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<div class="pre-content"><div><div class="bk_prnt"><p class="small">NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health.</p><p>Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-. </p></div><div class="iconblock clearfix whole_rhythm no_top_margin bk_noprnt"><a class="img_link icnblk_img" title="Table of Contents Page" href="/books/n/mlprobe/"><img class="source-thumb" src="/corehtml/pmc/pmcgifs/bookshelf/thumbs/th-mlprobe-lrg.png" alt="Cover of Probe Reports from the NIH Molecular Libraries Program" height="100px" width="80px" /></a><div class="icnblk_cntnt eight_col"><h2>Probe Reports from the NIH Molecular Libraries Program [Internet].</h2><a data-jig="ncbitoggler" href="#__NBK47336_dtls__">Show details</a><div style="display:none" class="ui-widget" id="__NBK47336_dtls__"><div>Bethesda (MD): National Center for Biotechnology Information (US); 2010-.</div></div><div class="half_rhythm"><ul class="inline_list"><li style="margin-right:1em"><a class="bk_cntns" href="/books/n/mlprobe/">Contents</a></li></ul></div><div class="bk_noprnt"><form method="get" action="/books/n/mlprobe/" id="bk_srch"><div class="bk_search"><label for="bk_term" class="offscreen_noflow">Search term</label><input type="text" title="Search this book" id="bk_term" name="term" value="" data-jig="ncbiclearbutton" /> <input type="submit" class="jig-ncbibutton" value="Search this book" submit="false" style="padding: 0.1em 0.4em;" /></div></form></div></div><div class="icnblk_cntnt two_col"><div class="pagination bk_noprnt"><a class="active page_link prev" href="/books/n/mlprobe/ml085/" title="Previous page in this title">&lt; Prev</a><a class="active page_link next" href="/books/n/mlprobe/ml083/" title="Next page in this title">Next &gt;</a></div></div></div></div></div>
<div class="main-content lit-style" itemscope="itemscope" itemtype="http://schema.org/CreativeWork"><div class="meta-content fm-sec"><h1 id="_NBK47336_"><span class="title" itemprop="name">Identification of lipid storage modulators</span></h1><p class="contrib-group"><span itemprop="author">Mathias Beller</span>, <span itemprop="author">Craig Thomas</span>, <span itemprop="author">Min Shen</span>, and <span itemprop="author">Douglas Auld</span>.</p><p class="small">Received: <span itemprop="datePublished">April 16, 2009</span>; Last Update: <span itemprop="dateModified">September 2, 2010</span>.</p></div><div class="jig-ncbiinpagenav body-content whole_rhythm" data-jigconfig="allHeadingLevels: ['h2'],smoothScroll: false" itemprop="text"><div id="_abs_rndgid_" itemprop="description"><p>Storing lipids as a reservoir for energy or the anabolism of elementary metabolites is a common feature of life in organisms from bacteria to humans. The chemical probes yielded by this project should serve as useful tools for providing a better understanding of cellular and organismic lipid storage on a functional and evolutionary level. Furthermore, active substances might result in the identification of lead compounds for the treatment of emerging lipid storage-associated diseases, including atherosclerosis, diabetes, or obesity. Also, the present assay will establish a profile of compounds that modulate this ubiquitous area of biology. The probe described here came from initial screening of known bioactive compound collections within the MLSMR. By combining the small molecule screening results with lipid metabolism modulating gene functions identified in the genome-wide RNAi screen, we were able to identify and link the Golgi Type I coat proteins known as cotamer, COPI to lipid storage. The developed probe, ML084 (CID-310557; Exo1), was shown to induce a lipid storage phenotype in cells through modulation of the protein trafficking pathways.</p></div><div class="h2"></div><p>
<b>Assigned Assay Grant #:</b> 1 R03 MH085686-01 </p><p>
<b>Screening Center Name &#x00026; PI:</b> NIH Chemical Genomics Center,
Christopher Austin </p><p>
<b>Chemistry Center Name &#x00026; PI:</b> NIH Chemical Genomics Center,
Christopher Austin </p><p>
<b>Assay Submitter &#x00026; Institution:</b> Dr. Mathias Beller, Max Planck
Institute for Biophysical Chemistry </p><p><b>PubChem Primary Bioassay Identifier (AID):</b>
<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1519" ref="pagearea=body&amp;targetsite=entrez&amp;targetcat=link&amp;targettype=pubchem">AID-1519</a></p><div id="ml084.s1"><h2 id="_ml084_s1_">Probe Structure &#x00026; Characteristics</h2><div id="ml084.tu1" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK47336/table/ml084.tu1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml084.tu1_lrgtbl__"><table><tbody><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">PubChem CID</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CID-310557</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Molecular Weight</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">273.76</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Molecular Formula</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">C<sub>15</sub>H<sub>12</sub>FNO<sub>3</sub></td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">XLogP</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">3.4</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">H-Bond Donor</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">1</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">H-Bond Acceptor</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">4</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Rotatable Bond Count</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">4</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Exact Mass</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">273.08</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Topological Polar Surface Area</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">55.4</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Heavy Atom Count</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">20</td></tr></tbody></table></div></div><div id="ml084.fu1" class="figure"><div class="graphic"><img src="/books/NBK47336/bin/ml084fu1.jpg" alt="Image ml084fu1" /></div></div><div id="ml084.tu2" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK47336/table/ml084.tu2/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml084.tu2_lrgtbl__"><table><thead><tr><th id="hd_h_ml084.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">CID/ML</th><th id="hd_h_ml084.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Target Name</th><th id="hd_h_ml084.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">IC<sub>50</sub>/EC<sub>50</sub>
