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<title>High Throughput Screening for Small Molecule Inhibitors of Heparin-induced Tau Fibril Formation - 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="High Throughput Screening for Small Molecule Inhibitors of Heparin-induced Tau Fibril Formation" /><meta name="citation_publisher" content="National Center for Biotechnology Information (US)" /><meta name="citation_date" content="2010/09/02" /><meta name="citation_author" content="Ronald L Johnson" /><meta name="citation_author" content="Wenwei Huang" /><meta name="citation_author" content="Ruili Huang" /><meta name="citation_author" content="Alex Crowe" /><meta name="citation_author" content="Carlo Ballatore" /><meta name="citation_author" content="John Q Trojanowski" /><meta name="citation_author" content="Kurt R Brunden" /><meta name="citation_author" content="Virginia M-Y Lee" /><meta name="citation_pmid" content="21433364" /><meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK47348/" /><link rel="schema.DC" href="http://purl.org/DC/elements/1.0/" /><meta name="DC.Title" content="High Throughput Screening for Small Molecule Inhibitors of Heparin-induced Tau Fibril Formation" /><meta name="DC.Type" content="Text" /><meta name="DC.Publisher" content="National Center for Biotechnology Information (US)" /><meta name="DC.Contributor" content="Ronald L Johnson" /><meta name="DC.Contributor" content="Wenwei Huang" /><meta name="DC.Contributor" content="Ruili Huang" /><meta name="DC.Contributor" content="Alex Crowe" /><meta name="DC.Contributor" content="Carlo Ballatore" /><meta name="DC.Contributor" content="John Q Trojanowski" /><meta name="DC.Contributor" content="Kurt R Brunden" /><meta name="DC.Contributor" content="Virginia M-Y Lee" /><meta name="DC.Date" content="2010/09/02" /><meta name="DC.Identifier" content="https://www.ncbi.nlm.nih.gov/books/NBK47348/" /><meta name="description" content="The microtubule-associated protein tau contributes directly or indirectly to key structural and regulatory cellular functions. Under pathological conditions, tau becomes sequestered into insoluble neurofibrillary tangles that promote axonal transport deficits, ultimately lead to synaptic dysfunction and neuronal loss. Beta-amyloid protein undergoes a similar type of aggregation as tau, and is implicated in the pathology of Alzheimer's disease. Recent reports describing screening of libraries of small molecules for their ability to inhibit tau fibrillization have shown that some classes of compounds, such as anthraquinones, phenothiazines, and porphyrins have the ability to inhibit tau fibril formation. While these hits may be unlikely to be developed into novel therapeutics for the treatment of tauopathies, the results suggest that in vitro assays used for these screenings are capable of detecting inhibitors of tau fibrillization. Thus, further qHTS for small molecule inhibitors of heparin-induced tau fibril formation promises to provide the identification novel and more tractable hits. HTS screening identified the probe ML103 (CID-9795907) as a member of a series of tau oligomerization/fibrillization inhibitors that can be used to study tau protein aggregation in vitro, as well as a starting point for drug development for the treatment of Alzheimer's Disease (AD) and other tauopathies." /><meta name="og:title" content="High Throughput Screening for Small Molecule Inhibitors of Heparin-induced Tau Fibril Formation" /><meta name="og:type" content="book" /><meta name="og:description" content="The microtubule-associated protein tau contributes directly or indirectly to key structural and regulatory cellular functions. Under pathological conditions, tau becomes sequestered into insoluble neurofibrillary tangles that promote axonal transport deficits, ultimately lead to synaptic dysfunction and neuronal loss. Beta-amyloid protein undergoes a similar type of aggregation as tau, and is implicated in the pathology of Alzheimer's disease. Recent reports describing screening of libraries of small molecules for their ability to inhibit tau fibrillization have shown that some classes of compounds, such as anthraquinones, phenothiazines, and porphyrins have the ability to inhibit tau fibril formation. While these hits may be unlikely to be developed into novel therapeutics for the treatment of tauopathies, the results suggest that in vitro assays used for these screenings are capable of detecting inhibitors of tau fibrillization. Thus, further qHTS for small molecule inhibitors of heparin-induced tau fibril formation promises to provide the identification novel and more tractable hits. HTS screening identified the probe ML103 (CID-9795907) as a member of a series of tau oligomerization/fibrillization inhibitors that can be used to study tau protein aggregation in vitro, as well as a starting point for drug development for the treatment of Alzheimer's Disease (AD) and other tauopathies." /><meta name="og:url" content="https://www.ncbi.nlm.nih.gov/books/NBK47348/" /><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/ml103/" /><link rel="canonical" href="https://www.ncbi.nlm.nih.gov/books/NBK47348/" /><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="#__NBK47348_dtls__">Show details</a><div style="display:none" class="ui-widget" id="__NBK47348_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/ml104/" title="Previous page in this title">< Prev</a><a class="active page_link next" href="/books/n/mlprobe/ml102/" 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="_NBK47348_"><span class="title" itemprop="name">High Throughput Screening for Small Molecule Inhibitors of Heparin-induced Tau
|
||
Fibril Formation</span></h1><p class="contrib-group"><span itemprop="author">Ronald L Johnson</span>, <span itemprop="author">Wenwei Huang</span>, and <span itemprop="author">Ruili Huang</span>. <span itemprop="author">Alex Crowe</span>, <span itemprop="author">Carlo Ballatore</span>, <span itemprop="author">John Q Trojanowski</span>, <span itemprop="author">Kurt R Brunden</span>, and <span itemprop="author">Virginia M-Y Lee</span>.</p><a data-jig="ncbitoggler" href="#__NBK47348_ai__" style="border:0;text-decoration:none">Author Information and Affiliations</a><div style="display:none" class="ui-widget" id="__NBK47348_ai__"><p class="contrib-group"><h4>Authors</h4><span itemprop="author">Ronald L Johnson</span>, <span itemprop="author">Wenwei Huang</span>, and <span itemprop="author">Ruili Huang</span>.<sup>1</sup> <span itemprop="author">Alex Crowe</span>, <span itemprop="author">Carlo Ballatore</span>, <span itemprop="author">John Q Trojanowski</span>, <span itemprop="author">Kurt R Brunden</span>, and <span itemprop="author">Virginia M-Y Lee</span>.<sup>2</sup></p><h4>Affiliations</h4><div class="affiliation"><sup>1</sup> NIH Chemical Genomics Center</div><div class="affiliation"><sup>2</sup> Center for Neurodegenerative Disease Research, University of
|
||
Pennsylvania School of Medicine</div></div><p class="small">Received: <span itemprop="datePublished">May 18, 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>The microtubule-associated protein tau contributes directly or indirectly to key structural and regulatory cellular functions. Under pathological conditions, tau becomes sequestered into insoluble neurofibrillary tangles that promote axonal transport deficits, ultimately lead to synaptic dysfunction and neuronal loss. Beta-amyloid protein undergoes a similar type of aggregation as tau, and is implicated in the pathology of Alzheimer's disease. Recent reports describing screening of libraries of small molecules for their ability to inhibit tau fibrillization have shown that some classes of compounds, such as anthraquinones, phenothiazines, and porphyrins have the ability to inhibit tau fibril formation. While these hits may be unlikely to be developed into novel therapeutics for the treatment of tauopathies, the results suggest that in vitro assays used for these screenings are capable of detecting inhibitors of tau fibrillization. Thus, further qHTS for small molecule inhibitors of heparin-induced tau fibril formation promises to provide the identification novel and more tractable hits. HTS screening identified the probe ML103 (CID-9795907) as a member of a series of tau oligomerization/fibrillization inhibitors that can be used to study tau protein aggregation in vitro, as well as a starting point for drug development for the treatment of Alzheimer's Disease (AD) and other tauopathies.</p></div><div class="h2"></div><p><b>Assigned Assay Grant #:</b> X01 MH083262-01</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> Carlo Ballatore, University of
|
||
Pennsylvania</p><p><b>PubChem Summary Bioassay Identifier (AID):</b>
|
||
<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1475" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1475</a></p><div id="ml103.s2"><h2 id="_ml103_s2_">Probe Structure & Characteristics</h2><div id="ml103.tu1" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK47348/table/ml103.tu1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml103.tu1_lrgtbl__"><table><tbody><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">PubChem CID</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CID-9795907</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Chemical Formula</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">C13H9N3O3S</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;">287.29</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Log P</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">1.89</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">tPSA</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">96</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Number of hydrogen donors</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">2</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Number of hydrogen acceptors:</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">5</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Rotatable bond</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">2</td></tr></tbody></table></div></div><div id="ml103.fu1" class="figure"><div class="graphic"><img src="/books/NBK47348/bin/ml103fu1.jpg" alt="Image ml103fu1" /></div></div><div id="ml103.tu2" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK47348/table/ml103.tu2/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml103.tu2_lrgtbl__"><table class="no_bottom_margin"><thead><tr><th id="hd_h_ml103.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CID/ML</th><th id="hd_h_ml103.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Target Name</th><th id="hd_h_ml103.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">IC50/EC50 (nM) [SID,
|
||
AID]</th><th id="hd_h_ml103.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_ml103.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">IC50/EC50 (nM) [SID,
|
||
AID]</th><th id="hd_h_ml103.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Selectivity</th><th id="hd_h_ml103.tu2_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Secondary Assay(s) %
|
||
Inhibition Name: [SID, AID]</th></tr></thead><tbody><tr><td headers="hd_h_ml103.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CID-9795907/<a href="/pcsubstance/?term=ML103[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML103</a></td><td headers="hd_h_ml103.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Human tau</td><td headers="hd_h_ml103.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">6,300 (100%)<sup>1</sup>
|
||
[<a href="https://pubchem.ncbi.nlm.nih.gov/substance/57288397" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-57288397</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1558" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1558</a>]</td><td headers="hd_h_ml103.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Aβ(1–42)</td><td headers="hd_h_ml103.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">35,500 (24%)
|
||
[<a href="https://pubchem.ncbi.nlm.nih.gov/substance/57288397" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-57288397</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1712" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1712</a>]</td><td headers="hd_h_ml103.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">5.6 (4.2)</td><td headers="hd_h_ml103.tu2_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Tau Sedimentation Assay 84%
|
||
[<a href="https://pubchem.ncbi.nlm.nih.gov/substance/57288397" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-57288397</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1720" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1720</a>]</td></tr></tbody></table></div><div><div><dl class="temp-labeled-list small"><dt>***</dt><dd><div id="ml103.tfn1"><p class="no_margin">NCGC IDs and corresponding PubChem IDs are listed in <a class="figpopup" href="/books/NBK47348/table/ml103.t5/?report=objectonly" target="object" rid-figpopup="figml103t5" rid-ob="figobml103t5">Table 5</a>.</p></div></dd></dl></div></div></div></div><div id="ml103.s3"><h2 id="_ml103_s3_">Recommendations for the scientific use of this probe</h2><p>This probe is a member of a series of tau oligomerization/fibrillization inhibitors.
