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<meta name="ncbi_db" content="books" /><meta name="ncbi_pdid" content="book-part" /><meta name="ncbi_acc" content="NBK47358" /><meta name="ncbi_domain" content="mlprobe" /><meta name="ncbi_report" content="record" /><meta name="ncbi_type" content="fulltext" /><meta name="ncbi_objectid" content="" /><meta name="ncbi_pcid" content="/NBK47358/" /><meta name="ncbi_pagename" content="Identification of Functionally Selective Small Molecule Antagonists of the Neuropeptide-S Receptor: Naphthopyranopyrimidines - Probe Reports from the NIH Molecular Libraries Program - NCBI Bookshelf" /><meta name="ncbi_bookparttype" content="chapter" /><meta name="ncbi_app" content="bookshelf" />
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<title>Identification of Functionally Selective Small Molecule Antagonists of the Neuropeptide-S Receptor: Naphthopyranopyrimidines - Probe Reports from the NIH Molecular Libraries Program - NCBI Bookshelf</title>
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<meta name="robots" content="INDEX,FOLLOW,NOARCHIVE" /><meta name="citation_inbook_title" content="Probe Reports from the NIH Molecular Libraries Program [Internet]" /><meta name="citation_title" content="Identification of Functionally Selective Small Molecule Antagonists of the Neuropeptide-S Receptor: Naphthopyranopyrimidines" /><meta name="citation_publisher" content="National Center for Biotechnology Information (US)" /><meta name="citation_date" content="2010/09/02" /><meta name="citation_author" content="Juan Marugan" /><meta name="citation_author" content="Ke Liu" /><meta name="citation_author" content="Wei Zheng" /><meta name="citation_author" content="Robert Eskay" /><meta name="citation_author" content="Noel Southall" /><meta name="citation_author" content="Markus Heilig" /><meta name="citation_author" content="James Inglese" /><meta name="citation_author" content="Christopher Austin" /><meta name="citation_pmid" content="21433374" /><meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK47358/" /><link rel="schema.DC" href="http://purl.org/DC/elements/1.0/" /><meta name="DC.Title" content="Identification of Functionally Selective Small Molecule Antagonists of the Neuropeptide-S Receptor: Naphthopyranopyrimidines" /><meta name="DC.Type" content="Text" /><meta name="DC.Publisher" content="National Center for Biotechnology Information (US)" /><meta name="DC.Contributor" content="Juan Marugan" /><meta name="DC.Contributor" content="Ke Liu" /><meta name="DC.Contributor" content="Wei Zheng" /><meta name="DC.Contributor" content="Robert Eskay" /><meta name="DC.Contributor" content="Noel Southall" /><meta name="DC.Contributor" content="Markus Heilig" /><meta name="DC.Contributor" content="James Inglese" /><meta name="DC.Contributor" content="Christopher Austin" /><meta name="DC.Date" content="2010/09/02" /><meta name="DC.Identifier" content="https://www.ncbi.nlm.nih.gov/books/NBK47358/" /><meta name="description" content="Neuropeptide S receptor (NPSR), previously known as GPR154, is a recently de-orphanized G protein coupled receptor. Its endogenous ligand is the 20 amino acid peptide, Neuropeptide S (NPS). Activation of NPSR induces transient increases in intracellular calcium and cAMP, suggesting coupling of this receptor to both Gs and Gq G proteins. NPS and its receptor are found in various tissues. The receptor is highly expressed in brain areas that have been implicated in modulation of arousal, stress and anxiety. Central administration of NPS in mice produces an unusual profile of activity by inducing wakefulness and arousal, while at the same time suppressing anxiety. Therefore, NPSR may represent a novel drug target for the treatment of sleep and anxiety disorders. The relative importance of Gs and Gq G protein-coupled signaling to NPSR action is not known. The goal of the project is to generate a series of compounds that can selectively antagonize these signaling pathways. ML079 (CID-3719993) is one compound from a series of congeners satisfies this criteria and can be used to dissect the role these pathways play in NPSR biology." /><meta name="og:title" content="Identification of Functionally Selective Small Molecule Antagonists of the Neuropeptide-S Receptor: Naphthopyranopyrimidines" /><meta name="og:type" content="book" /><meta name="og:description" content="Neuropeptide S receptor (NPSR), previously known as GPR154, is a recently de-orphanized G protein coupled receptor. Its endogenous ligand is the 20 amino acid peptide, Neuropeptide S (NPS). Activation of NPSR induces transient increases in intracellular calcium and cAMP, suggesting coupling of this receptor to both Gs and Gq G proteins. NPS and its receptor are found in various tissues. The receptor is highly expressed in brain areas that have been implicated in modulation of arousal, stress and anxiety. Central administration of NPS in mice produces an unusual profile of activity by inducing wakefulness and arousal, while at the same time suppressing anxiety. Therefore, NPSR may represent a novel drug target for the treatment of sleep and anxiety disorders. The relative importance of Gs and Gq G protein-coupled signaling to NPSR action is not known. The goal of the project is to generate a series of compounds that can selectively antagonize these signaling pathways. ML079 (CID-3719993) is one compound from a series of congeners satisfies this criteria and can be used to dissect the role these pathways play in NPSR biology." /><meta name="og:url" content="https://www.ncbi.nlm.nih.gov/books/NBK47358/" /><meta name="og:site_name" content="NCBI Bookshelf" /><meta name="og:image" content="https://www.ncbi.nlm.nih.gov/corehtml/pmc/pmcgifs/bookshelf/thumbs/th-mlprobe-lrg.png" /><meta name="twitter:card" content="summary" /><meta name="twitter:site" content="@ncbibooks" /><meta name="bk-non-canon-loc" content="/books/n/mlprobe/ml079/" /><link rel="canonical" href="https://www.ncbi.nlm.nih.gov/books/NBK47358/" /><link rel="stylesheet" href="/corehtml/pmc/css/figpopup.css" type="text/css" media="screen" /><link rel="stylesheet" href="/corehtml/pmc/css/bookshelf/2.26/css/books.min.css" type="text/css" /><link rel="stylesheet" href="/corehtml/pmc/css/bookshelf/2.26/css/books_print.min.css" type="text/css" media="print" /><style type="text/css">p a.figpopup{display:inline !important} .bk_tt {font-family: monospace} .first-line-outdent .bk_ref {display: inline} .body-content h2, .body-content .h2 {border-bottom: 1px solid #97B0C8} .body-content h2.inline {border-bottom: none} a.page-toc-label , .jig-ncbismoothscroll a {text-decoration:none;border:0 !important} .temp-labeled-list .graphic {display:inline-block !important} .temp-labeled-list img{width:100%}</style><script type="text/javascript" src="/corehtml/pmc/js/jquery.hoverIntent.min.js"> </script><script type="text/javascript" src="/corehtml/pmc/js/common.min.js?_=3.18"> </script><script type="text/javascript" src="/corehtml/pmc/js/large-obj-scrollbars.min.js"> </script><script type="text/javascript">window.name="mainwindow";</script><script type="text/javascript" src="/corehtml/pmc/js/bookshelf/2.26/book-toc.min.js"> </script><script type="text/javascript" src="/corehtml/pmc/js/bookshelf/2.26/books.min.js"> </script><meta name="book-collection" content="NONE" />
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<div class="pre-content"><div><div class="bk_prnt"><p class="small">NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health.</p><p>Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-. </p></div><div class="iconblock clearfix whole_rhythm no_top_margin bk_noprnt"><a class="img_link icnblk_img" title="Table of Contents Page" href="/books/n/mlprobe/"><img class="source-thumb" src="/corehtml/pmc/pmcgifs/bookshelf/thumbs/th-mlprobe-lrg.png" alt="Cover of Probe Reports from the NIH Molecular Libraries Program" height="100px" width="80px" /></a><div class="icnblk_cntnt eight_col"><h2>Probe Reports from the NIH Molecular Libraries Program [Internet].</h2><a data-jig="ncbitoggler" href="#__NBK47358_dtls__">Show details</a><div style="display:none" class="ui-widget" id="__NBK47358_dtls__"><div>Bethesda (MD): National Center for Biotechnology Information (US); 2010-.</div></div><div class="half_rhythm"><ul class="inline_list"><li style="margin-right:1em"><a class="bk_cntns" href="/books/n/mlprobe/">Contents</a></li></ul></div><div class="bk_noprnt"><form method="get" action="/books/n/mlprobe/" id="bk_srch"><div class="bk_search"><label for="bk_term" class="offscreen_noflow">Search term</label><input type="text" title="Search this book" id="bk_term" name="term" value="" data-jig="ncbiclearbutton" /> <input type="submit" class="jig-ncbibutton" value="Search this book" submit="false" style="padding: 0.1em 0.4em;" /></div></form></div></div><div class="icnblk_cntnt two_col"><div class="pagination bk_noprnt"><a class="active page_link prev" href="/books/n/mlprobe/ml080/" title="Previous page in this title">< Prev</a><a class="active page_link next" href="/books/n/mlprobe/ml077/" title="Next page in this title">Next ></a></div></div></div></div></div>
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<div class="main-content lit-style" itemscope="itemscope" itemtype="http://schema.org/CreativeWork"><div class="meta-content fm-sec"><h1 id="_NBK47358_"><span class="title" itemprop="name">Identification of Functionally Selective Small Molecule Antagonists of the
|
||
Neuropeptide-S Receptor: Naphthopyranopyrimidines</span></h1><p class="contrib-group"><span itemprop="author">Juan Marugan</span>, <span itemprop="author">Ke Liu</span>, <span itemprop="author">Wei Zheng</span>, <span itemprop="author">Robert Eskay</span>, <span itemprop="author">Noel Southall</span>, <span itemprop="author">Markus Heilig</span>, <span itemprop="author">James Inglese</span>, and <span itemprop="author">Christopher Austin</span>.</p><a data-jig="ncbitoggler" href="#__NBK47358_ai__" style="border:0;text-decoration:none">Author Information and Affiliations</a><div style="display:none" class="ui-widget" id="__NBK47358_ai__"><p class="contrib-group"><h4>Authors</h4><span itemprop="author">Juan Marugan</span>,<sup>1</sup> <span itemprop="author">Ke Liu</span>,<sup>1</sup> <span itemprop="author">Wei Zheng</span>,<sup>1</sup> <span itemprop="author">Robert Eskay</span>,<sup>2</sup> <span itemprop="author">Noel Southall</span>,<sup>1</sup> <span itemprop="author">Markus Heilig</span>,<sup>2</sup> <span itemprop="author">James Inglese</span>,<sup>1</sup> and <span itemprop="author">Christopher Austin</span><sup>1</sup>.</p><h4>Affiliations</h4><div class="affiliation"><sup>1</sup>
