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<script type="text/javascript" src="/corehtml/pmc/jatsreader/ptpmc_3.22/js/jr.boots.min.js"> </script><title>Optimization and characterization of a triazole urea inhibitor for diacylglycerol lipase beta (DAGL-β) - 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="Optimization and characterization of a triazole urea inhibitor for diacylglycerol lipase beta (DAGL-β)">
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<meta name="citation_publisher" content="National Center for Biotechnology Information (US)">
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<meta name="citation_date" content="2013/02/25">
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<meta name="citation_author" content="Ku-Lung Hsu">
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<meta name="citation_author" content="Katsunori Tsuboi">
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<meta name="citation_author" content="Anna E Speers">
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<meta name="citation_author" content="Steven J Brown">
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<meta name="citation_author" content="Timothy Spicer">
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<meta name="citation_author" content="Virneliz Fernandez-Vega">
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<meta name="citation_author" content="Jill Ferguson">
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<meta name="citation_author" content="Benjamin F Cravatt">
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<meta name="citation_author" content="Peter Hodder">
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<meta name="citation_author" content="Hugh Rosen">
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<meta name="citation_pmid" content="23658950">
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<meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK133443/">
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<meta name="DC.Title" content="Optimization and characterization of a triazole urea inhibitor for diacylglycerol lipase beta (DAGL-β)">
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<meta name="DC.Type" content="Text">
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<meta name="DC.Publisher" content="National Center for Biotechnology Information (US)">
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<meta name="DC.Contributor" content="Ku-Lung Hsu">
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<meta name="DC.Contributor" content="Katsunori Tsuboi">
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<meta name="DC.Contributor" content="Anna E Speers">
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<meta name="DC.Contributor" content="Steven J Brown">
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<meta name="DC.Contributor" content="Timothy Spicer">
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<meta name="DC.Contributor" content="Virneliz Fernandez-Vega">
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<meta name="DC.Contributor" content="Jill Ferguson">
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<meta name="DC.Contributor" content="Benjamin F Cravatt">
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<meta name="DC.Contributor" content="Peter Hodder">
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<meta name="DC.Contributor" content="Hugh Rosen">
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<meta name="DC.Date" content="2013/02/25">
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<meta name="description" content="Endocannabinoids (ECs) are a unique group of lipids that function as chemical messengers in the nervous system. The two principle ECs thus far identified in mammals are N-arachidonoyl-ethanolamine (anandamide) and 2-arachidonoyl-glycerol (2-AG). These compounds have been implicated in various physiological and pathological functions including appetite, pain, sensation, memory, and addiction. Because ECs are synthesized and released on demand and then rapidly degraded to terminate signaling, the metabolic pathways that govern EC turnover directly influence the magnitude and duration of neuronal signaling events. There is strong evidence that two serine hydrolases, diacylglycerol lipase-alpha and -beta (DAGL-α and -β) function as 2-AG synthetic enzymes both in vitro and in vivo. However, because constitutive gene disruption, the only currently available means to investigate DAGL-α/β biology due to a lack of selective chemical inhibitors, can result in compensatory effects and network-wide changes, there is still uncertainty surrounding the extent to which DAGL-α/β contribute to 2-AG-mediated signaling. In an effort to provide chemical tools for manipulation of DAGL-β activity, we initiated a competitive activity-based protein profiling (ABPP) screen of triazole urea compounds to identify selective enzyme inhibitors. This campaign, made possible by previous inhibitor development efforts for LYPLA1/2 (ML211), PAFAH2 (ML225), and ABHD11 (ML226) based on the triazole urea scaffold, yielded the medchem optimized probe ML294 (SID 125269120). ML294 is highly potent against its target enzyme (IC50 = 56 nM in vitro; 12 nM in situ), and is active in vivo, showing both oral bioavailability and blood-brain barrier penetration. Out of more than 20 serine hydrolases (SHs) profiled by gel-based competitive ABPP, ML294 is observed to have one anti-target, alpha/beta hydrolase domain-containing protein 6 (ABHD6). Otherwise, ML294 is at least 35-fold selective for all other brain SHs (approximately 20) assessed by gel-based competitive ABPP and 7-fold selective vs. its closest homolog, DAGL-α. To control for ABHD6-directed activity in biological studies, we also developed a structurally related ABHD6-selective control “anti-probe”, ML295, also based on the triazole urea scaffold. The complete properties, characterization, and synthesis of ML294 are detailed in this report, and full details of ABHD6 inhibitors are detailed in the Probe Report for ML295 and ML296.">
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<meta name="og:description" content="Endocannabinoids (ECs) are a unique group of lipids that function as chemical messengers in the nervous system. The two principle ECs thus far identified in mammals are N-arachidonoyl-ethanolamine (anandamide) and 2-arachidonoyl-glycerol (2-AG). These compounds have been implicated in various physiological and pathological functions including appetite, pain, sensation, memory, and addiction. Because ECs are synthesized and released on demand and then rapidly degraded to terminate signaling, the metabolic pathways that govern EC turnover directly influence the magnitude and duration of neuronal signaling events. There is strong evidence that two serine hydrolases, diacylglycerol lipase-alpha and -beta (DAGL-α and -β) function as 2-AG synthetic enzymes both in vitro and in vivo. However, because constitutive gene disruption, the only currently available means to investigate DAGL-α/β biology due to a lack of selective chemical inhibitors, can result in compensatory effects and network-wide changes, there is still uncertainty surrounding the extent to which DAGL-α/β contribute to 2-AG-mediated signaling. In an effort to provide chemical tools for manipulation of DAGL-β activity, we initiated a competitive activity-based protein profiling (ABPP) screen of triazole urea compounds to identify selective enzyme inhibitors. This campaign, made possible by previous inhibitor development efforts for LYPLA1/2 (ML211), PAFAH2 (ML225), and ABHD11 (ML226) based on the triazole urea scaffold, yielded the medchem optimized probe ML294 (SID 125269120). ML294 is highly potent against its target enzyme (IC50 = 56 nM in vitro; 12 nM in situ), and is active in vivo, showing both oral bioavailability and blood-brain barrier penetration. Out of more than 20 serine hydrolases (SHs) profiled by gel-based competitive ABPP, ML294 is observed to have one anti-target, alpha/beta hydrolase domain-containing protein 6 (ABHD6). Otherwise, ML294 is at least 35-fold selective for all other brain SHs (approximately 20) assessed by gel-based competitive ABPP and 7-fold selective vs. its closest homolog, DAGL-α. To control for ABHD6-directed activity in biological studies, we also developed a structurally related ABHD6-selective control “anti-probe”, ML295, also based on the triazole urea scaffold. The complete properties, characterization, and synthesis of ML294 are detailed in this report, and full details of ABHD6 inhibitors are detailed in the Probe Report for ML295 and ML296.">
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match">◀</a><button id="jr-fip-matches">no matches yet</button><a id="jr-fip-next" class="wsprkl btn" title="Jump to next match">▶</a></nav></nav></div><div id="jr-epub-interstitial" class="hidden"></div><div id="jr-content"><article data-type="main"><div class="main-content lit-style" itemscope="itemscope" itemtype="http://schema.org/CreativeWork"><div class="meta-content fm-sec"><div class="fm-sec"><h1 id="_NBK133443_"><span class="title" itemprop="name">Optimization and characterization of a triazole urea inhibitor for diacylglycerol
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lipase beta (DAGL-β)</span></h1><p class="contribs">Hsu KL, Tsuboi K, Speers AE, et al.</p><p class="fm-aai"><a href="#_NBK133443_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>Endocannabinoids (ECs) are a unique group of lipids that function as chemical
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messengers in the nervous system. The two principle ECs thus far identified in
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mammals are <i>N</i>-arachidonoyl-ethanolamine (anandamide) and
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2-arachidonoyl-glycerol (2-AG). These compounds have been implicated in various
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physiological and pathological functions including appetite, pain, sensation,
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memory, and addiction. Because ECs are synthesized and released on demand and then
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rapidly degraded to terminate signaling, the metabolic pathways that govern EC
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turnover directly influence the magnitude and duration of neuronal signaling events.
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There is strong evidence that two serine hydrolases, diacylglycerol lipase-alpha and
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-beta (DAGL-α and -β) function as 2-AG synthetic enzymes both
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<i>in vitro</i> and <i>in vivo</i>. However, because
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constitutive gene disruption, the only currently available means to investigate
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DAGL-α/β biology due to a lack of selective chemical inhibitors, can
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result in compensatory effects and network-wide changes, there is still uncertainty
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surrounding the extent to which DAGL-α/β contribute to 2-AG-mediated
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signaling. In an effort to provide chemical tools for manipulation of DAGL-β
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activity, we initiated a competitive activity-based protein profiling (ABPP) screen
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of triazole urea compounds to identify selective enzyme inhibitors. This campaign,
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made possible by previous inhibitor development efforts for LYPLA1/2 (<a href="/pcsubstance/?term=ML211[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML211</a>), PAFAH2 (<a href="/pcsubstance/?term=ML225[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML225</a>), and ABHD11
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(<a href="/pcsubstance/?term=ML226[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML226</a>) based on the triazole urea scaffold, yielded the medchem
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optimized probe <a href="/pcsubstance/?term=ML294[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML294</a> (<a href="https://pubchem.ncbi.nlm.nih.gov/substance/125269120" ref="pagearea=abstract&targetsite=entrez&targetcat=link&targettype=pubchem">SID 125269120</a>). <a href="/pcsubstance/?term=ML294[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML294</a> is highly potent against its target enzyme (IC50 = 56
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nM <i>in vitro</i>; 12 nM <i>in situ</i>), and is active
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<i>in vivo</i>, showing both oral bioavailability and blood-brain
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barrier penetration. Out of more than 20 serine hydrolases (SHs) profiled by
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gel-based competitive ABPP, <a href="/pcsubstance/?term=ML294[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML294</a> is observed to have one anti-target,
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alpha/beta hydrolase domain-containing protein 6 (ABHD6). Otherwise, <a href="/pcsubstance/?term=ML294[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML294</a> is at least 35-fold selective for all other brain SHs
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(approximately 20)
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assessed by gel-based competitive ABPP and 7-fold selective vs. its closest homolog,
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DAGL-α. To control for ABHD6-directed activity in biological studies, we
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also developed a structurally related ABHD6-selective control
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“anti-probe”, <a href="/pcsubstance/?term=ML295[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML295</a>, also based on the
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triazole urea scaffold. The complete properties, characterization, and synthesis of
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<a href="/pcsubstance/?term=ML294[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML294</a> are detailed in this report, and full details of ABHD6
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inhibitors are detailed in the Probe Report for <a href="/pcsubstance/?term=ML295[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML295</a> and <a href="/pcsubstance/?term=ML296[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML296</a>.</p></div><div class="h2"></div><p><b>Assigned Assay Grant #:</b> 1 R01 DA025285</p><p><b>Screening Center Name & PI:</b> The Scripps Research Institute Molecular
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Screening Center (SRIMSC), H Rosen</p><p><b>Chemistry Center Name & PI:</b> SRIMSC, H Rosen</p><p><b>Assay Submitter & Institution:</b> BF Cravatt, TSRI, La Jolla</p><p><b>PubChem Summary Bioassay Identifier (AID):</b>
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<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504420" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">504420</a></p><div id="ml294.s1"><h2 id="_ml294_s1_">Probe Structure & Characteristics</h2><div id="ml294.fu1" class="figure"><div class="graphic"><img src="/books/NBK133443/bin/ml294fu1.jpg" alt="Image ml294fu1" /></div></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml294tu1"><a href="/books/NBK133443/table/ml294.tu1/?report=objectonly" target="object" title="Table" class="img_link icnblk_img figpopup" rid-figpopup="figml294tu1" rid-ob="figobml294tu1"><img class="small-thumb" src="/books/NBK133443/table/ml294.tu1/?report=thumb" src-large="/books/NBK133443/table/ml294.tu1/?report=previmg" alt="Image " /></a><div class="icnblk_cntnt"><h4 id="ml294.tu1"><a href="/books/NBK133443/table/ml294.tu1/?report=objectonly" target="object" rid-ob="figobml294tu1">Table</a></h4></div></div></div><div id="ml294.s2"><h2 id="_ml294_s2_">1. Recommendations for Scientific Use of the Probe</h2><p>The endocannabinoid 2-arachidonoylglycerol (2-AG) plays a pivotal role in synaptic
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signaling events and has been postulated to function as a retrograde messenger that
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suppresses neurotransmitter release. 2-AG has been implicated in various
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physiological and pathological functions including appetite, pain, sensation,
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memory, and addiction [<a class="bibr" href="#ml294.r1" rid="ml294.r1">1</a>]. Upon activation of postsynaptic neurons, ECs are produced from
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membrane-derived lipid precursors, traverse the synaptic cleft, and activate
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specific G-protein-coupled cannabinoid receptors present on presynaptic termini. The
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activated cannabinoid receptors initiate a series of signal transduction cascades
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that ultimately leads to the suppression of neurotransmitter release. Depending on
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the type of neurotransmitter released (excitatory vs. inhibitory), this feedback
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|
mechanism can attenuate or prolong neurotransmission, and is hypothesized to
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modulate both short- and long-term synaptic plasticity [<a class="bibr" href="#ml294.r2" rid="ml294.r2">2</a>].</p><p>Unlike traditional neurotransmitters, which are stored in vesicles, ECs are
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synthesized and released on demand, and then rapidly degraded to terminate
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signaling. Thus, the metabolic pathways that govern EC turnover are critical in
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determining the magnitude and duration of neuronal signaling events [<a class="bibr" href="#ml294.r3" rid="ml294.r3">3</a>]. The development of both
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genetic knockouts and selective inhibitors has facilitated our understanding of EC
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degradation at the molecular level, and allowed the principle anandamide- and
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2-AG-degrading enzymes (fatty acid amide hydrolase, FAAH [<a class="bibr" href="#ml294.r4" rid="ml294.r4">4</a>] and monoacylglycerol lipase
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[MAGL], respectively [<a class="bibr" href="#ml294.r5" rid="ml294.r5">5</a>]) to be characterized <i>in vivo.</i> In contrast,
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our understanding of EC biosynthesis has lagged considerably and the identities of
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the enzymes responsible for 2-AG biosynthesis remained uncertain. Recently, two
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serine hydrolases, DAGL-α and -β were cloned and found to
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selectively cleave <i>sn</i>-1 acyl chains from diacylglycerols (DAG) to
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generate 2-AG <i>in vitro</i>[<a class="bibr" href="#ml294.r6" rid="ml294.r6">6</a>]. Their function in the nervous system was validated
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<i>in vivo</i> by the generation of DAGL-α and -β
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knock-out mice [<a class="bibr" href="#ml294.r7" rid="ml294.r7">7</a>, <a class="bibr" href="#ml294.r8" rid="ml294.r8">8</a>]. These studies identified the
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key 2-AG biosynthetic enzymes and represent a major step towards advancing our
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understanding of EC biosynthesis in the nervous system. However, constitutive gene
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disruption can result in compensatory effects and network-wide changes that
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complicate direct interpretation of enzyme function. Thus, it is still unclear to
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what extent DAGL-α/β catalytic activity contributes to 2-AG-mediated
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signaling. The development of potent and selective inhibitors would offer a means to
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perturb DAGL-α/β activity in a selective, reversible, and
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temporally-controlled manner. Given the non-selective nature of current
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DAGL-α/β inhibitors [<a class="bibr" href="#ml294.r9" rid="ml294.r9">9</a>], specific chemical probes would serve as invaluable tools to
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delineate DAGL-α/β function in 2-AG signaling networks of the brain.