|
||
It can be used to study tau protein aggregation <i>in vitro</i>, as well
|
||
as a starting point for drug development for the treatment of
|
||
Alzheimer’s Disease (AD) and other tauopathies.</p><div id="ml103.s4"><h3>Specific Aim</h3><p>SPECIFIC AIM 1. Screen the compound libraries of the MLSMR in an heparin-induced
|
||
tau K18 protein fibrillization assay based on ThT fluorescence; generate
|
||
dose-response curves for all compounds, select hits that exhibit IC50s of 5
|
||
μM or less; and confirm the results in a secondary sedimentation
|
||
assay.</p><p>SPECIFIC AIM 2. Prioritize hits based on (a) inspection of the chemical structure
|
||
of each hit (b) examination of dose-response curves (c) interpretation of
|
||
structure-function relationships obtained by comparing hits with related
|
||
compounds present in the chemical libraries.</p><p>SPECIFIC AIM 3. Based on the data generated in Specific Aim 1–2,
|
||
validate the most representative hits in one or more assays designed to
|
||
segregate false positives: (a) generate dose response curves in
|
||
primary/secondary assay at different K18 concentration; (b) perform
|
||
primary/secondary assay with and without reducing agents in the reaction
|
||
mixture; (c) run primary/secondary assay using different anionic cofactor.</p></div><div id="ml103.s5"><h3>Significance</h3><p>Six soluble tau isoforms are expressed in the normal adult human brain from a
|
||
single gene by alternative splicing. Normally, tau binds to and stabilizes
|
||
microtubules (MTs), thereby maintaining the network of MTs essential for axonal
|
||
transport in neurons. While phosphorylation negatively regulates the binding of
|
||
tau to MTs, tau becomes pathologically hyperphosphorylated in AD, resulting in
|
||
reduced MT-binding, followed by aggregation and sequestration of tau as paired
|
||
helical filaments (PHFs or PHFtau) into neurofibrillary tangles (NFTs). This
|
||
loss of tau function leads to destabilization of the MT system, which is
|
||
essential for axonal transport of proteins and other cargo to and from the cell
|
||
body of neurons. By analogy with a railway, tau functions like the cross ties on
|
||
railroad tracks (MTs), upon which trains (molecular motors) convey cargo
|
||
(organelles, proteins) to and from nodes on the railway network (destinations in
|
||
neuronal perikarya and their processes); the loss of a critical number of cross
|
||
ties (the consequence of converting tau into PHFs) results in dissolution of the
|
||
railroad tracks, leading to the failure to deliver cargo to assigned
|
||
destinations (impaired axonal transport). The deleterious consequences of these
|
||
events are the dysfunction and subsequent death of affected neurons.
|
||
Importantly, recent advances in the development of <i>in vitro</i> tau
|
||
fibrillization assays enable HTS to be used for interrogating compound libraries
|
||
to identify fibrillization inhibitors. However, clinical drug candidates have
|
||
not yet been identified. In addition, it is notable that the fibrillization
|
||
assays reported to date differ in important ways, such as the tau isoforms or
|
||
anionic cofactors employed, or concentrations of reducing agents in the reaction
|
||
mixture. While the significance of such differences is not known, it is likely
|
||
these differences will be reflected in the types and numbers of hits generated
|
||
from HTS using different assays. Hence, it is timely to conduct further HTS for
|
||
small-molecule inhibitors of tau fibrillization using more than one assay, and
|
||
to compare results from different assays.</p></div><div id="ml103.s6"><h3>Rationale</h3><p>Specific Aim 1: Recent reports describing screening of libraries of small
|
||
molecules for their ability to inhibit tau fibrillization have shown that some
|
||
classes of compounds, such as anthraquinones (<a class="bk_pop" href="#ml103.r11">Pickhardt, Gazova et al. 2005</a>), phenothiazines (<a class="bk_pop" href="#ml103.r15">Wischik, Edwards et al. 1996</a>; <a class="bk_pop" href="#ml103.r13">Taniguchi, Suzuki et al. 2005</a>) and
|
||
porphyrins (<a class="bk_pop" href="#ml103.r13">Taniguchi, Suzuki et al.
|
||
2005</a>), have the ability to inhibit tau fibril formation. While these
|
||
molecules are unlikely to be developed into novel therapeutics for the treatment
|
||
of tauopathies, they have importantly demonstrated that the <i>in
|
||
vitro</i> assays used in the screenings were indeed capable of
|
||
detecting inhibitors of tau fibrillization. Therefore, further HTS holds the
|
||
promise of identifying novel and more tractable hits.</p><p>Specific Aim 2: In order to accurately assign a priority to the compounds, or
|
||
classes of compounds populating Group III (see <a class="figpopup" href="/books/NBK47348/table/ml103.t4/?report=objectonly" target="object" rid-figpopup="figml103t4" rid-ob="figobml103t4">Table 4</a>), an analysis of the data and the chemical
|
||
structures will be essential at this point. Indeed, simple visual inspection of
|
||
the chemical structure could rapidly highlight potential liabilities. For
|
||
example, compounds containing reactive aldehydes or
|
||
α,β-unsaturated systems that could easily react with the
|
||
ɛ-amino group of lysines could be readily identified. Likewise,
|
||
examination of the concentration-response curve will be helpful in identifying
|
||
promiscuous inhibitors that act by forming large aggregates in solution. These
|
||
compounds typically produce a steep concentration-response curve (<a class="bk_pop" href="#ml103.r12">Shoichet 2006</a>) Last but not least, a
|
||
first assessment of structure-activity relationship should be made at this
|
||
point, if possible, by comparing hits with structurally related compounds in
|
||
Group I or Group II (<a class="figpopup" href="/books/NBK47348/table/ml103.t4/?report=objectonly" target="object" rid-figpopup="figml103t4" rid-ob="figobml103t4">Table 4</a>).</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml103t4"><a href="/books/NBK47348/table/ml103.t4/?report=objectonly" target="object" title="Table 4" class="img_link icnblk_img figpopup" rid-figpopup="figml103t4" rid-ob="figobml103t4"><img class="small-thumb" src="/books/NBK47348/table/ml103.t4/?report=thumb" src-large="/books/NBK47348/table/ml103.t4/?report=previmg" alt="Table 4. Structure-activity relationships of the aminothienopyridazine series." /></a><div class="icnblk_cntnt"><h4 id="ml103.t4"><a href="/books/NBK47348/table/ml103.t4/?report=objectonly" target="object" rid-ob="figobml103t4">Table 4</a></h4><p class="float-caption no_bottom_margin">Structure-activity relationships of the aminothienopyridazine
|
||
series. </p></div></div><p>Specific Aim 3: The rationale for Specific Aim 3 is to screen the most
|
||
representative hits emerging from Specific Aim 1–2 against
|
||
additional assays that, based on our previous experiences (see Preliminary
|
||
Studies; Section 4) and our current understanding of the assay, are designed to
|
||
accurately test mechanisms of non-specific inhibition. For example, since
|
||
non-specific binding of aggregates of small molecules onto proteins is often the
|
||
source of false positives in HTS, concentration-response curves will be
|
||
generated at different concentrations of K18 tau (<a class="bk_pop" href="#ml103.r9">McGovern, Caselli et al. 2002</a>; <a class="bk_pop" href="#ml103.r10">McGovern, Helfand et al. 2003</a>). It is expected that
|
||
IC50s of compounds that exhibit inhibitory activity through the formation of
|
||
aggregates will be independent of protein concentration. In addition, since it
|
||
was previously demonstrated that compound-DTT interactions could lead to
|
||
non-specific inhibition of tau filament formation via peroxide formation (see
|
||
<a class="figpopup" href="/books/NBK47348/table/ml103.t1/?report=objectonly" target="object" rid-figpopup="figml103t1" rid-ob="figobml103t1">Table 1</a>; Preliminary Studies),
|
||
HTS will be conducted in the absence of DTT. However, secondary assays will be
|
||
conducted in the presence of DTT to determine whether active compounds
|
||
identified from HTS are also active in the presence of reducing agent.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml103t1"><a href="/books/NBK47348/table/ml103.t1/?report=objectonly" target="object" title="Table 1" class="img_link icnblk_img figpopup" rid-figpopup="figml103t1" rid-ob="figobml103t1"><img class="small-thumb" src="/books/NBK47348/table/ml103.t1/?report=thumb" src-large="/books/NBK47348/table/ml103.t1/?report=previmg" alt="Table 1. Final 1536-well assay protocol." /></a><div class="icnblk_cntnt"><h4 id="ml103.t1"><a href="/books/NBK47348/table/ml103.t1/?report=objectonly" target="object" rid-ob="figobml103t1">Table 1</a></h4><p class="float-caption no_bottom_margin">Final 1536-well assay protocol. </p></div></div></div></div><div id="ml103.s7"><h2 id="_ml103_s7_">Assay Implementation and Screening</h2><div id="ml103.s8"><h3>PubChem Bioassay Name</h3><p>Quantitative High-Throughput Screen for Inhibitors of Tau Fibril Formation</p></div><div id="ml103.s9"><h3>List of PubChem bioassay identifiers generated for this screening project
|
||
(AIDs)</h3><div id="ml103.tu4" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK47348/table/ml103.tu4/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml103.tu4_lrgtbl__"><table><thead><tr><th id="hd_h_ml103.tu4_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">AID</th><th id="hd_h_ml103.tu4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Target</th><th id="hd_h_ml103.tu4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Concentration</th><th id="hd_h_ml103.tu4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Bioassay type</th></tr></thead><tbody><tr><td headers="hd_h_ml103.tu4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1475" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1475</a></td><td headers="hd_h_ml103.tu4_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Tau</td><td headers="hd_h_ml103.tu4_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"></td><td headers="hd_h_ml103.tu4_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Summary</td></tr><tr><td headers="hd_h_ml103.tu4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1460" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1460</a></td><td headers="hd_h_ml103.tu4_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Tau</td><td headers="hd_h_ml103.tu4_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">19 nM-57 µM</td><td headers="hd_h_ml103.tu4_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Primary qHTS (ThT)</td></tr><tr><td headers="hd_h_ml103.tu4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1468" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1468</a></td><td headers="hd_h_ml103.tu4_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Tau</td><td headers="hd_h_ml103.tu4_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">19 nM-57 µM</td><td headers="hd_h_ml103.tu4_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Primary qHTS (FP, mP)</td></tr><tr><td headers="hd_h_ml103.tu4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1463" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1463</a></td><td headers="hd_h_ml103.tu4_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Tau</td><td headers="hd_h_ml103.tu4_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">19 nM-57 µM</td><td headers="hd_h_ml103.tu4_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Primary Counterscreen qHTS (FP, Total