|
||
NIH Chemical Genomics Center, 9800 Medical Center Dr.,
|
||
Building B, Bethesda, MD, 20892-3370</div><div class="affiliation"><sup>2</sup>
|
||
National Institute on Alcohol Abuse and Alcoholism,
|
||
Bethesda, MD 20892</div></div><p class="small">Received: <span itemprop="datePublished">February 13, 2009</span>; Last Update: <span itemprop="dateModified">September 2, 2010</span>.</p></div><div class="jig-ncbiinpagenav body-content whole_rhythm" data-jigconfig="allHeadingLevels: ['h2'],smoothScroll: false" itemprop="text"><div id="_abs_rndgid_" itemprop="description"><p>Neuropeptide S receptor (NPSR), previously known as GPR154, is a recently de-orphanized G protein coupled receptor. Its endogenous ligand is the 20 amino acid peptide, Neuropeptide S (NPS). Activation of NPSR induces transient increases in intracellular calcium and cAMP, suggesting coupling of this receptor to both Gs and Gq G proteins. NPS and its receptor are found in various tissues. The receptor is highly expressed in brain areas that have been implicated in modulation of arousal, stress and anxiety. Central administration of NPS in mice produces an unusual profile of activity by inducing wakefulness and arousal, while at the same time suppressing anxiety. Therefore, NPSR may represent a novel drug target for the treatment of sleep and anxiety disorders. The relative importance of Gs and Gq G protein-coupled signaling to NPSR action is not known. The goal of the project is to generate a series of compounds that can selectively antagonize these signaling pathways. ML079 (CID-3719993) is one compound from a series of congeners satisfies this criteria and can be used to dissect the role these pathways play in NPSR biology.</p></div><div class="h2"></div><p><b>Assigned Assay Grant #:</b> X01-DA026210-01</p><p><b>Screening Center Name & PI:</b> NIH Chemical Genomics Center,
|
||
Christopher Austin</p><p><b>Chemistry Center Name & PI:</b> NIH Chemical Genomics Center,
|
||
Christopher Austin</p><p><b>Assay Submitter & Institution:</b> Markus Heilig, National Institute on
|
||
Alcohol Abuse and Alcoholism</p><p><b>PubChem Summary Bioassay Identifier (AID):</b>
|
||
<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1461" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1461</a></p><div id="ml079.s2"><h2 id="_ml079_s2_">Probe Structure & Characteristics</h2><div id="ml079.tu1" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK47358/table/ml079.tu1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml079.tu1_lrgtbl__"><table><tbody><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;">PubChem CID</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">3719993</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Molecular Weight</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">498.573[g/mol]</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Molecular Formula</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">C29H30N4O4</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">XLogP</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">3.8</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">H-Bond Donor</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">1</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">H-Bond Acceptor</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">8</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Rotatable Bond Count</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">6</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Exact Mass</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">498.227</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Topological Polar Surface Area</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">79.6</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Heavy Atom Count</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">37</td></tr></tbody></table></div></div><div id="ml079.fu1" class="figure"><div class="graphic"><img src="/books/NBK47358/bin/ml079fu1.jpg" alt="Image ml079fu1" /></div></div><div id="ml079.tu2" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK47358/table/ml079.tu2/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml079.tu2_lrgtbl__"><table><thead><tr><th id="hd_h_ml079.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">CID/ML</th><th id="hd_h_ml079.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Target Name</th><th id="hd_h_ml079.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">IC50/EC 50 (nM) [SID,
|
||
AID]</th><th id="hd_h_ml079.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Anti-target Name(s)</th><th id="hd_h_ml079.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">IC50/EC50 (μM)
|
||
[SID, AID]</th><th id="hd_h_ml079.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Selectivity</th><th id="hd_h_ml079.tu2_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Secondary Assay(s) Name: IC50/EC50 (nM)
|
||
[SID, AID]</th></tr></thead><tbody><tr><td headers="hd_h_ml079.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">CID-3719993/<a href="/pcsubstance/?term=ML079[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML079</a></td><td headers="hd_h_ml079.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">NPSR1</td><td headers="hd_h_ml079.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">1585 nM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/14741035" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-14741035</a>,
|
||
<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1491" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1491</a>]</td><td headers="hd_h_ml079.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Muscarinic acetylcholine receptor M1</td><td headers="hd_h_ml079.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">>100</td><td headers="hd_h_ml079.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">>30</td><td headers="hd_h_ml079.tu2_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">[125I]Y10-hNPS
|
||
displacement: 190 nM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/56431665" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-56431665</a>,
|
||
<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1493" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1493</a>]</td></tr></tbody></table></div></div></div><div id="ml079.s3"><h2 id="_ml079_s3_">Recommendations for the scientific use of this probe</h2><p>This probe exhibits functional antagonism equally for cAMP and intraceullar calcium
|
||
signaling pathways stimulated by NPS in the cell-based assays, as opposed to the
|
||
only other reported antagonist of NPS, SHA68, which selectively inhibits calcium
|
||
signaling. The complete blockade of both cAMP and calcium signaling pathways may be
|
||
necessary to fully antagonize NPSR1 function in animal models. As such, this probe
|
||
should be used as a research tool to further study the functions of NPS receptor
|
||
<i>ex vivo</i> and in animal models. In addition, this probe could
|
||
serve as the starting point of drug development for treatment of sleep or anxiety
|
||
disorders such as post-traumatic stress disorder, addictive disorders, and primarily
|
||
alcoholism. The goal of the project was to generate a selective inhibitor of NPS,
|
||
with a potency better than 1 micromolar in a displacement assay against the target;
|
||
CID-3719993 satisfies these criteria. CID-3719993 was the most potent molecule
|
||
tested in this displacement assay.</p><p>Throughout this probe report, MLSMR and NCGC identification numbers are used to
|
||
identify compounds. A complete listing of corresponding PubChem CIDs can be found in
|
||
table 9.</p></div><div id="ml079.s4"><h2 id="_ml079_s4_">1. Scientific Rationale for Project</h2><p>Neuropeptide S receptor (NPSR), previously known as GPR154, is a recently
|
||
de-orphanized G protein coupled receptor. Its endogenous ligand is the 20 amino acid
|
||
peptide Neuropeptide S (NPS). Activation of NPSR induces transient increases in
|
||
intracellular calcium and cAMP, suggesting coupling of this receptor to both Gs and
|
||
Gq G proteins. NPS and its receptor are found in various tissues. The receptor is
|
||
highly expressed in brain areas that have been implicated in modulation of arousal,
|
||
stress and anxiety. Central administration of NPS in mice produces an unusual
|
||
profile of activity by inducing wakefulness and arousal, while at the same time
|
||
suppressing anxiety. Therefore, NPSR may represent a novel drug target for the
|
||
treatment of sleep and anxiety disorders.</p><p>Recent work from Dr. Heilig’s laboratory has indicated that a class of
|
||
stress-related neuropeptides constitutes a novel, promising category of candidate
|
||
targets for the treatment of alcoholism (<a class="bk_pop" href="#ml079.r1">1</a>,<a class="bk_pop" href="#ml079.r2">2</a>).
|
||
Corticotropin-releasing Hormone (CRH) and neuropeptide Y (NPY) are key mediators
|
||
that belong to this category. New, unpublished data from the Heilig laboratory
|
||
indicates that NPS may also belong to this circuitry. For example,
|
||
cerebroventricular administration of NPS mimics stress and CRH in inducing
|
||
relapse-like behavior in an animal model of alcohol seeking behavior.</p><p>Prompted by these data, the main hypothesis is that NPS receptors may be candidate
|
||
targets for relapse prevention in alcoholism. A major roadblock for research aimed
|
||
at establishing this role of the NPS system is the lack of pharmacological tools.