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<a href="/pcsubstance/?term=ML294[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML294</a>, along with control probe <a href="/pcsubstance/?term=ML295[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML295</a><b>,</b> is recommended for use in primary research
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studies aimed at elucidating the patho/physiological roles of DAGL-β and its
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contribution to 2-AG-mediated signaling, as well as other structurally related lipid
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transmitters such as eicosanoids and diacylglycerols.</p></div><div id="ml294.s3"><h2 id="_ml294_s3_">2. Materials and Methods</h2><p>All reagents for chemical synthesis were obtained from ThermoFisher or SigmaAldrich.
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All other protocols are summarized below.</p><div id="ml294.s4"><h3>2.1. Assays</h3><div id="ml294.s5"><h4>Probe Characterization Assays</h4><p><b>Solubility in PBS:</b> The solubility of compounds are tested in
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triplicate in phosphate buffered saline (PBS), pH 7.4. per well, 198
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μl PBS is added to a Millipore Solvinert Hydrophilic PTFE 96 well
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filter plate: pore size: 0.45μm (MSRLN0450). Test compounds are
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introduced from 10 mM DMSO stock solutions (2 μl). The final
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concentration of DMSO was 1 percent. Samples are allowed to incubate at
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22°C for 18 hours. In the morning the plate is centrifuged where the
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soluble portion passes through the filter and is collected in a capture
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plate. Clotrimazole is included as a control to assure the assay is working
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properly. The samples are analyzed by HPLC. Peak area is compared to a
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standard of known concentration. In cases when the concentration was too low
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for UV analysis or when the compound did not possess a good chromophore,
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LC-MS-MS analysis is used.</p><p><b>Solubility in Media:</b> The solubility of compounds are tested in
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triplicate in complete media (DMEM + 10% FBS). Per well, 198
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μl PBS is added to a Millipore Solvinert Hydrophilic PTFE 96 well
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filter plate: pore size: 0.45μm (MSRLN0450). Test compounds are
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introduced from 10 mM DMSO stock solutions (2 μl). The final
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concentration of DMSO was 1 percent. Samples are allowed to incubate at
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22°C for 18 hours. In the morning the plate is centrifuged where the
|
|
soluble portion passes through the filter and is collected in a capture
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plate. The samples are analyzed by HPLC (Agilent 1100 with diode-array
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detector). Peak area is compared to a standard of known concentration. In
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cases when the concentration was too low for UV analysis or when the
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compound did not possess a good chromophore, LC-MS-MS analysis is used.</p><p><b>Stability in PBS:</b> Demonstration of stability in PBS was
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conducted by addition of 10 μM compound from a DMSO stock to PBS in
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HPLC autosampler vials. Samples are held in the HPLC autosampler at ambient
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temperature. At approximately 0, 1, 2, 4, 8, 24, and 48 hours the samples
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are injected on the HPLC. Peak area and retention time are compared between
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injections. Data is log transformed and represented as half-life. DMSO is
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added as a co-solvent as needed for solubility.</p><p><b>Determination of Glutathione reactivity:</b> Compound (10
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μM) is incubated at 37°C for 6 hours in the presence of 50
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μM freshly prepared reduced glutathione. At 0 and 6 hours the
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samples are injected on the HPLC. Peak area and retention time are compared
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between injections. Samples are evaluated for a glutathione dependent
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decrease in compound concentration. DMSO is added as a co-solvent as needed
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for solubility.</p></div><div id="ml294.s6"><h4>Primary Assays</h4><div id="ml294.s7"><h5>Primary uHTS assay to identify DAGL-β inhibitors (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504411" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID
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504411</a>)</h5><p><b>Assay Overview:</b> The purpose of this assay is to identify
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|
compounds that inhibit the activity of human diacylglycerol lipase-beta
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|
(DAGL-β). In this assay, compounds are preincubated with
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membranes derived from mouse DAGL-β-transfected HEK293T cells
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|
(Open Biosystems Accession <a href="/nuccore/16359288" class="bk_tag" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=nuccore">BC016105</a>), followed by incubation with the
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fluorogenic lipase substrate EnzChek. Fluorescence is determined at a
|
|
specific time point. As designed, compounds that act as DAGL-β
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|
inhibitors will slow the rate of substrate hydrolysis, resulting in a
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|
decrease in well fluorescence. Compounds are tested in singlicate at a
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final nominal concentration of 5.17 μM.</p><p><b>Protocol Summary:</b> Prior to the start of the assay,
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|
DAGL-β membrane lysate was sonicated three times for 5 seconds,
|
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1 minute between each pulse using a microtip. Then, 4.0 μL of
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Assay Buffer (50 mM HEPES pH7.2, 100 mM NaCl, 5 mM CaCl2, 0.1%
|
|
Triton X-100, 10% DMSO and 0.5 mM DTT) containing 0.375 mg/mL of
|
|
DAGL-β membrane lysate were dispensed into 1536 microtiter
|
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plates. Next, 26 nL of test compound in DMSO or DMSO alone
|
|
(0.52% final concentration) were added to the appropriate wells
|
|
and incubated for 60 minutes at 25°C. The assay was started by
|
|
dispensing 1.0 μL of 5 μM EnzChek lipase substrate in
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|
Assay Buffer to all wells. Plates were centrifuged and incubated for 60
|
|
minutes at 25°C. Fluorescence was read on a Viewlux microplate
|
|
reader (PerkinElmer, Turku, Finland) using a FITC filter set (excitation
|
|
= 480 nm, emission = 540 nm) and a FITC dichroic mirror
|
|
for 1 second. <b>Assay Cutoff:</b> Compounds that inhibited
|
|
DAGL-β ≥34.1% were considered active.</p></div><div id="ml294.s8"><h5>Confirmation uHTS assay to identify DAGL-β inhibitors
|
|
(<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504445" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504445</a>)</h5><p><b>Assay Overview:</b> The purpose of this assay is to confirm
|
|
activity of compounds identified as active in the primary uHTS pPAFAH
|
|
screen (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504411" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504411</a>). In this assay, the
|
|
fluorogenic lipase substrate EnzChek is used to assess inhibition of
|
|
DAGL-β in a complex membrane preparation as described above
|
|
(<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504411" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504411</a>). Compounds were tested in
|
|
singlicate at a final nominal concentration of 5.17 μM.</p><p><b>Protocol Summary:</b> The assay was performed as described above
|
|
(<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504411" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504411</a>), except that compounds were
|
|
tested in triplicate. <b>Assay Cutoff:</b> Compounds that
|
|
inhibited DAGL-β ≥34.1% were considered
|
|
active.</p></div></div><div id="ml294.s9"><h4>Secondary Assays</h4><div id="ml294.s10"><h5>Gel-based competitive ABPP inhibition of overexpressed DAGL-β
|
|
<i>in vitro</i>: triazole urea library (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602303" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID
|
|
602303</a>)</h5><p><b>Assay Overview</b>: The purpose of this assay is to determine
|
|
whether test compounds belonging to the triazole urea scaffold can
|
|
inhibit DAGL-β in a complex proteome lysate using a gel-based
|
|
competitive activity-based proteomic profiling (ABPP) assay. In this
|
|
assay, the target enzyme DAGL-β is incubated with test compound
|
|
followed by reaction with a fluorescently-tagged serine-hydrolase
|
|
specific activity-based probe, fluorophosphonate-rhodamine (FP-Rh,
|
|
Thermo #88318). For this assay and all other gel-based ABPP
|
|
assays described below, the reaction products are separated by SDS-PAGE
|
|
and visualized in-gel using a flatbed fluorescence scanner. The
|
|
percentage activity remaining is determined by measuring the integrated
|
|
optical density (IOD) of the bands. As designed, test compounds that act
|
|
as DAGL-β inhibitors will prevent enzyme-probe interactions,
|
|
thereby decreasing the proportion of bound fluorescent probe, giving
|
|
lower fluorescence intensity in the band in the gel. See also ref.
|
|
[<a class="bibr" href="#ml294.r9" rid="ml294.r9">9</a>]</p><p><b>Protocol Summary</b>: Membrane proteome of transiently
|
|
transfected 293T Hek cells overexpressing mouse DAGL-β (25
|
|
μL of 0.3 mg/mL) in Dulbecco’s PBS (DPBS) was treated
|
|
with test compound (0.5 μL of a 50× stock in DMSO; 500
|
|
nM final concentration) or DMSO (0.5 μL) for 30 minutes at
|
|
37°C. The activity-based probe FP-Rh (0.5 μL of a
|
|
50× stock in DMSO; 5 μM final concentration) was added,
|
|
and the reaction was incubated for 30 minutes at 37°C, quenched
|
|
with an equal volume of 2× SDS-PAGE loading buffer (reducing),
|
|
separated by SDS-PAGE, and visualized by in-gel fluorescent scanning.
|
|
The percentage of DAGL-β activity remaining was determined by
|
|
measuring the integrated optical density of the individual protein bands
|
|
relative to the DMSO-only (no compound) control. <b>Assay
|
|
Cutoff</b>: Compounds with greater than or equal to 50%
|
|
inhibition were considered active.</p></div><div id="ml294.s11"><h5>Gel-based competitive ABPP inhibition of overexpressed DAGL-β
|
|
<i>in vitro</i>: SAR compounds in Table 3.4-1 (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602302" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID
|
|
602302</a>) and Table 3.4-2 (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602299" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID
|
|
602299</a>)</h5><p><b>Assay Overview</b>: The purpose of this assay is to determine
|
|
whether test compounds can inhibit DAGL-β in a complex proteome
|
|
lysate using a gel-based ABPP assay. In this assay, the target enzyme
|
|
DAGL-β is incubated with test compound followed by reaction with
|
|
a fluorescently-labeled activity-based probe, HT-01 [<a class="bibr" href="#ml294.r9" rid="ml294.r9">9</a>], which selectively
|
|
labels several serine hydrolases including DAGL-β. The reaction
|
|
products are analyzed as described for <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602303" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 602303</a>.</p><p><b>Protocol Summary</b>: Membrane proteome of transiently
|
|
transfected 293T Hek cells overexpressing mouse DAGL-β (25
|
|
μL of 0.3 mg/mL) in Dulbecco’s PBS (DPBS) was treated
|
|
with test compound (0.5 μL of a 50× stock in DMSO; 100
|
|
nM [<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602302" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 602302</a>] or 10 μM
|
|
[<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602299" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 602299</a>] final concentration)
|
|
or DMSO (0.5 μL) for 30 minutes at 37°C. The
|
|
activity-based probe HT-01 (0.5 μL of a 50× stock in
|
|
DMSO; 1 μM final concentration) was added, and the reaction was
|
|
incubated for 30 minutes at 37°C, quenched with an equal volume
|
|
of 2× SDS-PAGE loading buffer (reducing), separated by SDS-PAGE,
|
|
and visualized by in-gel fluorescent scanning. The percentage of
|
|
DAGL-β activity remaining was determined by measuring the
|
|
integrated optical density of the individual protein bands relative to
|
|
the DMSO-only (no compound) control. <b>Assay Cutoff</b>:
|
|
Compounds with greater than or equal to 50% inhibition were
|
|
considered active.</p></div><div id="ml294.s12"><h5>Gel-based competitive ABPP inhibition of DAGL-β and ABHD6
|
|
<i>in vitro</i>: assessment of enantiomeric potency of
|
|
KT116 (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/624472" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 624472</a>)</h5><p><b>Assay Overview:</b> The purpose of this assay is to determine
|
|
whether test compounds can inhibit DAGL-β and ABHD6 in a
|
|
gel-based competitive activity-based proteomic profiling (ABPP) assay.
|
|
In this assay, a complex proteome containing DAGL-β and ABHD6 is
|
|
incubated with test compound followed by reaction with a
|
|
fluorescently-labeled activity-based probe, HT-01 [<a class="bibr" href="#ml294.r9" rid="ml294.r9">9</a>], which selectively
|
|
labels several serine hydrolases including DAGL-β and ABHD6. The
|
|
reaction products are analyzed as described for <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602303" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID
|
|
602303</a>.</p><p><b>Protocol Summary:</b> Membrane proteome of Neuro-2A murine
|
|
neuroblastoma cells (25 μL of 1 mg/mL) in Dulbecco’s PBS
|
|
(DPBS) was treated with test compound (0.5 μL of a 50×
|
|
stock in DMSO; 1 nM, 10 nM, 100 nM, 1000 nM, or 10000 nM final
|
|
concentration) or DMSO (0.5 μL) for 30 minutes at 37 degrees
|
|
Celsius. The activity-based probe HT-01 (0.5 μL of a 50×
|
|
stock in DMSO; 1 μM final concentration) was added and the
|
|
reaction was incubated for 30 minutes at 37 degrees Celsius, quenched
|
|
with an equal volume of 2× SDS-PAGE loading buffer (reducing),
|
|
separated by SDS-PAGE, and visualized by in-gel fluorescent scanning.