|
||
Fluorescence)</td></tr><tr><td headers="hd_h_ml103.tu4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1558" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1558</a></td><td headers="hd_h_ml103.tu4_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Tau</td><td headers="hd_h_ml103.tu4_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">49 nM-100 µM</td><td headers="hd_h_ml103.tu4_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Secondary (ThT)</td></tr><tr><td headers="hd_h_ml103.tu4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1559" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1559</a></td><td headers="hd_h_ml103.tu4_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Tau</td><td headers="hd_h_ml103.tu4_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">49 nM-100 µM</td><td headers="hd_h_ml103.tu4_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Secondary (FP, mP)</td></tr><tr><td headers="hd_h_ml103.tu4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1694" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1694</a></td><td headers="hd_h_ml103.tu4_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Tau</td><td headers="hd_h_ml103.tu4_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">49 nM-100 µM</td><td headers="hd_h_ml103.tu4_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Secondary Counterscreen (FP, Total
|
||
Fluorescence)</td></tr><tr><td headers="hd_h_ml103.tu4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1709" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1709</a></td><td headers="hd_h_ml103.tu4_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Tubulin</td><td headers="hd_h_ml103.tu4_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">50 µM</td><td headers="hd_h_ml103.tu4_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Secondary</td></tr><tr><td headers="hd_h_ml103.tu4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1711" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1711</a></td><td headers="hd_h_ml103.tu4_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Caspase-1</td><td headers="hd_h_ml103.tu4_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">7 pM-41 µM</td><td headers="hd_h_ml103.tu4_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Selectivity</td></tr><tr><td headers="hd_h_ml103.tu4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1712" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1712</a></td><td headers="hd_h_ml103.tu4_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Beta-amyloid</td><td headers="hd_h_ml103.tu4_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">16 nM-80 µM</td><td headers="hd_h_ml103.tu4_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Selectivity</td></tr><tr><td headers="hd_h_ml103.tu4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1719" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1719</a></td><td headers="hd_h_ml103.tu4_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Tau</td><td headers="hd_h_ml103.tu4_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">100 µM</td><td headers="hd_h_ml103.tu4_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Secondary</td></tr><tr><td headers="hd_h_ml103.tu4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1720" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1720</a></td><td headers="hd_h_ml103.tu4_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Tau</td><td headers="hd_h_ml103.tu4_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">100 µM</td><td headers="hd_h_ml103.tu4_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Secondary</td></tr></tbody></table></div></div></div><div id="ml103.s10"><h3>Primary Assay Description as Defined in PubChem</h3><div id="ml103.s11"><h4>Overview</h4><p>The microtubule-associated protein tau is an abundant protein in the axons of
|
||
neurons that stabilizes microtubules. With its ability to modulate
|
||
microtubule dynamics, tau contributes directly or indirectly to key
|
||
structural and regulatory cellular functions. Particularly important is the
|
||
influence tau exerts on axonal transport, which allows signaling molecules,
|
||
trophic factors and other essential cellular constituents to travel along
|
||
the axons. Under pathological conditions, tau becomes sequestered into
|
||
insoluble aggregates called neurofibrillary tangles. This phenomenon is
|
||
believed to have pathological consequences by promoting axonal transport
|
||
deficits that ultimately lead to synaptic dysfunction and neuronal loss. To
|
||
identify inhibitors of tau aggregation, a heparin-induced tau fibril
|
||
formation assay was used that employed a recombinantly expressed fragment of
|
||
tau, K18 (Q242-E372), bearing a P301L mutation (<a class="bk_pop" href="#ml103.r4">Gustke, Trinczek et al. 1994</a>; <a class="bk_pop" href="#ml103.r5">Hong, Zhukareva et al. 1998</a>). This
|
||
assay monitors tau fibrillization through the two complementary readouts of
|
||
Thioflavin T (ThT) fluorescence and fluorescence polarization (FP) of Alexa
|
||
594-labeled K18 P301L which incorporates into growing multimers of unlabeled
|
||
tau.</p></div><div id="ml103.s12"><h4>Protocol</h4><p>Two K18 mutants were produced: P301L (K18PL), which fibrillizes faster than
|
||
the wild-type form (<a class="bk_pop" href="#ml103.r14">von Bergen,
|
||
Barghorn et al. 2001</a>) and K311D (K19KD), which does not
|
||
fibrillize (<a class="bk_pop" href="#ml103.r8">Khlistunova, Biernat et al.
|
||
2006</a>) and was thus used as non-fibrillizing control in the assay.
|
||
For screening, 2 μL/well human K18 P301L (15 μM
|
||
unlabeled and 0.24 μM Alexa 594-labeled K18PL tau, final
|
||
concentrations) in reagent buffer (100 mM sodium acetate pH 7) was dispensed
|
||
into black solid 1536-well plates (Grenier) using a solenoid-based
|
||
dispenser. Following transfer of 23 nL compound or DMSO vehicle by a pin
|
||
tool, 2 μL/well heparin (20 μM final concentration)
|
||
in reagent buffer was added and the plate centrifuged 15 s at 1000 RPM.
|
||
Plates were incubated 6 hr at 37 °C and then 1
|
||
μL/well ThT (30 μM final concentration) was added.
|
||
After a 1 hr ambient incubation, the plates were read twice by an Envision
|
||
(Perkin Elmer) plate reader to sequentially monitor ThT fluorescence (450/8
|
||
nm excitation and 510/23 nm emission) and Alexa 594 FP (555 nm excitation
|
||
and 632 nm emission).</p></div></div><div id="ml103.s13"><h3>Summary of the Primary Screen</h3><div id="ml103.s14"><h4>Assay principle and protocol</h4><p>The assay principle and protocol are indicated in <a class="figpopup" href="/books/NBK47348/figure/ml103.f1/?report=objectonly" target="object" rid-figpopup="figml103f1" rid-ob="figobml103f1">Figure 1</a> and <a class="figpopup" href="/books/NBK47348/table/ml103.t1/?report=objectonly" target="object" rid-figpopup="figml103t1" rid-ob="figobml103t1">Table 1</a>, respectively.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml103f1" co-legend-rid="figlgndml103f1"><a href="/books/NBK47348/figure/ml103.f1/?report=objectonly" target="object" title="Figure 1" class="img_link icnblk_img figpopup" rid-figpopup="figml103f1" rid-ob="figobml103f1"><img class="small-thumb" src="/books/NBK47348/bin/ml103f1.gif" src-large="/books/NBK47348/bin/ml103f1.jpg" alt="Figure 1. Schematic of Tau fibrillization assay." /></a><div class="icnblk_cntnt" id="figlgndml103f1"><h4 id="ml103.f1"><a href="/books/NBK47348/figure/ml103.f1/?report=objectonly" target="object" rid-ob="figobml103f1">Figure 1</a></h4><p class="float-caption no_bottom_margin">Schematic of Tau fibrillization assay. Upon the addition of heparin, tau monomers oligomerize into fibrils.
|
||
The fluorescent dye, Thioflavine T, binds to tau oligomers, which
|
||
results in increased fluorescence intensity upon light excitation.
|
||
Tau oligomerization <a href="/books/NBK47348/figure/ml103.f1/?report=objectonly" target="object" rid-ob="figobml103f1">(more...)</a></p></div></div></div><div id="ml103.s15"><h4>qHTS summary of assay results</h4><p>The combined ThT/FP assay was optimized and validated using 1536-well test
|
||
plates to ensure that automated procedures would provide adequate signal to
|
||
background (S:B) ratios, and Z factor values exceeding 0.5 (<a class="figpopup" href="/books/NBK47348/table/ml103.t2/?report=objectonly" target="object" rid-figpopup="figml103t2" rid-ob="figobml103t2">Table 2</a>). Subsequently, a
|
||
~292,000 compound library underwent qHTS at six concentrations, ranging from
|
||
56 μM to 18 nM for each compound. The combination of good
|
||
signal-to-background and low CV values resulted in average plate Z-scores of
|
||
0.85 ± 0.12 and 0.79 ± 0.03 for the ThT and FP
|
||
analyses, respectively (text adapted from (<a class="bk_pop" href="#ml103.r2">Crowe, Huang et al. Submitted</a>)).</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml103t2"><a href="/books/NBK47348/table/ml103.t2/?report=objectonly" target="object" title="Table 2" class="img_link icnblk_img figpopup" rid-figpopup="figml103t2" rid-ob="figobml103t2"><img class="small-thumb" src="/books/NBK47348/table/ml103.t2/?report=thumb" src-large="/books/NBK47348/table/ml103.t2/?report=previmg" alt="Table 2. Assay optimization, validation, and screen performance." /></a><div class="icnblk_cntnt"><h4 id="ml103.t2"><a href="/books/NBK47348/table/ml103.t2/?report=objectonly" target="object" rid-ob="figobml103t2">Table 2</a></h4><p class="float-caption no_bottom_margin">Assay optimization, validation, and screen performance. </p></div></div></div><div id="ml103.s16"><h4>Identification of tau inhibitors</h4><p>Following the screen, the concentration-response curve (CRC) data were
|
||