|
||
Development of non-peptide NPS antagonists through this project will provide the
|
||
pharmacological tools necessary to establish the role of endogenous NPS transmission
|
||
in relapse to alcohol seeking behavior, and allow validation of the NPS receptor as
|
||
a candidate treatment target.</p><p>During the process of our probe discovery for the NPS receptor, one structural series
|
||
of antagonist were reported (<a class="bk_pop" href="#ml079.r3">3</a>,<a class="bk_pop" href="#ml079.r4">4</a>), represented by SHA68,
|
||
1-diphenyl-tetrahydro-oxazolo[3,4-a]pyrazine-7-carboxylicacid4-fluoro-benzylamide,
|
||
CAS#= 847555-75-3). We have found that this compound
|
||
selectively inhibited the intracellular calcium signaling stimulated by NPS (25nM
|
||
IC50) compared with that of cAMP signaling pathway (583nM IC50), yielding a
|
||
>20-fold functional selectivity for this compound.</p></div><div id="ml079.s5"><h2 id="_ml079_s5_">2. Project Description</h2><p>The process of NPSR antagonist discovery is summarized and shown in <a class="figpopup" href="/books/NBK47358/figure/ml079.f1/?report=objectonly" target="object" rid-figpopup="figml079f1" rid-ob="figobml079f1">Figure 1</a>.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml079f1" co-legend-rid="figlgndml079f1"><a href="/books/NBK47358/figure/ml079.f1/?report=objectonly" target="object" title="Figure 1" class="img_link icnblk_img figpopup" rid-figpopup="figml079f1" rid-ob="figobml079f1"><img class="small-thumb" src="/books/NBK47358/bin/ml079f1.gif" src-large="/books/NBK47358/bin/ml079f1.jpg" alt="Figure 1. Overview of screening process." /></a><div class="icnblk_cntnt" id="figlgndml079f1"><h4 id="ml079.f1"><a href="/books/NBK47358/figure/ml079.f1/?report=objectonly" target="object" rid-ob="figobml079f1">Figure 1</a></h4><p class="float-caption no_bottom_margin">Overview of screening process. </p></div></div><div id="ml079.t1" class="table"><h3><span class="label">Table 1</span><span class="title">Summary of PubChem data depositions for the NPS project</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK47358/table/ml079.t1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml079.t1_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml079.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">PubChem AID</th><th id="hd_h_ml079.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Type</th><th id="hd_h_ml079.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Target</th><th id="hd_h_ml079.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Conc. Range</th><th id="hd_h_ml079.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Samples Tested</th></tr></thead><tbody><tr><td headers="hd_h_ml079.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1461" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1461</a></td><td headers="hd_h_ml079.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Primary DR</td><td headers="hd_h_ml079.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">NPSR1; cAMP</td><td headers="hd_h_ml079.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0.5nM–46μM</td><td headers="hd_h_ml079.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">221,370</td></tr><tr><td headers="hd_h_ml079.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1491" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1491</a></td><td headers="hd_h_ml079.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">DR</td><td headers="hd_h_ml079.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">NPSR1; cAMP</td><td headers="hd_h_ml079.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0.4nM–38μM</td><td headers="hd_h_ml079.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">114</td></tr><tr><td headers="hd_h_ml079.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1489" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1489</a></td><td headers="hd_h_ml079.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">DR</td><td headers="hd_h_ml079.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">NPSR1; Ca++</td><td headers="hd_h_ml079.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0.4nM–38μM</td><td headers="hd_h_ml079.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">114</td></tr><tr><td headers="hd_h_ml079.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1492" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1492</a></td><td headers="hd_h_ml079.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Counterscreen</td><td headers="hd_h_ml079.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Muscarinic M1;
|
||
Ca++</td><td headers="hd_h_ml079.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0.4nM–38μM</td><td headers="hd_h_ml079.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">85</td></tr><tr><td headers="hd_h_ml079.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1493" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1493</a></td><td headers="hd_h_ml079.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Secondary</td><td headers="hd_h_ml079.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">[125I]NPSR
|
||
displacement</td><td headers="hd_h_ml079.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0.01nM–22μM</td><td headers="hd_h_ml079.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">16</td></tr><tr><td headers="hd_h_ml079.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1464" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1464</a></td><td headers="hd_h_ml079.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Summary</td><td headers="hd_h_ml079.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">.</td><td headers="hd_h_ml079.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">N/A</td><td headers="hd_h_ml079.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">15</td></tr></tbody></table></div></div><div id="ml079.s6"><h3>a. Overall Goal of Project</h3><p>To identify antagonists for neuropeptide S receptor as research probes for
|
||
further studies on the function of NPS receptor in animal models. In addition,
|
||
the antagonists of NPS receptor identified from this project would also serve as
|
||
the starting point of drug development to treat anxiety disorders, including
|
||
post-traumatic stress disorder, and addictive disorders, and primarily
|
||
alcoholism. An additional focus is to obtain structurally distinct probes with
|
||
new structural features, emphasizing appropriate LogP and low polar surface area
|
||
(tPSA≤ 95) for increasing the probability of crossing the blood-brain-barrier.
|
||
Finally, the molecule should have an association constant below 1 µM in the NPS
|
||
binding assay.</p></div><div id="ml079.s7"><h3>b. Assay implementation and screening</h3><div id="ml079.s8"><h4>qHTS Assay for Antagonists of the Neuropeptide S Receptor: cAMP Signal
|
||
Transduction [<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1461" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1461</a>; Primary DR]</h4><div id="ml079.s9"><h5>Assay Rationale & Description</h5><p>It is known that NPS stimulates the cAMP signaling pathway and increases
|
||
the cAMP level in cells expressing NPS receptor. This change in
|
||
intracellular cAMP level can be detected by a cAMP HTRF assay (<a class="figpopup" href="/books/NBK47358/figure/ml079.f2/?report=objectonly" target="object" rid-figpopup="figml079f2" rid-ob="figobml079f2">Fig 2</a>), which is based on
|
||
TR-FRET (time resolved fluorescence resonance energy transfer) between a
|
||
cAMP-specific antibody labeled with europium (Eu) cryptate (Ab-cAMP-Eu),
|
||
and a cAMP analog conjugated to the fluorescent dye d2 (cAMP-d2). Light
|
||
pulse at 320 nm excites the europium of Ab-cAMP-Eu and the energy
|
||
emitted is transferred to the cAMP-d2 bound to the antibody, generating
|
||
a TR-FRET signal at 665 nm. Residual energy from the Eu-cryptate will
|
||
produce a light at 620 nm. The native unlabeled cAMP from cell lysates
|
||
competes with the cAMP-d2 for Ab-cAMP-Eu binding and reversely reduces
|
||
the emission signal of cAMP-d2 by interrupting FRET between the two
|
||
labeled molecules. Both emission signals from the FRET donor (620 nm)
|
||
and the acceptor (665 nm) can be measured by a plate reader. Expression
|
||
of result in fluorescence ratio (665 nm/620 nm) helps to normalize
|
||
differences due to cell density and reagent dispensing.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml079f2" co-legend-rid="figlgndml079f2"><a href="/books/NBK47358/figure/ml079.f2/?report=objectonly" target="object" title="Figure 2" class="img_link icnblk_img figpopup" rid-figpopup="figml079f2" rid-ob="figobml079f2"><img class="small-thumb" src="/books/NBK47358/bin/ml079f2.gif" src-large="/books/NBK47358/bin/ml079f2.jpg" alt="Figure 2. Schematic illustration of the assay principle of the HTRF cAMP assay." /></a><div class="icnblk_cntnt" id="figlgndml079f2"><h4 id="ml079.f2"><a href="/books/NBK47358/figure/ml079.f2/?report=objectonly" target="object" rid-ob="figobml079f2">Figure 2</a></h4><p class="float-caption no_bottom_margin">Schematic illustration of the assay principle of the HTRF
|
||
cAMP assay. </p></div></div></div><div id="ml079.s10"><h5>Assay Protocol</h5><p>All the reagents used for the NPSR cAMP assay and their resources are
|
||
listed in <a class="figpopup" href="/books/NBK47358/table/ml079.t2/?report=objectonly" target="object" rid-figpopup="figml079t2" rid-ob="figobml079t2">Table 2</a>. A
|
||
schematic illustration of the assay principle is shown in <a class="figpopup" href="/books/NBK47358/figure/ml079.f2/?report=objectonly" target="object" rid-figpopup="figml079f2" rid-ob="figobml079f2">figure 2</a>. The assay protocol is
