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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="Scaffold Hopping Affords a Highly Selective in vitro and in vivo T-Type Calcium Inhibitor Probe Free From IP Issues" /><meta name="citation_publisher" content="National Center for Biotechnology Information (US)" /><meta name="citation_date" content="2013/03/22" /><meta name="citation_author" content="Xinmin Xie" /><meta name="citation_author" content="John T. Brogan" /><meta name="citation_author" content="Michael L. Schulte" /><meta name="citation_author" content="Debbie Mi" /><meta name="citation_author" content="Haibo Yu" /><meta name="citation_author" content="Eric S. Dawson" /><meta name="citation_author" content="Min Li" /><meta name="citation_author" content="Owen McManus" /><meta name="citation_author" content="Julie Engers" /><meta name="citation_author" content="L. Michelle Lewis" /><meta name="citation_author" content="Analisa Thompson" /><meta name="citation_author" content="Carrie K. Jones" /><meta name="citation_author" content="C. David Weaver" /><meta name="citation_author" content="Craig W. Lindsley" /><meta name="citation_pmid" content="23762962" /><meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK143195/" /><link rel="schema.DC" href="http://purl.org/DC/elements/1.0/" /><meta name="DC.Title" content="Scaffold Hopping Affords a Highly Selective in vitro and in vivo T-Type Calcium Inhibitor Probe Free From IP Issues" /><meta name="DC.Type" content="Text" /><meta name="DC.Publisher" content="National Center for Biotechnology Information (US)" /><meta name="DC.Contributor" content="Xinmin Xie" /><meta name="DC.Contributor" content="John T. Brogan" /><meta name="DC.Contributor" content="Michael L. Schulte" /><meta name="DC.Contributor" content="Debbie Mi" /><meta name="DC.Contributor" content="Haibo Yu" /><meta name="DC.Contributor" content="Eric S. Dawson" /><meta name="DC.Contributor" content="Min Li" /><meta name="DC.Contributor" content="Owen McManus" /><meta name="DC.Contributor" content="Julie Engers" /><meta name="DC.Contributor" content="L. Michelle Lewis" /><meta name="DC.Contributor" content="Analisa Thompson" /><meta name="DC.Contributor" content="Carrie K. Jones" /><meta name="DC.Contributor" content="C. David Weaver" /><meta name="DC.Contributor" content="Craig W. Lindsley" /><meta name="DC.Date" content="2013/03/22" /><meta name="DC.Identifier" content="https://www.ncbi.nlm.nih.gov/books/NBK143195/" /><meta name="description" content="A scaffold hopping approach afforded ML218 (CID 45115620) a selective T-Type Ca2+ (Cav3.1, Cav3.2, Cav3.3) inhibitor (Cav3.2, IC50 = 150 nM in Ca2+ flux; Cav3.2 IC50 = 310 nM and Cav3.3 IC50 = 270 nM in patch clamp electrophysiology) suitable for in vitro and in vivo studies. ML218 possess excellent dystrophia myotonica protein kinase (DMPK) properties, acceptable in vivo rat PK, excellent brain levels and was efficacious upon oral dosing in a preclinical Parkinsonian model. Thus, ML218 is a powerful new probe to study T-Type Ca2+ function in vitro and in vivo." /><meta name="og:title" content="Scaffold Hopping Affords a Highly Selective in vitro and in vivo T-Type Calcium Inhibitor Probe Free From IP Issues" /><meta name="og:type" content="book" /><meta name="og:description" content="A scaffold hopping approach afforded ML218 (CID 45115620) a selective T-Type Ca2+ (Cav3.1, Cav3.2, Cav3.3) inhibitor (Cav3.2, IC50 = 150 nM in Ca2+ flux; Cav3.2 IC50 = 310 nM and Cav3.3 IC50 = 270 nM in patch clamp electrophysiology) suitable for in vitro and in vivo studies. ML218 possess excellent dystrophia myotonica protein kinase (DMPK) properties, acceptable in vivo rat PK, excellent brain levels and was efficacious upon oral dosing in a preclinical Parkinsonian model. 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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="#__NBK143195_dtls__">Show details</a><div style="display:none" class="ui-widget" id="__NBK143195_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/ml220/" title="Previous page in this title">< Prev</a><a class="active page_link next" href="/books/n/mlprobe/ml217/" 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="_NBK143195_"><span class="title" itemprop="name">Scaffold Hopping Affords a Highly Selective <i>in vitro</i> and <i>in vivo</i> T-Type Calcium Inhibitor Probe Free From IP Issues</span></h1><p class="contrib-group"><span itemprop="author">Xinmin Xie</span>, <span itemprop="author">John T. Brogan</span>, <span itemprop="author">Michael L. Schulte</span>, <span itemprop="author">Debbie Mi</span>, <span itemprop="author">Haibo Yu</span>, <span itemprop="author">Eric S. Dawson</span>, <span itemprop="author">Min Li</span>, <span itemprop="author">Owen McManus</span>, <span itemprop="author">Julie Engers</span>, <span itemprop="author">L. Michelle Lewis</span>, <span itemprop="author">Analisa Thompson</span>, <span itemprop="author">Carrie K. Jones</span>, <span itemprop="author">C. David Weaver</span>, and <span itemprop="author">Craig W. Lindsley</span>.