(&#x003bc;M) [SID, AID]</th><th id="hd_h_ml084.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Anti-target Name(s)</th><th id="hd_h_ml084.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">IC<sub>50</sub>/EC<sub>50</sub>
(&#x003bc;M) [SID, AID]</th><th id="hd_h_ml084.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Selectivity</th><th id="hd_h_ml084.tu2_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Selectivity Assay(s) Name:
IC<sub>50</sub>/EC<sub>50</sub> (nM) [SID,
AID]<sup>&#x000a7;</sup></th></tr></thead><tbody><tr><td headers="hd_h_ml084.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">CID-310557/<a href="/pcsubstance/?term=ML084[synonym]" ref="pagearea=body&amp;targetsite=entrez&amp;targetcat=term&amp;targettype=pubchem">ML084</a></td><td headers="hd_h_ml084.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">COPI</td><td headers="hd_h_ml084.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">5 &#x000b1; 2.5<br />
[<a href="https://pubchem.ncbi.nlm.nih.gov/substance/11114231" ref="pagearea=body&amp;targetsite=entrez&amp;targetcat=link&amp;targettype=pubchem">SID-11114231</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1519" ref="pagearea=body&amp;targetsite=entrez&amp;targetcat=link&amp;targettype=pubchem">AID-1519</a>]</td><td headers="hd_h_ml084.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Cytotoxicity</td><td headers="hd_h_ml084.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Inactive @40 &#x003bc;M,
[<a href="https://pubchem.ncbi.nlm.nih.gov/substance/11114231" ref="pagearea=body&amp;targetsite=entrez&amp;targetcat=link&amp;targettype=pubchem">SID-11114231</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1561" ref="pagearea=body&amp;targetsite=entrez&amp;targetcat=link&amp;targettype=pubchem">AID-1561</a>]</td><td headers="hd_h_ml084.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">&#x0003e;10-fold</td><td headers="hd_h_ml084.tu2_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Cell-Titer Glo [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/11114231" ref="pagearea=body&amp;targetsite=entrez&amp;targetcat=link&amp;targettype=pubchem">SID-11114231</a>,
<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1561" ref="pagearea=body&amp;targetsite=entrez&amp;targetcat=link&amp;targettype=pubchem">AID-1561</a>]</td></tr></tbody></table></div></div></div><div id="ml084.s3"><h2 id="_ml084_s3_">Recommendations for the scientific use of this probe</h2><p>The probe Exo-1 is a modulator of protein trafficking. Our work has linked the Golgi
Type I coat proteins known as cotamer, COPI to lipid storage. Exo-1 can be used to
induce a lipid storage phenotype in cells through modulation of the protein
trafficking pathways.</p><div id="ml084.s4"><h3>Specific Aim</h3><p>We have developed a cell-based assay in <i>Drosophila melanogaster</i>
embryonic Kc167 and S3 cells capable of analyzing the effects of alterations in
lipid metabolism. We have recently used a similar assay successfully in a
genome-wide RNAi screen to identify gene functions regulating cellular lipid
storage. This assay was adapted to laser-scanning microplate cytometry suitable
for a 1,536-well microtiter plate format, which will allow the assay to be
screened against the 300K MLSMR collection. The goal is to identify chemical
probes affecting lipid storage regulation.</p></div><div id="ml084.s5"><h3>Significance</h3><p>The chemical probes yielded by this project should be useful tools for providing
a better understanding of cellular and organismic lipid storage on a functional
and evolutionary level. Furthermore, active substances might result in the
identification of lead compounds for the treatment of emerging lipid
storage-associated diseases, including atherosclerosis, diabetes or obesity.
Also, the present assay will establish a profile of compounds within the MLSMR
that modulate this ubiquitous area of biology. The probe described here came
from initial screening of known bioactive compound collections. By combining the
small molecule screening results with lipid metabolism modulating gene functions
identified in the genome-wide RNAi screen, we were able to identify COPI
proteins as negative regulators of lipid storage and the probe Exo1 as chemical
probe to modulate lipid storage.</p></div><div id="ml084.s6"><h3>Rationale</h3><p>Storing lipids as a reservoir for energy or the anabolism of elementary
metabolites is a common feature of life in organisms from bacteria to humans.
The universal cellular lipid storage organelle is the so-called lipid storage
droplet (LD). Despite their ubiquitous nature, LDs share a simple, stereotyped
structure of a hydrophobic core harboring the storage lipids, which is shielded
by a droplet-specific phospholipid monolayer to which proteins are attached. The
current model of LD biogenesis involves an incorporation of the lipid core into
the membrane leaflets of the endoplasmic reticulum (ER), followed by a
subsequent budding-like maturation of a LD, which ultimately pinches off. Once
released, LD volume can increase by localized lipogenesis or fusion of existing
droplets. Storage lipids are re-mobilized enzymatically by lipase activity.
Lipase regulation in the adipocyte is heavily studied and involves multiple
components including catecholamine signaling (<a class="bk_pop" href="#ml084.r1">1</a>), the LD-associated proteins Perilipin and
comparative gene identification 58 (CGI-58) (<a class="bk_pop" href="#ml084.r2">2</a>), and at least two lipases named hormone sensitive
lipase (HSL) (<a class="bk_pop" href="#ml084.r3">3</a>) and adipocyte
triglyceride lipase (ATGL) (<a class="bk_pop" href="#ml084.r4" data-bk-pop-others="ml084.r5 ml084.r6">4&#x02013;6</a>). LD biogenesis, turnover and
mobilization are poorly understood and only few components are known. However,
there is an urgent need to learn more about ectopic fat depots as mislocalized
storage of lipids, for example in the liver or muscle, is an eminent health
problem associated with insulin resistance or the metabolic syndrome (<a class="bk_pop" href="#ml084.r7">7</a>).</p></div></div><div id="ml084.s7"><h2 id="_ml084_s7_">Assay Implementation and Screening</h2><div id="ml084.s8"><h3>PubChem Bioassay Name</h3><p><i>Dmel</i> lipid storage</p></div><div id="ml084.s9"><h3>List of PubChem bioassay identifiers generated for this screening project