|
||
described below, and in <a class="figpopup" href="/books/NBK47358/table/ml079.t3/?report=objectonly" target="object" rid-figpopup="figml079t3" rid-ob="figobml079t3">table
|
||
3</a>.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml079t2"><a href="/books/NBK47358/table/ml079.t2/?report=objectonly" target="object" title="Table 2" class="img_link icnblk_img figpopup" rid-figpopup="figml079t2" rid-ob="figobml079t2"><img class="small-thumb" src="/books/NBK47358/table/ml079.t2/?report=thumb" src-large="/books/NBK47358/table/ml079.t2/?report=previmg" alt="Table 2. Reagents and resources for the HTRP cAMP assay." /></a><div class="icnblk_cntnt"><h4 id="ml079.t2"><a href="/books/NBK47358/table/ml079.t2/?report=objectonly" target="object" rid-ob="figobml079t2">Table 2</a></h4><p class="float-caption no_bottom_margin">Reagents and resources for the HTRP cAMP assay. </p></div></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml079t3"><a href="/books/NBK47358/table/ml079.t3/?report=objectonly" target="object" title="Table 3" class="img_link icnblk_img figpopup" rid-figpopup="figml079t3" rid-ob="figobml079t3"><img class="small-thumb" src="/books/NBK47358/table/ml079.t3/?report=thumb" src-large="/books/NBK47358/table/ml079.t3/?report=previmg" alt="Table 3. cAMP assay protocol for the CHO-NPSR cells in 1536-well plate format." /></a><div class="icnblk_cntnt"><h4 id="ml079.t3"><a href="/books/NBK47358/table/ml079.t3/?report=objectonly" target="object" rid-ob="figobml079t3">Table 3</a></h4><p class="float-caption no_bottom_margin">cAMP assay protocol for the CHO-NPSR cells in 1536-well plate
|
||
format. </p></div></div><p>A Chinese hamster ovary cell line stably expressing the NPSR was
|
||
generated in Dr. Heilig’s lab. The cells were maintained in
|
||
F12 medium containing 10 % FBS, 100 units/ml Penicillin, 100
|
||
μg/ml Streptomycin, and 200 μg/ml Geneticin at
|
||
37 ºC, 5% CO2.</p><p>Suspended CHO-NPSR cells were seeded into 1536-well tissue
|
||
culture-treated white plates at a density of 1800 cells/well in 4
|
||
μl media without Geneticin and incubated at 37
|
||
ºC, 5 % CO<sub>2</sub> for overnight. After
|
||
1μl of stimulation buffer (1X PBS buffer, 0.1%
|
||
BSA, 0.05% Tween-20, 500 μM Ro 20-1724, EC80 of
|
||
NPS) was added to each well, cells were incubated at 37 ºC,
|
||
5 % CO<sub>2</sub> for 30 min. 1.25 μl of d2
|
||
conjugated cAMP and 1 μl of cryptate conjugated anti-cAMP
|
||
antibody were then added. D2 conjugated cAMP and cryptate conjugated
|
||
anti-cAMP antibody were both prepared in cell lysis buffer according to
|
||
the manufacturer’s instruction. After 30 minutes, plates
|
||
were then read in Viewlux plate reader (Perkin Elmer) using the TRF
|
||
detection mode optimized for HTRF.</p></div><div id="ml079.s11"><h5>Summary of Results</h5><p>NCGC tested 1284 1536-well plates in the primary screen. The average
|
||
Z’ for the screen was 0.68 +/− 0.10,
|
||
excluding 34 plates which failed visual QC. Results are reported for
|
||
221,370 samples. 2,309 compounds gave significant
|
||
concentration-responses in the primary screen. Compounds were
|
||
deprioritized for confirmation if they were suspected to interfere with
|
||
the readout of the assay platform, or to affect GPCR signal transduction
|
||
at a point in the pathway beyond the neuropeptide S receptor using
|
||
internal NCGC SAR data from other related assays.</p></div></div><div id="ml079.confcon"><h4>Confirmation Concentration-Response Assay for Antagonists of the
|
||
Neuropeptide S Receptor: cAMP Signal Transduction [<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1491" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1491</a>;
|
||
Confirmatory DR]</h4><div id="ml079.s12"><h5>Assay Description</h5><p>Select samples active in the primary screen were obtained in DMSO
|
||
solution from the MLSMR and/or as powders from compound vendors to
|
||
confirm activity in the original assay.</p></div><div id="ml079.s13"><h5>Assay Protocol</h5><p>The assay protocol and reagents are identical to: qHTS Assay for
|
||
Antagonists of the Neuropeptide S Receptor: cAMP Signal Transduction
|
||
[<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1651" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1651</a>; Primary]</p></div><div id="ml079.s14"><h5>Summary of Results</h5><p>Prioritized compounds had a high confirmation rate, but the most potent
|
||
compounds from the MLSMR were only in the micromolar range using the
|
||
HTRF assay format.</p></div></div><div id="ml079.s15"><h4>qHTS Assay for Antagonists of the Neuropeptide S Receptor: Calcium
|
||
Mobilization [<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1489" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1489</a>; DR]</h4><div id="ml079.s16"><h5>Assay Description</h5><p>The fluorescent calcium dyes Fluo-4 AM has been widely used in GPCR
|
||
studies to visualize and measure changes in intracellular calcium. It is
|
||
able to enter the cell by passive diffuse and is de-esterified by
|
||
endogenous esterases in the cytosol that is not fluorescent. The dye
|
||
becomes fluorescent upon binding of calcium, resulting in fluorescent
|
||
signals proportional to the cytosol free calcium concentration that is
|
||
dramatically increased by the stimulation of certain GPCRs. Because
|
||
intracellular calcium responses are transient with a fast kinetic (a
|
||
half-life in the range of seconds to minutes), its detection requires
|
||
special instruments that combine both the automated reagent dispenser
|
||
and the kinetic fluorescence reader. A FDSS-7000 system (FDSS,
|
||
Hamamatsu, Hamamatsu City, Japan) was used at NCGC to measure the
|
||
intracellular calcium changes in response to agonist stimulation on
|
||
GPCRs.</p></div><div id="ml079.s17"><h5>Assay Protocol</h5><p>All the reagents used for this assay and their resources are listed in
|
||
<a class="figpopup" href="/books/NBK47358/table/ml079.t4/?report=objectonly" target="object" rid-figpopup="figml079t4" rid-ob="figobml079t4">Table 4</a>. The assay
|
||
protocol is described below, and in <a class="figpopup" href="/books/NBK47358/table/ml079.t5/?report=objectonly" target="object" rid-figpopup="figml079t5" rid-ob="figobml079t5">table 5</a>.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml079t4"><a href="/books/NBK47358/table/ml079.t4/?report=objectonly" target="object" title="Table 4" class="img_link icnblk_img figpopup" rid-figpopup="figml079t4" rid-ob="figobml079t4"><img class="small-thumb" src="/books/NBK47358/table/ml079.t4/?report=thumb" src-large="/books/NBK47358/table/ml079.t4/?report=previmg" alt="Table 4. Reagents and resources for the calcium mobilization assay." /></a><div class="icnblk_cntnt"><h4 id="ml079.t4"><a href="/books/NBK47358/table/ml079.t4/?report=objectonly" target="object" rid-ob="figobml079t4">Table 4</a></h4><p class="float-caption no_bottom_margin">Reagents and resources for the calcium mobilization
|
||
assay. </p></div></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml079t5"><a href="/books/NBK47358/table/ml079.t5/?report=objectonly" target="object" title="Table 5" class="img_link icnblk_img figpopup" rid-figpopup="figml079t5" rid-ob="figobml079t5"><img class="small-thumb" src="/books/NBK47358/table/ml079.t5/?report=thumb" src-large="/books/NBK47358/table/ml079.t5/?report=previmg" alt="Table 5. Calcium mobilization assay protocol for the CHO-NPSR cells in 1536-well plate format." /></a><div class="icnblk_cntnt"><h4 id="ml079.t5"><a href="/books/NBK47358/table/ml079.t5/?report=objectonly" target="object" rid-ob="figobml079t5">Table 5</a></h4><p class="float-caption no_bottom_margin">Calcium mobilization assay protocol for the CHO-NPSR cells in
|
||
1536-well plate format. </p></div></div><p>The CHO-NPSR cell line used in the cAMP assay was also used in this
|
||
calcium mobilization assay. The suspended cells were plated at 3
|
||
μl/well with 2000 cells in the black, tissue culture
|
||
treated, clear bottom 1536-well plates. After overnight incubation at 37
|
||
ºC, 5% CO<sub>2</sub>, 3 μl of the
|
||
calcium dye (no wash High Performance PBX Calcium Assay Kit, BD
|
||
Biosciences) was loaded to each well and the plates were incubated at 37
|
||
ºC, 5 % CO<sub>2</sub> for 1 hour followed by
|
||
10-min incubation with 23 nl compound prepared in DMSO solution. The
|
||
assay plates were then placed onto the FDSS-7000 kinetic fluorescence
|
||
plate reader for measuring the changes of intracellular free calcium.
|
||
The basal fluorescence signal was recorded for 6 sec at 1 Hz followed by
|
||
an addition of 1 μl of NPS stimulation buffer (1X PBS
|
||
buffer, 0.1% BSA, 0.05% Tween-20, 500
|
||
μM Ro 20-1724, EC80 of NPS) and 4-minute continuously
|
||
recording at 1 Hz.</p></div><div id="ml079.s18"><h5>Summary of Results</h5><p>Most compounds tested were more potent against the calcium mobilization
|
||
assay than the cAMP assay, indicating potential antagonist functional
|
||
selectivity for the Gs and Gq signaling pathways. The literature
|
||
compound SHA 68 is a potent antagonist of Gq signaling.</p></div></div><div id="ml079.s19"><h4>Counterscreen for Antagonists of the Neuropeptide S Receptor: Muscarinic
|
||
Acetylcholine M1 Receptor Antagonism [<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1492" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1492</a>;
|
||
Counterscreen]</h4><div id="ml079.s20"><h5>Assay Description</h5><p>Muscarinic acetylcholine M1 receptor is a G protein-coupled receptor
|
||
found in the plasma membranes of certain neurons and other cells.