</p><a data-jig="ncbitoggler" href="#__NBK143195_ai__" style="border:0;text-decoration:none">Author Information and Affiliations</a><div style="display:none" class="ui-widget" id="__NBK143195_ai__"><p class="contrib-group"><h4>Authors</h4><span itemprop="author">Xinmin Xie</span>,<sup>3</sup> <span itemprop="author">John T. Brogan</span>,<sup>2</sup> <span itemprop="author">Michael L. Schulte</span>,<sup>2</sup> <span itemprop="author">Debbie Mi</span>,<sup>2</sup> <span itemprop="author">Haibo Yu</span>,<sup>1</sup> <span itemprop="author">Eric S. Dawson</span>,<sup>2</sup> <span itemprop="author">Min Li</span>,<sup>1</sup> <span itemprop="author">Owen McManus</span>,<sup>1</sup> <span itemprop="author">Julie Engers</span>,<sup>2</sup> <span itemprop="author">L. Michelle Lewis</span>,<sup>2</sup> <span itemprop="author">Analisa Thompson</span>,<sup>2</sup> <span itemprop="author">Carrie K. Jones</span>,<sup>2</sup> <span itemprop="author">C. David Weaver</span>,<sup>2</sup> and <span itemprop="author">Craig W. Lindsley</span><sup>2*</sup><sup>,<img src="/corehtml/pmc/pmcgifs/corrauth.gif" alt="corresponding author" /></sup>.</p><h4>Affiliations</h4><div class="affiliation"><sup>1</sup>
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Johns Hopkins Ion Channel Center, Nashville, TN, 37232, USA</div><div class="affiliation"><sup>2</sup>
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Vanderbilt Specialized Chemistry Center for Accelerated Probe Development, Nashville, TN, 37232, USA</div><div class="affiliation"><sup>3</sup>
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AfaSci Research, Stanford, CA 94305</div><div class="affiliation"><sup><img src="/corehtml/pmc/pmcgifs/corrauth.gif" alt="corresponding author" /></sup><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="ude.tlibrednav@yelsdnil.giarc" class="oemail">ude.tlibrednav@yelsdnil.giarc</a></div></div><p class="small">Received: <span itemprop="datePublished">March 31, 2011</span>; Last Update: <span itemprop="dateModified">March 22, 2013</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>A scaffold hopping approach afforded <a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a> (CID 45115620) a selective T-Type Ca<sup>2+</sup> (Ca<sub>v</sub>3.1, Ca<sub>v</sub>3.2, Ca<sub>v</sub>3.3) inhibitor (Ca<sub>v</sub>3.2, IC<sub>50</sub> = 150 nM in Ca<sup>2+</sup> flux; Ca<sub>v</sub>3.2 IC<sub>50</sub> = 310 nM and Ca<sub>v</sub>3.3 IC<sub>50</sub> = 270 nM in patch clamp electrophysiology) suitable for <i>in vitro</i> and <i>in vivo</i> studies. <a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a> possess excellent dystrophia myotonica protein kinase (DMPK) properties, acceptable <i>in vivo</i> rat PK, excellent brain levels and was efficacious upon oral dosing in a preclinical Parkinsonian model. Thus, <a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a> is a powerful new probe to study T-Type Ca<sup>2+</sup> function <i>in vitro</i> and <i>in vivo</i>.</p></div><div class="h2"></div><p><b>Assigned Assay Grant #:</b> NS050771-01</p><p><b>Screening Center Name & PI:</b> Vanderbilt Screening Center for GPCRs, Ion Channels and Transporters (MLSCN), David Weaver; Follow-up by Johns Hopkins Ion Channel Center, Min Li</p><p><b>Chemistry Center Name & PI:</b> Vanderbilt Specialized Chemistry Center, Craig Lindsley</p><p><b>Assay Submitter & Institution:</b> Xinmin (Simon) Xie, SRI International</p><p><b>PubChem Summary Bioassay Identifier</b> (AID): <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463807" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">463807</a></p><div id="ml218.rp"><h2 id="_ml218_rp_">Resulting Publications</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="ml218.rpr1">Xiang Z, Thompson AD, Brogan JT, Schulte ML, Melancon BJ, Mi D, Lewis LM, Zou B, Yang L, Morrison R, Santomango T, Byers F, Brewer K, Aldrich JS, Yu H, Dawson ES, Li M, McManus O, Jones CK, Daniels JS, Hopkins CR, Xie XS, Conn PJ, Weaver CD, Lindsley CW. The Discovery and Characterization of ML218: A Novel,
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Centrally Active T-Type Calcium Channel Inhibitor with Robust Effects in
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STN Neurons and in a Rodent Model of Parkinson's Disease. <span><span class="ref-journal">ACS chemical neuroscience. </span>2011 Dec 21;<span class="ref-vol">2</span>(12):730–42.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/22368764" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 22368764</span></a>]</div></dd></dl></div><div id="ml218.s1"><h2 id="_ml218_s1_">Probe Structure & Characteristics</h2><div id="ml218.fu1" class="figure bk_fig"><div class="graphic"><img src="/books/NBK143195/bin/ml218fu1.jpg" alt="3,5-dichloro-N-(((1R,5S,6S)-3-(3,3-dimethylbutyl)-3-azabicylco[3.1.0]hexan-6-yl)methyl)benzamide, ML218." /></div><h3><span class="title">3,5-dichloro-N-(((1<i>R</i>,5<i>S</i>,6<i>S</i>)-3-(3,3-dimethylbutyl)-3-azabicylco[3.1.0]hexan-6-yl)methyl)benzamide, ML218</span></h3></div><div id="ml218.tu1" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK143195/table/ml218.tu1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml218.tu1_lrgtbl__"><table><thead><tr><th id="hd_h_ml218.tu1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">CID/ML#</th><th id="hd_h_ml218.tu1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Target Name</th><th id="hd_h_ml218.tu1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">EC<sub>50</sub> (nM) [SID, AID]</th><th id="hd_h_ml218.tu1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Anti-target Name(s)</th><th id="hd_h_ml218.tu1_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">IC<sub>50</sub>/EC<sub>50</sub> (μM) [SID, AID]</th><th id="hd_h_ml218.