(AIDs)</h3><div id="ml084.tu3" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK47336/table/ml084.tu3/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml084.tu3_lrgtbl__"><table><thead><tr><th id="hd_h_ml084.tu3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">AID</th><th id="hd_h_ml084.tu3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Target</th><th id="hd_h_ml084.tu3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Concentration</th><th id="hd_h_ml084.tu3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Bioassay type</th></tr></thead><tbody><tr><td headers="hd_h_ml084.tu3_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1519" ref="pagearea=body&amp;targetsite=entrez&amp;targetcat=link&amp;targettype=pubchem">1519</a></td><td headers="hd_h_ml084.tu3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">S3 cells</td><td headers="hd_h_ml084.tu3_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">40 &#x003bc;M to 0.26 nM</td><td headers="hd_h_ml084.tu3_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Primary qHTS</td></tr><tr><td headers="hd_h_ml084.tu3_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&amp;targetsite=entrez&amp;targetcat=link&amp;targettype=pubchem">1569</a></td><td headers="hd_h_ml084.tu3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">S3 cells</td><td headers="hd_h_ml084.tu3_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">40 &#x003bc;M to 0.26 nM</td><td headers="hd_h_ml084.tu3_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Confirmatory</td></tr><tr><td headers="hd_h_ml084.tu3_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1561" ref="pagearea=body&amp;targetsite=entrez&amp;targetcat=link&amp;targettype=pubchem">1561</a></td><td headers="hd_h_ml084.tu3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Cytotoxicity</td><td headers="hd_h_ml084.tu3_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">40 &#x003bc;M to 0.26 nM</td><td headers="hd_h_ml084.tu3_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Selectivity</td></tr><tr><td headers="hd_h_ml084.tu3_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1547" ref="pagearea=body&amp;targetsite=entrez&amp;targetcat=link&amp;targettype=pubchem">1547</a></td><td headers="hd_h_ml084.tu3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">NEFA incorporation/release</td><td headers="hd_h_ml084.tu3_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">5 &#x003bc;M</td><td headers="hd_h_ml084.tu3_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Secondary</td></tr><tr><td headers="hd_h_ml084.tu3_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&amp;targetsite=entrez&amp;targetcat=link&amp;targettype=pubchem">1623</a></td><td headers="hd_h_ml084.tu3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Lipid over-storage</td><td headers="hd_h_ml084.tu3_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">N/A</td><td headers="hd_h_ml084.tu3_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Summary</td></tr></tbody></table></div></div></div><div id="ml084.s10"><h3>Primary Assay Description as defined in PubChem</h3><div id="ml084.s11"><h4>Overview</h4><p>The primary goal of this project is to identify chemical probes that either
increase or decrease the lipid content of cells. Most cells are capable of
storing energy rich lipids (mainly triacylglycerols), which are generated on
basis of <i>de novo</i> synthesized fatty acids or non-esterified
free fatty acids (NEFA) taken up from the environment. There are only few
drugs for treating metabolic diseases and a very limited number of chemical
probes to study lipid storage <i>in vitro</i>. We have found that
embryonic <i>Drosophila</i> S3 and Kc167 cells are capable of
depositing LDs, and we developed an assay to measure lipid storage amounts
by fluorescent staining of LDs in S3 cells, using laser-scanning microplate
cytometry for detection. Importantly, most results obtained in the
<i>Drosophila</i> cell systems could be successfully
translated into different mammalian cell culture models, opening up the
possibility to test for evolutionary conservation of mechanism.</p></div><div id="ml084.s12"><h4>Protocol</h4><p>Detection of lipid droplets was performed using <i>Drosophila</i>
S3 (obtained from NIDDK) cells, following 400 &#x003bc;M oleic acid
feeding in growth media. In the assay 4 &#x003bc;L of S3 cells at 1.25 x
10<sup>6</sup> cells/mL in growth media was dispensed into LoBase Aurora
1,536-well plates (black walled, clear bottom COP plastic; Aurora) (<a class="bk_pop" href="#ml084.r8">8</a>) using a bottle-valve
solenoid-based dispenser (<a class="bk_pop" href="#ml084.r9">9</a>) to
give 5,000 cells/well. Twenty-three nL of compound solution was transferred
to the assay plates using a Kalypsys pin tool (San Diego, CA) equipped with
a 1,536-pin array containing 10 nL slotted pins (FP1S10, 0.457 mm diameter,
50.8 mm long; V&#x00026;P Scientific5). Next, 1 &#x003bc;l of oleic acid
(400 &#x003bc;M) was added and the plates were lidded with stainless
steel rubber gasket-lined lids containing pin-holes for gas exchange and
incubated for 18&#x02013;24 hr at 24 &#x000b0;C, 95%
humidity. Detection was performed by the addition of the lipid-droplet
specific dye BODIPY 493/503 (Molecular Probes), and CellTracker&#x02122;
Red CMTPX (Invitrogen) was used to enumerate cell number. Lipid droplet
accumulation was measured using the Acumen Explorer (TTP LapTech) (<a class="bk_pop" href="#ml084.r10">10</a>) using a 488 nm laser. The
total intensity in channel 1 (500&#x02013;530 nm) was used to measure
lipid droplet accumulation with cell objects defined using channel 3
(575&#x02013;640 nm) and a 5 &#x003bc;m width and 100 &#x003bc;m
depth filters. The ratio of the total intensity in PMT channel 1 over total
intensity channel 3 was also calculated.</p></div></div><div id="ml084.s13"><h3>Summary of the Primary Screen</h3><div id="ml084.s14"><h4>Assay principle and protocol</h4><div id="ml084.f1" class="figure bk_fig"><div class="graphic"><img src="/books/NBK47336/bin/ml084f1.jpg" alt="Figure 1. Top, distribution of the fluorescent signal in PMT 1 (lipid specific channel) without (left) and with (right) oleic acid (OA) feeding." /></div><h3><span class="label">Figure 1</span></h3><div class="caption"><p>Top, distribution of the fluorescent signal in PMT 1 (lipid specific
channel) without (left) and with (right) oleic acid (OA) feeding.