|
||
Because this receptor is coupled to the same G proteins as NPSR, it
|
||
provides a good counterscreen for compounds that antagonize signal
|
||
transduction through receptor-independent mechanisms (such as direct
|
||
modulators of cellular cAMP levels).</p></div><div id="ml079.s21"><h5>Assay Protocol</h5><p>All the reagents used for this assay and their resources are listed in
|
||
<a class="figpopup" href="/books/NBK47358/table/ml079.t6/?report=objectonly" target="object" rid-figpopup="figml079t6" rid-ob="figobml079t6">Table 6</a>. The assay
|
||
protocol is described below, and in <a class="figpopup" href="/books/NBK47358/table/ml079.t7/?report=objectonly" target="object" rid-figpopup="figml079t7" rid-ob="figobml079t7">table 7</a>. A Chinese hamster ovary (CHO) cell
|
||
line stably expressing muscarinic acetylcholine M1 receptor (CHO-M1) was
|
||
obtained from ATCC and maintained in F-12 Kaighn’s media
|
||
(Invitrogen, Carlsbad, CA, 21127) supplemented with 10 %
|
||
FBS, 100 units/ml penicillin, 100 ug/ml streptomycin and 250 ug/ml
|
||
geneticin at 37C, 5% CO2 in a humidified atmosphere. Before
|
||
the assay, aliquots of cells were frozen and stored at
|
||
−135C. The assay was performed on a FDSS-7000 kinetic plate
|
||
reader in 1536-well format. The maximums of kinetic fluorescence
|
||
responses were converted into text files using the
|
||
instrument’s software data export utility. Data for
|
||
antagonist response were normalized to the controls for basal activity
|
||
(DMSO only) and 100% inhibition. AC50 values were determined
|
||
from concentration-response data modeled with the standard Hill
|
||
equation.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml079t6"><a href="/books/NBK47358/table/ml079.t6/?report=objectonly" target="object" title="Table 6" class="img_link icnblk_img figpopup" rid-figpopup="figml079t6" rid-ob="figobml079t6"><img class="small-thumb" src="/books/NBK47358/table/ml079.t6/?report=thumb" src-large="/books/NBK47358/table/ml079.t6/?report=previmg" alt="Table 6. Reagents and resources for the calcium mobilization assay." /></a><div class="icnblk_cntnt"><h4 id="ml079.t6"><a href="/books/NBK47358/table/ml079.t6/?report=objectonly" target="object" rid-ob="figobml079t6">Table 6</a></h4><p class="float-caption no_bottom_margin">Reagents and resources for the calcium mobilization
|
||
assay. </p></div></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml079t7"><a href="/books/NBK47358/table/ml079.t7/?report=objectonly" target="object" title="Table 7" class="img_link icnblk_img figpopup" rid-figpopup="figml079t7" rid-ob="figobml079t7"><img class="small-thumb" src="/books/NBK47358/table/ml079.t7/?report=thumb" src-large="/books/NBK47358/table/ml079.t7/?report=previmg" alt="Table 7. Calcium mobilization assay protocol for the CHO-M1 cells in 1536-well plate format." /></a><div class="icnblk_cntnt"><h4 id="ml079.t7"><a href="/books/NBK47358/table/ml079.t7/?report=objectonly" target="object" rid-ob="figobml079t7">Table 7</a></h4><p class="float-caption no_bottom_margin">Calcium mobilization assay protocol for the CHO-M1 cells in
|
||
1536-well plate format. </p></div></div><p>Frozen CHO-M1 cells were thawed, washed once with fresh media and
|
||
resuspended in F-12 Kaighn’s media supplemented with 10
|
||
% FBS, 100 units/ml penicillin and 100 ug/ml streptomycin.
|
||
Cells were plated at 3 ul/well (1200 cells) to black, clear-bottom,
|
||
tissue-culture treated 1536-well plates, and then cultured at 37C, 5
|
||
% CO2 for 16 to 30 hours. 3 ul of calcium dye (from High
|
||
Performance PBX Calcium Assay Kit, BD Biosciences) was added. The
|
||
calcium dye was prepared according to the manufactory’s
|
||
instruction.</p><p>Plates were incubated at 37C, 5 % CO2 for 1 hour. Add 23
|
||
nl/well of compound in DMSO solution was added. The final titration for
|
||
each compound was between 0.6 nM and 46 µM. Plates were then loaded onto
|
||
the FDSS-7000. The following steps were performed on the FDSS-7000.
|
||
(<a class="bk_pop" href="#ml079.r1">1</a>) Record fluorescent
|
||
background (Ex 480 nm, Em 520–560 nm) for 10 s. (<a class="bk_pop" href="#ml079.r2">2</a>) Add 2 μl of
|
||
stimulation buffer (1X HBSS buffer, 0.1% BSA, 60 nM
|
||
carbachol). (<a class="bk_pop" href="#ml079.r3">3</a>) Record
|
||
antagonist response (Ex 480 nm, Em 520–560 nm) for 180
|
||
s.</p></div><div id="ml079.s22"><h5>Summary of Results</h5><p>Validated molecules did not antagonize M1 stimulation by carbachol,
|
||
indicating some selectivity of the molecules for NPSR.</p></div></div><div id="ml079.s23"><h4>qHTS Assay for Antagonists of the Neuropeptide S Receptor: Radioactive
|
||
Ligand Displacement [<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1493" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1493</a>; Secondary]</h4><div id="ml079.s24"><h5>Assay Description</h5><p>Select samples was tested in a direct ligand binding assay, measuring
|
||
[<sup>125</sup>I] Y<sup>10</sup>-hNPS
|
||
displacement as previously described (<a class="bk_pop" href="#ml079.r5">5</a>).</p></div><div id="ml079.s25"><h5>Assay Protocol</h5><p>All the reagents used for this assay and their resources are listed in
|
||
<a class="figpopup" href="/books/NBK47358/table/ml079.t8/?report=objectonly" target="object" rid-figpopup="figml079t8" rid-ob="figobml079t8">Table 8</a>. The assay was
|
||
carried out as described (<a class="bk_pop" href="#ml079.r5">5</a>)
|
||
with minor modification. Y10-NPS labeled with 125I was bought from NEN
|
||
Perkin Elmer (Boston, MA). CHO cells stably expressing human NPSR were
|
||
seeded into 24-well plates and cultured until reaching
|
||
90–95% confluency. Cells were washed with 1ml
|
||
PBS once and then incubated with radioligand with or without compounds
|
||
or in DMEM medium containing 0.1% bovine serum albumin at
|
||
20C for 1.5 hr. Increasing concentrations of compounds or unlabeled
|
||
human NPS were used to compete with 0.15 nM
|
||
[125I] Y10-NPS. Nonspecific binding was
|
||
determined in the presence of 1 µM unlabeled human NPS. Cells were
|
||
washed twice with cold PBS and lysed with 1 N NaOH. Bound radioactivity
|
||
was counted in a liquid scintillation counter.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml079t8"><a href="/books/NBK47358/table/ml079.t8/?report=objectonly" target="object" title="Table 8" class="img_link icnblk_img figpopup" rid-figpopup="figml079t8" rid-ob="figobml079t8"><img class="small-thumb" src="/books/NBK47358/table/ml079.t8/?report=thumb" src-large="/books/NBK47358/table/ml079.t8/?report=previmg" alt="Table 8. Reagents and resources for the radioactive ligand binding assay." /></a><div class="icnblk_cntnt"><h4 id="ml079.t8"><a href="/books/NBK47358/table/ml079.t8/?report=objectonly" target="object" rid-ob="figobml079t8">Table 8</a></h4><p class="float-caption no_bottom_margin">Reagents and resources for the radioactive ligand binding
|
||
assay. </p></div></div></div><div id="ml079.s26"><h5>Summary of Results</h5><p>Validated molecules displaced radioactive NPS ligand, typically at
|
||
concentrations well below that required for antagonism of the receptor
|
||
in the cell-based assays. It is possible that molecules which did not
|
||
displace radioactive NPS ligand bind allosterically to the receptor, but
|
||
the project team had no assay with which to verify this possibility.</p></div></div></div><div id="ml079.s27"><h3>b. Probe optimization (Naphthopyranopyrimidine)</h3><div id="ml079.s28"><h4>Initial results of lead series</h4><p>MLS-000558527 antagonized an NPS response with an IC50 of 2μM in
|
||
the primary screening; in the confirmatory cAMP and calcium assays, it
|
||
exhibited 250nM activity in a direct binding, displacement assay, and was
|
||
the most potent molecule identified during screening. It did not exhibit any
|
||
agonism towards the NPS receptor (not shown; data given in PubChem <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1491" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1491</a>). The activity of MLS-000558527 was confirmed for the both a sample
|
||
re-ordered from the MLSMR repository, as well as for a sample synthesized
|
||
in-house.</p><div id="ml079.f3" class="figure bk_fig"><div class="graphic"><img src="/books/NBK47358/bin/ml079fu1.jpg" alt="Figure 3. Structure of MLS-000558527." /></div><h3><span class="label">Figure 3</span><span class="title">Structure of MLS-000558527</span></h3></div></div><div id="ml079.s29"><h4>Development of lead series</h4><p>During the process of this RO3 application, two small molecule antagonists of
|
||
NPS receptor were reported (<a class="bk_pop" href="#ml079.r3">3</a>,
|
||
<a class="bk_pop" href="#ml079.r4">4</a>, <a class="bk_pop" href="#ml079.r6">6</a>). They are SHA66
|
||
(3-oxo-1,1-diphenyl-tetrahydro-ox-azolo[3,4-a]pyrazine-7-carboxylicacidbenzylamide,
|
||
CAS# 847553-81-5) and SHA68,
|
||
1-diphenyl-tetrahydro-oxazolo[3,4-a]pyrazine-7-carboxylicacid4-fluoro-benzylamide,
|
||
CAS# 847555-75-3), two closely related bi-cyclicpiperazines with
|
||