|
|
The percentage of DAGL-β and ABHD6 activity remaining was
|
|
determined by measuring the integrated optical density of the individual
|
|
protein bands relative to the DMSO-only (no compound) control.</p></div><div id="ml294.s13"><h5>Gel-based competitive ABPP inhibition of overexpressed ABHD11
|
|
<i>in vitro</i> (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602301" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 602301</a>)</h5><p><b>Assay Overview</b>: The purpose of this assay is to determine
|
|
whether test compounds can inhibit anti-target ABHD11 in a complex
|
|
proteome lysate using a gel-based competitive ABPP assay. In this assay,
|
|
the anti-target enzyme ABHD11 is spiked into a complex proteome and
|
|
incubated with test compound followed by reaction with the ABPP probe
|
|
FP-Rh. The reaction products are analyzed as described for <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602303" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID
|
|
602303</a>.</p><p><b>Protocol Summary</b>: Membrane proteome of transiently
|
|
transfected 293T Hek cells overexpressing mouse DAGL-β (25
|
|
μL of 0.3 mg/mL; Open Biosystems Accession <a href="/nuccore/16359288" class="bk_tag" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=nuccore">BC016105</a>) with added
|
|
recombinant mouse ABHD11 (0.050 μM, Open Biosystems Accession
|
|
<a href="/nuccore/47682715" class="bk_tag" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=nuccore">BC069866</a>) in Dulbecco’s PBS (DPBS) was treated with test
|
|
compound (0.5 μL of a 50× stock in DMSO; 10 μM
|
|
final concentration) or DMSO (0.5 μL) for 30 minutes at
|
|
37°C. The activity-based probe FP-Rh (0.5 μL of a
|
|
50× stock in DMSO; 5 μM final concentration) was added,
|
|
and the reaction was incubated for 30 minutes at 37°C, quenched
|
|
with an equal volume of 2× SDS-PAGE loading buffer (reducing),
|
|
separated by SDS-PAGE, and visualized by in-gel fluorescent scanning.
|
|
The percentage of ABHD11 activity remaining was determined by measuring
|
|
the integrated optical density of the individual protein bands relative
|
|
to the DMSO-only (no compound) control. Note: DAGL-β inhibition
|
|
was not quantified in this experiment do to an overlapping protein band.
|
|
<b>Assay Cutoff</b>: Compounds with ≥50%
|
|
inhibition were considered active.</p></div><div id="ml294.s14"><h5>Gel-based competitive ABPP inhibition of overexpressed DAGL-α
|
|
<i>in vitro</i> (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602403" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 602403</a>)</h5><p><b>Assay Overview</b>: The purpose of this assay is to determine
|
|
whether test compounds can inhibit anti-target DAGL-α in a
|
|
complex proteome lysate using a gel-based competitive ABPP assay. In
|
|
this assay, the anti-target overexpressed DAGL-α is incubated
|
|
with test compound followed by reaction with the ABPP probe HT-01. The
|
|
reaction products are analyzed as described for <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602303" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID
|
|
602303</a>.</p><p><b>Protocol Summary</b>: Membrane proteome of transiently
|
|
transfected 293T Hek cells overexpressing mouse DAGL-α (50
|
|
μL of 2 mg/mL; Open Biosystems Accession <a href="/nuccore/159155871" class="bk_tag" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=nuccore">BC148308</a>) in
|
|
Dulbecco’s PBS (DPBS) was treated with test compound (1
|
|
μL of a 50× stock in DMSO; 10 μM, 2 μM,
|
|
0.4 μM, 0.08 μM, or 0.016 μM final
|
|
concentration) or DMSO (1 μL) for 30 minutes at 37°C.
|
|
The activity-based probe HT-01 (1 μL of a 50× stock in
|
|
DMSO; 1 μM final concentration) was added, and the reaction was
|
|
incubated for 30 minutes at 37°C, quenched with an equal volume
|
|
of 2× SDS-PAGE loading buffer (reducing), separated by SDS-PAGE,
|
|
and visualized by in-gel fluorescent scanning. The percentage of
|
|
DAGL-α activity remaining was determined by measuring the
|
|
integrated optical density of the individual protein bands relative to
|
|
the DMSO-only (no compound) control. <b>Assay Cutoff</b>:
|
|
Compounds with less than or equal to 50% inhibition at 0.4
|
|
μM compound concentration were considered active.</p></div><div id="ml294.s15"><h5>Gel-based competitive ABPP inhibition of SHs in mouse brain membrane
|
|
with FP-Rh (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602311" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 602311</a>) and HT-01 (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602323" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID
|
|
602323</a>)</h5><p><b>Assay Overview</b>: The purpose of this assay is to assay test
|
|
compound inhibition of serine hydrolases (SHs) in a complex proteome
|
|
lysate using a gel-based competitive ABPP assay. In this assay, a
|
|
complex proteome is incubated with test compound followed by reaction
|
|
with the ABPP probe FP-Rh or HT-01. The reaction products are analyzed
|
|
as described for <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602303" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 602303</a>.</p><p><b>Protocol Summary</b>: Mouse brain membrane proteome (25
|
|
μL of 1 mg/mL in Dulbecco’s PBS [DPBS])
|
|
was treated with test compound (0.5 μ of a 50× stock in
|
|
DMSO; 10 μM final concentration for FP-Rh assay
|
|
[<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602311" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 602311</a>], 1 μM final
|
|
concentration for HT-01 assay [<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602323" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 602323</a>])
|
|
or DMSO (0.5 μL) for 30 minutes at 37°C. The
|
|
activity-based probe FP-Rh (0.5 μL of a 50× stock in
|
|
DMSO; 1 μM final concentration) or HT-01 (0.5 μL of a
|
|
50× stock in DMSO; 1 μM final concentration) was added,
|
|
and the reaction was incubated for 30 minutes at 37°C, quenched
|
|
with an equal volume of 2× SDS-PAGE loading buffer (reducing),
|
|
separated by SDS-PAGE, and visualized by in-gel fluorescent scanning.
|
|
The percentage activity remaining for each SH was determined by
|
|
measuring the integrated optical density of the individual protein bands
|
|
relative to the DMSO-only (no compound) control. SHs with at least
|
|
50% inhibition by one or more compounds (FP-Rh: ABHD6, APEH,
|
|
FAAH, KIAA1363, ABHD12, MAGL, LYPLA1, and LYPLA2; HT-01: DAGL-β,
|
|
ABHD6, PLA2G7, and a SH with MW of 80kDa) are reported. <b>Assay
|
|
Cutoff</b>: Compounds with greater than or equal to 50%
|
|
inhibition for a given SH were considered active.</p></div><div id="ml294.s16"><h5>Gel-based competitive ABPP inhibition of DAGL-β for IC50
|
|
determination <i>in vitro</i> (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602320" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID
|
|
602320</a>)</h5><p><b>Assay Overview</b>: The purpose of this assay is to determine
|
|
the IC50 values of powder samples of test compounds for DAGL-β
|
|
inhibition in a complex proteome lysate using a competitive ABPP assay.
|
|
In this assay, the ABPP probe HT-01 is used to label DAGL-β in
|
|
the presence of test compounds. The reaction products are analyzed as
|
|
described for <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602303" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 602303</a>.</p><p><b>Protocol Summary</b>: Membrane proteome of transiently
|
|
transfected 293T Hek cells overexpressing mouse DAGL-β (25
|
|
μL of 0.3 mg/mL; Open Biosystems Accession <a href="/nuccore/16359288" class="bk_tag" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=nuccore">BC016105</a>) in
|
|
Dulbecco’s PBS (DPBS) was treated with test compound (0.5
|
|
μL of a 50× stock in DMSO) or DMSO (0.5 μL) for
|
|
30 minutes at 37°C. The activity-based probe HT-01 (0.5
|
|
μL of a 50× stock in DMSO; 1 μM final
|
|
concentration) was added, and the reaction was incubated for 30 minutes
|
|
at 37°C, quenched with an equal volume of 2× SDS-PAGE
|
|
loading buffer (reducing), separated by SDS-PAGE, and visualized by
|
|
in-gel fluorescent scanning. The percentage activity remaining for
|
|
DAGL-β was determined by measuring the integrated optical
|
|
density of the individual protein bands relative to the DMSO-only (no
|
|
compound) control. IC50 values for inhibition of DAGL-β were
|
|
determined from dose-response curves from three replicates at each
|
|
inhibitor concentration (10,000 nM and 8-point 1:3 dilution series from
|
|
1000 nM to 0.33 nM). <b>Assay Cutoff</b>: Compounds with an IC50
|
|
less than or equal to 500 nM were considered active.</p></div><div id="ml294.s17"><h5>Gel-based competitive ABPP inhibition of ABHD6 for IC50 determination
|
|
<i>in vitro</i> (AIDs <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602322" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">602322</a> and <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/624039" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">624039</a>)</h5><p><b>Assay Overview</b>: The purpose of this assay is to determine
|
|
the IC50 values of powder samples of test compounds for anti-target
|
|
ABHD6 inhibition in a complex proteome lysate using a competitive ABPP
|
|
assay. In this assay, the ABPP probe HT-01 is used to label ABHD6 in the
|
|
presence of test compounds. The reaction products are analyzed as
|
|
described for <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602303" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 602303</a>.</p><p><b>Protocol Summary</b>: Membrane proteome of Neuro-2A murine
|
|
neuroblastoma cells (25 μL of 1 mg/mL) in Dulbecco’s PBS
|
|
(DPBS) was treated with test compound (0.5 μL of a 50×
|
|
stock in DMSO) or DMSO (0.5 μL) for 30 minutes at 37°C.
|
|
The activity-based probe HT-01 (0.5 μL of a 50× stock in
|
|
DMSO; 1 μM final concentration) was added, and the reaction was
|
|
incubated for 30 minutes at 37°C, quenched with an equal volume
|
|
of 2× SDS-PAGE loading buffer (reducing), separated by SDS-PAGE,
|
|
and visualized by in-gel fluorescent scanning. The percentage activity
|
|
remaining for anti-target ABHD6 was determined by measuring the
|
|
integrated optical density of the individual protein bands relative to
|
|
the DMSO-only (no compound) control. IC50 values were determined from
|
|
dose-response curves from three replicates at each inhibitor
|
|
concentration (7-point 1:3 dilution series from either 3333 nM to 3.3
|
|
nM, 100 nM to 0.1 nM, or 33 nM to 0.033 nM). Note: inhibition of
|
|
DAGL-β at each test concentration is also reported. <b>Assay
|
|
Cutoff</b>: Compounds with an IC50 for ABHD6 less than or equal
|
|
to 50 nM were considered active.</p></div><div id="ml294.s18"><h5>Gel-based competitive ABPP inhibition of SHs anti-targets <i>in
|
|
vitro</i> (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602355" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 602355</a>)</h5><p><b>Assay Overview</b>: The purpose of this assay is to assay test
|
|
compound inhibition of SHs in a complex proteome using a gel-based
|
|
competitive ABPP assay. In this assay, a complex proteome is incubated
|
|
with test compound followed by reaction with the ABPP probe FP-Rh or
|
|
HT-01. The reaction products are analyzed as described for <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602303" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID
|
|
602303</a>.</p><p><b>Protocol Summary</b>: The following proteome sources, prepared
|
|
in Dulbecco’s PBS (DPBS), were used:
|
|
<b>DAGL-β</b>: Membrane proteome of transiently
|
|
transfected 293T Hek cells overexpressing mouse DAGL-β (25
|
|
μL of 0.3 mg/mL; Open Biosystems Accession <a href="/nuccore/16359288" class="bk_tag" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=nuccore">BC016105</a>).