The populations are defined using width and depth filters using the
fluorescent signal from PMT 3 (cell- specific channel) to define
cell-objects. Cell objects with low PMT 1 fluorescence are defined
in the absence of OA feeding (&#x0201c;unfed
population&#x0201d;, orange data), and a shift to brighter
fluorescence is observed following OA feeding that is used to define
lipid-containing cells (&#x0201c;fed population&#x0201d;,
green data at top right). Unclassified objects not used in the
calculation are shown as red. Middle pictures show false color (same
color code as in histograms) plate well images from the Acumen for
unfed (left) and fed (right) cells. The bottom two panels represent
images from the InCell1000 using the same cell stains. Cells are
treated with 400 &#x003bc;M OA in the presence (top) or absence
(bottom) of the inhibitor Triacsin C. In the fused image, the InCell
software swaps the colors so that the red color represent the lipid
stain and the green represent the cell stain.</p></div></div><p>The optimized 1536-well protocol is given in <a class="figpopup" href="/books/NBK47336/table/ml084.t1/?report=objectonly" target="object" rid-figpopup="figml084t1" rid-ob="figobml084t1">Table 1</a>.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml084t1"><a href="/books/NBK47336/table/ml084.t1/?report=objectonly" target="object" title="Table 1" class="img_link icnblk_img figpopup" rid-figpopup="figml084t1" rid-ob="figobml084t1"><img class="small-thumb" src="/books/NBK47336/table/ml084.t1/?report=thumb" src-large="/books/NBK47336/table/ml084.t1/?report=previmg" alt="Table 1. Final 1536-well assay protocol." /></a><div class="icnblk_cntnt"><h4 id="ml084.t1"><a href="/books/NBK47336/table/ml084.t1/?report=objectonly" target="object" rid-ob="figobml084t1">Table 1</a></h4><p class="float-caption no_bottom_margin">Final 1536-well assay protocol. </p></div></div><p>Following adaptation of the lipid droplet assay to 1536-well plates, we used
the assay to screen several libraries. The libraries were screened at
between seven and fifteen concentrations using quantitative HTS (qHTS)
(<a class="bk_pop" href="#ml084.r11">11</a>). A total of 98
1536-well plates were screened (<a class="figpopup" href="/books/NBK47336/figure/ml084.f2/?report=objectonly" target="object" rid-figpopup="figml084f2" rid-ob="figobml084f2">Figure
2</a>). This included 27 DMSO blanks, a 15 point titration of the
LOPAC library (Sigma), a 13 point titration of the Prestwick library, seven
point titrations of two combinatorial libraries targeted to GPCRs and
kinases (PTL1 and PTL2), seven point titrations of two libraries from
Spectrum/Micorsource and a fifteen point titration of the Tocris library.
Overall the assay showed excellent performance with a Z&#x02032;
= 0.77 &#x000b1; 0.23, S/B = 25 &#x000b1;
68, CV = 3 &#x000b1; 3%. Three plates failed QC
(arrows and low Z-factor plates in <a class="figpopup" href="/books/NBK47336/figure/ml084.f2/?report=objectonly" target="object" rid-figpopup="figml084f2" rid-ob="figobml084f2">Figure 2c,d</a>) due to a clogged tip.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml084f2" co-legend-rid="figlgndml084f2"><a href="/books/NBK47336/figure/ml084.f2/?report=objectonly" target="object" title="Figure 2" class="img_link icnblk_img figpopup" rid-figpopup="figml084f2" rid-ob="figobml084f2"><img class="small-thumb" src="/books/NBK47336/bin/ml084f2.gif" src-large="/books/NBK47336/bin/ml084f2.jpg" alt="Figure 2. Validation of the 1536-well Acumen-based lipid droplet assay in Drosophila cells." /></a><div class="icnblk_cntnt" id="figlgndml084f2"><h4 id="ml084.f2"><a href="/books/NBK47336/figure/ml084.f2/?report=objectonly" target="object" rid-ob="figobml084f2">Figure 2</a></h4><p class="float-caption no_bottom_margin">Validation of the 1536-well Acumen-based lipid droplet assay in
<i>Drosophila</i> cells. a) heatmap representation of fed cell objects. Four control columns
are present at left containing untreated cells (column 1, no OA), a
titration of Triacsin C (column 2), <a href="/books/NBK47336/figure/ml084.f2/?report=objectonly" target="object" rid-ob="figobml084f2">(more...)</a></p></div></div><div id="ml084.s15"><h5>Identification of Exo1</h5><p>Following the qHTS, the CRC data was subjected to a classification scheme
to rank the quality of the CRCs as described by Inglese and co-workers
(<a class="bk_pop" href="#ml084.r11">11</a>) (see <a class="figpopup" href="/books/NBK47336/figure/ml084.f3/?report=objectonly" target="object" rid-figpopup="figml084f3" rid-ob="figobml084f3">scheme 1</a>). Briefly, CRCs are
placed into four classes. Class 1 contains complete CRCs showing both
upper and lower asymptotes and r<sup>2</sup> values &#x0003e; 0.9. Class
2 contains incomplete CRCs lacking the lower asymptote and shows
r<sup>2</sup> values greater than 0.9. Class 3 curves are of the
lowest confidence because they are defined by a single concentration
point where the minimal acceptable activity is set at 3 SD of the mean
activity calculated from the lowest tested concentration. Finally, class
4 contains compounds that do not show any CRCs and are therefore
classified as inactive.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml084f3" co-legend-rid="figlgndml084f3"><a href="/books/NBK47336/figure/ml084.f3/?report=objectonly" target="object" title="Scheme 1" class="img_link icnblk_img figpopup" rid-figpopup="figml084f3" rid-ob="figobml084f3"><img class="small-thumb" src="/books/NBK47336/bin/ml084f3.gif" src-large="/books/NBK47336/bin/ml084f3.jpg" alt="Scheme 1. Example qHTS data and classification scheme for assignment of resulting curve-fit data into classes." /></a><div class="icnblk_cntnt" id="figlgndml084f3"><h4 id="ml084.f3"><a href="/books/NBK47336/figure/ml084.f3/?report=objectonly" target="object" rid-ob="figobml084f3">Scheme 1</a></h4><p class="float-caption no_bottom_margin">Example qHTS data and classification scheme for assignment of
resulting curve-fit data into classes. <i>Top</i>, qHTS curve-fit data from AID-361 binned into
curve classifications 1&#x02013;4 based classification
criteria. <i>Below</i>, Examples of curves fitting the
following <a href="/books/NBK47336/figure/ml084.f3/?report=objectonly" target="object" rid-ob="figobml084f3">(more...)</a></p></div></div></div><div id="ml084.s16"><h5>qHTS summary of assay results</h5><p>Compounds showing decreasing lipid storage were generally associated with
cytotoxicity, except for the positive control Triacsin C as identified
in the red cell stain channel. However, we found many compounds that
induced lipid overstorage (<a class="figpopup" href="/books/NBK47336/figure/ml084.f4/?report=objectonly" target="object" rid-figpopup="figml084f4" rid-ob="figobml084f4">Figure
3</a>). In the Tocris library, we noted a small molecule Arf1
modulator (Exo1) (<a class="bk_pop" href="#ml084.r12">12</a>) that
increased lipid droplet accumulation (EC<sub>50</sub> = 5