classified to rank the curve quality, as described (<a class="bk_pop" href="#ml103.r7">Inglese, Auld et al. 2006</a>). Briefly, CRCs were
|
||
placed into four classes. Class 1 contains complete CRCs showing both upper
|
||
and lower asymptotes and r<sup>2</sup> values > 0.9. Class 2 contains
|
||
incomplete CRCs lacking the lower asymptote and shows r<sup>2</sup> values
|
||
> 0.9. Class 3 curves are of the lowest confidence because they are
|
||
poorly fit or based on activity at a single concentration point. Class 4 are
|
||
inactives having either no curve-fit or an efficacy below threshold activity
|
||
(3 SD of the mean activity). While both activators and inhibitors were
|
||
recovered from the qHTS, activators were not considered further, as their
|
||
activity likely arose from compound fluorescence. The qHTS resulted in
|
||
17,911 FP and 51,266 ThT actives (Class 1–3) of which 1,011 FP
|
||
and 7,092 ThT actives were scored high quality (Class 1 and 2.1, <a class="figpopup" href="/books/NBK47348/figure/ml103.f2/?report=objectonly" target="object" rid-figpopup="figml103f2" rid-ob="figobml103f2">Figures 2</a> and <a class="figpopup" href="/books/NBK47348/figure/ml103.f3/?report=objectonly" target="object" rid-figpopup="figml103f3" rid-ob="figobml103f3">3</a>).</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml103f2" co-legend-rid="figlgndml103f2"><a href="/books/NBK47348/figure/ml103.f2/?report=objectonly" target="object" title="Figure 2" class="img_link icnblk_img figpopup" rid-figpopup="figml103f2" rid-ob="figobml103f2"><img class="small-thumb" src="/books/NBK47348/bin/ml103f2.gif" src-large="/books/NBK47348/bin/ml103f2.jpg" alt="Figure 2. Titration-response plots of tau inhibitors." /></a><div class="icnblk_cntnt" id="figlgndml103f2"><h4 id="ml103.f2"><a href="/books/NBK47348/figure/ml103.f2/?report=objectonly" target="object" rid-ob="figobml103f2">Figure 2</a></h4><p class="float-caption no_bottom_margin">Titration-response plots of tau inhibitors. Inhibitors from the ThT binding (top panel) and fluorescence
|
||
polarization (bottom panel) measures are shown for class 1 (left),
|
||
class 2 (middle), and class 3 (right). For classes 1 and-2, blue and
|
||
orange curves <a href="/books/NBK47348/figure/ml103.f2/?report=objectonly" target="object" rid-ob="figobml103f2">(more...)</a></p></div></div><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml103f3" co-legend-rid="figlgndml103f3"><a href="/books/NBK47348/figure/ml103.f3/?report=objectonly" target="object" title="Figure 3" class="img_link icnblk_img figpopup" rid-figpopup="figml103f3" rid-ob="figobml103f3"><img class="small-thumb" src="/books/NBK47348/bin/ml103f3.gif" src-large="/books/NBK47348/bin/ml103f3.jpg" alt="Figure 3. Classification of tau actives." /></a><div class="icnblk_cntnt" id="figlgndml103f3"><h4 id="ml103.f3"><a href="/books/NBK47348/figure/ml103.f3/?report=objectonly" target="object" rid-ob="figobml103f3">Figure 3</a></h4><p class="float-caption no_bottom_margin">Classification of tau actives. A) Pie charts indicating overall FP and ThT activity of screened
|
||
compounds. Inhibitors (grey) and activators (maroon) comprised
|
||
4.9% and 9.4%, respectively, of the
|
||
collection while the remainders were inactive (yellow). <a href="/books/NBK47348/figure/ml103.f3/?report=objectonly" target="object" rid-ob="figobml103f3">(more...)</a></p></div></div><p>To derive nascent structure-activity relationships, an in-house auto scaffold
|
||
detection program was used to cluster 2989 class 1 and 2 FP actives
|
||
(including class 2.2 < −50% efficacy),
|
||
yielding 514 structural series and 755 singletons. Each series contained at
|
||
least three compounds, of which at least one of which was active. In
|
||
addition, compounds could occupy multiple series. The series were then
|
||
flagged for potential liabilities using activities from the tau and other
|
||
assays (<a class="figpopup" href="/books/NBK47348/table/ml103.t3/?report=objectonly" target="object" rid-figpopup="figml103t3" rid-ob="figobml103t3">Table 3</a>), resulting in
|
||
42 series with no liabilities.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml103t3"><a href="/books/NBK47348/table/ml103.t3/?report=objectonly" target="object" title="Table 3" class="img_link icnblk_img figpopup" rid-figpopup="figml103t3" rid-ob="figobml103t3"><img class="small-thumb" src="/books/NBK47348/table/ml103.t3/?report=thumb" src-large="/books/NBK47348/table/ml103.t3/?report=previmg" alt="Table 3. Criteria for identifying liabilities of tau active series." /></a><div class="icnblk_cntnt"><h4 id="ml103.t3"><a href="/books/NBK47348/table/ml103.t3/?report=objectonly" target="object" rid-ob="figobml103t3">Table 3</a></h4><p class="float-caption no_bottom_margin">Criteria for identifying liabilities of tau active series. </p></div></div><p>Series were filtered and prioritized further using the following
|
||
criteria:</p><ol><li class="half_rhythm"><div>Active selection- Compounds with increasing total Alexa Red
|
||
fluorescence in the FP assay were excluded, and only compounds
|
||
active in both the FP and ThT assays (class 1 and 2; AC50 <
|
||
20 μM and efficacy <−40%)
|
||
were included for further consideration.</div></li><li class="half_rhythm"><div>Known scaffolds- Compounds containing previously identified scaffolds
|
||
(<a class="bk_pop" href="#ml103.r1">Crowe, Ballatore et al.
|
||
2007</a>; <a class="bk_pop" href="#ml103.r6">Honson,
|
||
Johnson et al. 2007</a>), including pyrimidotriazines,
|
||
depsidones, anthraquinones, phenothiazines, porphyrins, sulfonated
|
||
dyes, and cyanine dyes, were excluded.</div></li><li class="half_rhythm"><div>Undesirable functional groups- Compounds containing undesirable
|
||
functional groups with known chemical liabilities, such as quinones,
|
||
metal complexes, reactive halides, imines (except hydrazones),
|
||
aldehydes, aliphatic thiols, and potential Michael acceptors (except
|
||
rhodanines), were excluded.</div></li><li class="half_rhythm"><div>Lipinski’s rule of five (Ro5)- Compounds having more than
|
||
two Ro5 violations were excluded. In general, only compounds with
|
||
molecular weight <600 and AlogP <6 were included.</div></li></ol><p>Twenty three series passed this filtering process and were chosen for retest.
|
||
An additional six series were selected, where the most potent compound in
|
||
each series showed low efficacy in the FP readout, but showed
|
||
50% or greater efficacy in the ThT readout. For retest, 98
|
||
compounds (actives, inactive, and analogs) were chosen and of these, 85 were
|
||
available for testing. Of the 85 compounds, 70 were identified from the
|
||
screen (70 actives, 2 inconclusives, and 6 inactives) and 7 were analogs.
|
||
When checked by LCMS, 75% of the compounds passed (correct mass
|
||
and 90% or greater purity). The compounds were tested at both
|
||
the NCGC and CNDR, and 94% and 73% of the qHTS
|
||
actives retested positive in the ThT and FP assays, respectively. The
|
||
compounds were tested in a sedimentation assay that measures the percentage
|
||
of soluble and insoluble tau (<a class="bk_pop" href="#ml103.r1">Crowe,
|
||
Ballatore et al. 2007</a>), where 46% of the 76 qHTS
|
||
actives and analogs were scored as active (40% or greater
|
||
inhibition) and 11% as inconclusive
|
||
(20–40% inhibition). All of the 47 samples scored
|
||
active and inconclusive in the sedimentation assay were examined by
|
||
transmission electron microscopy; 66% were scored active,
|
||
showing a lack of fibrillar clumps, with only single filaments or pieces
|
||
evident (data not shown). Following the retest studies, an
|
||
aminothienopyridazine series that showed good performance in the follow-up
|
||
assays and suitable chemical tractability was chosen for probe
|
||
optimization.</p></div></div></div><div id="ml103.s17"><h2 id="_ml103_s17_">Probe Characterization</h2><p>The ability of aminothienopyridazines (ATPZ’s) to inhibit tau fibril
|
||
formation was evaluated in the K18PL fluorescence and sedimentation assays to
|
||
determine their AC50 values and percent maximal inhibition (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1558" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1558</a>;<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1720" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1720</a>). As
|
||
shown in <a class="figpopup" href="/books/NBK47348/table/ml103.t4/?report=objectonly" target="object" rid-figpopup="figml103t4" rid-ob="figobml103t4">Table 4</a>, N-acylations (R1;
|
||
entries 3–5) as well as substitution in the C-5 position (R2; entries 5
|
||
and 6) result in a dramatic loss of activity. Structural modifications in the
|
||
fragment linked at C-4 (R3; entries 7–12) appear to be generally well
|
||
tolerated. Indeed, several compounds bearing R3 modifications (entries
|
||
7–8, and 11–12) appear to cause greater maximal inhibition
|
||
than the original hits identified during qHTS (entries 1 and 2). Furthermore,
|
||
although the correlations between biological activity and the stereo-electronic
|
||
properties of phenyl moieties (R4) are not entirely clear, the nature, number and
|
||
position of the substituents in the aromatic ring appeared to modulate ATPZ activity
|
||
(entries 1–2 and 13–18). Although previous studies have
|
||
demonstrated that tau fragments such as K18PL form fibrils which resemble those
|
||
observed in tauopathies (<a class="bk_pop" href="#ml103.r1">Crowe, Ballatore et
|
||
al. 2007</a>), it was important to demonstrate that the compounds could
|
||
disrupt assembly of full-length tau40. Two ATPZ’s (NCGC-00053250 and
|
||
NCGC-00031883) were tested at 50 µM concentration and caused at least a
|
||
60% inhibition of ThT fluorescence in the tau40 assay. This result thus
|
||
confirms that ATPZ’s affect full-length tau in addition to the truncated
|
||
tau fragment. Since an important function of tau protein is to stabilize
|
||
microtubules (MTs), we evaluated whether these inhibitors interfered with the
|
||
ability of tau to promote tubulin polymerization into MTs. Unlike methylene blue, a
|
||
promiscuous compound active in a high percentage of PubChem screens (data not
|
||
shown), the ATPZ’s had no effect on tau-mediated tubulin polymerization
|
||
(<a class="figpopup" href="/books/NBK47348/figure/ml103.f4/?report=objectonly" target="object" rid-figpopup="figml103f4" rid-ob="figobml103f4">Figure 4</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1709" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1709</a>; text adapted
|
||
from (<a class="bk_pop" href="#ml103.r2">Crowe, Huang et al.