|
|
<b>ABHD11</b>: recombinant mouse ABHD11 (0.050 μM,
|
|
Open Biosystems Accession <a href="/nuccore/47682715" class="bk_tag" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=nuccore">BC069866</a>) doped into membrane proteome of 293T
|
|
Hek cells (0.3 mg/mL). <b>PAFAH2</b>: Soluble proteome (1 mg/mL)
|
|
of BW5147-derived murine T-cells. <b>FAAH, MAGL, ABHD6, ABHD12,
|
|
LYPLA1, LYPLA2, PLA2G7, PNPLA6</b>: Mouse brain membrane proteome
|
|
(1 mg/mL). Proteome (25 μL) was treated with test compound (0.5
|
|
μL of a 50× stock in DMSO, 10, 2, 0.4, 0.08, 0.016, or
|
|
0.003 μM final concentration) or DMSO (0.5 μL) for 30
|
|
minutes at 37°C. The activity-based probe FP-Rh (0.5 μL
|
|
of a 50× stock in DMSO; 1 μM final concentration) or
|
|
HT-01 (0.5 μL of a 50× stock in DMSO; 1 μM final
|
|
concentration) was added. HT-01 was utilized to detect DAGL-β
|
|
and PLA2G7, for all other proteins, FP-Rh was used. The reaction was
|
|
incubated for 30 minutes at 37°C, quenched with an equal volume
|
|
of 2× SDS-PAGE loading buffer (reducing), separated by SDS-PAGE,
|
|
and visualized by in-gel fluorescent scanning. The percentage activity
|
|
remaining for each SH was determined by measuring the integrated optical
|
|
density of the individual protein bands relative to the DMSO-only (no
|
|
compound) control. <b>Assay Cutoff</b>: Compounds with greater
|
|
than or equal to 50% inhibition for a given SH at 2 μM
|
|
test compound concentration were considered active.</p></div><div id="ml294.s19"><h5>Gel-based competitive ABPP inhibition of DAGL-β and ABHD6 for
|
|
IC50 determination <i>in situ</i> (AIDs <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602354" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">602354</a> and <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602335" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">602335</a>)</h5><p><b>Assay Overview</b>: The purpose of this assay is to determine
|
|
the IC50 values of powder samples of test compounds for DAGL-β
|
|
and anti-target ABHD6 inhibition <i>in situ</i> using a
|
|
competitive ABPP assay. In this assay, the ABPP probe HT-01 is used to
|
|
label DAGL-β and ABHD6 in the presence of test compounds. The
|
|
reaction products are analyzed as described for <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602303" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID
|
|
602303</a>.</p><p><b>Protocol Summary</b>: Cultured Neuro-2A murine neuroblastoma
|
|
cells (5 mL DMEM medium with 10% FCS) were treated with test
|
|
compound (1 μL of 5000× stock in DMSO) or DMSO only (1
|
|
μL) and incubated for 4 hours at 37°C. Cells were washed
|
|
with DPBS (4x), harvested, and homogenized by sonication. The protein
|
|
concentration was adjusted to 2 mg/mL with Dulbecco’s PBS
|
|
(DPBS), and an aliquot (50 μL) was reacted with HT-01 (1
|
|
μL of a 50× stock in DMSO; 1 μM final
|
|
concentration) for 30 minutes at 37°C. Samples were quenched
|
|
with an equal volume of 2× SDS-PAGE loading buffer, separated by
|
|
SDS-PAGE and visualized by in-gel fluorescent scanning. The percentage
|
|
activity remaining was determined by measuring the integrated optical
|
|
density of the DAGL-β and anti-target ABHD6 bands relative to
|
|
the DMSO-only (no compound) control. IC50 values were determined from
|
|
dose-response curves from three replicates at each inhibitor
|
|
concentration (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602354" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 602354</a>: 9-point 1:3 dilution series
|
|
from 333 nM to 0.033 nM; <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602335" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 602335</a>: 8-point
|
|
1:3 dilution series from 100 nM to 0.033 nM). <b>Assay Cutoff</b>:
|
|
Compounds with an IC50 less than or equal to 50 nM were considered
|
|
active.</p></div><div id="ml294.s20"><h5>LCMS-based DAGL-β substrate (SAG) assay for IC50
|
|
determination <i>in vitro</i> (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/624468" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID
|
|
624468</a>)</h5><p><b>Assay Overview:</b> This substrate-based IC50 assay is based on
|
|
hydrolysis of an endogenous DAG substrate,
|
|
1-stearoyl-2-arachidonoylglycerol (SAG), and quantification of
|
|
2-arachidonoylglycerol (2-AG) production by LC-MS. In this assay,
|
|
compounds are preincubated with DAGL-β-transfected HEK293T cell
|
|
membranes for 30 min, followed by incubation with SAG substrate for 30
|
|
min. The production of 2-AG is quantified by LC-MS. As designed,
|
|
compounds that act as DAGL-β inhibitors will slow the rate of
|
|
enzyme hydrolysis, resulting in a decreased production of 2-AG.</p><p><b>Protocol Summary:</b> Membrane proteome of transiently
|
|
transfected 293T Hek cells overexpressing mouse DAGL-β (70
|
|
μL of 0.3 mg/mL; Open Biosystems Accession <a href="/nuccore/16359288" class="bk_tag" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=nuccore">BC016105</a>) in DAGL
|
|
buffer (5 mM CaCl2, 100 mM NaCl, 50 mM HEPES) was treated with test
|
|
compound (1.4 μL of a 50× stock in DMSO; 10 μM
|
|
final concentration) or DMSO (1.4 μL) for 30 minutes at 37
|
|
degrees Celsius. The diglyceride substrate, SAG, was added to sample
|
|
reaction (30 μL, 500 μM final [SAG]) and
|
|
incubated for 30 minutes at 37 degrees Celsius. The reaction was
|
|
quenched with 2:1 chloroform/methanol doped with C15:0 monoacylglycerol
|
|
(MAG) lipid standard (1 nmol per 300 μL of quenching solution).
|
|
The organic (bottom) layer was extracted 30 μl of the organic
|
|
phase was injected onto an Agilent 1100 series LC-MSD SL instrument for
|
|
analysis. LC separation was achieved with a Gemini reversed-phase C18
|
|
column (5 μm, 4.6 mm × 50 mm). Mobile phase A was
|
|
composed of 95:5 v/v H2O:MeOH, and mobile phase B was composed of
|
|
60:35:5 v/v/v i-PrOH:MeOH:H2O, each containing 0.1% formic acid.
|
|
The flow rate was 0.5 ml/min and the gradient consisted of 5 min
|
|
0% B, a linear increase to 100% B over 15 min, followed
|
|
by an isocratic gradient of 100% B for 12 min before
|
|
equilibrating for 5 min at 0% B (37 min total). MS analysis, in
|
|
scanning mode from scanning from m/z = 200–1200, was
|
|
performed with a positive electrospray ionization (ESI) source. The
|
|
hydrolysis product 2-AG (379 m/z) was quantified by measuring the area
|
|
under the peak in comparison with the C15:0 MAG standard (317 m/z). The
|
|
specific activity was calculated by measuring the pmoles of product 2-AG
|
|
formed per minute per mg of HEK293T-DAGL-β proteome used for
|
|
analysis. The % inhibition for each compound was determined by
|
|
comparing the specific activity of inhibitor-treated proteomes with
|
|
DMSO-treated proteomes. IC50 values were determined from two replicates
|
|
at each compound concentration (6 point dilution series starting at a
|
|
nominal concentration of 10 μM). <b>Assay Cutoff</b>:
|
|
Compounds with an IC50 less than or equal to 500 nM were considered
|
|
active.</p></div><div id="ml294.s21"><h5>Analysis of triazole urea cytotoxicity (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602337" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID
|
|
602337</a>)</h5><p><b>Assay Overview</b>: The purpose of this assay is to determine
|
|
cytotoxicity of powder samples of synthetic inhibitor compounds
|
|
belonging to the triazole urea scaffold. In this assay, Neuro-2A murine
|
|
neuroblastoma cells in either serum-free medium (Assay 1) or medium
|
|
containing fetal calf serum (FCS) (Assay 2) are incubated with test
|
|
compounds, followed by determination of cell viability. The assay
|
|
utilizes the WST-1 substrate which is converted into colorimetric
|
|
formazan dye by the metabolic activity of viable cells. The amount of
|
|
formed formazan directly correlates to the number of metabolically
|
|
active cells in the culture. As designed, compounds that reduce cell
|
|
viability will result in decreased absorbance of the dye.</p><p><b>Protocol Summary</b>: This assay was started by dispensing
|
|
Neuro-2A murine neuroblastoma cells in DMEM medium supplemented with
|
|
10% FCS (100 μL, 15,000 cells/well) into a 96-well
|
|
plate. Cells were incubated for 24 hours at 37°C in a humidified
|
|
incubator, medium was removed, and 100 μL of fresh, serum-free
|
|
medium (Assay 1) or medium supplemented with 10% FCS (Assay 2)
|
|
was added. Compound (10 μL of 11× stocks in medium
|
|
containing 10% DMSO) or an equal volume medium containing
|
|
10% DMSO only was added to each well. Cells were incubated for
|
|
48 hours at 37°C in a humidified incubator and cell viability
|
|
was determined by the WST-1 assay (Roche) according to manufacturer
|
|
instructions. CC50 values were determined from dose-response curves from
|
|
six replicates at each inhibitor concentration (100, 50, 10, 1, 0.1, and
|
|
0.01 μM). <b>Assay Cutoff</b>: Compounds with CC50 values
|
|
less than or equal to 1 μM were considered active
|
|
(cytotoxic).</p></div><div id="ml294.s22"><h5>LC-MS/MS-based ABPP-SILAC analysis of selectivity <i>in
|
|
situ</i> for <a href="/pcsubstance/?term=ML294[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML294</a> (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602339" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID
|
|
602339</a>) and <a href="/pcsubstance/?term=ML295[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML295</a> (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602341" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID
|
|
602341</a>)</h5><p><b>Assay Overview</b>: The purpose of this assay is to determine
|
|
the selectivity profile of powder samples of test compounds using ABPP
|
|
in combination with stable isotope labeling with amino acids in cell
|
|
culture (SILAC) [<a class="bibr" href="#ml294.r10" rid="ml294.r10">10</a>] as described [<a class="bibr" href="#ml294.r9" rid="ml294.r9">9</a>, <a class="bibr" href="#ml294.r11" rid="ml294.r11">11</a>]. In this assay, cultured Neuro-2A cells are
|
|
metabolically labeled with heavy or light amino acids. Heavy and light
|
|
cells are treated with test compound and DMSO, respectively, <i>in
|
|
situ</i>. Cells are lysed, proteomes are treated with the
|
|
serine-hydrolase-specific activity-based fluorophosphonate-biotin
|
|
(FP-biotin, [<a class="bibr" href="#ml294.r12" rid="ml294.r12">12</a>]) affinity probe, and combined in a 1:1 (w/w) ratio.
|
|
Biotinylated proteins are enriched, trypsinized, and analyzed by
|
|
multi-dimensional liquid chromatography tandem mass spectrometery
|
|
LC/LC-MS/MS (MudPIT) [<a class="bibr" href="#ml294.r13" rid="ml294.r13">13</a>, <a class="bibr" href="#ml294.r14" rid="ml294.r14">14</a>]. Inhibition of target and anti-target activity is
|
|
quantified by comparing intensities of light and heavy peptide peaks. As
|
|
designed, compounds that act as inhibitors will block FP-biotin
|
|
labeling, reducing enrichment in the inhibitor-treated (heavy) sample
|
|
relative to the DMSO-treated (light) sample, giving a smaller
|
|
heavy/light ratio for each protein. Proteins not targeted by inhibitors
|
|
would be expected to have a ratio of 1.</p><p><b>Protocol Summary</b>: <i>Sample Preparation</i>.
|
|
Neuro-2A murine neuroblastoma cells were initially grown for 10 passages
|
|
in either light or heavy SILAC DMEM medium supplemented with 10%
|
|
dialyzed FCS and 2 mM L-glutamine. Light medium was supplemented with
|
|
100 μg/mL L-arginine and 100 ug/mL L-lysine. Heavy medium was
|
|
supplemented with 100 μg/mL
|
|
[<sup>13</sup>C<sub>6</sub><sup>15</sup>N<sub>4</sub>]-L-Arginine
|
|
and 100 μg/mL
|
|
[<sup>13</sup>C<sub>6</sub><sup>15</sup>N<sub>2</sub>]-L-Lysine.
|
|
Heavy cells (in 10 mL medium) were treated with test compound (10
|
|
μL of a 1000× stock in DMSO; 25 nM final concentration)
|
|
and light cells were treated with DMSO (10 μL) for 4 hours at
|
|
37°C. Cells were washed with DPBS (4x), harvested, and
|
|
homogenized by sonication in DPBS. The soluble and membrane fractions
|
|
were isolated by centrifugation (100K × g, 45 minutes) and the
|
|
protein concentration for each fraction was adjusted to 2 mg/mL with
|
|
DPBS. The light and heavy proteomes were labeled with the activity-based
|
|
affinity probe FP-biotin (500 μL total reaction volume, 10
|
|
μM final concentration) for 2 hours at 25°C. After
|
|
incubation, light and heavy proteomes were mixed in 1:1 ratio, and the
|
|
membrane proteomes were additionally solubilized with 1%
|
|
Triton-X100. Samples were desalted over PD10 columns (GE Healthcare) in
|
|
DPBS, and biotinylated proteins enriched with streptavidin beads (50
|
|
μL beads; conditions: 1 hour, 25°C 0.5% SDS in
|
|
DPBS). The beads were washed with 1% SDS in DPBS (1x), 6 M urea
|
|
(1x), and DPBS (2x), then resuspended in in 6 M urea (150 μL),
|
|
reduced with 5 mM TCEP for 20 minutes, and alkylated with 10 mM
|
|
iodoacetamide for 30 minutes at 25°C in the dark. The urea
|
|
concentration was reduced to 2 M with 2× volume DPBS. On-bead
|
|
digestions were performed for 12 hours at 37°C with
|
|
sequence-grade modified trypsin (Promega; 2 μg) in the presence
|
|
of 2 mM CaCl<sub>2</sub>. Peptide samples were acidified to a final
|
|
concentration of 5% (v/v) formic acid and stored at
|
|
−80°C prior to analysis.</p><p><i>LC-MS/MS analysis</i>. Samples were analyzed by
|
|
multidimensional liquid chromatography tandem mass spectrometry (MudPIT)
|
|
using an Agilent 1200-series quaternary pump and Thermo Scientific
|
|
LTQ-Orbitrap Velos ion trap mass spectrometer. Peptides were eluted in a
|
|
5-step MudPIT experiment using 0%, 25%, 50%,
|
|
80%, and 100% salt bumps of 500 mM aqueous ammonium
|
|
acetate and data were collected in data-dependent acquisition mode with
|
|
dynamic exclusion turned on (20 seconds, repeat of 1). Specifically, one
|
|
full MS (MS1) scan (400–1800 m/z) was followed by 30 MS2 scans
|
|
of the most abundant ions. The MS2 spectra data were extracted from the
|
|
raw file using RAW Xtractor (version 1.9.9.2; publicly available at
|
|
<a href="http://fields.scripps.edu/researchtools.php" ref="pagearea=body&targetsite=external&targetcat=link&targettype=uri">http://fields.scripps.edu/researchtools.php</a>). MS2
|
|
spectra data were searched using the ProLuCID algorithm (publicly
|
|
available at <a href="http://fields.scripps.edu/downloads.php" ref="pagearea=body&targetsite=external&targetcat=link&targettype=uri">http://fields.scripps.edu/downloads.php</a>) against the
|
|
latest version of the mouse IPI database concatenated with the reversed
|
|
database for assessment of false-discovery rates. ProLucid searches
|
|
allowed for static modification of cysteine residues (+57.02146
|
|
due to alkylation), methionine oxidation (+15.9949), mass shifts
|
|
of labeled amino acids (+10.0083 R, +8.0142 K) and no
|
|
enzyme specificity. The resulting MS2 spectra matches were assembled
|
|
into protein identifications and filtered using DTASelect (version
|
|
2.0.41, <a href="http://fields.scripps.edu/researchtools.php" ref="pagearea=body&targetsite=external&targetcat=link&targettype=uri">http://fields.scripps.edu/researchtools.php</a>) using the
|
|
--modstat, --mass, and --trypstat options (applies different statistical
|
|
models for the analysis of high resolution masses, peptide digestion
|
|
state, and methionine oxidation state respectively). Ratios of
|
|
heavy/light (test compound/DMSO) peaks were calculated using in-house
|
|
software and normalized at the peptide level to the average ratio of all
|
|
non-serine hydrolase peptides. Reported ratios represent the mean of all
|
|
unique, quantified peptides per protein and do not include peptides that
|
|
were >3 standard deviations from the median peptide value.