&#x003bc;M) in S3 cells (<a class="figpopup" href="/books/NBK47336/figure/ml084.f5/?report=objectonly" target="object" rid-figpopup="figml084f5" rid-ob="figobml084f5">Figure
4</a>). This compound is known to inhibit the vesicle-mediated
Coat Protein complex I (COPI) transport complex. Further, we have
identified key <i>Drosophila</i> candidate genes for lipid
droplet regulation by RNA interference (RNAi) screening that included
the COPI transport complex, which was found to be required for limiting
lipid storage.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml084f4" co-legend-rid="figlgndml084f4"><a href="/books/NBK47336/figure/ml084.f4/?report=objectonly" target="object" title="Figure 3" class="img_link icnblk_img figpopup" rid-figpopup="figml084f4" rid-ob="figobml084f4"><img class="small-thumb" src="/books/NBK47336/bin/ml084f4.gif" src-large="/books/NBK47336/bin/ml084f4.jpg" alt="Figure 3. Example CRCs (fed objects) for lipids storage activators from the qHTS of S3 cells." /></a><div class="icnblk_cntnt" id="figlgndml084f4"><h4 id="ml084.f4"><a href="/books/NBK47336/figure/ml084.f4/?report=objectonly" target="object" rid-ob="figobml084f4">Figure 3</a></h4><p class="float-caption no_bottom_margin">Example CRCs (fed objects) for lipids storage activators from
the qHTS of S3 cells. </p></div></div></div></div></div></div><div id="ml084.s17"><h2 id="_ml084_s17_">Probe Characterization</h2><p>We found that interference with COPI function by RNAi in <i>Drosophila</i>
Kc167 cells, as well as in mouse 3T3-L1 or AML12 cells, results in increased lipid
storage (see <a class="figpopup" href="/books/NBK47336/figure/ml084.f5/?report=objectonly" target="object" rid-figpopup="figml084f5" rid-ob="figobml084f5">Figure 4</a> and Beller et al.,
(<a class="bk_pop" href="#ml084.r13">13</a>)). In order to characterize the
COPI knockdown effects on lipid storage in greater detail, and to confirm the
independently identified effect of the Exo1 compound, we utilized different
secondary assays. In addition to Exo1, we used BFA, another compound that has been
implicated in the modulation of COPI-mediated trafficking in mammalian cells (<a class="bk_pop" href="#ml084.r14">14</a>). In order to support our finding of
evolutionary conservation of COPI effects on lipid storage, we also utilized Exo1
and BFA in the mammalian cell system. Both compounds reduced NEFA release to the
same extent as the siRNAs targeting COPI subunit encoding mRNAs (<a class="figpopup" href="/books/NBK47336/figure/ml084.f6/?report=objectonly" target="object" rid-figpopup="figml084f6" rid-ob="figobml084f6">Figure 5</a> and see Ref [<a class="bk_pop" href="#ml084.r13">13</a>]). Mimicking genetic
epistasis experiments by combining RNAi and compound treatment, we furthermore
obtained results supporting the hypothesis that COPI-functions in the same pathway
as ATGL and might be involved in its activation (see <a class="figpopup" href="/books/NBK47336/figure/ml084.f6/?report=objectonly" target="object" rid-figpopup="figml084f6" rid-ob="figobml084f6">Figure 5</a> and Ref[<a class="bk_pop" href="#ml084.r13">13</a>]), opening up new possibilities to study
this currently heavily studied question. Positive regulation of lipolysis by the
COPI retrograde vesicle trafficking pathway was the most striking and unexpected
result of both the RNAi and small molecule compound screen. We propose that COPI is
likely to function directly at the lipid droplet surface and rather than indirectly
through the Golgi. See Beller et al. (<a class="bk_pop" href="#ml084.r13">13</a>) for a full description of these findings.</p><div id="ml084.s18"><h3>Mode of action for Exo1</h3><div id="ml084.f5" class="figure bk_fig"><div class="graphic"><img src="/books/NBK47336/bin/ml084f5.jpg" alt="Figure 4. The COPI-Mediated Retrograde Trafficking Pathway is a negative regulator of lipid storage." /></div><h3><span class="label">Figure 4</span><span class="title">The COPI-Mediated Retrograde Trafficking Pathway is a
negative regulator of lipid storage</span></h3><div class="caption"><p>(A&#x02013;F) Drosophila cells with or without oleic acid
stained with BODIPY493/503 to detect lipid. Control cells not
treated with dsRNA (A and D) or cells incubated with dsRNAs
targeting Arf79F (an Arf1 homolog) (B and E) or alphaCop (C and
F) are shown. (G) All COPI, and several COPII members as well as
additional Arfs, were retested using independent dsRNAs and gave
similar results. Results and number of dsRNAs present in the
primary screen (including oleic acid) and retests are given. (H)
Dose response of Drosophila S3 cells to Exo1 (structure inset)
showing the %-activity derived either from the lipid
specific signal (filled circles) or the lipid/cell ratio (open
circles). Percent activity refers hereby to the changes of lipid
storage relative to Triacsin C treatment, which decreases lipid
storage by blocking TG synthesis. Increased activity indicates
increased lipid storage, which increased with concentration.
Scale bar in (A) represents 10. Adopted from Beller et al. 2008.
(<a class="bk_pop" href="#ml084.r13">13</a>)</p></div></div><div id="ml084.f6" class="figure bk_fig"><div class="graphic"><img src="/books/NBK47336/bin/ml084f6.jpg" alt="Figure 5. NEFA Incorporation and NEFA Release Measured in AML12 Cells after compound treatment." /></div><h3><span class="label">Figure 5</span><span class="title">NEFA Incorporation and NEFA Release Measured in AML12 Cells after
compound treatment</span></h3><div class="caption"><p>Relative activity [(experimental/ALLStars negative control
(control) and DMSO) Radiolabel assays for NEFA release (nM) relative to
total protein concentration in cells treated with siRNAs targeting the
indicated transcripts in the presence of DMSO only (open bar), BFA (5
&#x003bc;M) in DMSO (light-grey bar), or Exo1 (5 &#x003bc;M) in DMSO
(dark-grey bar). Significance at p&#x0003c;0.01, unpaired t-test, is shown
(indicated by an asterisk [*]). Standard
error is indicated by the bars.</p></div></div></div><div id="ml084.s19"><h3>Synthesis of Exo1 analogs</h3><div id="ml084.s20"><h4>General procedure for the synthesis of Exo-1 (CID-310557) and its
analogues</h4><p>To a solution of the appropriate aryl acid (220 mg, 1.45 mmol) in DCM (20 ml)
was added SOCl<sub>2</sub> (2.12 ml, 29.0 mmol) and several drops of DMF,
and the mixture was stirred at room temperature for 0.5 h. The solvent and
excess SOCl<sub>2</sub> were removed under reduced pressure and the
resulting film was reconstituted in DCM (10 ml) followed by the addition of
Et<sub>3</sub>N (2.42 ml, 1.74 mmol, 1.2 eq.) and the appropriate
analine (253 mg, 1.60 mmol, 1.1 eq). The resulting mixture was stirred for 3
h and water (10 ml) was added, and the organic layer was separated. The
aqueous layer was further extracted with DCM (20 ml). The combined organic
layers were washed with brine and dried over Na<sub>2</sub>SO<sub>4</sub>.