|
||
Submitted</a>)).</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml103f4" co-legend-rid="figlgndml103f4"><a href="/books/NBK47348/figure/ml103.f4/?report=objectonly" target="object" title="Figure 4" class="img_link icnblk_img figpopup" rid-figpopup="figml103f4" rid-ob="figobml103f4"><img class="small-thumb" src="/books/NBK47348/bin/ml103f4.gif" src-large="/books/NBK47348/bin/ml103f4.jpg" alt="Figure 4. Effects of ATPZ tau fibrilization inhibitors on tau-mediated MT assembly." /></a><div class="icnblk_cntnt" id="figlgndml103f4"><h4 id="ml103.f4"><a href="/books/NBK47348/figure/ml103.f4/?report=objectonly" target="object" rid-ob="figobml103f4">Figure 4</a></h4><p class="float-caption no_bottom_margin">Effects of ATPZ tau fibrilization inhibitors on tau-mediated MT
|
||
assembly. Representative traces of tau-induced microtubule assembly in the presence of
|
||
50 μM test compounds or DMSO, as described in the assay methods
|
||
below. </p></div></div><p>The ATPZ’s were also tested for their ability to block the fibrillization
|
||
of Aβ(1–42). We found that these compounds were less
|
||
effective in blocking Aβ(1–42) fibril formation, as judged
|
||
by ThT fluorescence, than they were in inhibiting tau fibrillization (<a class="figpopup" href="/books/NBK47348/figure/ml103.f5/?report=objectonly" target="object" rid-figpopup="figml103f5" rid-ob="figobml103f5">Figure 5</a>). Among the tested compounds, none
|
||
caused >45% inhibition of Aβ(1–42) fibril
|
||
formation when tested at 80 µM (AID: 1712).</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml103f5" co-legend-rid="figlgndml103f5"><a href="/books/NBK47348/figure/ml103.f5/?report=objectonly" target="object" title="Figure 5" class="img_link icnblk_img figpopup" rid-figpopup="figml103f5" rid-ob="figobml103f5"><img class="small-thumb" src="/books/NBK47348/bin/ml103f5.gif" src-large="/books/NBK47348/bin/ml103f5.jpg" alt="Figure 5. Comparison of K18PL tau and Aβ(1–42) fibrillization in the presence of CID-9795907 (NCGC-00183199)." /></a><div class="icnblk_cntnt" id="figlgndml103f5"><h4 id="ml103.f5"><a href="/books/NBK47348/figure/ml103.f5/?report=objectonly" target="object" rid-ob="figobml103f5">Figure 5</a></h4><p class="float-caption no_bottom_margin"><i>Comparison of K18PL tau and Aβ(1–42)
|
||
fibrillization in the presence of</i> CID-9795907
|
||
(<i>NCGC-00183199)</i>. CID-9795907 (NCGC-00183199) was tested at the indicated concentrations in ThT
|
||
binding assays for K18PL tau and Aβ(1–42) <a href="/books/NBK47348/figure/ml103.f5/?report=objectonly" target="object" rid-ob="figobml103f5">(more...)</a></p></div></div></div><div id="ml103.s18"><h2 id="_ml103_s18_">Synthesis of analogs</h2><p>CID-647821 (NCGC-00031883) (<b>8a</b>), CID-719449 (NCGC-00053250)
|
||
(<b>8b</b>), CID-798725 (NCGC-00183206) (<b>8c</b>), CID-25181381
|
||
(NCGC-00183204) (<b>8d</b>), CID-25181382 (NCGC-00183205) (<b>8e</b>),
|
||
CID-25181375 (NCGC-00183195) (<b>8f</b>), and CID-9795907 (NCGC-00183199)
|
||
(<b>9</b>) were synthesized (<a class="figpopup" href="/books/NBK47348/figure/ml103.f6/?report=objectonly" target="object" rid-figpopup="figml103f6" rid-ob="figobml103f6">Scheme
|
||
1</a>) following a modified literature procedure (<a class="bk_pop" href="#ml103.r3">Ferguson, Valant et al. 2008</a>). Commercially available
|
||
anilines <b>4a–e</b> were converted to aryl diazonium salts
|
||
<b>5</b>, which reacted with β-ketoesters to form hydrazones
|
||
<b>6</b> as a mixture of E/Z isomers. Condensation reactions of the
|
||
hydrazones with ethyl cyanoacetate gave pyridazines <b>7</b>. Subsequently
|
||
reactions of pyridazines <b>7</b> with sulfur under Gewald conditions
|
||
generated <b>8a–f</b>. Saponification of <b>8a</b> gave the
|
||
corresponding <b>9</b>.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml103f6" co-legend-rid="figlgndml103f6"><a href="/books/NBK47348/figure/ml103.f6/?report=objectonly" target="object" title="Scheme 1" class="img_link icnblk_img figpopup" rid-figpopup="figml103f6" rid-ob="figobml103f6"><img class="small-thumb" src="/books/NBK47348/bin/ml103f6.gif" src-large="/books/NBK47348/bin/ml103f6.jpg" alt="Scheme 1. Reagents and conditions: (i) X = BF4; HBF4, NaNO2; X = Cl; isoamyl nitrile; (ii) NaOAc, EtOH, R2CH2COCH2CO2Et; (iii) 4-aminobutyric acid, ethyl cyanoacetate; (iv) S8, morpholine, MW, 150 °C, 10 min; (v) NaOH." /></a><div class="icnblk_cntnt" id="figlgndml103f6"><h4 id="ml103.f6"><a href="/books/NBK47348/figure/ml103.f6/?report=objectonly" target="object" rid-ob="figobml103f6">Scheme 1</a></h4><p class="float-caption no_bottom_margin">Reagents and conditions: (<i>i</i>) X =
|
||
BF<sub>4</sub>; HBF<sub>4</sub>, NaNO<sub>2</sub>; X = Cl;
|
||
isoamyl nitrile; (<i>ii</i>) NaOAc, EtOH,
|
||
R<sub>2</sub>CH<sub>2</sub>COCH<sub>2</sub>CO<sub>2</sub>Et;
|
||
(<i>iii</i>) 4-aminobutyric acid, ethyl cyanoacetate;
|
||
(<i>iv</i>) S<sub>8</sub>, morpholine, MW, 150 °C,
|
||
10 min; (<i>v</i>) NaOH. </p></div></div><p>CID-25181378 (NCGC-00183201) (<b>11a</b>), CID-25181379 (NCGC-00183202)
|
||
(<b>10b</b>), and CID-25181383 (NCGC-00183207) (<b>10c</b>) were
|
||
prepared by a reaction sequence starting from <b>7a</b> as depicted in <a class="figpopup" href="/books/NBK47348/figure/ml103.f7/?report=objectonly" target="object" rid-figpopup="figml103f7" rid-ob="figobml103f7">Scheme 2</a>. Saponification of <b>7a</b>
|
||
under acidic condition yielded <b>10a</b>. Amide <b>10b</b> was formed
|
||
by reacting <b>7a</b> with ammonium. Coupling reactions of acid
|
||
<b>10a</b> with appropriate amines followed by Gewald reaction yielded the
|
||
desired amides <b>11a–c</b>.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml103f7" co-legend-rid="figlgndml103f7"><a href="/books/NBK47348/figure/ml103.f7/?report=objectonly" target="object" title="Scheme 2" class="img_link icnblk_img figpopup" rid-figpopup="figml103f7" rid-ob="figobml103f7"><img class="small-thumb" src="/books/NBK47348/bin/ml103f7.gif" src-large="/books/NBK47348/bin/ml103f7.jpg" alt="Scheme 2. Reagents and conditions: (i) X = OH: HCl/HOAc, MW, 150 °C, 15 min; X = NH2: NH3.H2O, DMF, MW, 150 °C, 10 min; (ii) DMC, iPr2NEt, DCM, r.t. o/n; (iii) S8, morpholine, MW, 150 °C, 10 min;." /></a><div class="icnblk_cntnt" id="figlgndml103f7"><h4 id="ml103.f7"><a href="/books/NBK47348/figure/ml103.f7/?report=objectonly" target="object" rid-ob="figobml103f7">Scheme 2</a></h4><p class="float-caption no_bottom_margin">Reagents and conditions: (<i>i</i>) X = OH:
|
||
HCl/HOAc, MW, 150 °C, 15 min; X = NH<sub>2</sub>:
|
||
NH<sub>3</sub>.H<sub>2</sub>O, DMF, MW, 150 °C, 10 min; (ii)
|
||
DMC, <i>i</i>Pr<sub>2</sub>NEt, DCM, r.t. o/n; (iii)
|
||
S<sub>8</sub>, morpholine, MW, 150 °C, 10 min;. </p></div></div><p>CID-4185185 (NCGC-00182502) (<b>12a</b>) and CID-2814251 (NCGC-00183197)
|
||
(<b>12b</b>) were prepared by reactions of <b>8a</b> with
|
||
appropriate acid chlorides, <a class="figpopup" href="/books/NBK47348/figure/ml103.f8/?report=objectonly" target="object" rid-figpopup="figml103f8" rid-ob="figobml103f8">Scheme 3</a>.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml103f8" co-legend-rid="figlgndml103f8"><a href="/books/NBK47348/figure/ml103.f8/?report=objectonly" target="object" title="Scheme 3" class="img_link icnblk_img figpopup" rid-figpopup="figml103f8" rid-ob="figobml103f8"><img class="small-thumb" src="/books/NBK47348/bin/ml103f8.gif" src-large="/books/NBK47348/bin/ml103f8.jpg" alt="Scheme 3" /></a><div class="icnblk_cntnt" id="figlgndml103f8"><h4 id="ml103.f8"><a href="/books/NBK47348/figure/ml103.f8/?report=objectonly" target="object" rid-ob="figobml103f8">Scheme 3</a></h4></div></div><p>The preparation of CID-25181383 (NCGC-00183207) (<b>18</b>) is illustrated in
|
||
<a class="figpopup" href="/books/NBK47348/figure/ml103.f9/?report=objectonly" target="object" rid-figpopup="figml103f9" rid-ob="figobml103f9">Scheme 4</a>. EDC-mediated coupling
|
||
reaction of acid <b>10a</b> with <i>tert</i>-butylcarbazate followed
|
||
by Boc-deprotection gave hydrazide <b>14</b>. The hydrazide reacted with
|
||
nitrous acid in acetic acid produced carboazide <b>15</b>. Curtius
|
||
rearrangement of <b>15</b> in ethanol gave ethyl carbamate <b>17</b>.