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Fold Selective</th><th id="hd_h_ml218.tu1_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Secondary Assay(s) Name: IC<sub>50</sub>/EC<sub>50</sub> (nM) [SID, AID]</th></tr></thead><tbody><tr><td headers="hd_h_ml218.tu1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">CID 45115620/<a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a></td><td headers="hd_h_ml218.tu1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Ca<sub>v</sub>3.2 (Ca<sup>2+</sup>flux)</td><td headers="hd_h_ml218.tu1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">150, [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/92310249" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 92310249</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/449739" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 449739</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/489005" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 489005</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/493021" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 493021</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/493022" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 493022</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/493023" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 493023</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/493041" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 493041</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504619" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504619</a>]</td><td headers="hd_h_ml218.tu1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">L/N-Type Ca<sup>2+</sup><br /><br />hERG<br /><br />Na<sup>+</sup>/K<sup>+</sup> current in rat DRG</td><td headers="hd_h_ml218.tu1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">>10, [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/92310249" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 92310249</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504584" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504584</a>]<br /><br />>10 [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/92310249" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 92310249</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504584" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504584</a>]<br /><br />>10 [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/92310249" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 92310249</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504579" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504579</a>]</td><td headers="hd_h_ml218.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">>60<br /><br />>60<br /><br />>60</td><td headers="hd_h_ml218.tu1_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Ca<sub>v</sub>3.2_IWS: 310 [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/92310249" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 92310249</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504425" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504425</a>]<br /><br />Ca<sub>v</sub>3.3_IWS: 270 [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/92310249" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 92310249</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/504425" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 504425</a>]</td></tr></tbody></table></div></div></div><div id="ml218.s2"><h2 id="_ml218_s2_">1. Recommendations for Scientific Use of the Probe</h2><p>This probe (<a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a>, CID 45115620) can be used to investigate the role of selective T-Type Ca<sup>2+</sup> (Ca<sub>v</sub>3.1, Ca<sub>v</sub>3.2, Ca<sub>v</sub>3.3) inhibition <i>in vitro</i> and <i>in vivo</i>. There are many known T-Type Ca<sup>2+</sup> inhibitors, but the potent and selective ones are patented. This was known prior to running the HTS screen, and NIH desired we develop a T-Type Ca<sup>2+</sup> inhibitor free of IP issues so that the biomedical community could possess a potent and selective probe without concern over use restrictions. <a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a> is a potent inhibitor (Ca<sub>v</sub>3.2, IC<sub>50</sub> = 150 nM in Ca<sup>2+</sup> flux; Ca<sub>v</sub>3.2 IC<sub>50</sub> = 310 nM and Ca<sub>v</sub>3.3 IC<sub>50</sub> = 270 nM in patch clamp electrophysiology) and selective against the other members of the calcium family of ion channels (L- and N-type calcium channels) as well as multiple sodium and potassium channels. Lastly, <a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a> was tested at Ricerca’s (formerly MDS Pharma’s) Lead Profiling Screen (binding assay panel of 68 GPCRs, ion channels and transporters screened at 10 μM) and was found to significantly bind to only 2 of the 68 targets; importantly, <a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a> was found to be selective against hERG and many other ion channels. <a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a> possess excellent DMPK properties, acceptable <i>in vivo</i> rat PK, excellent brain levels and was efficacious in a haloperidol-induced catalepsy assay, a preclinical Parkinsonian model. Thus, <a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a> is a powerful new probe to study T-Type Ca<sup>2+</sup> function <i>in vitro</i> and <i>in vivo</i>.