After the removal of solvent, the residue was purified by column
chromatography (EtOAc/DCM= 1/40) to afford desired product (297
mg, 75%).</p><div id="ml084.fu2" class="figure"><div class="graphic"><img src="/books/NBK47336/bin/ml084fu2.jpg" alt="Image ml084fu2" /></div></div><p><sup>1</sup>H NMR (400 MHz, CDCl<sub>3</sub>) &#x003b4; 12.03 (brs, 1H),
8.92 (dd, <i>J</i><sub>HH</sub>= 8.6, 1.0 Hz, 1H),
8.04&#x02013;8.15 (m, 3H), 7.61 (td,
<i>J</i><sub>HH</sub>= 8.0, 1.6 Hz, 1H),
7.20&#x02013;7.43 (m, 3H), 3.97 (s, 3H); (TOFMS) <i>m/z</i>
274.0878 (M+H<sup>+</sup>) (calculated for
C<sub>15</sub>H<sub>13F</sub>NO<sub>3</sub>+) 274.0879.</p><div id="ml084.fu3" class="figure"><div class="graphic"><img src="/books/NBK47336/bin/ml084fu3.jpg" alt="Image ml084fu3" /></div></div><p><sup>1</sup>H NMR (400 MHz, CDCl<sub>3</sub>) &#x003b4; 8.71 (brs, 1H),
8.60 (dd, <i>J</i><sub>HH</sub>= 8.0, 1.6 Hz, 1H),
8.42 (d, <i>J</i><sub>HH</sub>= 0.4 Hz, 1H),
7.96&#x02013;8.02 (m, 3H), 7.87&#x02013;7.92 (m, 1H),
7.55&#x02013;7.62 (m, 2H), 7.03&#x02013;7.14 (m, 2H), 6.95 (dd,
<i>J</i><sub>HH</sub>= 8.0 Hz, 1.6 Hz, 1H), 3.95
(s, 3H); (TOFMS) <i>m/z</i>
(M+H<sup>+</sup>) 278.1196 (calculated for
C<sub>18</sub>H<sub>16</sub>NO<sub>2</sub>+) 278.1181</p><div id="ml084.fu4" class="figure"><div class="graphic"><img src="/books/NBK47336/bin/ml084fu4.jpg" alt="Image ml084fu4" /></div></div><p><sup>1</sup>H NMR (400 MHz, CDCl<sub>3</sub>) &#x003b4; 12.21 (brs, 1H),
8.89 (d, <i>J</i><sub>HH</sub>= 8.2 Hz, 2H), 8.15 (d,
<i>J</i><sub>HH</sub>= 8.4 Hz, 1H), 8.11 (dd,
<i>J</i><sub>HH</sub>= 8.0, 1.6 Hz, 1H), 7.82 (d,
<i>J</i><sub>HH</sub>= 8.2 Hz, 2H), 7.63 (td,
<i>J</i><sub>HH</sub>= 7.8, 1.6 Hz, 1H), 7.17 (m,
1H), 3.98 (s, 3H); (TOFMS) <i>m/z</i> 281.0925
(M+H<sup>+</sup>) (calculated for
C<sub>16</sub>H<sub>13</sub>N<sub>2</sub>O<sub>3</sub>+)
281.0926.</p><div id="ml084.fu5" class="figure"><div class="graphic"><img src="/books/NBK47336/bin/ml084fu5.jpg" alt="Image ml084fu5" /></div></div><p><sup>1</sup>H NMR (400 MHz, CDCl<sub>3</sub>) &#x003b4; 11.82 (d,
<i>J</i><sub>HF</sub>= 7.2 Hz, 1H), 8.92 (dd,
<i>J</i><sub>HH</sub>= 8.4, 0.8 Hz, 1H),
8.04&#x02013;8.15 (m, 2H), 7.60 (td,
<i>J</i><sub>HH</sub>= 7.0, 1.6 Hz, 1H), 7.14 (td,
<i>J</i><sub>HH</sub>= 7.6, 0.8 Hz, 1H),
6.90&#x02013;7.06 (m, 2H), 3.94 (s, 3H); (TOFMS) <i>m/z</i>
292.0781 (M+H<sup>+</sup>) (calculated for
C<sub>15</sub>H<sub>12</sub>F<sub>2</sub>NO<sub>3</sub>+)
292.0785.</p></div></div><div id="ml084.s21"><h3>SAR of Exo1 analogs</h3><div id="ml084.fu6" class="figure"><div class="graphic"><img src="/books/NBK47336/bin/ml084fu6.jpg" alt="Image ml084fu6" /></div></div><p>NCGC-000168461 shows the weakest activity in agreement with the SAR derived from
characterization of Exo1 analogs in mammalian cells by Feng et al. (<a class="bk_pop" href="#ml084.r12">12</a>)</p><p>This compound has been provided to the MLSMR: MLS-000722799</p><p><i>Canonical SMILES:</i>
COC(=O)C1=CC=CC=C1NC(=O)C2=CC=C(C=C2)F</p><p><i>InChI:</i>
InChI=1/C15H12FNO3/c1-20-15(19)12-4-2-3-5-13(12)17-14(18)10-6-8-11(16)9-7-10/h2-9H,1H3,(H,17,18)/f/h17H</p></div><div id="ml084.s23"><h3>Description of secondary assays used in probe characterization</h3><div id="ml084.s24"><h4>Lipolysis and lipogenesis measurements in AML12 cells</h4><p>Measurements of NEFA released from lipid droplets or incorporated into the TG
fraction were performed as previously described (<a class="bk_pop" href="#ml084.r13">13</a>). Briefly, AML12 cells treated with or without
specific siRNAs (10 nM) for 4 d were incubated overnight with growth medium,
supplemented with 400 &#x003bc;M oleic acid complexed to
0.4% bovine serum albumin, to promote triacylglycerol deposition
and [3H] oleic acid, at 1 x 10<sup>6</sup> dpm/well,
was included as a tracer. In lipolysis experiments, re-esterification of
fatty acids in AML12 cells was prevented by including 10 &#x003bc;M
Triacsin C (Biomol), an inhibitor of acyl coenzyme A synthetase, in the
medium. Quadruplicate wells were tested for each condition. Lipolysis was
determined by measuring radioactivity released into the media in 1 h. For
the lipid extraction and thin layer chromatography, the cell monolayer was
washed with ice-cold PBS and scraped into 1 ml of PBS. Lipids were extracted
by the Bligh-Dyer method [84], and 10%
of the total lipid was analyzed by thin layer chromatography. AML12 cells
treated with or without specific siRNAs were additionally incubated with
either vehicle (DMSO), 5 &#x003bc;M of Exo1 (12.5 mg/ml DMSO), or BFA
(10 mg/ml DMSO) during the time of radioactivity release into the media (2
h). NEFA incorporation into the TG fraction and NEFA release are calculated
as nanomoles/milligram protein. Protein measurements were performed using a