|
||
Under basic condition, the carbamate was converted to the desired amine
|
||
<b>18</b>.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml103f9" co-legend-rid="figlgndml103f9"><a href="/books/NBK47348/figure/ml103.f9/?report=objectonly" target="object" title="Scheme 4" class="img_link icnblk_img figpopup" rid-figpopup="figml103f9" rid-ob="figobml103f9"><img class="small-thumb" src="/books/NBK47348/bin/ml103f9.gif" src-large="/books/NBK47348/bin/ml103f9.jpg" alt="Scheme 4. Reagents and conditions: (i) EDC, NH2NHBoc; (ii) MeOH, HCl/dioxane; (iii) HOAc, NaNO2; (iv) EtOH, MW, 150 °C, 10 min; (v) S8, morpholine, MW, 150 °C, 10 min; (vi) Et3N, EtOH/H2O, MW, 170 °C, 30 min." /></a><div class="icnblk_cntnt" id="figlgndml103f9"><h4 id="ml103.f9"><a href="/books/NBK47348/figure/ml103.f9/?report=objectonly" target="object" rid-ob="figobml103f9">Scheme 4</a></h4><p class="float-caption no_bottom_margin">Reagents and conditions: (<i>i</i>) EDC, NH<sub>2</sub>NHBoc;
|
||
(ii) MeOH, HCl/dioxane; (iii) HOAc, NaNO<sub>2</sub>; (<i>iv</i>)
|
||
EtOH, MW, 150 °C, 10 min; (v) S<sub>8</sub>, morpholine, MW, 150
|
||
°C, 10 min; (vi) Et<sub>3</sub>N, EtOH/H<sub>2</sub>O, MW, 170
|
||
°C, 30 min. </p></div></div><p>CID-25181376 (NCGC-00183196) (<b>19</b>) was prepared by LiAlH<sub>4</sub>
|
||
reduction of ester <b>8a</b>, <a class="figpopup" href="/books/NBK47348/figure/ml103.f10/?report=objectonly" target="object" rid-figpopup="figml103f10" rid-ob="figobml103f10">Scheme
|
||
5</a>.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml103f10" co-legend-rid="figlgndml103f10"><a href="/books/NBK47348/figure/ml103.f10/?report=objectonly" target="object" title="Scheme 5" class="img_link icnblk_img figpopup" rid-figpopup="figml103f10" rid-ob="figobml103f10"><img class="small-thumb" src="/books/NBK47348/bin/ml103f10.gif" src-large="/books/NBK47348/bin/ml103f10.jpg" alt="Scheme 5" /></a><div class="icnblk_cntnt" id="figlgndml103f10"><h4 id="ml103.f10"><a href="/books/NBK47348/figure/ml103.f10/?report=objectonly" target="object" rid-ob="figobml103f10">Scheme 5</a></h4></div></div><div id="ml103.s19"><h3>Probe</h3><div id="ml103.s20"><h4>a. Chemical name of probe compound</h4><p>5-amino-4-oxo-3-phenylthieno[3,4-d]pyridazine-1-carboxylic
|
||
acid (<a href="/pcsubstance/?term=ML103[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML103</a>)</p></div><div id="ml103.s21"><h4>b. Probe chemical structure including stereochemistry</h4><div id="ml103.fu2" class="figure"><div class="graphic"><img src="/books/NBK47348/bin/ml103fu15.jpg" alt="Image ml103fu15" /></div></div></div><div id="ml103.s22"><h4>c. Structural Verification Information of probe (<a href="https://pubchem.ncbi.nlm.nih.gov/substance/57288397" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-57288397</a>)</h4><p>Structural verification and purity quantification were performed by
|
||
<sup>1</sup>H NMR analysis using a Varian spectrometer and by LC-MS
|
||
analysis using a Waters Acquity UPLC in the following conditions:</p><ul class="simple-list"><li class="half_rhythm"><div>Column: Phenomenex 2.5um Luna C18(2)-HST 100x2mm</div></li><li class="half_rhythm"><div>Column Temperature: 45 degrees C</div></li><li class="half_rhythm"><div>Flow: 0.5mL/min</div></li><li class="half_rhythm"><div>Solvent A: 0.05% TFA in Water</div></li><li class="half_rhythm"><div>Solvent B: 0.025% TFA in Acetonitrile</div></li><li class="half_rhythm"><div>Gradient: 2% to 100% Solvent B over 2.2
|
||
minutes.</div></li><li class="half_rhythm"><div>Run Time: 3.0 min</div></li><li class="half_rhythm"><div>Detectors: PDA, ELSD, and MS (ESI<sup>+</sup>).</div></li></ul><p>Both NMR and LC-MS analyses showed purity greater than 95% for
|
||
CID-9795907 (NCGC-000183199-01; <a href="https://pubchem.ncbi.nlm.nih.gov/substance/57288397" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-57288397</a>). <sup>1</sup>H NMR (400 MHz,
|
||
DMSO-d6) δ 7.12 (s, 1H), 7.35–7.39 (m, 1H),
|
||
7.44–7.54 (m, 4H), 7.60 (bs, 2H), 13.31 (bs, 1H); HPLC:
|
||
t<sub>R</sub> = 1.89 min, ELSD =
|
||
100%, UV<sub>220</sub> = 100%; HRMS
|
||
(ESI): <i>m/z</i> calcd for
|
||
C<sub>13</sub>H<sub>9</sub>N<sub>3</sub>O<sub>3</sub>S
|
||
[M+1]+ 288.0437, found
|
||
288.0441.</p></div><div id="ml103.s23"><h4>d. PubChem CID (corresponding to the SID)</h4><p>CID-9795907</p></div><div id="ml103.s24"><h4>e. Availability from a vendor</h4><p>N/A.</p></div><div id="ml103.s25"><h4>f. Mode of action for biological activity of probe</h4><p>The probe is a member of a series of Tau Oligomerization/Fibrillization
|
||
inhibitors.</p></div><div id="ml103.s26"><h4>g. Detailed synthetic pathway for making probe</h4><p>CID-9795907 (NCGC00183199) (<b>9</b>) was synthesized (<a class="figpopup" href="/books/NBK47348/figure/ml103.f11/?report=objectonly" target="object" rid-figpopup="figml103f11" rid-ob="figobml103f11">Scheme 3</a>) following a modified
|
||
literature procedure (<a class="bk_pop" href="#ml103.r3">Ferguson, Valant
|
||
et al. 2008</a>).</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml103f11" co-legend-rid="figlgndml103f11"><a href="/books/NBK47348/figure/ml103.f11/?report=objectonly" target="object" title="Scheme 3" class="img_link icnblk_img figpopup" rid-figpopup="figml103f11" rid-ob="figobml103f11"><img class="small-thumb" src="/books/NBK47348/bin/ml103f11.gif" src-large="/books/NBK47348/bin/ml103f11.jpg" alt="Scheme 3. Reagents and conditions: (i) HBF4, NaNO2; (ii) NaOAc, EtOH, CH3COCH2CO2Et; (iii) 4-aminobutyric acid, ethyl cyanoacetate; (iv) S8, morpholine, MW, 150 °C, 10 min; (v) NaOH." /></a><div class="icnblk_cntnt" id="figlgndml103f11"><h4 id="ml103.f11"><a href="/books/NBK47348/figure/ml103.f11/?report=objectonly" target="object" rid-ob="figobml103f11">Scheme 3</a></h4><p class="float-caption no_bottom_margin">Reagents and conditions: (<i>i</i>) HBF<sub>4</sub>,
|
||
NaNO<sub>2</sub>; (<i>ii</i>) NaOAc, EtOH,
|
||
CH<sub>3</sub>COCH<sub>2</sub>CO<sub>2</sub>Et;
|
||
(<i>iii</i>) 4-aminobutyric acid, ethyl cyanoacetate;
|
||
(<i>iv</i>) S<sub>8</sub>, morpholine, MW, 150
|
||
°C, 10 min; (<i>v</i>) NaOH. </p></div></div><p>This compound has been made available through the MLSMR (MLS-002472423).</p><p><i>Canonical SMILES:</i>
|
||
C1=CC=C(C=C1)N2C(=O)C3=C(SC=C3C(=N2)C(=O)O)N</p><p><i>InChI:</i>
|
||
InChI=1S/C13H9N3O3S/c14-11-9-8(6-20-11)10(13(18)19)15–16(12(9)17)7-4-2-1-3-5-7/h1-6H,14H2,(H,18,19)</p></div></div><div id="ml103.s27"><h3>Description of secondary assays used to optimize/characterize probe
|
||
structure</h3><div id="ml103.s28"><h4>Assay: Tau Sedimentation</h4><p>Overview: The microtubule-associated protein tau is an abundant protein in
|
||
the axons of neurons that stabilizes microtubules. With its ability to
|
||
modulate microtubule dynamics, tau contributes directly or indirectly, to
|
||
key structural and regulatory cellular functions. Particularly important is
|
||
the influence tau exerts on axonal transport, which allows signaling
|
||
molecules, trophic factors and other essential cellular constituents to
|
||
travel along the axons. Under pathological conditions, tau becomes
|
||
sequestered into insoluble aggregates called neurofibrillary tangles. This
|
||
phenomenon is believed to have pathological consequences by promoting axonal
|
||
transport deficits that ultimately lead to synaptic dysfunction and neuronal
|
||
loss. The ability of compounds to block tau fibrillization was assessed
|
||
using a sedimentation assay (<a class="bk_pop" href="#ml103.r1">Crowe,
|
||
Ballatore et al. 2007</a>). Unlike tau monomers, tau fibrils readily
|
||
sediment upon centrifugation. Samples from the tau ThT confirmation assay
|
||
treated with 100 µM compound (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1558" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1558</a>) were centrifuged and the amount of
|
||
tau present in the supernatant and pellet was determined.</p><p>Protocol: Human K18PL (15 µM) in assay buffer (20 µM heparin, 12.5 µM
|
||
Thioflavine T, 100 mM sodium acetate pH 7) was centrifuged at 186,000 x g
|
||
for 30 min and the supernatant removed from the pellet. Pellets were
|
||
resuspended in a volume equal to the supernatant and equal amounts of
|
||
supernatants and pellet were analyzed by SDS–PAGE using a
|
||
12.5% acrylamide gel. The gel was stained with Coomassie Blue
|
||
and the percentage of tau in each fraction was determined by
|
||
densitometry.</p></div><div id="ml103.s29"><h4>Assay: Transmission Electron Microscopy</h4><p>Overview: The microtubule-associated protein tau is an abundant protein in
|
||
the axons of neurons that stabilizes microtubules. With its ability to
|
||
modulate microtubule dynamics, tau contributes directly or indirectly, to
|
||
key structural and regulatory cellular functions. Particularly important is
|
||
the influence tau exerts on axonal transport, which allows signaling
|
||
molecules, trophic factors and other essential cellular constituents to
|
||
travel along the axons. Under pathological conditions, tau becomes
|
||
sequestered into insoluble aggregates called neurofibrillary tangles. This
|
||
phenomenon is believed to have pathological consequences by promoting axonal
|
||
transport deficits that ultimately lead to synaptic dysfunction and neuronal
|
||
loss. The ability of compounds to block tau fibrillization was visualized
|
||
using transmission electron microscopy (TEM). Samples from the tau ThT
|
||
confirmation assay (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1558" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1558</a>) were centrifuged and imaged using TEM.