antagonistic properties at the NPS receptor (NPSR).</p><div id="ml079.f4" class="figure bk_fig"><div class="graphic"><img src="/books/NBK47358/bin/ml079f4.jpg" alt="Figure 4. Structure of SHA-66 and SHA-68." /></div><h3><span class="label">Figure 4</span><span class="title">Structure of SHA-66 and SHA-68</span></h3></div><p>This report changed our initial SAR strategy, which was directed towards
|
||
systematic substitution. We decided to use molecular modeling to compare the
|
||
energy minimized structures of the two series. Chemical Computing
|
||
Group’s MOE was used to flexibly align the molecules, with
|
||
MMFF94x parameterization and without hydrogen bonding penalty. <a class="figpopup" href="/books/NBK47358/figure/ml079.f5/?report=objectonly" target="object" rid-figpopup="figml079f5" rid-ob="figobml079f5">Figures 5</a> and <a class="figpopup" href="/books/NBK47358/figure/ml079.f6/?report=objectonly" target="object" rid-figpopup="figml079f6" rid-ob="figobml079f6">6</a> show two different ways of superimposing the
|
||
minimized structures. The stereochemistry of the active enantiomer of
|
||
MLS-000558527 is not known. Alternate stereochemistries were used to
|
||
generate alternate models shown in <a class="figpopup" href="/books/NBK47358/figure/ml079.f5/?report=objectonly" target="object" rid-figpopup="figml079f5" rid-ob="figobml079f5">figure 5</a> (R) and <a class="figpopup" href="/books/NBK47358/figure/ml079.f6/?report=objectonly" target="object" rid-figpopup="figml079f6" rid-ob="figobml079f6">6</a> (S).</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml079f5" co-legend-rid="figlgndml079f5"><a href="/books/NBK47358/figure/ml079.f5/?report=objectonly" target="object" title="Figure 5" class="img_link icnblk_img figpopup" rid-figpopup="figml079f5" rid-ob="figobml079f5"><img class="small-thumb" src="/books/NBK47358/bin/ml079f5.gif" src-large="/books/NBK47358/bin/ml079f5.jpg" alt="Figure 5. Superimposition of NCGC lead series with SHA 68." /></a><div class="icnblk_cntnt" id="figlgndml079f5"><h4 id="ml079.f5"><a href="/books/NBK47358/figure/ml079.f5/?report=objectonly" target="object" rid-ob="figobml079f5">Figure 5</a></h4><p class="float-caption no_bottom_margin">Superimposition of NCGC lead series with SHA 68. </p></div></div><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml079f6" co-legend-rid="figlgndml079f6"><a href="/books/NBK47358/figure/ml079.f6/?report=objectonly" target="object" title="Figure 6" class="img_link icnblk_img figpopup" rid-figpopup="figml079f6" rid-ob="figobml079f6"><img class="small-thumb" src="/books/NBK47358/bin/ml079f6.gif" src-large="/books/NBK47358/bin/ml079f6.jpg" alt="Figure 6. Alternate superimposition of NCGC lead series with SHA 68. 2D depiction exaggerated to highlight spatial alignment." /></a><div class="icnblk_cntnt" id="figlgndml079f6"><h4 id="ml079.f6"><a href="/books/NBK47358/figure/ml079.f6/?report=objectonly" target="object" rid-ob="figobml079f6">Figure 6</a></h4><p class="float-caption no_bottom_margin">Alternate superimposition of NCGC lead series with SHA 68. 2D
|
||
depiction exaggerated to highlight spatial alignment. </p></div></div><p>Both models present a good overlapping of the core cyclic rings as well as
|
||
the adjacent aromatic regions. The main difference between these two series
|
||
is the conformation and structure of the ureic benzyl amine functional
|
||
group. This group sticking out from the main core of SHA68, and it is
|
||
absence in the NCGC series. In a recent publication, it has been shown that
|
||
the introduction of the ureic benzyl amine functional group in the main core
|
||
is fundamental for the activity of the series. For these reasons, we decided
|
||
to develop several exploratory series introducing an aromatic ring at
|
||
different regions and distances of our series. <a class="figpopup" href="/books/NBK47358/figure/ml079.f7/?report=objectonly" target="object" rid-figpopup="figml079f7" rid-ob="figobml079f7">Figure 7</a> shows some of our analogues synthesized
|
||
for this strategy.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml079f7" co-legend-rid="figlgndml079f7"><a href="/books/NBK47358/figure/ml079.f7/?report=objectonly" target="object" title="Figure 7" class="img_link icnblk_img figpopup" rid-figpopup="figml079f7" rid-ob="figobml079f7"><img class="small-thumb" src="/books/NBK47358/bin/ml079f7.gif" src-large="/books/NBK47358/bin/ml079f7.jpg" alt="Figure 7. Introduction of a new aromatic ring in several points of the molecule." /></a><div class="icnblk_cntnt" id="figlgndml079f7"><h4 id="ml079.f7"><a href="/books/NBK47358/figure/ml079.f7/?report=objectonly" target="object" rid-ob="figobml079f7">Figure 7</a></h4><p class="float-caption no_bottom_margin">Introduction of a new aromatic ring in several points of the
|
||
molecule. </p></div></div><p>As it can be seen on <a class="figpopup" href="/books/NBK47358/table/ml079.t9/?report=objectonly" target="object" rid-figpopup="figml079t9" rid-ob="figobml079t9">Table 9</a>,
|
||
the potency of the series did not improve with this first round of
|
||
chemistry. However, all the compounds in this round were synthesized without
|
||
the methoxy substituents in the top phenyl ring, which were thought to be
|
||
beneficial for binding. Interestingly, NCGC-00181361 exhibited activity
|
||
within experimental error of MLS-000558527, and along with NCGC-00182492 and
|
||
NCGC-00182493, give us insight into the relationship between the SHA68
|
||
series and this one (<a class="figpopup" href="/books/NBK47358/figure/ml079.f8/?report=objectonly" target="object" rid-figpopup="figml079f8" rid-ob="figobml079f8">Figure
|
||
8</a>).</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml079t9"><a href="/books/NBK47358/table/ml079.t9/?report=objectonly" target="object" title="Table 9" class="img_link icnblk_img figpopup" rid-figpopup="figml079t9" rid-ob="figobml079t9"><img class="small-thumb" src="/books/NBK47358/table/ml079.t9/?report=thumb" src-large="/books/NBK47358/table/ml079.t9/?report=previmg" alt="Table 9. Activity of probe series in NPS cAMP assay, and radioactive ligand displacement (for select analogs), along with corresponding PubChem CIDs." /></a><div class="icnblk_cntnt"><h4 id="ml079.t9"><a href="/books/NBK47358/table/ml079.t9/?report=objectonly" target="object" rid-ob="figobml079t9">Table 9</a></h4><p class="float-caption no_bottom_margin">Activity of probe series in NPS cAMP assay, and radioactive
|
||
ligand displacement (for select analogs), along with corresponding
|
||
PubChem CIDs. </p></div></div><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml079f8" co-legend-rid="figlgndml079f8"><a href="/books/NBK47358/figure/ml079.f8/?report=objectonly" target="object" title="Figure 8" class="img_link icnblk_img figpopup" rid-figpopup="figml079f8" rid-ob="figobml079f8"><img class="small-thumb" src="/books/NBK47358/bin/ml079f8.gif" src-large="/books/NBK47358/bin/ml079f8.jpg" alt="Figure 8. Explanation for the lack of activity of NCGC-00182492 and NCGC-00182493." /></a><div class="icnblk_cntnt" id="figlgndml079f8"><h4 id="ml079.f8"><a href="/books/NBK47358/figure/ml079.f8/?report=objectonly" target="object" rid-ob="figobml079f8">Figure 8</a></h4><p class="float-caption no_bottom_margin">Explanation for the lack of activity of NCGC-00182492 and
|
||
NCGC-00182493. </p></div></div><p>The next round of synthesis explored the need di-methoxy substitution on the
|
||
phenyl ring. <a class="figpopup" href="/books/NBK47358/figure/ml079.f9/?report=objectonly" target="object" rid-figpopup="figml079f9" rid-ob="figobml079f9">Figure 9</a> shows some
|
||
of the analogues initially synthesized for evaluating the di-methoxy
|
||
functional groups. <a class="figpopup" href="/books/NBK47358/table/ml079.t9/?report=objectonly" target="object" rid-figpopup="figml079t9" rid-ob="figobml079t9">Table 9</a>
|
||
shows that the analogue with meta and para di-methoxy substitution is five
|
||
times more active than the unsubstituted phenyl ring. Although
|
||
mono-para-methoxy substitution showed some effect improving the activity, it
|
||
is the meta-methoxy substituent the one that provide the main increment on
|
||
activity, showing that the mono-meta-methoxy substitution is equally potent
|
||
that the para-meta di-methoxy substitution. Focusing on the imidine
|
||
substitution comparing NCGC00181382 and NCGC00183143, it can be seen that
|
||
the phenyl substitution is close to five times better than the ethyl
|
||
morpholino substitution.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml079f9" co-legend-rid="figlgndml079f9"><a href="/books/NBK47358/figure/ml079.f9/?report=objectonly" target="object" title="Figure 9" class="img_link icnblk_img figpopup" rid-figpopup="figml079f9" rid-ob="figobml079f9"><img class="small-thumb" src="/books/NBK47358/bin/ml079f9.gif" src-large="/books/NBK47358/bin/ml079f9.jpg" alt="Figure 9. Exploration of the substitution in the phenyl ring." /></a><div class="icnblk_cntnt" id="figlgndml079f9"><h4 id="ml079.f9"><a href="/books/NBK47358/figure/ml079.f9/?report=objectonly" target="object" rid-ob="figobml079f9">Figure 9</a></h4><p class="float-caption no_bottom_margin">Exploration of the substitution in the phenyl ring. </p></div></div><p>Activation of NPSR induces transient increases in intracellular calcium and
|
||
cAMP, suggesting coupling of this receptor to both Gs and Gq G proteins.