</p></div><div id="ml218.s3"><h2 id="_ml218_s3_">2. Materials and Methods</h2><ul><li class="half_rhythm"><div>HEK-293 cell line expressing T-type (CaV3.2) channels</div></li><li class="half_rhythm"><div>Native HEK-293 cell line</div></li><li class="half_rhythm"><div>Fluo-4 AM dye (Invitrogen, Cat #<a href="/nuccore/860755" class="bk_tag" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=nuccore">F14202</a>)</div></li><li class="half_rhythm"><div>KK-3-118 (positive control T-type inhibitor)</div></li><li class="half_rhythm"><div>Cell plates: BD Biocoat, Poly-D-Lysine coated, black/clear bottom (cat # 356936) 384 well assay plates</div></li><li class="half_rhythm"><div>Compound daughter plates: 384-well Greiner round bottom, clear, custom barcoded (cat# 781281)</div></li><li class="half_rhythm"><div>BioTek ELx405 plate washer</div></li><li class="half_rhythm"><div>Thermo Fisher Combi</div></li><li class="half_rhythm"><div>Labcyte Echo555</div></li><li class="half_rhythm"><div>Hammamatsu FDSS 6000 kinetic imaging plate reader’</div></li><li class="half_rhythm"><div>HEK293 cell lines stably expressing Cav3.2 and Cav3.3 channels</div></li><li class="half_rhythm"><div>Parental HEK cell line</div></li><li class="half_rhythm"><div>Population Patch Clamp patch plates from Molecular Devices</div></li></ul><div id="ml218.s4"><h3>2.1. Assays</h3><div id="ml218.s5"><h4>T-type calcium channel Cell-based FDSS Ca<sup>2+</sup> flux Assay</h4><div id="ml218.s6"><h5>T Type Primary (AID 449739)</h5><ol><li class="half_rhythm"><div>Cell culture: Cells are routinely cultured in D-MEM/F12 (Gibco 11330-032) supplemented with 10% heat-inactivated FBS (Sigma F2442), 0.5% Sodium Pyruvate (Gibco 11360), and 1mg/ml G418 (Cellgro 30-234-CR)</div></li><li class="half_rhythm"><div>Cell plating: Add 20 μl/well of 1000 cells/μl suspended in D-MEM/F12 (Gibco 11330-032) supplemented with 10% heat-inactivated FBS (Sigma F2442) and 0.5% Sodium Pyruvate (Gibco 11360).</div></li><li class="half_rhythm"><div>Incubate overnight at 37 °C and 5% CO<sub>2</sub>.</div></li><li class="half_rhythm"><div>Remove medium with ELx405 and replace with 20 μl/well of Assay Buffer (140 mM NaCl, 5 mM KCl, 1 mM MgCl<sub>2</sub>, 0.5 mM CaCl<sub>2</sub>, 10 mM glucose, 10 mM HEPES pH 7.3).</div></li><li class="half_rhythm"><div>Add 20 μl/well Dye Loading Solution (4.6 μM Fluo-4 AM with 0.02% w/v Pluronic F-127 in Assay Buffer) with Combi</div></li><li class="half_rhythm"><div>Incubate 60 minutes at room temperature (RT)</div></li><li class="half_rhythm"><div>Prepare 2X compound plates by transferring 80 nL/well from stock plates containing compounds at 10 mM in DMSO to compound daughter plates and add 40μl/well Assay Buffer using Combi.</div></li><li class="half_rhythm"><div>Negative (vehicle) control and positive control wells are 0.2% DMSO in Assay Buffer and EC<sub>80</sub> of KK-3-118 in Assay Buffer, respectively in every other well of columns 1–2, 23–24.</div></li><li class="half_rhythm"><div>Replace Dye Loading Solution with 20 μL/well of Assay Buffer using ELx405.</div></li><li class="half_rhythm"><div>Load cell plates to Hamamatsu FDSS 6000 kinetic imaging plate reader.</div></li><li class="half_rhythm"><div>Measure fluorescence for 10 seconds at 1Hz to establish baseline.</div></li><li class="half_rhythm"><div>Add 20 μl/well test compounds from daughter plates and continue to collect data for 50 seconds at 1Hz.</div></li><li class="half_rhythm"><div>Remove plates from FDSS and incubate for 20 min at RT</div></li><li class="half_rhythm"><div>Reload plates to FDSS and resume collecting data at 1Hz</div></li><li class="half_rhythm"><div>After 10 seconds add 10 μl/well 5 × Stimulus Buffer (140 mM NaCl, 5 mM KCl, 1 mM MgCl<sub>2</sub>, 50 mM CaCl<sub>2</sub>, 5 mM glucose, 10 mM HEPES pH 7.3) and continue collecting data for 2 min. Calculate ratio readout as F/F<sub>0</sub> to normalize the data and reduce the waveform data to reflect the maximum normalized fluorescence/well. Calculate the average and standard deviation for negative and positive controls in each plate, as well as Z′.</div></li><li class="half_rhythm"><div>Calculate Z and B scores for test compounds using ratios calculated in Step 15.</div></li><li class="half_rhythm"><div>Outcome assignment: If the Z or B score of the test compound was more than 3 standard deviations below the average of the test population the compounds were denoted as “Active”. If compounds were less than 3 standard deviations from the average of the test population they were labeled as “Inactive”</div></li><li class="half_rhythm"><div>Score assignment: “Active” compounds were assigned a score of “100”. “Inactive” assigned a score of “0”.