|
||
Representative fields from electron micrographs of objects were imaged to
|
||
judge fibril content using grading scale from 0–4: 0, all
|
||
fibrils in clumps, no fragments; 1, small fibril clumps, free filaments and
|
||
some pieces; 2, filaments and pieces but no clumps; 3, short filaments and
|
||
tiny pieces; 4, only tiny pieces.</p><p>Protocol: Human K18PL tau (15 µM) in assay buffer (20 µM heparin, 30 µM
|
||
Thioflavine T, 100 mM sodium acetate pH 7) was centrifuged at 186,000 g for
|
||
30 min through a 25% sucrose cushion and the supernatant removed
|
||
from the pellet. Pellets were resuspended, stained with uranyl acetate, and
|
||
imaged by electron microscopy</p></div><div id="ml103.s30"><h4>Assay: Aβ1-42 Fibrillization</h4><p>Overview: The microtubule-associated protein tau is an abundant protein in
|
||
the axons of neurons that stabilizes microtubules. With its ability to
|
||
modulate microtubule dynamics, tau contributes directly or indirectly, to
|
||
key structural and regulatory cellular functions. Particularly important is
|
||
the influence tau exerts on axonal transport, which allows signaling
|
||
molecules, trophic factors and other essential cellular constituents to
|
||
travel along the axons. Under pathological conditions, tau becomes
|
||
sequestered into insoluble aggregates called neurofibrillary tangles. This
|
||
phenomenon is believed to have pathological consequences by promoting axonal
|
||
transport deficits that ultimately lead to synaptic dysfunction and neuronal
|
||
loss. Beta-amyloid protein undergoes a similar type of aggregation as tau
|
||
and is implicated in the pathology of Alzheimer’s disease. To
|
||
examine the selectivity of tau fibrillization inhibitors, compounds were
|
||
tested in an <i>in vitro</i> assay for fibrillization of the
|
||
Beta-amyloid fragment, Aβ<sub>1–42</sub>.</p><p>Protocol: Synthetic Aβ<sub>1–42</sub> aliquots were
|
||
reconstituted to 2 mg/ml in DMSO and then diluted to 15 μM in 25
|
||
mM Tris, pH 7.0 buffer to which test compound was added at 50 μM
|
||
final concentration, or at several concentrations ranging from
|
||
0.16–40 μM. The reaction mixtures were dispensed at
|
||
25 μl/well into a 384-well plate and then incubated at
|
||
37°C for 4 hrs. Upon completion of the reaction, 25
|
||
μl of 25 μM ThT was added to each well followed by
|
||
ThT fluorescence readings as described above for K18PL.</p></div></div><div id="ml103.s31"><h3>Known probe properties</h3><div id="ml103.tu5" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK47348/table/ml103.tu5/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml103.tu5_lrgtbl__"><table><thead><tr><th id="hd_h_ml103.tu5_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;">CID</th><th id="hd_h_ml103.tu5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">9795907</th></tr></thead><tbody><tr><td headers="hd_h_ml103.tu5_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">ID</td><td headers="hd_h_ml103.tu5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NCGC00183199</td></tr><tr><td headers="hd_h_ml103.tu5_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Chemical Formula</td><td headers="hd_h_ml103.tu5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">C<sub>13</sub>H<sub>g</sub>N<sub>3</sub>O<sub>3</sub>S</td></tr><tr><td headers="hd_h_ml103.tu5_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Molecular Weight</td><td headers="hd_h_ml103.tu5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">287.29</td></tr><tr><td headers="hd_h_ml103.tu5_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Log P</td><td headers="hd_h_ml103.tu5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">1.89</td></tr><tr><td headers="hd_h_ml103.tu5_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">tPSA</td><td headers="hd_h_ml103.tu5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">96</td></tr><tr><td headers="hd_h_ml103.tu5_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Number of hydrogen donors</td><td headers="hd_h_ml103.tu5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">2</td></tr><tr><td headers="hd_h_ml103.tu5_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Number of hydrogen acceptors:</td><td headers="hd_h_ml103.tu5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">5</td></tr><tr><td headers="hd_h_ml103.tu5_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Rotatable bond</td><td headers="hd_h_ml103.tu5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">2</td></tr></tbody></table></div></div></div><div id="ml103.sum1"><h3>Summary of probe properties (solubility, absorbance/fluorescence, reactivity,
|
||
toxicity, etc.) and recommendations for the scientific use of probe as research
|
||
tool</h3><p>CID-9795907 (NCGC-000183199) is a yellowish solid and dissolves well in DMSO. It
|
||
is stable at room temperature.</p></div><div id="ml103.s32"><h3>Compound preparation</h3><p>Compound is prepared in DMSO at 10 mM stock concentration. Assays described above
|
||
have 0.6% DMSO final concentration in buffer.</p></div><div id="ml103.s33"><h3>Probe availability</h3><p>Aliquots of 10 mM DMSO solution are available from the NCGC upon request. In
|
||
addition, compound has been made available through the MLSMR
|
||
(MLS-002472423).</p></div></div><div id="ml103.s34"><h2 id="_ml103_s34_">Appendices</h2><div id="ml103.s35"><h3>Comparative data on probe, similar compound structures and prior
|
||
probes</h3><div id="ml103.s36"><h4>Prior State-of-the-Art for Probe Development</h4><div id="ml103.tu6" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK47348/table/ml103.tu6/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml103.tu6_lrgtbl__"><table><thead><tr><th id="hd_h_ml103.tu6_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Structure</th><th id="hd_h_ml103.tu6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Name</th><th id="hd_h_ml103.tu6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Class</th><th id="hd_h_ml103.tu6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Stage</th><th id="hd_h_ml103.tu6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CID</th><th id="hd_h_ml103.tu6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Source</th><th id="hd_h_ml103.tu6_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Reference</th></tr></thead><tbody><tr><td headers="hd_h_ml103.tu6_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
|
||
<div class="graphic"><img src="/books/NBK47348/bin/ml103fu16.jpg" alt="Image ml103fu16.jpg" /></div>
|
||
</td><td headers="hd_h_ml103.tu6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BF-170</td><td headers="hd_h_ml103.tu6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Quinoline</td><td headers="hd_h_ml103.tu6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">ex vivo</td><td headers="hd_h_ml103.tu6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">16219029</td><td headers="hd_h_ml103.tu6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Sigma-Aldrich cat. no.
|
||
B4311</td><td headers="hd_h_ml103.tu6_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Okamura et al 2005</td></tr><tr><td headers="hd_h_ml103.tu6_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
|
||
<div class="graphic"><img src="/books/NBK47348/bin/ml103fu17.jpg" alt="Image ml103fu17.jpg" /></div>
|
||
</td><td headers="hd_h_ml103.tu6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Methylene blue</td><td headers="hd_h_ml103.tu6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Phenothiazine</td><td headers="hd_h_ml103.tu6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">phase II</td><td headers="hd_h_ml103.tu6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">6099</td><td headers="hd_h_ml103.tu6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Sigma-Aldrich cat. no.
|
||
03978</td><td headers="hd_h_ml103.tu6_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
|
||
<a class="bk_pop" href="#ml103.r15">Wischik et al
|
||
1996</a>
|
||
</td></tr><tr><td headers="hd_h_ml103.tu6_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
|
||
<div class="graphic"><img src="/books/NBK47348/bin/ml103fu18.jpg" alt="Image ml103fu18.jpg" /></div>
|
||
</td><td headers="hd_h_ml103.tu6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Emodin</td><td headers="hd_h_ml103.tu6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Anthraquinone</td><td headers="hd_h_ml103.tu6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">in vitro</td><td headers="hd_h_ml103.tu6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3220</td><td headers="hd_h_ml103.tu6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Sigma-Aldrich cat. no.
|
||
45170</td><td headers="hd_h_ml103.tu6_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
|
||
<a class="bk_pop" href="#ml103.r11">Pickhardt et al
|
||
2005</a>
|
||
</td></tr><tr><td headers="hd_h_ml103.tu6_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
|
||
<div class="graphic"><img src="/books/NBK47348/bin/ml103fu19.jpg" alt="Image ml103fu19.jpg" /></div>
|
||
</td><td headers="hd_h_ml103.tu6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">NCGC00065256</td><td headers="hd_h_ml103.tu6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Quinoxaline</td><td headers="hd_h_ml103.tu6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">in vitro</td><td headers="hd_h_ml103.tu6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">663281</td><td headers="hd_h_ml103.tu6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Ambinter 4S-27438</td><td headers="hd_h_ml103.tu6_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
|
||
<a class="bk_pop" href="#ml103.r6">Honson et al
|
||
2007</a>
|
||
</td></tr><tr><td headers="hd_h_ml103.tu6_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
|
||
<div class="graphic"><img src="/books/NBK47348/bin/ml103fu20.jpg" alt="Image ml103fu20.jpg" /></div>
|
||
</td><td headers="hd_h_ml103.tu6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">N744</td><td headers="hd_h_ml103.tu6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Cyanine Dye</td><td headers="hd_h_ml103.tu6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">in vitro</td><td headers="hd_h_ml103.tu6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1103407</td><td headers="hd_h_ml103.tu6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Commercially unavailable</td><td headers="hd_h_ml103.tu6_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Congdon et al 2007</td></tr><tr><td headers="hd_h_ml103.tu6_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
|
||
<div class="graphic"><img src="/books/NBK47348/bin/ml103fu21.jpg" alt="Image ml103fu21.jpg" /></div>
|
||
</td><td headers="hd_h_ml103.tu6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">113F08</td><td headers="hd_h_ml103.tu6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Quinoxaline</td><td headers="hd_h_ml103.tu6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">in vitro</td><td headers="hd_h_ml103.tu6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2301472</td><td headers="hd_h_ml103.tu6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Chembridge cat. No.</td><td headers="hd_h_ml103.tu6_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
|
||
<a class="bk_pop" href="#ml103.r1">Crowe et al 2007</a>
|
||
</td></tr><tr><td headers="hd_h_ml103.tu6_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
|
||
<div class="graphic"><img src="/books/NBK47348/bin/ml103fu22.jpg" alt="Image ml103fu22.jpg" /></div>
|
||
</td><td headers="hd_h_ml103.tu6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">19</td><td headers="hd_h_ml103.tu6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Rodaline</td><td headers="hd_h_ml103.tu6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">in vitro</td><td headers="hd_h_ml103.tu6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">24756229</td><td headers="hd_h_ml103.tu6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Commercially unavailable</td><td headers="hd_h_ml103.tu6_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Bulic et al 2009</td></tr><tr><td headers="hd_h_ml103.tu6_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
|
||
<div class="graphic"><img src="/books/NBK47348/bin/ml103fu23.jpg" alt="Image ml103fu23.jpg" /></div>
|
||
</td><td headers="hd_h_ml103.tu6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Gossypetin</td><td headers="hd_h_ml103.tu6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Polyphenol</td><td headers="hd_h_ml103.tu6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">in vitro</td><td headers="hd_h_ml103.tu6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">5280647</td><td headers="hd_h_ml103.tu6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Microsource cat. No.