|
|
Proteins with less than three peptides per protein ID were not included
|
|
in the analysis. <b>Assay Cutoff</b>: A compound was considered
|
|
active for a particular target/anti-target with a light/heavy ratio of
|
|
less than or equal to 0.5.</p></div><div id="ml294.s23"><h5>Gel-based competitive ABPP inhibition of DAGL-β and ABHD6
|
|
<i>in vivo</i> (AIDs <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602347" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">602347</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602345" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">602345</a>, and <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602343" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">602343</a>)</h5><p><b>Assay Overview</b>: The purpose of this assay is to determine
|
|
whether or not powder samples of test compounds are active <i>in
|
|
vivo</i> (brain [<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602347" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 602347</a>]
|
|
and macrophages [<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602345" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">602345</a>])
|
|
using a competitive ABPP assay. In this assay, test compounds are
|
|
administered to mice. Mice are sacrificed, and their brain tissue and
|
|
peritoneal macrophages harvested, homogenized, and the membrane fraction
|
|
isolated and reacted with either the ABPP probe FP-Rh or HT-01. The
|
|
reaction products are analyzed as described for <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602303" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID
|
|
602303</a> and in ref. [<a class="bibr" href="#ml294.r9" rid="ml294.r9">9</a>].</p><p><b>Protocol Summary</b>: Purpose-bred C57-black laboratory mice
|
|
were administered test compound either by intraperitonial injection
|
|
(i.p.) (AIDs <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602347" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">602347</a> and <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602345" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">602345</a>; 20, 10, or 5 mg/kg; n = 2 per group) or
|
|
oral administration (p.o.) (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602343" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 602343</a>; 20, 10,
|
|
or 5 mg/kg; n = 1 per group) or vehicle only (18:1:1
|
|
saline:PEG300:EtOH). Note: for macrophage analysis, mice were injected 4
|
|
days prior with thioglycollate. After 4 hours, mice were humanely
|
|
sacrificed (anesthetized with isoflurane and decapitated) and tissue
|
|
(brain or peritoneal macrophages) was removed and snap frozen in liquid
|
|
nitrogen. Tissues were homogenized and the membrane fraction isolated by
|
|
centrifugation (45 min, 100K × g) and adjusted to 1 mg/mL in
|
|
DPBS. Aliquots (50 μL) were reacted with the activity-based
|
|
probes HT-01 or FP-Rh (1 μL of a 50× stock in DMSO, 1
|
|
μM final concentration) for 30 minutes at 37°C. The
|
|
reactions were quenched with an equal volume of 2× SDS-PAGE
|
|
loading buffer (reducing), separated by SDS-PAGE and visualized by
|
|
in-gel fluorescent scanning. The percentage activity remaining was
|
|
determined by measuring the integrated optical density of test compound
|
|
bands relative to vehicle bands. For this assay, anti-target ABHD6 was
|
|
used as a diagnostic for observing inhibition due to the weak and
|
|
diffuse nature of the DAGL-β band. To assess selectivity, the
|
|
number of other anti-targets (with at least 50% inhibition) for
|
|
each ABPP probe is reported. <b>Assay Cutoff</b>: Compounds with
|
|
greater than or equal to 50% inhibition at 5 mg/kg compound
|
|
(i.p. or 10 mg/kg compound (p.o.) and fewer than 2 other anti-targets
|
|
were considered active.</p></div><div id="ml294.s24"><h5>LC-MS/MS-based ABPP-MudPIT analysis of selectivity <i>in
|
|
vivo</i> for <a href="/pcsubstance/?term=ML294[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML294</a> (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602353" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID
|
|
602353</a>) and <a href="/pcsubstance/?term=ML295[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML295</a> (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602351" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID
|
|
602351</a>)</h5><p><b>Assay Overview</b>: The purpose of this assay is to determine
|
|
the selectivity profile of powder samples of test compounds <i>in
|
|
vivo</i> by ABPP in combination with the multidimensional
|
|
protein identification technology (MudPIT) liquid chromatography tandem
|
|
mass spectrometry (LC-M/MS) analysis platform [<a class="bibr" href="#ml294.r9" rid="ml294.r9">9</a>, <a class="bibr" href="#ml294.r15" rid="ml294.r15">15</a>]. In this assay, mice are
|
|
administered test compound or vehicle only. Mice are sacrificed, and
|
|
peritoneal macrophages removed, lysed, and reacted with the serine
|
|
hydrolase specific activity-based fluorophosphonate-biotin (FP-biotin)
|
|
affinity probe. Biotinylated proteins are enriched, trypsinized, and
|
|
analyzed by MudPIT. Inhibition of target and anti-target activity is
|
|
quantified by comparing spectra counts between the test compound- and
|
|
DMSO-treated samples. As designed, compounds that act as inhibitors will
|
|
block FP-biotin labeling, reducing enrichment in the inhibitor-treated
|
|
sample relative to the DMSO-treated sample, thus giving a lower number
|
|
of smaller spectra counts in the inhibitor treated sample. Proteins not
|
|
targeted by inhibitors would be expected to have a spectra count ratio
|
|
similar to DMSO control.</p><p><b>Protocol Summary</b>: <i>Sample Preparation.</i>
|
|
Purpose-bred C57-black laboratory mice, injected 4 days prior with
|
|
thioglycollate, were administered test compound (5 mg/kg in 18:1:1
|
|
saline:PEG300:EtOH vehicle solution, i.p.) or vehicle only (n=3
|
|
per group). After 4 hours, mice were humanely sacrificed (anesthetized
|
|
with isoflurane and decapitated) and peritoneal macrophages were removed
|
|
and snap frozen in liquid nitrogen. Tissues were homogenized and the
|
|
protein concentration for each lysate was adjusted to 2 mg/mL with DPBS.
|
|
The macrophage proteomes were labeled with the activity-based affinity
|
|
probe FP-biotin (500 μL total reaction volume, 10 μM
|
|
final concentration) for 2 hours at 25°C. After incubation, the
|
|
proteomes were additionally solubilized with 1% Triton-X100.
|
|
Samples were desalted over PD10 columns (GE Healthcare) in DPBS, and
|
|
biotinylated proteins enriched with streptavidin beads (50 μL
|
|
beads; conditions: 1 hour, 25°C 0.5% SDS in DPBS). The
|
|
beads were washed with 1% SDS in DPBS (1x), 6 M urea (1x), and
|
|
DPBS (2x), then resuspended in 6 M urea (150 μL), reduced with 5
|
|
mM TCEP for 20 minutes, and alkylated with 10 mM iodoacetamide for 30
|
|
minutes at 25°C in the dark. The urea concentration was reduced
|
|
to 2 M with 2× volume DPBS. On-bead digestions were performed
|
|
for 12 hours at 37°C with sequence-grade modified trypsin
|
|
(Promega; 2 μg) in the presence of 2 mM CaCl<sub>2</sub>.
|
|
Peptide samples were acidified to a final concentration of 5%
|
|
(v/v) formic acid and stored at −80°C prior to
|
|
analysis.</p><p><i>LC-MS/MS analysis.</i> Digested and acidified peptide
|
|
mixtures were analyzed by two-dimensional liquid chromatography/tandem
|
|
mass spectrometry (MudPIT) using an Agilent 1200-series quaternary pump
|
|
and Thermo Scientific LTQ ion trap mass spectrometer. Peptides were
|
|
eluted in a 5-step MudPIT experiment using 0%, 25%,
|
|
50%, 80%, and 100% salt bumps of 500 mM aqueous
|
|
ammonium acetate, with chromatographic elution following each salt pulse
|
|
accomplished using an increasing gradient of aqueous acetonitrile
|
|
containing 0.1% formic acid over 125 minutes. Data were
|
|
collected in data-dependent acquisition mode with dynamic exclusion
|
|
turned on (90 s, repeat of 1). Specifically, one full MS (MS1) scan
|
|
(400–1800 m/z) was followed by 7 MS2 scans of the most abundant
|
|
ions. The MS2 spectra data were extracted from the raw file using RAW
|
|
Xtractor (version 1.9.1; publically available at <a href="http://fields.scripps.edu/downloads.php" ref="pagearea=body&targetsite=external&targetcat=link&targettype=uri">http://fields.scripps.edu/downloads.php</a>). MS2 spectra
|
|
data were searched using the PROLUCID algorithm (publically available at
|
|
<a href="http://fields.scripps.edu/downloads.php" ref="pagearea=body&targetsite=external&targetcat=link&targettype=uri">http://fields.scripps.edu/downloads.php</a>) against the
|
|
latest version of the mouse UniProt database concatenated with the
|
|
reversed database for assessment of false-discovery rates. PROLUCID
|
|
searches allowed for variable oxidation of methionine (+16),
|
|
static modification of cysteine (+57 due to alkylation), and no
|
|
enzyme specificity. The resulting MS2 spectra matches were assembled
|
|
into protein identifications and filtered using DTASelect (version
|
|
2.0.41) using the –trypstat and –modstat options, which
|
|
apply different statistical models for the analysis of tryptic,
|
|
half-tryptic, non-tryptic, and modified peptides. Spectra count
|
|
abundance ratios (test compound/DMSO) were calculated for all serine
|
|
hydrolases with an average of 10 or more spectra counts in the DMSO
|
|
control samples, as well as for anti-target ABHD6, which had fewer
|
|
spectra counts than the 10-count cutoff. A Student’s T-test
|
|
(unpaired, equal variances) was utilized to determine statistical
|
|
significance between the average spectra counts in compound vs. DMSO
|
|
samples. <b>Assay Cutoff</b>: A compound was considered active for
|
|
a particular target/anti-target if there was a statistical difference
|
|
between the average spectra counts in the test compound- and
|
|
DMSO-treated samples (P < 0.01).</p></div></div></div><div id="ml294.s25"><h3>2.2. Probe Chemical Characterization</h3><div id="ml294.fu2" class="figure bk_fig"><div class="graphic"><img src="/books/NBK133443/bin/ml294fu2.jpg" alt="ML294 SID 125269120 CID 53364485." /></div><h3><span class="title"><a href="/pcsubstance/?term=ML294[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML294</a><br /><a href="https://pubchem.ncbi.nlm.nih.gov/substance/125269120" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID
|
|
125269120</a><br />CID 53364485</span></h3></div><p>The probe structure was verified by <sup>1</sup>H NMR (see <a href="#ml294.s26">Section 2.3</a>) and high resolution MS
|
|
(<i>m/z</i> calculated for
|
|
C<sub>28</sub>H<sub>29</sub>N<sub>4</sub>O<sub>2</sub>
|
|
[M+H]<sup>+</sup>: 453.2285, found
|
|
453.2297). Purity was assessed to be greater than 95% by NMR. See
|
|
<a href="/pcsubstance/?term=ML295[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML295</a> Report for synthesis and structural
|
|
characterization of the anti-probe <a href="/pcsubstance/?term=ML295[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML295</a>.</p><p>It should be noted that <a href="/pcsubstance/?term=ML294[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML294</a> was synthesized in racemic form, and
|
|
the independent activity of each enantiomer has not yet been tested. However, we
|
|
have resolved by chiral HPLC the respective enantiomers of the related analog
|
|
KT116 (<a href="https://pubchem.ncbi.nlm.nih.gov/substance/125269114" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 125269114</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/624472" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 624472</a>). Independent
|
|
assessment of potency revealed the (−)enantiomer (<a href="https://pubchem.ncbi.nlm.nih.gov/substance/136946704" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID
|
|
136946704</a>) to be approximately 100× more potent than the
|
|
(+)enantiomer (<a href="https://pubchem.ncbi.nlm.nih.gov/substance/136946703" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 136946703</a>) (~50% inhibition of
|
|
DAGL-β achieved at 100 nM vs. 10 μM compound concentration). As
|
|
expected, the racemic form (<a href="https://pubchem.ncbi.nlm.nih.gov/substance/125269114" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 125269114</a>) is slightly
|
|
less potent (~2-fold) than (−)KT116, suggesting that the combination of
|
|
the two enantiomers does not give rise to any synergistic or antagonistic
|
|
inhibitory effects. Given the structural similarity of KT116 and <a href="/pcsubstance/?term=ML294[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML294</a>, it is anticipated that the respective enantiomers of the
|
|
Probe will have analogous properties.</p><p>Solubility (room temperature) for <a href="/pcsubstance/?term=ML294[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML294</a> and <a href="/pcsubstance/?term=ML295[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML295</a> was determined to be <1 μM in PBS and DMEM
|
|
medium alone, but much higher (6.4 μM and 3.2 μM, respectively)
|
|
in DMEM medium containing 10% fetal calf serum. The latter buffer
|
|
conditions are more relevant to biological experiments (complex proteome or
|
|
cell-based studies in serum-containing medium), and we have not found the modest
|
|
solubility to be a hindrance for biological application. Stability in PBS for
|
|
<a href="/pcsubstance/?term=ML294[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML294</a> and <a href="/pcsubstance/?term=ML295[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML295</a> was determined to be >48 hours.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml294t1"><a href="/books/NBK133443/table/ml294.t1/?report=objectonly" target="object" title="Table 1" class="img_link icnblk_img figpopup" rid-figpopup="figml294t1" rid-ob="figobml294t1"><img class="small-thumb" src="/books/NBK133443/table/ml294.t1/?report=thumb" src-large="/books/NBK133443/table/ml294.t1/?report=previmg" alt="Table 1. Compounds submitted to the SMR collection (04-09-2012)." /></a><div class="icnblk_cntnt"><h4 id="ml294.t1"><a href="/books/NBK133443/table/ml294.t1/?report=objectonly" target="object" rid-ob="figobml294t1">Table 1</a></h4><p class="float-caption no_bottom_margin">Compounds submitted to the SMR collection (04-09-2012). </p></div></div></div><div id="ml294.s26"><h3>2.3. Probe Preparation</h3><div id="ml294.fu3" class="figure"><div class="graphic"><img src="/books/NBK133443/bin/ml294fu3.jpg" alt="Image ml294fu3" /></div></div><p><b>Synthesis of Compound 8 (KT116):</b> A solution of racemic 2-benzyl
|
|
piperidine (<b>A</b>, 0.32 g, 1.8 mmol) in THF (15 ml) was treated with
|
|
iPr<sub>2</sub>NEt (0.95 ml, 5.4 mmol) and triphosgene (0.27 g, 0.9 mmol),
|
|
and the reaction mixture was stirred for 30 minutes at 4°C. The mixture
|
|
was poured into water and extracted with ethyl acetate. The organic layer was
|
|
washed with water and brine, dried over Na<sub>2</sub>SO<sub>4</sub> and
|
|
concentrated under reduced pressure. The intermediate was dissolved in THF (20
|
|
ml), and iPr<sub>2</sub>NEt (0.95 ml, 5.4 mmol), DMAP (218 mg, 1.8 mmol) and
|
|
4-(4-bromophenyl)-1H-1,2,3-triazole (0.40 g, 1.8 mmol) were added to the
|
|
solution. The mixture was stirred for 2 hours at 60°C and poured into
|
|
saturated aqueous NH<sub>4</sub>Cl solution. The mixture was extracted with
|
|
ethyl acetate, washed with water and brine, dried over
|
|
Na<sub>2</sub>SO<sub>4</sub> and concentrated under reduced pressure.