</div></li></ol></div></div><div id="ml218.s7"><h4>Ca<sub>v</sub>3.2 and Ca<sub>v</sub>3.3 calcium channel IonWorks Electrophysiology Assays</h4><p>Ca<sub>v</sub>3 channel activity was examined in electrophysiological assays using the population patch clamp mode on the Ionworks Quattro (MDC, Sunnyvale, CA), an automated patch clamp instrument. The HEK293 cells stably expressing Ca<sub>v</sub>3.2 and Ca<sub>v</sub>3.3 channels were freshly dislodged from flasks and dispensed into a 384-well population patch clamp (PPC) plate, separately. The cell plating density was 7,000 cells/well suspended in the extracellular solution, composed of (in mM): 137 NaCl, 4 KCl, 1 MgCl<sub>2</sub>, 1.8 CaCl<sub>2</sub>, 10 HEPES, and 10 glucose, pH 7.4 adjusted with NaOH.</p><p>After dispensing, seal resistance of cells was measured for each well and cells were perforated by incubation with 50μg/mL amphotericin B (Sigma, St. Louis, MO) on the intracellular side, which was dissolved in the internal solution composed of (in mM): 40 KCl, 100 K-Gluconate, 1 MgCl<sub>2</sub>, 5 HEPES, 2 mM CaCl2 pH 7.2 adjusted with KOH. The channel activity was then measured with the recording protocol as described below. Leak currents were linearly subtracted by extrapolating the current elicited by a 100-ms step to −110 mV from a holding potential of −100 mV. During the voltage pulse protocol, cells were held at −100 mV, followed by a depolarization step to −30mV for 1 s, and then back to −90mV. The currents were measured at the peak of the inward currents before and after the application of compounds for 3 minutes. Only cells with a current amplitude greater than 200 pA at −30 mV and a seal resistance >30 MΩ were included in the data analysis.</p><p>Compound effects were assessed by the percentage changes in the inward currents, which were calculated by dividing the difference of current amplitude between pre- and post-compound recordings by the respective pre-compound currents in the same well. No corrections for liquid junction potentials were applied. The current signal was sampled at 0.625 kHz.</p></div><div id="ml218.s8"><h4>DMPK Methods</h4><p><i><u>In vitro</u></i>: The metabolism of <a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a> was investigated in rat hepatic microsomes (BD Biosciences, Billerica, MA) using substrate depletion methodology (% test article remaining). A potassium phosphate-buffered reaction mixture (0.1 M, pH 7.4) of test article (1 μM) and microsomes (0.5 mg/mL) was pre-incubated (5 min) at 37°C prior to the addition of NADPH (1 μM). The incubations, performed in 96-well plates, were continued at 37 °C under ambient oxygenation and aliquots (80 μL) were removed at selected time intervals (0, 3, 7, 15, 25 and 45 min). Protein was precipitated by the addition of chilled acetonitrile (160 μL), containing glyburide as an internal standard (50 ng/mL), and centrifuged at 3000 rpm (4°C) for 10 minutes. Resulting supernatants were transferred to new 96-well plates in preparation for LC/MS/MS analysis. The in vitro half-life (<i>t</i><sub>1/2</sub>, min, <a href="#ml218.eq1">Eq. 1</a>), intrinsic clearance (CL<sub>int</sub>, mL/min/kg, <a href="#ml218.eq2">Eq. 2</a>) and subsequent predicted hepatic clearance (CL<sub>hep</sub>, mL/min/kg, <a href="#ml218.eq3">Eq. 3</a>) was determined employing the following equations:</p><div class="pmc_disp_formula whole_rhythm clearfix" id="ml218.eq1"><div class="inline_block pmc_inline_block pmc_va_middle pmc_hide_overflow eleven_col">
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<i>t</i><sub>1/2</sub> = Ln(2) / <i>k</i> ; where <i>k</i> represents the slope from linear regression analysis (% test article remaining)</div><div class="inline_block pmc_inline_block pmc_va_middle pmc_hide_overflow last bk_equ_label "><div><span class="nowrap">1</span></div></div></div><div class="pmc_disp_formula whole_rhythm clearfix" id="ml218.eq2"><div class="inline_block pmc_inline_block pmc_va_middle pmc_hide_overflow eleven_col">
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CL<sub>int</sub> = (0.693 / <i>t</i><sub>1/2</sub>) (rxn volume / mg of microsomes) (45 mg microsomes / gram of liver) (20<sup><i>a</i></sup> gm of liver / kg body weight); <sup><i>a</i></sup>scale-up factors of 20 (human) and 45 (rat)</div><div class="inline_block pmc_inline_block pmc_va_middle pmc_hide_overflow last bk_equ_label "><div><span class="nowrap">2</span></div></div></div><div class="pmc_disp_formula whole_rhythm clearfix" id="ml218.eq3"><div class="inline_block pmc_inline_block pmc_va_middle pmc_hide_overflow eleven_col">
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<math id="ml218.eq4" display="block"><semantics><mrow><mi>C</mi><mi>L</mi><mi>h</mi><mi>e</mi><mi>p</mi><mo>=</mo><mfrac><mrow><mi>Q</mi><mo>·</mo><mi>C</mi><mi>L</mi><mi> </mi><mtext>int</mtext></mrow><mrow><mi>Q</mi><mo>+</mo><mi>C</mi><mi>L</mi><mi> </mi><mtext>int</mtext></mrow></mfrac></mrow></semantics></math></div><div class="inline_block pmc_inline_block pmc_va_middle pmc_hide_overflow last bk_equ_label "><div><span class="nowrap">3</span></div></div></div><p><i><u>In vivo</u></i>: Male Sprague-Dawley rats (n=2) weighing around 300g were purchased from Harlon laboratories (Indianapolis, IN) and implanted with catheters in the carotid artery and jugular vein. The cannulated animals were acclimated to their surroundings for approximately one week before dosing and provided food and water <i>ad libitum</i>. <a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a> was administered intravenously (IV) to rats via the jugular vein catheter in 20% DMSO/80% saline at a dose of 1 mg/kg and a dose volume of 1 mL/kg. Blood collections via the carotid artery were performed at pre-dose, at 2, 7, 15, and 30 minutes, and at 1, 2, 4, 7 and 24 hrs post dose. Samples were collected into chilled, EDTA-fortified tubes, centrifuged for 10 minutes at 3000 rpm (4 °C), and resulting plasma aliquoted into 96-well plates for LC/MS/MS analysis. All pharmacokinetic analysis was performed employing noncompartmental analysis. For oral exposure studies, measuring both systemic plasma and CNS tissue exposure, <a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a> was administered (oral gavage) to fasted rats (n=2) as suspensions in 10% tween 80/0.5% methylcellulose at a dose of 10 mg/kg and in a dosing volume of 10 mL/kg; blood and whole brain samples were collected at 1.5h post dose. Blood was collected into chilled, EDTA-fortified tubes, centrifuged for 10 minutes at 3000 rpm (4 °C) and stored at −80 °C until LC/MS/MS analysis. The brain samples were rinsed in PBS, snap frozen and stored at −80 °C. Prior to LC/MS/MS analysis, brain samples were thawed to room temperature and subjected to mechanical homogenation employing a Mini-Beadbeater™ and 1.0 mm Zirconia/Silica Beads (BioSpec Products). All animal studies were approved by the Vanderbilt University Medical Center Institutional Animal Care and Use Committee. The animal care and use program is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International.</p><div id="ml218.s9"><h5>Plasma Protein Binding</h5><p>Protein binding of <a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a> was determined in rat plasma via equilibrium dialysis employing Single-Use RED Plates with inserts (ThermoFisher Scientific, Rochester, NY). Briefly, plasma (220 μL) was added to the 96 well plate containing test article (5 μL) and mixed thoroughly. Subsequently, 200 μL of the plasma-test article mixture was transferred to the <i>cis</i> chamber (red) of the RED plate, with an accompanying 350 μL of phosphate buffer (25 mM, pH 7.4) in the <i>trans</i> chamber. The RED plate was sealed and incubated 4 h at 37 °C with shaking. At completion, 50 μL aliquots from each chamber were diluted 1:1 (50 μL) with either plasma (<i>cis</i>) or buffer (<i>trans</i>) and transferred to a new 96 well plate, at which time ice-cold acetonitrile (2 volumes) was added to extract the matrices. The plate was centrifuged (3000 rpm, 10 min) and supernatants transferred to a new 96 well plate. The sealed plate was stored at −20°C until LC/MS/MS analysis.</p></div></div><div id="ml218.s10"><h4>Liquid Chromatography/Mass Spectrometry Analysis</h4><p><i>In vivo experiments:</i>
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<a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a> was analyzed via electrospray ionization (ESI) on an AB Sciex API-4000 (Foster City, CA) triple-quadrupole instrument that was coupled with Shimadzu LC-10AD pumps (Columbia, MD) and a Leap Technologies CTC PAL auto-sampler (Carrboro, NC). Analytes were separated by gradient elution using a Fortis C18 2.1 × 50 mm, 3.5 μm column (Fortis Technologies Ltd, Cheshire, UK) thermostated at 40°C. HPLC mobile phase A was 0.1% NH<sub>4</sub>OH (pH unadjusted), mobile phase B was acetonitrile. The gradient started at 30% B after a 0.2 min hold and was linearly increased to 90% B over 0.8 min; held at 90% B for 0.5 min and returned to 30% B in 0.1 min followed by a re-equilibration (0.9 min). The total run time was 2.5 min and the HPLC flow rate was 0.5 mL/min. The source temperature was set at 500°C and mass spectral analyses were performed using multiple reaction monitoring (MRM) utilizing a Turbo-Ionspray® source in positive ionization mode (5.0 kV spray voltage). All data were analyzed using AB Sciex Analyst 1.4.2 software.</p><p><i>In vitro experiments.</i>
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<a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a> was analyzed similarly to that described above (<i>In vivo</i>) with the following exceptions: LC/MS/MS analysis was performed employing a TSQ Quantum<sup>ULTRA</sup> that was coupled to a ThermoSurveyor LC system (Thermoelectron Corp., San Jose, CA) and a Leap Technologies CTC PAL auto-sampler (Carrboro, NC). Chromatographic separation of analytes was achieved with an Acquity BEH C18 2.1 × 50 mm, 1.7 μm column (Waters, Taunton, MA).</p></div><div id="ml218.s11"><h4>Haloperidol-Induced Catalepsy Methods</h4><p><b>Animals.</b> Male Sprague-Dawley rats weighing between 275 and 299 grams (Harlan Laboratories, Inc Indianapolis, IN) were used for the behavioral studies and were housed under a 12-h light/dark cycle (lights on at 6 AM, lights off at 6 PM) with free access to food and water. The experimental protocols, which were performed during the light cycle, were approved by the Institutional Animal Care and Use Committee of Vanderbilt University and conformed to the guidelines established by the National Research Council Guide for the Care and Use of Laboratory Animals. Rats were administered haloperidol (0.75mg/kg, i.p., dissolved in 8.5% lactic acid) 60 minutes prior to vehicle (10% Tween 80 in 0.5% methylcellulose) or <a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a> (0.1–30mg/kg). After an additional 30 minute pretreatment interval, all rats were assessed in the catalepsy model. Catalepsy was measured by placing the forepaws of each rat gently onto a horizontal placed 6cm from the testing surface with the body positioned at an angle of ~45° to the testing surface. The latency in seconds required for the rat to remove one or both forepaws from the bar was measured with a testing cutoff of 30 seconds.