commercial BCA assay kit (Pierce Biotechnology) according to the
manufacturer&#x02019;s instructions. Statistical significance was tested
by impaired Student t test (GraphPad software).</p></div></div></div><div id="ml084.s25"><h2 id="_ml084_s25_">Probe</h2><div id="ml084.s26"><h3>a. Chemical name</h3><p>Methyl 2-(4-fluorobenzamido)benzoate (<a href="/pcsubstance/?term=ML084[synonym]" ref="pagearea=body&amp;targetsite=entrez&amp;targetcat=term&amp;targettype=pubchem">ML084</a>)</p></div><div id="ml084.s27"><h3>b. Probe chemical structure</h3><div id="ml084.fu7" class="figure"><div class="graphic"><img src="/books/NBK47336/bin/ml084fu7.jpg" alt="Image ml084fu7" /></div></div></div><div id="ml084.s28"><h3>c. Structural Verification Information of probe SID
(11114231)</h3><p><sup>1</sup>H NMR (400 MHz, CDCl<sub>3</sub>) &#x003b4; 12.03
(brs, 1H), 8.92 (dd, <i>J</i><sub>HH</sub>=
8.6, 1.0 Hz, 1H), 8.04&#x02013;8.15 (m, 3H), 7.61 (td,
<i>J</i><sub>HH</sub>= 8.0, 1.6 Hz, 1H),
7.20&#x02013;7.43 (m, 3H), 3.97 (s, 3H); (TOFMS)
<i>m/z</i> 274.0878
(M+H<sup>+</sup>) (calculated for
C<sub>15</sub>H<sub>13F</sub>NO<sub>3</sub>+)
274.0879.</p></div><div id="ml084.s29"><h3>d. PubChem CID (corresponding to the SID)</h3><p>310557</p></div><div id="ml084.s30"><h3>e. Availability from a vendor</h3><p>Exo1 is sold by Tocris Biosciences (Tocris-1850).</p></div><div id="ml084.s31"><h3>f. Mode of action for biological activity of probe</h3><p>Increases lipid droplet storage by inhibition of COPI function.</p></div><div id="ml084.s32"><h3>g. Detailed synthetic pathway for making probe</h3><p>See <a href="#ml084.s20">page 11</a></p></div><div id="ml084.s33"><h3>h. Summary of probe properties</h3><p>Exo1 is not fluorescent using 488 nm excitation. Possess adequate
solubility for in vitro use. Probe is not toxic to cells at 60 &#x000b5;M
testing concentration (24 hr incubation). Probe properties such as
solubility are available at Tocris Biosciences.</p></div></div><div id="ml084.s34"><h2 id="_ml084_s34_">Compound preparation</h2><p>Compound is prepared in DMSO at 10 mM stock concentration. Assays described above
have 0.6% DMSO final concentration in buffer.</p></div><div id="ml084.bib"><h2 id="_ml084_bib_">Bibliography</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="ml084.r1">Langin D. Adipose tissue lipolysis as a metabolic pathway to define
pharmacological strategies against obesity and the metabolic
syndrome. <span><span class="ref-journal">Pharmacol Res. </span>2006;<span class="ref-vol">53</span>:48291.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/16644234" ref="pagearea=cite-ref&amp;targetsite=entrez&amp;targetcat=link&amp;targettype=pubmed">PubMed<span class="bk_prnt">: 16644234</span></a>]</div></dd><dt>2.</dt><dd><div class="bk_ref" id="ml084.r2">Subramanian V, Rothenberg A, Gomez C, Cohen AW, Garcia A, Bhattacharyya S, Shapiro L, Dolios G, Wang R, Lisanti MP, Brasaemle DL. Perilipin A mediates the reversible binding of CGI-58 to
lipid droplets in 3T3-L1 adipocytes. <span><span class="ref-journal">J Biol Chem. </span>2004;<span class="ref-vol">279</span>:4206271.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/15292255" ref="pagearea=cite-ref&amp;targetsite=entrez&amp;targetcat=link&amp;targettype=pubmed">PubMed<span class="bk_prnt">: 15292255</span></a>]</div></dd><dt>3.</dt><dd><div class="bk_ref" id="ml084.r3">Vaughan M, Berger JE, Steinberg D. Hormone-Sensitive Lipase and Monoglyceride Lipase Activities
in Adipose Tissue. <span><span class="ref-journal">J Biol Chem. </span>1964;<span class="ref-vol">239</span>:4019.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/14169138" ref="pagearea=cite-ref&amp;targetsite=entrez&amp;targetcat=link&amp;targettype=pubmed">PubMed<span class="bk_prnt">: 14169138</span></a>]</div></dd><dt>4.</dt><dd><div class="bk_ref" id="ml084.r4">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>:32330.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/16054079" ref="pagearea=cite-ref&amp;targetsite=entrez&amp;targetcat=link&amp;targettype=pubmed">PubMed<span class="bk_prnt">: 16054079</span></a>]</div></dd><dt>5.</dt><dd><div class="bk_ref" id="ml084.r5">Haemmerle G, Lass A, Zimmermann R, Gorkiewicz G, Meyer C, Rozman J, Heldmaier G, Maier R, Theussl C, Eder S, Kratky D, Wagner EF, Klingenspor M, Hoefler G, Zechner R. Defective lipolysis and altered energy metabolism in mice
lacking adipose triglyceride lipase. <span><span class="ref-journal">Science. </span>2006;<span class="ref-vol">312</span>:7347.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/16675698" ref="pagearea=cite-ref&amp;targetsite=entrez&amp;targetcat=link&amp;targettype=pubmed">PubMed<span class="bk_prnt">: 16675698</span></a>]</div></dd><dt>6.</dt><dd><div class="bk_ref" id="ml084.r6">Zimmermann R, Strauss JG, Haemmerle G, Schoiswohl G, Birner-Gruenberger R, Riederer M, Lass A, Neuberger G, Eisenhaber F, Hermetter A, Zechner R. Fat mobilization in adipose tissue is promoted by adipose