|
||
Appendix 3 shows the values of MLS-000558527 in both functional assays. It
|
||
can be seen that our probe is equally potent antagonizing both biological
|
||
pathways. In contrast, SHA68 has a 20 times selectivity towards calcium
|
||
activation that cAMP activation.</p><p>The activity of all these analogues and others can be seen in the Appendix.
|
||
MLS-000558527 is the most thoroughly characterized and among the most potent
|
||
compounds from this series, and has been selected as the probe molecule.
|
||
Gram scale synthesis of the compounds has been completed to facilitate
|
||
<i>ex vivo</i> and <i>in vivo</i> characterization
|
||
of this compound’s activity by other investigators. Potencies of
|
||
series analogs are presented in <a class="figpopup" href="/books/NBK47358/table/ml079.t9/?report=objectonly" target="object" rid-figpopup="figml079t9" rid-ob="figobml079t9">Table
|
||
9</a>.</p></div></div></div><div id="ml079.s30"><h2 id="_ml079_s30_">3. Probe</h2><div id="ml079.s31"><h3>a. Chemical name</h3><p>10-(2-morpholinoethyl)-11-imino-12-(m,p-dimethoxyphenyl)-10,11-dihydro-12H-naphtho[1',2':5,6]-pyrano
|
||
[2,3-d]pyrimidine (MLS-000558527)
|
||
<b>[<a href="/pcsubstance/?term=ML079[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML079</a>]</b></p></div><div id="ml079.s33"><h3>b. Probe chemical structure</h3><div id="ml079.fu2" class="figure"><div class="graphic"><img src="/books/NBK47358/bin/ml079fu1.jpg" alt="Image ml079fu1" /></div></div><p>Sample is an isomeric mixture.</p></div><div id="ml079.s34"><h3>c. Structural Verification Information of probe SID</h3><p>Structural verification and initial purity quantification was performed by
|
||
<sup>1</sup>H NMR analysis using a Varian spectrometer dissolving the sample
|
||
in Cl<sub>3</sub>CD. In addition, further analysis was carried out by LC/MS
|
||
using an Agilent system in the following conditions:</p><ul class="simple-list"><li class="half_rhythm"><div>Column: 3 x 75 mm Luna C18, 3 micron</div></li><li class="half_rhythm"><div>Run time: 4.5 minutes</div></li><li class="half_rhythm"><div>Gradient: 4% to 100% over 2.8 minutes</div></li><li class="half_rhythm"><div>Mobile phase: acetonitrile (0.025% TFA), water
|
||
(0.05% TFA)</div></li><li class="half_rhythm"><div>Flow rate: 0.8 to 1.0 mL/min</div></li><li class="half_rhythm"><div>Temperature: 50 C</div></li><li class="half_rhythm"><div>UV Wavelength: 220 nm, 254 nm</div></li></ul><p>The retention time of MLS-000558527 in those conditions was of 2.98 minutes. The
|
||
purity of the Mass spectra was recorded in the positive ionization mode using an
|
||
electrospray (API-ES) ionizing source with nitrogen as drying gas. Both NMR and
|
||
LC/MS analysis showed purity greater than 99% for those batches of
|
||
MLS-000558527 use in the biological evaluation.</p></div><div id="ml079.s35"><h3>d. PubChem CID (corresponding to the SID)</h3><p>CID-3719993</p></div><div id="ml079.s36"><h3>e. Availability from vendor</h3><p>MLS-000558527 can be purchased from the following vendors</p><div id="ml079.tu3" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK47358/table/ml079.tu3/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml079.tu3_lrgtbl__"><table><tbody><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Company Info:</td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Aurora Fine Chemicals LLC</td></tr><tr><td colspan="2" rowspan="1" style="text-align:left;vertical-align:top;">Email:
|
||
<a href="mailto:dev@null" data-email="moc.slacimehcenifarorua@arorua" class="oemail">moc.slacimehcenifarorua@arorua</a></td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Catalog Name:</td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Aurora Screening Library</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Order Number:</td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">kbsa-0079914</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Company Info:</td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Ambinter</td></tr><tr><td colspan="2" rowspan="1" style="text-align:left;vertical-align:top;">Email:
|
||
<a href="mailto:dev@null" data-email="moc.retnibma@tcatnoc" class="oemail">moc.retnibma@tcatnoc</a></td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Catalog Name:</td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Ambinter Stock Screening
|
||
Collection</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Order Number:</td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">STOCK4S-75882</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Company Info:</td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Scientific Exchange, Inc.</td></tr><tr><td colspan="2" rowspan="1" style="text-align:left;vertical-align:top;">Email:
|
||
<a href="mailto:dev@null" data-email="moc.sdnuopmocsth@selas" class="oemail">moc.sdnuopmocsth@selas</a></td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Catalog Name:</td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Scientific Exchange Product List</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Order Number:</td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">F-055679</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Company Info:</td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Ryan Scientific, Inc.</td></tr><tr><td colspan="2" rowspan="1" style="text-align:left;vertical-align:top;">Email:
|
||
<a href="mailto:dev@null" data-email="moc.icsnayr@selas" class="oemail">moc.icsnayr@selas</a></td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Catalog Name:</td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Ryan Scientific Screening Library</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Order Number:</td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">PHAR116579</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Company Info:</td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Interbioscreen Ltd.</td></tr><tr><td colspan="2" rowspan="1" style="text-align:left;vertical-align:top;">Email:
|
||
<a href="mailto:dev@null" data-email="ur.ghc.neercsbi@neercs" class="oemail">ur.ghc.neercsbi@neercs</a></td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Catalog Name:</td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Interbioscreen Compound Library</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Order Number:</td><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">STOCK4S-75882</td></tr></tbody></table></div></div></div><div id="ml079.s37"><h3>f. Mode of action for biological activity of probe</h3><p>Neuropeptide S receptor antagonist</p></div><div id="ml079.s38"><h3>g. Detailed synthetic pathway for making probe</h3><div id="ml079.fu3" class="figure"><div class="graphic"><img src="/books/NBK47358/bin/ml079fu35.jpg" alt="Image ml079fu35" /></div></div></div><div id="ml079.s39"><h3>h. Summary of probe properties</h3><p>MLS-000558527 is a very lypophilic compound ease to dissolve in organic solvents
|
||
such us MeOH, Cl2CH2 or acetone. As pure compound, MLS-000558527 is a white
|
||
powder at room temperature, chemically stable and with no apparent reactivity
|
||
with air.</p></div><div id="ml079.s40"><h3>i. Properties Computed from Structure</h3><p>Chemical Formula: C29H30N4O4</p><p>Molecular Weight: 498.57</p><p>Log P: 4.17</p><p>tPSA: 79.61</p><p>Number of hydrogen donors: 1</p><p>Number of hydrogen acceptors: 7</p></div></div><div id="ml079.s41"><h2 id="_ml079_s41_">4. Appendices</h2><div id="ml079.s42"><h3>a. Summary of activities of SHA 68 and MLS-000558527</h3><div id="ml079.tu4" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK47358/table/ml079.tu4/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml079.tu4_lrgtbl__"><table><thead><tr><th id="hd_h_ml079.tu4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"></th><th id="hd_h_ml079.tu4_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">[125I]NPS
|
||
Displacement</th><th id="hd_h_ml079.tu4_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">cAMP IC50 (nM)</th><th id="hd_h_ml079.tu4_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Calcium Mobilization IC50 (nM)</th><th id="hd_h_ml079.tu4_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Functional Selectivity for
|
||
Ca++:cAMP</th></tr></thead><tbody><tr><td headers="hd_h_ml079.tu4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">SHA 68</td><td headers="hd_h_ml079.tu4_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">6 nM</td><td headers="hd_h_ml079.tu4_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">583 nM</td><td headers="hd_h_ml079.tu4_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">28.9 nM</td><td headers="hd_h_ml079.tu4_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">20:1</td></tr><tr><td headers="hd_h_ml079.tu4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">MLS-000558527</td><td headers="hd_h_ml079.tu4_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">256 nM</td><td headers="hd_h_ml079.tu4_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">2990 nM</td><td headers="hd_h_ml079.tu4_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">2666 nM</td><td headers="hd_h_ml079.tu4_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">1:1</td></tr></tbody></table></div></div><p>SHA 68, while more potent in the radioligand experiment, only partially blocked
|
||
NPS-induced activity when injected at 50 mg/kg i.p. (<a class="bk_pop" href="#ml079.r3">3</a>) This dosing yielded single-digit micromolar
|
||