</p></div></div><div id="ml218.s12"><h3>2.2. Probe Chemical Characterization</h3><div id="ml218.fu2" class="figure"><div class="graphic"><img src="/books/NBK143195/bin/ml218fu2.jpg" alt="Image ml218fu2" /></div></div><p>Probe compound <a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a> (CID: 45115620, SID: <a href="https://pubchem.ncbi.nlm.nih.gov/substance/92310249" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">92310249</a>) was prepared according to the above scheme and provided the following characterization data: <sup>1</sup>H NMR (400 MHz, CDCl<sub>3</sub>) δ (ppm): 7.78 (d, <i>J</i> = 1.82 Hz, 2H); 7.57 (br, 1H); 7.42 (ap. t, <i>J</i> = 1.76 Hz, 1H); 3.64 (d, <i>J</i> = 10.56 Hz, 2H); 3.32 (t, <i>J</i> = 5.98 Hz, 2H); 2.95–2.83 (m,4H); 1.95 (m, 1H); 1.74 (s, 2H); 1.62 (m, 2H); 0.88 (s, 9H). <sup>13</sup>C NMR (100 MHz, CD<sub>3</sub>OD) δ (ppm): 165.41, 137.46, 135.50, 131.46, 126.36, 55.69, 53.11, 41.94, 39.42, 30.07, 29.44, 21.71; HRMS calc’d for C<sub>19</sub>H<sub>26</sub>Cl<sub>2</sub>N<sub>2</sub>O, 369.1500 [M+H]; found 369.1501.</p><p><b>Solubility.</b> Solubility (kinetic solubility) in PBS was determined to be 542 μM.</p><p><b>GSH Conjugates.</b> No glutathione conjugates detected.</p><p><b>Stability.</b> Stability was determined for <a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a> at 23 °C in PBS (no antioxidants or other protectorants and DMSO concentration below 0.1%) and is shown in <a class="figpopup" href="/books/NBK143195/table/ml218.t1/?report=objectonly" target="object" rid-figpopup="figml218t1" rid-ob="figobml218t1">Table 1</a>. We were surprised by the lack of stability in phosphate buffer, and Merck’s <b>7</b><sup><a class="bk_pop" href="#ml218.r24">24</a></sup> behaved similarly. Due to the high logP (clogP 5.2, experimentally derived logP 4.9), we proposed that over time, the thermodynamic nature of this assay, the compound precipitates out of solution. In the solubility assay in pH 7.4 phosphate buffer, <a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a> shows high solubility, but this is kinetic versus thermodynamic conditions. Upon closer inspection, it does appear that <a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a>, as well as <b>7</b>,<sup><a class="bk_pop" href="#ml218.r24">24</a></sup> do precipitate out of solution over the time course in the 48 hour PBS assay. Moreover, the <a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a> is stable in rat plasma and shows good stability <i>in vivo</i>. Therefore, the physical properties of <a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a> appear to be the culprit for the apparent lack of stability in the 48 hour PBS assay, which is actually a lack of solubility and not instability. <i>In vivo</i>, <a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a> is stable.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml218t1"><a href="/books/NBK143195/table/ml218.t1/?report=objectonly" target="object" title="Table 1" class="img_link icnblk_img figpopup" rid-figpopup="figml218t1" rid-ob="figobml218t1"><img class="small-thumb" src="/books/NBK143195/table/ml218.t1/?report=thumb" src-large="/books/NBK143195/table/ml218.t1/?report=previmg" alt="Table 1. Stability in Phosphate Buffer." /></a><div class="icnblk_cntnt"><h4 id="ml218.t1"><a href="/books/NBK143195/table/ml218.t1/?report=objectonly" target="object" rid-ob="figobml218t1">Table 1</a></h4><p class="float-caption no_bottom_margin">Stability in Phosphate Buffer. </p></div></div><div id="ml218.t2" class="table"><h3><span class="label">Table 2</span><span class="title">Calculated Property Comparison with MDDR Compounds</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK143195/table/ml218.t2/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml218.t2_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml218.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Property</th><th id="hd_h_ml218.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"><a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a></th><th id="hd_h_ml218.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">MDDR Phase I</th><th id="hd_h_ml218.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">MDDR Launched</th></tr></thead><tbody><tr><td headers="hd_h_ml218.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MW</td><td headers="hd_h_ml218.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">369.33</td><td headers="hd_h_ml218.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">438.98</td><td headers="hd_h_ml218.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">415.20</td></tr><tr><td headers="hd_h_ml218.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">cLogP</td><td headers="hd_h_ml218.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">5.28</td><td headers="hd_h_ml218.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">3.21</td><td headers="hd_h_ml218.