triglyceride lipase. <span><span class="ref-journal">Science. </span>2004;<span class="ref-vol">306</span>:13836.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/15550674" ref="pagearea=cite-ref&amp;targetsite=entrez&amp;targetcat=link&amp;targettype=pubmed">PubMed<span class="bk_prnt">: 15550674</span></a>]</div></dd><dt>7.</dt><dd><div class="bk_ref" id="ml084.r7">Frayn KN, Arner P, Yki-Jarvinen H. Fatty acid metabolism in adipose tissue, muscle and liver in
health and disease. <span><span class="ref-journal">Essays Biochem. </span>2006;<span class="ref-vol">42</span>:89103.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/17144882" ref="pagearea=cite-ref&amp;targetsite=entrez&amp;targetcat=link&amp;targettype=pubmed">PubMed<span class="bk_prnt">: 17144882</span></a>]</div></dd><dt>8.</dt><dd><div class="bk_ref" id="ml084.r8">Niles WD, Coassin PJ. Cyclic olefin polymers: innovative materials for high-density
multiwell plates. <span><span class="ref-journal">Assay Drug Dev Technol. </span>2008;<span class="ref-vol">6</span>:57790.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/18537466" ref="pagearea=cite-ref&amp;targetsite=entrez&amp;targetcat=link&amp;targettype=pubmed">PubMed<span class="bk_prnt">: 18537466</span></a>]</div></dd><dt>9.</dt><dd><div class="bk_ref" id="ml084.r9">Niles WD, Coassin PJ. Piezo- and Solenoid Valve-Based Liquid Dispensing for
Miniaturized Assays. <span><span class="ref-journal">Assay Drug. Devel. Technol. </span>2005;<span class="ref-vol">3</span>:189202.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/15871693" ref="pagearea=cite-ref&amp;targetsite=entrez&amp;targetcat=link&amp;targettype=pubmed">PubMed<span class="bk_prnt">: 15871693</span></a>]</div></dd><dt>10.</dt><dd><div class="bk_ref" id="ml084.r10">Bowen WP, Wylie PG. Application of Laser-Scanning Fluorescence Microplate
Cytometry in High Content Screening. <span><span class="ref-journal">Assay Drug Dev Technol. </span>2006;<span class="ref-vol">4</span>:209221.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/16712425" ref="pagearea=cite-ref&amp;targetsite=entrez&amp;targetcat=link&amp;targettype=pubmed">PubMed<span class="bk_prnt">: 16712425</span></a>]</div></dd><dt>11.</dt><dd><div class="bk_ref" id="ml084.r11">Inglese J, Auld DS, Jadhav A, Johnson RL, Simeonov A, Yasgar A, Zheng W, Austin CP. Quantitative high-throughput screening: A titration-based
approach that efficiently identifies biological activities in large
chemical libraries. <span><span class="ref-journal">Proc Natl Acad Sci U S A. </span>2006;<span class="ref-vol">103</span>:114738.</span> [<a href="/pmc/articles/PMC1518803/" ref="pagearea=cite-ref&amp;targetsite=entrez&amp;targetcat=link&amp;targettype=pmc">PMC free article<span class="bk_prnt">: PMC1518803</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/16864780" ref="pagearea=cite-ref&amp;targetsite=entrez&amp;targetcat=link&amp;targettype=pubmed">PubMed<span class="bk_prnt">: 16864780</span></a>]</div></dd><dt>12.</dt><dd><div class="bk_ref" id="ml084.r12">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
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homeostasis. <span><span class="ref-journal">PLoS Biol. </span>2008;<span class="ref-vol">6</span>:25302549.</span> [<a href="/pmc/articles/PMC2586367/" ref="pagearea=cite-ref&amp;targetsite=entrez&amp;targetcat=link&amp;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&amp;targetsite=entrez&amp;targetcat=link&amp;targettype=pubmed">PubMed<span class="bk_prnt">: 19067489</span></a>]</div></dd><dt>14.</dt><dd><div class="bk_ref" id="ml084.r14">Lippincott-Schwartz J, Yuan L, Tipper C, Amherdt M, Orci L, Klausner RD. Brefeldin A&#x02019;s effects on endosomes, lysosomes,
and the TGN suggest a general mechanism for regulating organelle
structure and membrane traffic. <span><span class="ref-journal">Cell. </span>1991;<span class="ref-vol">67</span>:60116.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/1682055" ref="pagearea=cite-ref&amp;targetsite=entrez&amp;targetcat=link&amp;targettype=pubmed">PubMed<span class="bk_prnt">: 1682055</span></a>]</div></dd></dl></div><div style="display:none"><div style="display:none" id="figml084f5"><img alt="Image ml084f5" src-large="/books/NBK47336/bin/ml084f5.jpg" /></div><div style="display:none" id="figml084f6"><img alt="Image ml084f6" src-large="/books/NBK47336/bin/ml084f6.jpg" /></div></div><div id="bk_toc_contnr"></div></div></div>
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