|
|
Chromatography (70 g, ethyl acetate:hexane=1:6~1:5) afforded KT116 (320
|
|
mg, 42%). <sup>1</sup>H NMR (CDCl<sub>3</sub>, 400 MHz) δ
|
|
7.72-7.54 (m, 4H), 7.45-6.89 (m, 6H), 5.29 (br, 1H), 4.34 (brd, 1H, J =
|
|
13.5 Hz), 3.42-3.10 (m, 2H), 2.67 (br, 1H), 2.04-1.60 (m, 6H). HRMS calculated
|
|
for C<sub>21</sub>H<sub>22</sub>BrN<sub>4</sub>O
|
|
[M+H]<sup>+</sup> 425.0971, found 425.0976.
|
|
The triazole substitution of KT116 as the 1,4-isomer was determined by X-ray
|
|
crystallography, as reported in ref. [<a class="bibr" href="#ml294.r9" rid="ml294.r9">9</a>].</p><div id="ml294.fu4" class="figure"><div class="graphic"><img src="/books/NBK133443/bin/ml294fu4.jpg" alt="Image ml294fu4" /></div></div><p><b>Synthesis of <a href="/pcsubstance/?term=ML294[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML294</a> (KT172):</b> A solution of
|
|
KT116 (30 mg, 0.071 mmol) in dioxane (2 ml) and water (0.1 ml) was treated with
|
|
phenyl bronic acid (17 mg, 0.13 mmol), K<sub>2</sub>CO<sub>3</sub> (30 mg, 0.22
|
|
mmol) and PdCl<sub>2</sub>(dppf) (8 mg, 0.011mmol), and the reaction mixture was
|
|
stirred for 2 hours at 80°C under N<sub>2</sub>. The mixture was poured
|
|
into water and extracted with ethyl acetate. The organic layer was washed with
|
|
water and brine, dried over Na<sub>2</sub>SO<sub>4</sub> and concentrated under
|
|
reduced pressure. The residue was purified by pTLC (ethyl
|
|
acetate:hexane=1:4) to afford <a href="/pcsubstance/?term=ML294[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML294</a> (27 mg,
|
|
85%).<sup>1</sup>H NMR (CDCl<sub>3</sub>, 400 MHz) δ 7.84
|
|
(br, 2H), 7.63 (d, 2H, J = 8.4 Hz), 7.50-6.95 (m, 9H), 4.87 (br, 1H),
|
|
4.37 (brd, 1H, J = 13.8 Hz), 3.42-3.10 (m, 2H), 2.71 (br, 1H), 2.03-1.65
|
|
(m, 6H). Purity > 95% by NMR. HRMS calculated for
|
|
C<sub>28</sub>H<sub>29</sub>N<sub>4</sub>O<sub>2</sub>
|
|
[M+H]<sup>+</sup> 453.2285, found
|
|
453.2297.</p></div></div><div id="ml294.s27"><h2 id="_ml294_s27_">3. Results</h2><div id="ml294.s28"><h3>3.1. Dose Response Curves for Probe</h3><p>Target and anti-target IC50 values for <a href="/pcsubstance/?term=ML294[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML294</a> and anti-probe
|
|
<a href="/pcsubstance/?term=ML295[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML295</a> were obtained from gel-based
|
|
competitive-ABPP data using the HT-01 activity-based probe [<a class="bibr" href="#ml294.r9" rid="ml294.r9">9</a>] both <i>in
|
|
vitro</i> and <i>in situ</i> (<a class="figpopup" href="/books/NBK133443/figure/ml294.f1/?report=objectonly" target="object" rid-figpopup="figml294f1" rid-ob="figobml294f1">Figure 1</a>; see caption for AID list). <a href="/pcsubstance/?term=ML294[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML294</a> showed high potency for both DAGL-β and
|
|
anti-target ABHD6 <i>in vitro</i> (IC50 values of 56 nM and ~1 nM,
|
|
respectively) and <i>in situ</i> (IC50 values of 12 nM and 0.48 nM,
|
|
respectively). In comparison, anti-probe <a href="/pcsubstance/?term=ML295[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML295</a> has IC50 values
|
|
for ABHD6 of 38 nM <i>in vitro</i> and 1.3 nM <i>in situ</i>
|
|
and high selectivity vs. DAGL-β (>75-fold). One of the primary
|
|
reasons <a href="/pcsubstance/?term=ML295[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML295</a> was chosen as the ABHD6 control
|
|
anti-probe was its similar potency for ABHD6 as compared to that of <a href="/pcsubstance/?term=ML294[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML294</a> for DAGL-β (IC50s of 38 vs. 56 nM <i>in
|
|
vitro</i>, 1.3 vs. 12 nM <i>in situ</i>). This property
|
|
allows administration of the compounds at comparable doses in biological
|
|
experiments, thus controlling for variables related to the overall amount of
|
|
compound introduced to the system.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml294f1" co-legend-rid="figlgndml294f1"><a href="/books/NBK133443/figure/ml294.f1/?report=objectonly" target="object" title="Figure 1" class="img_link icnblk_img figpopup" rid-figpopup="figml294f1" rid-ob="figobml294f1"><img class="small-thumb" src="/books/NBK133443/bin/ml294f1.gif" src-large="/books/NBK133443/bin/ml294f1.jpg" alt="Figure 1. IC50 curves for probe ML294 (SID 125269120) and anti-probe ML295 (SID 125269138)." /></a><div class="icnblk_cntnt" id="figlgndml294f1"><h4 id="ml294.f1"><a href="/books/NBK133443/figure/ml294.f1/?report=objectonly" target="object" rid-ob="figobml294f1">Figure 1</a></h4><p class="float-caption no_bottom_margin">IC50 curves for probe ML294 (SID
|
|
125269120) and anti-probe ML295 (SID 125269138). As
|
|
determined by gel-based competitive-ABPP with the activity-based probe
|
|
HT-01 against overexpressed DAGL-β in a complex proteome lysate
|
|
(left panels) and in cultured <a href="/books/NBK133443/figure/ml294.f1/?report=objectonly" target="object" rid-ob="figobml294f1">(more...)</a></p></div></div></div><div id="ml294.s29"><h3>3.2. Cellular Activity</h3><p><b><i>In situ Inhibition:</i></b> Probe <a href="/pcsubstance/?term=ML294[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML294</a> (<a href="https://pubchem.ncbi.nlm.nih.gov/substance/125269120" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 125269120</a>) and anti-probe <a href="/pcsubstance/?term=ML295[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML295</a> (<a href="https://pubchem.ncbi.nlm.nih.gov/substance/125269138" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 125269138</a>) are active <i>in
|
|
situ</i> (AIDs <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602354" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">602354</a> and <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602335" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">602335</a>) as assessed by
|
|
gel-based competitive ABPP following 4 hours of compound incubation with
|
|
Neuro-2A murine neuroblastoma cells (cultured in medium containing 10%
|
|
fetal calf serum) [<a class="bibr" href="#ml294.r9" rid="ml294.r9">9</a>].</p><p><b><i>In vivo Inhibition:</i></b> We also assessed the <i>in
|
|
vivo</i> inhibitory activity of probe <a href="/pcsubstance/?term=ML294[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML294</a> (<a href="https://pubchem.ncbi.nlm.nih.gov/substance/125269120" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 125269120</a>). For this experiment, mice
|
|
(n=2 per group) were administered test compound (5, 10 or 20 mg/kg,
|
|
i.p.) or vehicle only. After 4 hours, mice were sacrificed and their brain
|
|
tissue removed. The membrane fraction was isolated and subject to gel-based
|
|
competitive ABPP with either HT-01 or FP-Rh. The DAGL-β target band is
|
|
not visible under these conditions, as such, inhibition of anti-target ABHD6 a
|
|
key diagnostic of <i>in vivo</i> inhibitory activity in the brain. A
|
|
near-complete inhibition of ABHD6 is observed for <a href="/pcsubstance/?term=ML294[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML294</a> at all test concentrations.</p><p><b><i>Cytotoxicity:</i></b> The probe <a href="/pcsubstance/?term=ML294[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML294</a> (<a href="https://pubchem.ncbi.nlm.nih.gov/substance/125269120" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 125269120</a>) and anti-probe <a href="/pcsubstance/?term=ML295[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML295</a> (<a href="https://pubchem.ncbi.nlm.nih.gov/substance/125269138" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 125269138</a>) were evaluated for cytotoxicity
|
|
(<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602337" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID
|
|
602337</a>) in Neuro-2A murine neuroblastoma cells cultured in both
|
|
serum-free and serum-supplemented medium. The results of the two experiments
|
|
were quite different, with the serum-free conditions yielding 20-fold elevated
|
|
values (CC50s >100 μM) vs. the serum-supplemented experiment
|
|
(CC50s of 3–6 μM). Solubility for all compounds was determined
|
|
to be <1 μM in DMEM medium alone, but markedly improved in medium
|
|
supplemented with 10% serum (6.4 μM for <a href="/pcsubstance/?term=ML294[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML294</a> and 3.2 μM for <a href="/pcsubstance/?term=ML295[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML295</a>).</p></div><div id="ml294.s30"><h3>3.3. Profiling Assays</h3><p><b>Gel-based Competitive ABPP:</b> This medium-throughput proteome-wide
|
|
screening technique was instrumental in our medchem optimization of the probe
|
|
compound, allowing rapid assessment of potency and selectivity among SHs
|
|
(including lipases, esterases, proteases, and uncharacterized hydrolases), as
|
|
visualized by disappearance of bands in compound-treated lanes relative to the
|
|
DMSO-only control. It should be noted that gel-based profiling gives us access
|
|
to fairly low abundant proteins. For example, we have previously determined that
|
|
we could detect the SH fatty acid amide hydrolase (FAAH) at concentrations as
|
|
low as 0.0005% of the total proteome (~200 copies per cell) by gel-based
|
|
ABPP [<a class="bibr" href="#ml294.r16" rid="ml294.r16">16</a>].</p><p><b>HTS Analysis.</b> No HTS activity data are yet available for probe
|
|
<a href="/pcsubstance/?term=ML294[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML294</a> or any of the synthetic analogs, nor
|
|
have the probe compounds and analogs been submitted for commercial or
|
|
non-commercial broad panel screening. Rather, the reactivity of the triazole
|
|
urea compound class has been extensively characterized using competitive ABPP
|
|
methods, as described below.</p><p><b>Reactivity of triazole
|
|
ureas</b><b><i>outside</i></b><b>the SH enzyme
|
|
class.</b> As detailed in the Probe Report for <a href="/pcsubstance/?term=ML225[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML225</a> and in ref. [<a class="bibr" href="#ml294.r11" rid="ml294.r11">11</a>], we previously completed an analysis of triazole urea
|
|
reactivity outside of the SH class by gel-based competitive ABPP using
|
|
alkyne-functionalized triazole urea compounds. For these experiments, we
|
|
assessed whether or not the proteome reactivity profiles of the alkynyl triazole
|
|
ureas of moderate electrophilicity (akin to the SAR series in Section 3.4) could
|
|
be blocked by pre-incubation with the non-fluorescent SH-directed activity based
|
|
probe FP-biotin [<a class="bibr" href="#ml294.r12" rid="ml294.r12">12</a>] with visualization of labeled proteins achieved by click
|
|
chemistry conjugation of the alkyne-functionalized triazole ureas to an
|
|
azido-conjugated rhodamine reporter tag [<a class="bibr" href="#ml294.r17" rid="ml294.r17">17</a>]. FP-biotin was found to successfully
|
|
compete all proteins modified by the triazole ureas, indicating that the latter
|
|
reacted exclusively with members of the SH class; as such, anti-targets outside
|
|
of the SH class are not a primary concern for <a href="/pcsubstance/?term=ML294[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML294</a> and <a href="/pcsubstance/?term=ML295[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML295</a>.</p><p><b>Reactivity of triazole
|
|
ureas</b><b><i>within</i></b><b>the SH enzyme
|
|
class.</b> Probe <a href="/pcsubstance/?term=ML294[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML294</a> (<a href="https://pubchem.ncbi.nlm.nih.gov/substance/125269120" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 125269120</a>) and analogs
|
|
have been subject to gel-based competitive ABPP screening to assess SH
|
|
reactivity against more than 20 FP-sensitive SHs visible by 1D SDS-PAGE
|
|
separation and fluorescent detection in the mouse brain membrane proteome
|
|
(<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602311" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID
|
|
602311</a>). This proteome was chosen due to its relevance for future
|
|
study of DAGL-β in the nervous system as well as its diversity of SHs.
