/ml220.t6/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml220.t6_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml220.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">NCGC ID</th><th id="hd_h_ml220.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Probe/Analog</th><th id="hd_h_ml220.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">MLS ID</th><th id="hd_h_ml220.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">SID</th></tr></thead><tbody><tr><td headers="hd_h_ml220.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00241426-02</td><td headers="hd_h_ml220.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Probe (<a href="/pcsubstance/?term=ML219[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML219</a>)</td><td headers="hd_h_ml220.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS003221395</td><td headers="hd_h_ml220.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99460848" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99460848</a></td></tr><tr><td headers="hd_h_ml220.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00238551-02</td><td headers="hd_h_ml220.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Analog</td><td headers="hd_h_ml220.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS003224199</td><td headers="hd_h_ml220.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/143471935" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">143471935</a></td></tr><tr><td headers="hd_h_ml220.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00241426-02</td><td headers="hd_h_ml220.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Analog</td><td headers="hd_h_ml220.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS003221395</td><td headers="hd_h_ml220.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/160779721" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">160779721</a></td></tr><tr><td headers="hd_h_ml220.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00241438-01</td><td headers="hd_h_ml220.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Analog</td><td headers="hd_h_ml220.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS003221396</td><td headers="hd_h_ml220.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99460862" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99460862</a></td></tr><tr><td headers="hd_h_ml220.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00241435-01</td><td headers="hd_h_ml220.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Analog</td><td headers="hd_h_ml220.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS003221397</td><td headers="hd_h_ml220.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99460860" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99460860</a></td></tr><tr><td headers="hd_h_ml220.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00241443-01</td><td headers="hd_h_ml220.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Analog</td><td headers="hd_h_ml220.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS003221398</td><td headers="hd_h_ml220.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99460869" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99460869</a></td></tr><tr><td headers="hd_h_ml220.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00241420-01</td><td headers="hd_h_ml220.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Analog</td><td headers="hd_h_ml220.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS003221399</td><td headers="hd_h_ml220.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99460871" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99460871</a></td></tr><tr><td headers="hd_h_ml220.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00241419-01</td><td headers="hd_h_ml220.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Analog</td><td headers="hd_h_ml220.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS003221400</td><td headers="hd_h_ml220.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99460865" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99460865</a></td></tr></tbody></table></div></div></article><article data-type="fig" id="figobml220f4"><div id="ml220.f4" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%204.%20Stability%20of%20ML220%20in%20PBS%20buffer%20(pH%207.4%2C%2023%B0C)%20plotted%20as%20AUC%20vs.%20time%20for%20a%2048%20hr%20period%2C%20using%20LC%2FMS%20Method%201%20described%20in%20Section%202.&p=BOOKS&id=153217_ml220f4.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/NBK153217/bin/ml220f4.jpg" alt="Figure 4. Stability of ML220 in PBS buffer (pH 7.4, 23°C) plotted as AUC vs. time for a 48 hr period, using LC/MS Method 1 described in Section 2." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 4</span><span class="title">Stability of ML220 in PBS buffer (pH 7.4, 23°C) plotted as AUC vs. time for a 48 hr period, using LC/MS Method 1 described in <a href="#ml220.s3">Section 2</a></span></h3><div class="caption"><p>No instability was observed, and >90% of compound remained at 48 hours.</p></div></div></article><article data-type="table-wrap" id="figobml220t7"><div id="ml220.t7" class="table"><h3><span class="label">Table 7</span><span class="title">Probe and analogs submitted to the MLSMR</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK153217/table/ml220.t7/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml220.t7_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml220.t7_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">NCGC ID</th><th id="hd_h_ml220.t7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Probe/Analog</th><th id="hd_h_ml220.t7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">MLS ID</th><th id="hd_h_ml220.t7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">SID</th></tr></thead><tbody><tr><td headers="hd_h_ml220.t7_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">NCGC00241450-01</td><td headers="hd_h_ml220.t7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Probe (<a href="/pcsubstance/?term=ML220[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML220</a>)</td><td headers="hd_h_ml220.t7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS003390985</td><td headers="hd_h_ml220.t7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99460839" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99460839</a></td></tr><tr><td