|
||
MS-1505143</td><td headers="hd_h_ml103.tu6_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
|
||
<a class="bk_pop" href="#ml103.r13">Taniguchi et al
|
||
2005</a>
|
||
</td></tr><tr><td headers="hd_h_ml103.tu6_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
|
||
<div class="graphic"><img src="/books/NBK47348/bin/ml103fu24.jpg" alt="Image ml103fu24.jpg" /></div>
|
||
</td><td headers="hd_h_ml103.tu6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BSc3094</td><td headers="hd_h_ml103.tu6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">phenylthiazolyl-hydrazide</td><td headers="hd_h_ml103.tu6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">in vitro</td><td headers="hd_h_ml103.tu6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">25096749</td><td headers="hd_h_ml103.tu6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Commercially unavailable</td><td headers="hd_h_ml103.tu6_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Pickhardt et al 2007</td></tr><tr><td headers="hd_h_ml103.tu6_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
|
||
<div class="graphic"><img src="/books/NBK47348/bin/ml103fu25.jpg" alt="Image ml103fu25.jpg" /></div>
|
||
</td><td headers="hd_h_ml103.tu6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">B4A1</td><td headers="hd_h_ml103.tu6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Pyrimidine</td><td headers="hd_h_ml103.tu6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">in vitro</td><td headers="hd_h_ml103.tu6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">7908145</td><td headers="hd_h_ml103.tu6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Asinex BAS 00234164</td><td headers="hd_h_ml103.tu6_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
|
||
<a class="bk_pop" href="#ml103.r8">Khlistunova et al
|
||
2006</a>
|
||
</td></tr></tbody></table></div></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml103t5"><a href="/books/NBK47348/table/ml103.t5/?report=objectonly" target="object" title="Table 5" class="img_link icnblk_img figpopup" rid-figpopup="figml103t5" rid-ob="figobml103t5"><img class="small-thumb" src="/books/NBK47348/table/ml103.t5/?report=thumb" src-large="/books/NBK47348/table/ml103.t5/?report=previmg" alt="Table 5. MLS, NCGC and PubChem IDs of ATPZ series compounds." /></a><div class="icnblk_cntnt"><h4 id="ml103.t5"><a href="/books/NBK47348/table/ml103.t5/?report=objectonly" target="object" rid-ob="figobml103t5">Table 5</a></h4><p class="float-caption no_bottom_margin">MLS, NCGC and PubChem IDs of ATPZ series compounds. </p></div></div></div></div></div><div id="ml103.bib"><h2 id="_ml103_bib_">Bibliography</h2><ol><li><div class="bk_ref" id="ml103.r1">Crowe A, Ballatore C, et al. High throughput screening for small molecule inhibitors of
|
||
heparin-induced tau fibril formation. <span><span class="ref-journal">Biochem Biophys Res Commun. </span>2007;<span class="ref-vol">358</span>(1):1–6.</span> [<a href="/pmc/articles/PMC2646256/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2646256</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/17482143" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17482143</span></a>]</div></li><li><div class="bk_ref" id="ml103.r2">Crowe A, Huang W, et al. The Identification of Aminothienopyridazine Inhibitors of Tau
|
||
Assembly by Quantitative High Throughput Screening. <span></span> Submitted. [<a href="/pmc/articles/PMC2773749/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2773749</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/19580328" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19580328</span></a>]</div></li><li><div class="bk_ref" id="ml103.r3">Ferguson GN, Valant C, et al. 2-aminothienopyridazines as novel adenosine A1 receptor
|
||
allosteric modulators and antagonists. <span><span class="ref-journal">J Med Chem. </span>2008;<span class="ref-vol">51</span>(19):6165–72.</span> [<a href="/pmc/articles/PMC2605720/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2605720</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/18771255" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18771255</span></a>]</div></li><li><div class="bk_ref" id="ml103.r4">Gustke N, Trinczek B, et al. Domains of tau protein and interactions with
|
||
microtubules. <span><span class="ref-journal">Biochemistry. </span>1994;<span class="ref-vol">33</span>(32):9511–22.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/8068626" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 8068626</span></a>]</div></li><li><div class="bk_ref" id="ml103.r5">Hong M, Zhukareva V, et al. Mutation-specific functional impairments in distinct tau
|
||
isoforms of hereditary FTDP-17. <span><span class="ref-journal">Science. </span>1998;<span class="ref-vol">282</span>(5395):1914–7.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/9836646" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 9836646</span></a>]</div></li><li><div class="bk_ref" id="ml103.r6">Honson NS, Johnson RL, et al. Differentiating Alzheimer disease-associated aggregates with
|
||
small molecules. <span><span class="ref-journal">Neurobiol Dis. </span>2007;<span class="ref-vol">28</span>(3):251–60.</span> [<a href="/pmc/articles/PMC2194600/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2194600</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/17761424" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17761424</span></a>]</div></li><li><div class="bk_ref" id="ml103.r7">Inglese J, Auld DS, et al. Quantitative high-throughput screening: a titration-based
|
||
approach that efficiently identifies biological activities in large
|
||
chemical libraries. <span><span class="ref-journal">Proc Natl Acad Sci U S A. </span>2006;<span class="ref-vol">103</span>(31):11473–8.</span> [<a href="/pmc/articles/PMC1518803/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC1518803</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/16864780" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 16864780</span></a>]</div></li><li><div class="bk_ref" id="ml103.r8">Khlistunova I, Biernat J, et al. Inducible expression of Tau repeat domain in cell models of
|
||
tauopathy: aggregation is toxic to cells but can be reversed by
|
||
inhibitor drugs. <span><span class="ref-journal">J Biol Chem. </span>2006;<span class="ref-vol">281</span>(2):1205–14.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/16246844" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 16246844</span></a>]</div></li><li><div class="bk_ref" id="ml103.r9">McGovern SL, Caselli E, et al. A common mechanism underlying promiscuous inhibitors from
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virtual and high-throughput screening. <span><span class="ref-journal">J Med Chem. </span>2002;<span class="ref-vol">45</span>(8):1712–22.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/11931626" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 11931626</span></a>]</div></li><li><div class="bk_ref" id="ml103.r10">McGovern SL, Helfand BT, et al. A specific mechanism of nonspecific
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inhibition. <span><span class="ref-journal">J Med Chem. </span>2003;<span class="ref-vol">46</span>(20):4265–72.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/13678405" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 13678405</span></a>]</div></li><li><div class="bk_ref" id="ml103.r11">Pickhardt M, Gazova Z, et al. Anthraquinones inhibit tau aggregation and dissolve
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Alzheimer’s paired helical filaments in vitro and in
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cells. <span><span class="ref-journal">J Biol Chem. </span>2005;<span class="ref-vol">280</span>(5):3628–35.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/15525637" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 15525637</span></a>]</div></li><li><div class="bk_ref" id="ml103.r12">Shoichet BK. Interpreting steep dose-response curves in early inhibitor
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discovery. <span><span class="ref-journal">J Med Chem. </span>2006;<span class="ref-vol">49</span>(25):7274–7.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/17149857" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17149857</span></a>]</div></li><li><div class="bk_ref" id="ml103.r13">Taniguchi S, Suzuki N, et al. Inhibition of heparin-induced tau filament formation by
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phenothiazines, polyphenols, and porphyrins. <span><span class="ref-journal">J Biol Chem. </span>2005;<span class="ref-vol">280</span>(9):7614–23.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/15611092" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 15611092</span></a>]</div></li><li><div class="bk_ref" id="ml103.r14">von Bergen M, Barghorn S, et al. Mutations of tau protein in frontotemporal dementia promote
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aggregation of paired helical filaments by enhancing local
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aggregation by phenothiazines. <span><span class="ref-journal">Proc Natl Acad Sci U S A. </span>1996;<span class="ref-vol">93</span>(20):11213–8.</span> [<a href="/pmc/articles/PMC38310/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC38310</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/8855335" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 8855335</span></a>]</div></li></ol></div><div id="bk_toc_contnr"></div></div></div>
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<div class="post-content"><div><div class="half_rhythm"><a href="/books/about/copyright/">Copyright Notice</a></div><div class="small"><span class="label">Bookshelf ID: NBK47348</span><span class="label">PMID: <a href="https://pubmed.ncbi.nlm.nih.gov/21433364" title="PubMed record of this page" ref="pagearea=meta&targetsite=entrez&targetcat=link&targettype=pubmed">21433364</a></span></div><div style="margin-top:2em" class="bk_noprnt"><a class="bk_cntns" href="/books/n/mlprobe/">Contents</a><div class="pagination bk_noprnt"><a class="active page_link prev" href="/books/n/mlprobe/ml104/" title="Previous page in this title">< Prev</a><a class="active page_link next" href="/books/n/mlprobe/ml102/" title="Next page in this title">Next ></a></div></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/NBK47348/?report=reader">PubReader</a></li><li><a href="/books/NBK47348/?report=printable">Print View</a></li><li><a data-jig="ncbidialog" href="#_ncbi_dlg_citbx_NBK47348" data-jigconfig="width:400,modal:true">Cite this Page</a><div id="_ncbi_dlg_citbx_NBK47348" style="display:none" title="Cite this Page"><div class="bk_tt">Johnson RL, Huang W, Huang R, et al. High Throughput Screening for Small Molecule Inhibitors of Heparin-induced Tau Fibril Formation. 2009 May 18 [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/NBK47348/pdf/Bookshelf_NBK47348.pdf">PDF version of this page</a> (412K)</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="#ml103.s2" ref="log$=inpage&link_id=inpage">Probe Structure & Characteristics</a></li><li><a href="#ml103.s3" ref="log$=inpage&link_id=inpage">Recommendations for the scientific use of this probe</a></li><li><a href="#ml103.s7" ref="log$=inpage&link_id=inpage">Assay Implementation and Screening</a></li><li><a href="#ml103.s17" ref="log$=inpage&link_id=inpage">Probe Characterization</a></li><li><a href="#ml103.s18" ref="log$=inpage&link_id=inpage">Synthesis of analogs</a></li><li><a href="#ml103.s34" ref="log$=inpage&link_id=inpage">Appendices</a></li><li><a href="#ml103.bib" ref="log$=inpage&link_id=inpage">Bibliography</a></li></ul></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Related information</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="discovery_db_links" id="Shutter"></a></div><div class="portlet_content"><ul><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pmc&DbFrom=books&Cmd=Link&LinkName=books_pmc_refs&IdsFromResult=2359216" ref="log$=recordlinks">PMC</a><div class="brieflinkpop offscreen_noflow">PubMed Central citations</div></li><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pcassay&DbFrom=books&Cmd=Link&LinkName=books_pcassay_probe&IdsFromResult=2359216" ref="log$=recordlinks">PubChem BioAssay for Chemical Probe</a><div class="brieflinkpop offscreen_noflow">PubChem BioAssay records reporting screening data for the development of the chemical probe(s) described in this book chapter</div></li><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pcsubstance&DbFrom=books&Cmd=Link&LinkName=books_pcsubstance&IdsFromResult=2359216" ref="log$=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=2359216" ref="log$=recordlinks">PubMed</a><div class="brieflinkpop offscreen_noflow">Links to PubMed</div></li></ul></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Similar articles in PubMed</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="PBooksDiscovery_RA" id="Shutter"></a></div><div class="portlet_content"><ul><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/17482143" ref="ordinalpos=1&linkpos=1&log$=relatedarticles&logdbfrom=pubmed">High throughput screening for small molecule inhibitors of heparin-induced tau fibril formation.</a><span class="source">[Biochem Biophys Res Commun. 2007]</span><div class="brieflinkpop offscreen_noflow">High throughput screening for small molecule inhibitors of heparin-induced tau fibril formation.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Crowe A, Ballatore C, Hyde E, Trojanowski JQ, Lee VM. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Biochem Biophys Res Commun. 2007 Jun 22; 358(1):1-6. 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