|
|
Anti-target hits are identified by ≥50% disappearance of the
|
|
band in the gel relative to the DMSO control. (Note: DAGL-β is not
|
|
visible in these gels due to overlapping SH bands). Proteins are listed as
|
|
anti-targets if at least 50% inhibition is observed as quantified
|
|
relative to the DMSO control. For additional selectivity analysis, compounds
|
|
were also evaluated using the HT-01 probe in the mouse brain membrane proteome
|
|
by gel-based competitive ABPP (1 μM compound). <a href="/pcsubstance/?term=ML294[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML294</a> shows a clean selectivity profile by this analysis.</p><p><b>ABPP-SILAC:</b> To more comprehensively identify potential anti-targets,
|
|
we utilized an advanced quantitative mass spectrometry (MS)-based platform
|
|
termed competitive ABPP-SILAC. Competitive ABPP-SILAC [<a class="bibr" href="#ml294.r9" rid="ml294.r9">9</a>, <a class="bibr" href="#ml294.r11" rid="ml294.r11">11</a>] combines competitive ABPP [<a class="bibr" href="#ml294.r18" rid="ml294.r18">18</a>] with stable isotope
|
|
labeling of cells (SILAC) [<a class="bibr" href="#ml294.r10" rid="ml294.r10">10</a>], and allows for precise quantitation of enzyme
|
|
inhibition by calculating the isotopic ratios of peptides from inhibitor-treated
|
|
and control cells. We estimate that the sensitivity of a standard LC-MS/MS based
|
|
assay (ABPP-SILAC, ABPP-MudPIT) is at least 10-fold higher than our gel-based
|
|
assays (0.00005% of the total cell proteome, or 20 copies per cell). As
|
|
such, this method offers more sensitive detection of low abundance SHs for a
|
|
comprehensive selectivity analysis. The results demonstrate that both <a href="/pcsubstance/?term=ML294[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML294</a> and <a href="/pcsubstance/?term=ML295[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML295</a> display high
|
|
selectivity for their targets in Neuro-2A murine neuroblastoma cells <i>in
|
|
situ</i>, blocking >90% of activity of
|
|
DAGL-β/ABHD6 (<a href="/pcsubstance/?term=ML294[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML294</a>) and ABHD6 (<a href="/pcsubstance/?term=ML295[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML295</a>), while not affecting activity of 40+ other SHs.
|
|
These clean selectivity profiles highlight the value of specific chemotypes,
|
|
like the triazole ureas, from which it is possible to derive potent and
|
|
selective chemical probes for multiple enzyme targets.</p><p><b>ABPP-MudPIT:</b> To more comprehensively identify potential anti-targets
|
|
<i>in vivo</i>, we utilized a quantitative LC-MS/MS-based platform
|
|
termed competitive ABPP-MudPIT [<a class="bibr" href="#ml294.r15" rid="ml294.r15">15</a>]. ABPP-MudPIT allows for semi-quantitative determination
|
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of enzyme inhibition via comparison of the spectra counts of peptides from
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control vs. inhibitor-treated samples. Mice were administered <a href="/pcsubstance/?term=ML294[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML294</a> (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602353" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 602353</a>) or <a href="/pcsubstance/?term=ML295[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML295</a> (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602351" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 602351</a>). The results demonstrate that both
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<a href="/pcsubstance/?term=ML294[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML294</a> and <a href="/pcsubstance/?term=ML295[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML295</a> exhibit significant selectivity for their target enzymes
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(P<0.01).</p></div></div><div id="ml294.s31"><h2 id="_ml294_s31_">4. Discussion</h2><div id="ml294.s32"><h3>4.1. Comparison to Existing Art and How the New Probe is an Improvement</h3><p><a href="/pcsubstance/?term=ML294[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML294</a> is the first reported inhibitor with selectivity for
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DAGL-β vs. DAGL-α. Dual DAGL-α/β inhibitors
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tetrahydrolipstatin (THL) and <a href="/protein/1470879788/?report=GenPept" class="bk_tag" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=genpept">RHC80267</a> (see <a class="figpopup" href="/books/NBK133443/figure/ml294.f2/?report=objectonly" target="object" rid-figpopup="figml294f2" rid-ob="figobml294f2">Figure 2</a> for structures) have been reported in the literature;
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however, anti-target assessment of these compounds by competitive ABPP revealed
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that both compounds block several brain SHs with greater or equivalent potency
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vs. the DAGL enzymes [<a class="bibr" href="#ml294.r19" rid="ml294.r19">19</a>]. In contrast, <a href="/pcsubstance/?term=ML294[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML294</a> has one
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anti-target, ABHD6, and an available control “anti-probe”
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<a href="/pcsubstance/?term=ML295[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML295</a>, making these Probes, in concert, a
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significant improvement over prior art DAGL-β inhibitors and crucial
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chemical tools for the biological investigation of DAGL-β.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml294f2" co-legend-rid="figlgndml294f2"><a href="/books/NBK133443/figure/ml294.f2/?report=objectonly" target="object" title="Figure 2" class="img_link icnblk_img figpopup" rid-figpopup="figml294f2" rid-ob="figobml294f2"><img class="small-thumb" src="/books/NBK133443/bin/ml294f2.gif" src-large="/books/NBK133443/bin/ml294f2.jpg" alt="Figure 2. Structures of prior art dual DAGL-α/β inhibitors THL and RHC80267 and DAGL-b inhibitor ML294." /></a><div class="icnblk_cntnt" id="figlgndml294f2"><h4 id="ml294.f2"><a href="/books/NBK133443/figure/ml294.f2/?report=objectonly" target="object" rid-ob="figobml294f2">Figure 2</a></h4><p class="float-caption no_bottom_margin">Structures of prior art dual DAGL-α/β inhibitors THL
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and RHC80267 and DAGL-b inhibitor ML294. </p></div></div></div></div><div id="ml294.s33"><h2 id="_ml294_s33_">5. References</h2><dl class="temp-labeled-list"><dl class="bkr_refwrap"><dt>1.</dt><dd><div class="bk_ref" id="ml294.r1">Di Marzo V. Targeting the endocannabinoid system: to
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enhance or reduce? <span><span class="ref-journal">Nat. Rev. Drug
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Discov. </span>2008;<span class="ref-vol">7</span>(5):438–55.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/18446159" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18446159</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>2.</dt><dd><div class="bk_ref" id="ml294.r2">Chevaleyre V, Takahashi KA, Castillo PE. Endocannabinoid-mediated synaptic plasticity
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in the CNS. <span><span class="ref-journal">Annu. Rev.
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Neurosci. </span>2006;<span class="ref-vol">29</span>:37–76.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/16776579" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 16776579</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>3.</dt><dd><div class="bk_ref" id="ml294.r3">Ahn K, McKinney MK, Cravatt BF. Enzymatic pathways that regulate
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endocannabinoid signaling in the nervous
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system. <span><span class="ref-journal">Chem.
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Rev. </span>2008;<span class="ref-vol">108</span>(5):1687–707.</span> [<a href="/pmc/articles/PMC3150828/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3150828</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/18429637" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18429637</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>4.</dt><dd><div class="bk_ref" id="ml294.r4">Ahn K, et al. Mechanistic and pharmacological
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characterization of PF-04457845: a highly potent and selective fatty
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acid amide hydrolase inhibitor that reduces inflammatory and
|
|
noninflammatory pain. <span><span class="ref-journal">J. Pharmacol Exp.
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Ther. </span>2011;<span class="ref-vol">338</span>(1):114–24.</span> [<a href="/pmc/articles/PMC3126636/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3126636</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/21505060" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 21505060</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>5.</dt><dd><div class="bk_ref" id="ml294.r5">Blankman JL, Simon GM, Cravatt BF. A comprehensive profile of brain enzymes that
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hydrolyze the endocannabinoid
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2-arachidonoylglycerol. <span><span class="ref-journal">Chem.
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Biol. </span>2007;<span class="ref-vol">14</span>(12):1347–56.</span> [<a href="/pmc/articles/PMC2692834/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2692834</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/18096503" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18096503</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>6.</dt><dd><div class="bk_ref" id="ml294.r6">Bisogno T, et al. Cloning of the first sn1-DAG lipases points to
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the spatial and temporal regulation of endocannabinoid signaling in the
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brain. <span><span class="ref-journal">J. Cell
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Biol. </span>2003;<span class="ref-vol">163</span>(3):463–8.</span> [<a href="/pmc/articles/PMC2173631/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2173631</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/14610053" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 14610053</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>7.</dt><dd><div class="bk_ref" id="ml294.r7">Gao Y, et al. Loss of retrograde endocannabinoid signaling
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and reduced adult neurogenesis in diacylglycerol lipase knock-out
|
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mice. <span><span class="ref-journal">J.
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Neurosci. </span>2010;<span class="ref-vol">30</span>(6):2017–24.</span> [<a href="/pmc/articles/PMC6634037/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC6634037</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/20147530" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 20147530</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>8.</dt><dd><div class="bk_ref" id="ml294.r8">Tanimura A, et al. The endocannabinoid 2-arachidonoylglycerol
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produced by diacylglycerol lipase alpha mediates retrograde suppression
|
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of synaptic
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transmission. <span><span class="ref-journal">Neuron. </span>2010;<span class="ref-vol">65</span>(3):320–7.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/20159446" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 20159446</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>9.</dt><dd><div class="bk_ref" id="ml294.r9">Hsu KL, et al. DAGLB Regulates an Endocannabinoid-Eicosanoid
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Network Involved in Macrophage Inflammatory
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Responses. Submitted 2012.</div></dd></dl><dl class="bkr_refwrap"><dt>10.</dt><dd><div class="bk_ref" id="ml294.r10">Ong SE, et al. Stable isotope labeling by amino acids in cell
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culture, SILAC, as a simple and accurate approach to expression
|
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proteomics. <span><span class="ref-journal">Mol. Cell
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Proteomics. </span>2002;<span class="ref-vol">1</span>(5):376–86.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/12118079" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 12118079</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>11.</dt><dd><div class="bk_ref" id="ml294.r11">Adibekian A, et al. Click-generated triazole ureas as ultrapotent
|
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in vivo-active serine hydrolase inhibitors. <span><span class="ref-journal">Nat.
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Chem.
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Biol. </span>2011;<span class="ref-vol">7</span>(7):469–78.</span> [<a href="/pmc/articles/PMC3118922/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3118922</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/21572424" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 21572424</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>12.</dt><dd><div class="bk_ref" id="ml294.r12">Liu Y, Patricelli MP, Cravatt BF. Activity-based protein profiling: the serine
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hydrolases. <span><span class="ref-journal">Proc. Natl. Acad. Sci. U. S.
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A. </span>1999;<span class="ref-vol">96</span>(26):14694–9.</span> [<a href="/pmc/articles/PMC24710/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC24710</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/10611275" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 10611275</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>13.</dt><dd><div class="bk_ref" id="ml294.r13">Washburn MP, Wolters D, Yates JR 3rd. Large-scale analysis of the yeast proteome by
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multidimensional protein identification
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technology. <span><span class="ref-journal">Nat.
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Biotechnol. </span>2001;<span class="ref-vol">19</span>(3):242–7.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/11231557" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 11231557</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>14.</dt><dd><div class="bk_ref" id="ml294.r14">Wolters DA, Washburn MP, Yates JR 3rd. An automated multidimensional protein
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identification technology for shotgun
|
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proteomics. <span><span class="ref-journal">Anal.
|
|
Chem. </span>2001;<span class="ref-vol">73</span>(23):5683–90.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/11774908" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 11774908</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>15.</dt><dd><div class="bk_ref" id="ml294.r15">Jessani N, et al. A streamlined platform for high-content
|
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functional proteomics of primary human
|
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specimens. <span><span class="ref-journal">Nat.
|
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Methods. </span>2005;<span class="ref-vol">2</span>(9):691–7.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/16118640" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 16118640</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>16.</dt><dd><div class="bk_ref" id="ml294.r16">Jessani N, et al. Enzyme activity profiles of the secreted and
|
|
membrane proteome that depict cancer cell
|
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invasiveness. <span><span class="ref-journal">Proc. Natl. Acad. Sci. U. S.
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|
A. </span>2002;<span class="ref-vol">99</span>(16):10335–40.</span> [<a href="/pmc/articles/PMC124915/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC124915</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/12149457" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 12149457</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>17.</dt><dd><div class="bk_ref" id="ml294.r17">Speers AE, Cravatt BF. Profiling enzyme activities in vivo using
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click chemistry methods. <span><span class="ref-journal">Chem.
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Biol. </span>2004;<span class="ref-vol">11</span>(4):535–46.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/15123248" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 15123248</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>18.</dt><dd><div class="bk_ref" id="ml294.r18">Leung D, et al. Discovering potent and selective reversible
|
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inhibitors of enzymes in complex proteomes. <span><span class="ref-journal">Nat.
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Biotechnol. </span>2003;<span class="ref-vol">21</span>(6):687–91.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/12740587" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 12740587</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>19.</dt><dd><div class="bk_ref" id="ml294.r19">Hoover HS, et al. Selectivity of inhibitors of endocannabinoid
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biosynthesis evaluated by activity-based protein
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profiling. <span><span class="ref-journal">Bioorg. Med. Chem.