headers="hd_h_ml220.t7_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">NCGC00242556-01</td><td headers="hd_h_ml220.t7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Analog</td><td headers="hd_h_ml220.t7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS003370615</td><td headers="hd_h_ml220.t7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/110167689" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">110167689</a></td></tr><tr><td headers="hd_h_ml220.t7_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">NCGC00242559-01</td><td headers="hd_h_ml220.t7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Analog</td><td headers="hd_h_ml220.t7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS003370616</td><td headers="hd_h_ml220.t7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/110167692" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">110167692</a></td></tr><tr><td headers="hd_h_ml220.t7_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">NCGC00242560-01</td><td headers="hd_h_ml220.t7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Analog</td><td headers="hd_h_ml220.t7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS003370617</td><td headers="hd_h_ml220.t7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/110167693" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">110167693</a></td></tr><tr><td headers="hd_h_ml220.t7_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">NCGC00242553-01</td><td headers="hd_h_ml220.t7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Analog</td><td headers="hd_h_ml220.t7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS003370618</td><td headers="hd_h_ml220.t7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/110167686" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">110167686</a></td></tr><tr><td headers="hd_h_ml220.t7_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">NCGC00242554-01</td><td headers="hd_h_ml220.t7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Analog</td><td headers="hd_h_ml220.t7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS003370619</td><td headers="hd_h_ml220.t7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/110167687" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">110167687</a></td></tr><tr><td headers="hd_h_ml220.t7_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">NCGC00242557-01</td><td headers="hd_h_ml220.t7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Analog</td><td headers="hd_h_ml220.t7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS003370620</td><td headers="hd_h_ml220.t7_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/110167690" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">110167690</a></td></tr></tbody></table></div></div></article><article data-type="fig" id="figobml220f8"><div id="ml220.f8" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK153217/bin/ml220f8.jpg" alt="Scheme 1. Synthesis of enantiomerically pure ML206." /></div><h3><span class="label">Scheme 1</span><span class="title">Synthesis of enantiomerically pure ML206</span></h3></div></article><article data-type="fig" id="figobml220f10"><div id="ml220.f10" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK153217/bin/ml220f10.jpg" alt="Scheme 3. Synthesis of ML220." /></div><h3><span class="label">Scheme 3</span><span class="title">Synthesis of ML220</span></h3></div></article><article data-type="fig" id="figobml220f9"><div id="ml220.f9" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK153217/bin/ml220f9.jpg" alt="Scheme 2. Synthesis of ML219." /></div><h3><span class="label">Scheme 2</span><span class="title">Synthesis of ML219</span></h3></div></article><article data-type="fig" id="figobml220f5"><div id="ml220.f5" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%205.%20Potency%20confirmation.&p=BOOKS&id=153217_ml220f5.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/NBK153217/bin/ml220f5.jpg" alt="Figure 5. Potency confirmation." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 5</span><span class="title">Potency confirmation</span></h3><div class="caption"><p>Confirmatory data for the probe compounds in S3, Hela, HepG2 and AML12 cells, along with
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cytotoxicity determined in S3 cells. The lipid reduction % Activity plotted is from the
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lipid data. <b>A.</b> The racemic compound CID16192366. <b>B.</b> The enantiomerically
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pure compound where the absolute configuration was confirmed as (<i>R)</i> for <a href="/pcsubstance/?term=ML206[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML206</a>. <b>C.</b> The enantiomerically pure compound where the absolute configuration was confirmed as (<i>S</i>) for CID42288155. <b>D</b>. The probe compound <a href="/pcsubstance/?term=ML219[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML219</a> which also showed weak activity in AML12 cells. E. The probe compound <a href="/pcsubstance/?term=ML220[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML220</a> which also showed weak activity in AML12 cells.</p></div></div></article><article data-type="fig" id="figobml220f6"><div id="ml220.f6" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%206.%20BODIPY%20and%20Hoechst%20staining%20of%20S3%20cells%20treated%20with%20compounds.&p=BOOKS&id=153217_ml220f6.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/NBK153217/bin/ml220f6.jpg" alt="Figure 6. BODIPY and Hoechst staining of S3 cells treated with compounds." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 6</span><span class="title">BODIPY and Hoechst staining of S3 cells treated with compounds</span></h3><div class="caption"><p>Data was collected at the Max Planck Institute for Biophysical Chemistry, Department of Molecular Developmental Biology.</p></div></div></article><article data-type="fig" id="figobml220f7"><div id="ml220.f7" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK153217/bin/ml220f7.jpg" alt="Figure 7. Previous ML probes that modulate lipid storage in S3 cells." /></div><h3><span class="label">Figure 7</span><span class="title">Previous ML probes that modulate lipid storage in S3 cells</span></h3></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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