concentrations in brain. This is consistent with the hypothesis that antagonism
|
||
of NPS stimulation requires functional antagonism of either cAMP or both cAMP
|
||
and calcium signaling. MLS-000558527 provides a means of testing this hypothesis
|
||
because this series does not appear to be functionally selective for one pathway
|
||
or the other.</p></div><div id="ml079.s43"><h3>b. Concentration-Response data for MLS-000558527</h3><div id="ml079.fu4" class="figure"><div class="graphic"><img src="/books/NBK47358/bin/ml079fu36.jpg" alt="Image ml079fu36" /></div></div></div><div id="ml079.s44"><h3>c. Comparative data on probe, similar compound structures (establishing SAR) and
|
||
prior probes</h3><div id="ml079.s45"><h4>i. HTRF cAMP Assay</h4><div id="ml079.fu5" class="figure"><div class="graphic"><img src="/books/NBK47358/bin/ml079fu37.jpg" alt="Image ml079fu37" /></div></div></div><div id="ml079.s46"><h4>Calcium Mobilization Assay</h4><div id="ml079.fu6" class="figure"><div class="graphic"><img src="/books/NBK47358/bin/ml079fu38.jpg" alt="Image ml079fu38" /></div></div></div><div id="ml079.s47"><h4>ii. [125I]NPS Displacement</h4><div id="ml079.fu7" class="figure"><div class="graphic"><img src="/books/NBK47358/bin/ml079fu39.jpg" alt="Image ml079fu39" /></div></div></div></div><div id="ml079.s48"><h3>d. Performance of NPS standard in these assays</h3><div id="ml079.s49"><h4>i. Calcium mobilization assay</h4><div id="ml079.fu8" class="figure"><div class="graphic"><img src="/books/NBK47358/bin/ml079fu40.jpg" alt="Image ml079fu40" /></div></div></div><div id="ml079.s50"><h4>ii. cAMP assay</h4><div id="ml079.fu9" class="figure"><div class="graphic"><img src="/books/NBK47358/bin/ml079fu41.jpg" alt="Image ml079fu41" /></div></div></div><div id="ml079.s51"><h4>iii. Radioligand displacement</h4><p>See radioligand data plot above.</p></div></div></div><div id="ml079.bib"><h2 id="_ml079_bib_">5. Bibliography</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="ml079.r1">Heilig M, Egli M. Pharmacological treatment of alcohol dependence: target
|
||
symptoms and target mechanisms. <span><span class="ref-journal">Pharmacol Ther. </span>2006;<span class="ref-vol">111 </span>(3):855–876.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/16545872" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 16545872</span></a>]</div></dd><dt>2.</dt><dd><div class="bk_ref" id="ml079.r2">Heilig M, Koob GF. A key role for corticotropin-releasing factor in alcohol
|
||
dependence. <span><span class="ref-journal">Trends Neurosci. </span>2007;<span class="ref-vol">30 </span>(8):399–406.</span> [<a href="/pmc/articles/PMC2747092/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2747092</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/17629579" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17629579</span></a>]</div></dd><dt>3.</dt><dd><div class="bk_ref" id="ml079.r3">Okamura N, Habay SA, Zeng J, Chamberlin AR, Reinscheid RK. Synthesis and pharmacological in vitro and in vivo profile of
|
||
3-oxo-1,1-diphenyl-tetrahydro-oxazolo[3,4-a]pyrazine-7-carboxylic
|
||
acid 4-fluoro-benzylamide (SHA 68), a selective antagonist of the
|
||
neuropeptide S receptor. <span><span class="ref-journal">J Pharmacol Exp Ther. </span>2008;<span class="ref-vol">325 </span>(3):893–901.</span> [<a href="/pmc/articles/PMC2583099/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2583099</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/18337476" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18337476</span></a>]</div></dd><dt>4.</dt><dd><div class="bk_ref" id="ml079.r4">Fukatsu K, Nakayama Y, Tarui N, Mori M, Matsumoto H, Kurasawa O, Banno H, , inventors. WO/2005/021555. <span>2005 Mar 10;</span></div></dd><dt>5.</dt><dd><div class="bk_ref" id="ml079.r5">Xu YL, Reinscheid RK, Huitron-Resendiz S, Clark SD, Wang Z, Lin SH, Brucher FA, Zeng J, Ly NK, Henriksen SJ, de Lecea L, Civelli O. Neuropeptide S: a neuropeptide promoting arousal and
|
||
anxiolytic-like effects. <span><span class="ref-journal">Neuron. </span>2004;<span class="ref-vol">43 </span>(4):487–97.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/15312648" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 15312648</span></a>]</div></dd><dt>6.</dt><dd><div class="bk_ref" id="ml079.r6">Zhang Y, Gilmour BP, Navarro HA, Runyon SP. Identifying structural features on
|
||
1,1-diphenyl-hexahydro-oxazolo[3,4-a]pyrazin-3-ones
|
||
critical for Neuropeptide S antagonist activity. <span><span class="ref-journal">Bioorg Med Chem Lett. </span>2008;<span class="ref-vol">18 </span>(14):4064–7.</span> [<a href="/pmc/articles/PMC5390816/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC5390816</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/18555684" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18555684</span></a>]</div></dd></dl></div><div id="bk_toc_contnr"></div></div></div>
|
||
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<div xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance"></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Views</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="PDF_download" id="Shutter"></a></div><div class="portlet_content"><ul xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="simple-list"><li><a href="/books/NBK47358/?report=reader">PubReader</a></li><li><a href="/books/NBK47358/?report=printable">Print View</a></li><li><a data-jig="ncbidialog" href="#_ncbi_dlg_citbx_NBK47358" data-jigconfig="width:400,modal:true">Cite this Page</a><div id="_ncbi_dlg_citbx_NBK47358" style="display:none" title="Cite this Page"><div class="bk_tt">Marugan J, Liu K, Zheng W, et al. Identification of Functionally Selective Small Molecule Antagonists of the Neuropeptide-S Receptor: Naphthopyranopyrimidines. 2009 Feb 13 [Updated 2010 Sep 2]. 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></div></div></li><li><a href="/books/NBK47358/pdf/Bookshelf_NBK47358.pdf">PDF version of this page</a> (529K)</li></ul></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>In this Page</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="page-toc" id="Shutter"></a></div><div class="portlet_content"><ul xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="simple-list"><li><a href="#ml079.s2" ref="log$=inpage&link_id=inpage">Probe Structure & Characteristics</a></li><li><a href="#ml079.s3" ref="log$=inpage&link_id=inpage">Recommendations for the scientific use of this probe</a></li><li><a href="#ml079.s4" ref="log$=inpage&link_id=inpage">Scientific Rationale for Project</a></li><li><a href="#ml079.s5" ref="log$=inpage&link_id=inpage">Project Description</a></li><li><a href="#ml079.s30" ref="log$=inpage&link_id=inpage">Probe</a></li><li><a href="#ml079.s41" ref="log$=inpage&link_id=inpage">Appendices</a></li><li><a href="#ml079.bib" ref="log$=inpage&link_id=inpage">Bibliography</a></li></ul></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Related information</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="discovery_db_links" id="Shutter"></a></div><div class="portlet_content"><ul><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pmc&DbFrom=books&Cmd=Link&LinkName=books_pmc_refs&IdsFromResult=2360070" ref="log$=recordlinks">PMC</a><div class="brieflinkpop offscreen_noflow">PubMed Central citations</div></li><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pcsubstance&DbFrom=books&Cmd=Link&LinkName=books_pcsubstance&IdsFromResult=2360070" ref="log$=recordlinks">PubChem Substance</a><div class="brieflinkpop offscreen_noflow">Related PubChem Substances</div></li><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pubmed&DbFrom=books&Cmd=Link&LinkName=books_pubmed_refs&IdsFromResult=2360070" ref="log$=recordlinks">PubMed</a><div class="brieflinkpop offscreen_noflow">Links to PubMed</div></li></ul></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Similar articles in PubMed</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="PBooksDiscovery_RA" id="Shutter"></a></div><div class="portlet_content"><ul><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/21433397" ref="ordinalpos=1&linkpos=1&log$=relatedreviews&logdbfrom=pubmed"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> Identification of Small Molecule Antagonists of the Neuropeptide-S Receptor.</a><span class="source">[Probe Reports from the NIH Mol...]</span><div class="brieflinkpop offscreen_noflow"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> Identification of Small Molecule Antagonists of the Neuropeptide-S Receptor.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Patnaik S, Marugan J, Liu K, Zheng W, Thorsell A, Eskay R, Southall N, Heilig M, Inglese J, Austin C. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Probe Reports from the NIH Molecular Libraries Program. 2010</em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/27788040" ref="ordinalpos=1&linkpos=2&log$=relatedreviews&logdbfrom=pubmed"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> Neuropeptide S receptor ligands: a patent review (2005-2016).</a><span class="source">[Expert Opin Ther Pat. 2017]</span><div class="brieflinkpop offscreen_noflow"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> Neuropeptide S receptor ligands: a patent review (2005-2016).<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Ruzza C, Calò G, Di Maro S, Pacifico S, Trapella C, Salvadori S, Preti D, Guerrini R. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Expert Opin Ther Pat. 2017 Mar; 27(3):347-362. 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