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Lett. </span>2008;<span class="ref-vol">18</span>(22):5838–41.</span> [<a href="/pmc/articles/PMC2634297/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2634297</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/18657971" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18657971</span></a>]</div></dd></dl></dl></div><div id="bk_toc_contnr"></div></div></div><div class="fm-sec"><h2 id="_NBK133443_pubdet_">Publication Details</h2><h3>Author Information and Affiliations</h3><p class="contrib-group"><h4>Authors</h4><span itemprop="author">Ku-Lung Hsu</span>,<sup>1</sup> <span itemprop="author">Katsunori Tsuboi</span>,<sup>1</sup> <span itemprop="author">Anna E Speers</span>,<sup>1</sup> <span itemprop="author">Steven J Brown</span>,<sup>1</sup> <span itemprop="author">Timothy Spicer</span>,<sup>2</sup> <span itemprop="author">Virneliz Fernandez-Vega</span>,<sup>2</sup> <span itemprop="author">Jill Ferguson</span>,<sup>1</sup> <span itemprop="author">Benjamin F Cravatt</span>,<sup>1</sup> <span itemprop="author">Peter Hodder</span>,<sup>2</sup> and <span itemprop="author">Hugh Rosen</span><sup>1</sup><sup>,*</sup>.</p><h4>Affiliations</h4><div class="affiliation"><sup>1</sup>
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The Scripps Research Institute, La Jolla CA</div><div class="affiliation"><sup>2</sup>
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The Scripps Research Institute, Jupiter, FL.</div><div class="affiliation"><sup>*</sup> Corresponding author:
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<span class="before-email-separator"></span><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="ude.sppircs@nesorh" class="oemail">ude.sppircs@nesorh</a></div><h3>Publication History</h3><p class="small">Received: <span itemprop="datePublished">April 16, 2012</span>; Last Update: <span itemprop="dateModified">February 25, 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>Hsu KL, Tsuboi K, Speers AE, et al. Optimization and characterization of a triazole urea inhibitor for diacylglycerol lipase beta (DAGL-β) 2012 Apr 16 [Updated 2013 Feb 25]. 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/ml296/?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/ml293/?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="figobml294fu1"><div id="ml294.fu1" class="figure"><div class="graphic"><img data-src="/books/NBK133443/bin/ml294fu1.jpg" alt="Image ml294fu1" /></div></div></article><article data-type="table-wrap" id="figobml294tu1"><div id="ml294.tu1" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK133443/table/ml294.tu1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml294.tu1_lrgtbl__"><table class="no_bottom_margin"><thead><tr><th id="hd_h_ml294.tu1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CID/ML#</th><th id="hd_h_ml294.tu1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Target Name</th><th id="hd_h_ml294.tu1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Target IC50<sup>*</sup> (nM) [SID,
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AID]</th><th id="hd_h_ml294.tu1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Anti-target Name(s)</th><th id="hd_h_ml294.tu1_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Anti-target IC50<sup>*</sup> (nM)
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[SID, AID]</th><th id="hd_h_ml294.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Fold Selective<sup>†</sup></th><th id="hd_h_ml294.tu1_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Secondary Assay(s) Name:
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[SID, AID]</th></tr></thead><tbody><tr><td headers="hd_h_ml294.tu1_1_1_1_1" rowspan="3" colspan="1" style="text-align:center;vertical-align:middle;">CID 53364485/<a href="/pcsubstance/?term=ML294[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML294</a></td><td headers="hd_h_ml294.tu1_1_1_1_2" rowspan="3" colspan="1" style="text-align:center;vertical-align:middle;">DAGL-β</td><td headers="hd_h_ml294.tu1_1_1_1_3" rowspan="3" colspan="1" style="text-align:center;vertical-align:middle;">56 [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/125269120" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 125269120</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602320" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID
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602320</a>]</td><td headers="hd_h_ml294.tu1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">ABHD6</td><td headers="hd_h_ml294.tu1_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">~1 [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/125269120" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 125269120</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/624039" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID
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624039</a>]</td><td headers="hd_h_ml294.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0</td><td headers="hd_h_ml294.tu1_1_1_1_7" rowspan="3" colspan="1" style="text-align:left;vertical-align:middle;"><b>Inhibition
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Assay</b>: [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/125269120" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 125269120</a>,
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<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602320" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID
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602320</a>]<br /><b>Selectivity Assay
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ABHD6</b>: [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/125269120" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 125269120</a>,
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<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/624039" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID
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624039</a>]<br /><b>Selectivity Assay
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SHs</b>: [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/125269120" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 125269120</a>,
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<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602355" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID
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602355</a>]<br /><b>Selectivity Assay
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DAGL-</b>α: [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/125269120" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 125269120</a>, AIDs <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602403" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">602403</a><b>]</b><br /><b>Selectivity
|
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Assay ABPP-SILAC:</b> [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/125269120" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 125269120</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602339" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID
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602339</a>]<br /><b>Selectivity Assay
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ABPP-MudPIT</b>: [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/125269120" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 125269120</a>,
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<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602353" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID
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602353</a>]<br /><b><i>In
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situ</i></b><b>Assay</b>: IC50 = 12 nM
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[<a href="https://pubchem.ncbi.nlm.nih.gov/substance/125269120" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 125269120</a>,
|
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<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602354" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID
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602354</a>]<br /><b><i>In
|
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vivo</i></b><b>Assays</b>: [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/125269120" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 125269120</a>, AIDs <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602347" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">602347</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602345" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">602345</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602343" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">602343</a>]<br /><b>Cytotox assay</b>:
|
|
[<a href="https://pubchem.ncbi.nlm.nih.gov/substance/125269120" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 125269120</a>,
|
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<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602337" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 602337</a>]</td></tr><tr><td headers="hd_h_ml294.tu1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">20+ SH targets, including
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FAAH, MAGL, ABHD11, ABHD12, LYPLA1, LYPLA2, PLA2G7, PAFAH2</td><td headers="hd_h_ml294.tu1_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">> 2000<sup>**</sup> [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/125269120" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 125269120</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602355" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID
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|
602355</a>]</td><td headers="hd_h_ml294.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>35</td></tr><tr><td headers="hd_h_ml294.tu1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">DAGL-α (closest homolog)</td><td headers="hd_h_ml294.tu1_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>400<sup>**</sup> [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/125269120" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 125269120</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602403" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID
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602403</a>]</td><td headers="hd_h_ml294.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">7</td></tr></tbody></table></div><div class="tblwrap-foot"><div><dl class="temp-labeled-list small"><dl class="bkr_refwrap"><dt>*</dt><dd><div id="ml294.tfn1"><p class="no_margin">As assessed by gel-based competitive ABPP</p></div></dd></dl><dl class="bkr_refwrap"><dt>**</dt><dd><div id="ml294.tfn2"><p class="no_margin">IC50 of the anti-target is defined as greater than the test compound
|
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concentration at which less than or equal to 50% inhibition of
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the anti-target is observed, which is reported in <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602355" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID
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602355</a>. For <a href="https://pubchem.ncbi.nlm.nih.gov/substance/125269120" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 125269120</a>, no
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anti-targets were observed for all serine hydrolases (SHs) assayed at
|
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2000 nM concentration, so the IC50 is reported as >2000 nM.</p></div></dd></dl><dl class="bkr_refwrap"><dt>†</dt><dd><div id="ml294.tfn3"><p class="no_margin">Fold-selectivity was calculated as: >IC50 for anti-target/IC50 for
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DAGL-β</p></div></dd></dl></dl></div></div></div></article><article data-type="fig" id="figobml294fu2"><div id="ml294.fu2" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK133443/bin/ml294fu2.jpg" alt="ML294 SID 125269120 CID 53364485." /></div><h3><span class="title"><a href="/pcsubstance/?term=ML294[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML294</a><br /><a href="https://pubchem.ncbi.nlm.nih.gov/substance/125269120" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID
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|
125269120</a><br />CID 53364485</span></h3></div></article><article data-type="table-wrap" id="figobml294t1"><div id="ml294.t1" class="table"><h3><span class="label">Table 1</span><span class="title">Compounds submitted to the SMR collection (04-09-2012)</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK133443/table/ml294.t1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml294.t1_lrgtbl__"><table class="no_top_margin"><tbody><tr><th id="hd_b_ml294.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Designation</th><th id="hd_b_ml294.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CID</th><th id="hd_b_ml294.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SID</th><th id="hd_b_ml294.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SRID</th><th id="hd_b_ml294.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS</th></tr><tr><th id="hd_b_ml294.t1_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Probe</th><td headers="hd_b_ml294.t1_1_1_2_1 hd_b_ml294.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">53364485</td><td headers="hd_b_ml294.t1_1_1_2_1 hd_b_ml294.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/125269120" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">125269120</a></td><td headers="hd_b_ml294.t1_1_1_2_1 hd_b_ml294.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-02000001756-1</td><td headers="hd_b_ml294.t1_1_1_2_1 hd_b_ml294.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS004256809</td></tr><tr><th id="hd_b_ml294.t1_1_1_3_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Analog 1</th><td headers="hd_b_ml294.t1_1_1_3_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">53364509</td><td headers="hd_b_ml294.t1_1_1_3_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/125269102" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">125269102</a></td><td headers="hd_b_ml294.t1_1_1_3_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-02000001738-1</td><td headers="hd_b_ml294.t1_1_1_3_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS004256810</td></tr><tr><th id="hd_b_ml294.t1_1_1_4_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Analog 2</th><td headers="hd_b_ml294.t1_1_1_4_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">56643167</td><td headers="hd_b_ml294.t1_1_1_4_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/134420265" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">134420265</a></td><td headers="hd_b_ml294.t1_1_1_4_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-02000002085-1</td><td headers="hd_b_ml294.t1_1_1_4_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS004256811</td></tr><tr><th id="hd_b_ml294.t1_1_1_5_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Analog 3</th><td headers="hd_b_ml294.t1_1_1_5_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">53364486</td><td headers="hd_b_ml294.t1_1_1_5_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/125269106" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">125269106</a></td><td headers="hd_b_ml294.t1_1_1_5_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-02000001742-1</td><td headers="hd_b_ml294.t1_1_1_5_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS004256812</td></tr><tr><th id="hd_b_ml294.t1_1_1_6_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Analog 4</th><td headers="hd_b_ml294.t1_1_1_6_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">53364540</td><td headers="hd_b_ml294.t1_1_1_6_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/125269108" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">125269108</a></td><td headers="hd_b_ml294.t1_1_1_6_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-02000001744-1</td><td headers="hd_b_ml294.t1_1_1_6_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS004256813</td></tr><tr><th id="hd_b_ml294.t1_1_1_7_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Analog 5</th><td headers="hd_b_ml294.t1_1_1_7_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">53364489</td><td headers="hd_b_ml294.t1_1_1_7_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/125269114" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">125269114</a></td><td headers="hd_b_ml294.t1_1_1_7_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SR-02000001750-1</td><td headers="hd_b_ml294.t1_1_1_7_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS004256814</td></tr></tbody></table></div></div></article><article data-type="fig" id="figobml294fu3"><div id="ml294.fu3" class="figure"><div class="graphic"><img data-src="/books/NBK133443/bin/ml294fu3.jpg" alt="Image ml294fu3" /></div></div></article><article data-type="fig" id="figobml294fu4"><div id="ml294.fu4" class="figure"><div class="graphic"><img data-src="/books/NBK133443/bin/ml294fu4.jpg" alt="Image ml294fu4" /></div></div></article><article data-type="fig" id="figobml294f1"><div id="ml294.f1" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%201.%20IC50%20curves%20for%20probe%20ML294%20(SID%20125269120)%20and%20anti-probe%20ML295%20(SID%20125269138).&p=BOOKS&id=133443_ml294f1.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/NBK133443/bin/ml294f1.jpg" alt="Figure 1. IC50 curves for probe ML294 (SID 125269120) and anti-probe ML295 (SID 125269138)." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 1</span><span class="title">IC50 curves for probe ML294 (SID
|
|
125269120) and anti-probe ML295 (SID 125269138)</span></h3><div class="caption"><p>As
|
|
determined by gel-based competitive-ABPP with the activity-based probe
|
|
HT-01 against overexpressed DAGL-β in a complex proteome lysate
|
|
(left panels) and in cultured neuro-2A murine neuroblastoma cells grown
|
|
in medium supplemented with 10% FCS (right panels). Data are presented as means + s.e.m. for 3 independent
|
|
experiments. <i>AID information</i>: <a href="/pcsubstance/?term=ML294[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML294</a> vs. DAGL-β <i>in vitro</i>
|
|
(<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602320" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 602320</a>); <a href="/pcsubstance/?term=ML294[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML294</a> vs. ABHD6 <i>in vitro</i> (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/624039" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID
|
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624039</a>); <a href="/pcsubstance/?term=ML294[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML294</a> vs.
|
|
DAGL-β and ABHD6 <i>in situ</i> (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602354" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID
|
|
602354</a>); <a href="/pcsubstance/?term=ML295[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML295</a> vs.
|
|
DAGL-β and ABHD6 <i>in vitro</i> (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602322" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID
|
|
602322</a>); <a href="/pcsubstance/?term=ML295[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML295</a> vs.
|
|
DAGL-β and ABHD6 <i>in situ</i> (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/602335" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID
|
|
602335</a>).</p></div></div></article><article data-type="fig" id="figobml294f2"><div id="ml294.f2" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%202.%20Structures%20of%20prior%20art%20dual%20DAGL-%003B1%2F%003B2%20inhibitors%20THL%20and%20RHC80267%20and%20DAGL-b%20inhibitor%20ML294.&p=BOOKS&id=133443_ml294f2.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/NBK133443/bin/ml294f2.jpg" alt="Figure 2. Structures of prior art dual DAGL-α/β inhibitors THL and RHC80267 and DAGL-b inhibitor ML294." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 2</span><span class="title">Structures of prior art dual DAGL-α/β inhibitors THL
|
|
and RHC80267 and DAGL-b inhibitor <a href="/pcsubstance/?term=ML294[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML294</a></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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