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">2.21</td></tr><tr><td headers="hd_h_ml218.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">TPSA</td><td headers="hd_h_ml218.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">32.34</td><td headers="hd_h_ml218.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">97.06</td><td headers="hd_h_ml218.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">91.78</td></tr><tr><td headers="hd_h_ml218.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Hdon</td><td headers="hd_h_ml218.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">1.00</td><td headers="hd_h_ml218.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">2.12</td><td headers="hd_h_ml218.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">2.13</td></tr><tr><td headers="hd_h_ml218.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Hacc</td><td headers="hd_h_ml218.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">2.00</td><td headers="hd_h_ml218.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">7.06</td><td headers="hd_h_ml218.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">6.47</td></tr><tr><td headers="hd_h_ml218.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">LogS</td><td headers="hd_h_ml218.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">−5.43</td><td headers="hd_h_ml218.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">−4.96</td><td headers="hd_h_ml218.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">−3.73</td></tr><tr><td headers="hd_h_ml218.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">NrotB</td><td headers="hd_h_ml218.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">6.00</td><td headers="hd_h_ml218.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">7.08</td><td headers="hd_h_ml218.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">5.71</td></tr></tbody></table></div></div><p><b>Compounds added to the SMR collection (MLS#s):</b> 003431854 (<a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML 218</a>, CID 45115620, 20 mg), 003431850 (CID 44251479, 5.3 mg), 003431851 (CID 44251485, 5.0 mg), 003431852 (CID 45115609, 9.4 mg), 003431849 (CID 46943243, 5.5 mg), 003431853 (CID 46943258, 8.4 mg).</p></div><div id="ml218.s13"><h3>2.3. Probe Preparation</h3><div id="ml218.fu3" class="figure"><div class="graphic"><img src="/books/NBK143195/bin/ml218fu3.jpg" alt="Image ml218fu3" /></div></div><p><b><u>3,5-dichloro-<i>N</i>-((3-(3,3-dimethylbutyl)-3-azabicyclo[3.1.0]hexan-6-yl)methyl)benzamide (<a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a>)).</u></b>
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<i><u>tert</u></i><u>-butyl 6-((3,5-dichlorobenzamido)methyl)-3 azabicyclo[3.1.0]hexane-3-carboxylate</u> (<b>16</b>). To a solution of exo-3-Boc-6-aminomethyl-3-azabicyclo[3.1.0]hexane (1.0 eq.) and triethylamine (1.0 eq.) in CH<sub>2</sub>Cl<sub>2</sub> at 0 °C was added a solution of 3,5-dichlorobenzoyl chloride (1.0 eq.) in CH<sub>2</sub>Cl<sub>2</sub>. The reaction stirred at 0 °C for approximately 4 hrs. The mixture was then concentrated <i>in vacuo</i> and purified by reverse phase chromatography (MeCN/H<sub>2</sub>O/0.1% TFA) to afford the product as a white solid (93% yield). <sup>1</sup>H NMR (400 MHz, CD<sub>3</sub>OD) δ (ppm): 7.83 (d, <i>J</i> = 1.92 Hz, 2H); 7.66 (t, <i>J</i> = 1.88 Hz, 1H); 3.57 (m, 2H); 3.35 (m, 4H); 1.60 (m, 2H); 1.46 (s,9H); 0.90 (m, 1H). <sup>13</sup>C NMR (125 MHz, CD<sub>3</sub>OD) δ (ppm): 167.96, 157.61, 139.75, 137.33, 133.02, 127.99, 81.86, 43.65, 29.56, 24.46, 24.02, 23.23. To a vial containing tert-butyl 6-((3,5-dichlorobenzamido)methyl)-3-azabicyclo[3.1.0]hexane-3-carboxylate <b>16</b>, was added 4.0 M HCl in dioxane. This mixture was allowed to stir at room temp for 2 hrs. The reaction was then concentrated <i>in vacuo</i> and purified by reverse phase chromatography (MeCN/H<sub>2</sub>O/0.1% TFA) to afford the product as a white solid (95% yield). <sup>1</sup>H NMR (400 MHz, CD<sub>3</sub>OD) δ (ppm): 7.82 (d, <i>J</i> = 1.92 Hz, 2H); 7.68 (m, 1H); 3.45 (s, 4H); 3.34 (m, 2H); 1.90 (m, 2H); 1.21 (m,1H). <sup>13</sup>C NMR (100 MHz, CD<sub>3</sub>OD) δ (ppm): 168.04, 163.95, 139.57, 137.40, 133.17, 127.97, 43.02, 23.35, 22.48. <u>3,5-dichloro-</u><i><u>N</u></i><u>-((3-(3,3-dimethylbutyl)-3-azabicyclo[3.1.0]hexan-6-yl)methyl)benzamide</u> (<a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a>): To a solution of 3,3-dimethylbutyraldehyde (1.0 eq.) in CH<sub>2</sub>Cl<sub>2</sub> was added a solution of <i>N</i>-(3-azabicyclo[3.1.0]hexan-6-ylmethyl)-3,5-dichlorobenzamide (1.0 eq.) in CH<sub>2</sub>Cl<sub>2</sub> followed by addition of polymer-supported sodium triacetoxyborohydride (1.2 eq.). The reaction was left to stir overnight at room temperature after which the mixture was filtered through a pad of Celite eluting with CH<sub>2</sub>Cl<sub>2</sub> and concentrated <i>in vacuo</i> resulting in a crude oil which was then purified by reverse phase chromatography (MeCN/H<sub>2</sub>O/0.1% TFA) to afford the product as an off-white solid (88% yield). <sup>1</sup>H NMR (400 MHz, CDCl<sub>3</sub>) δ (ppm): 7.78 (d, <i>J</i> = 1.82 Hz, 2H); 7.57 (br, 1H); 7.42 (ap. t, <i>J</i> = 1.76 Hz, 1H); 3.64 (d, <i>J</i> = 10.56 Hz, 2H); 3.32 (t, <i>J</i> = 5.98 Hz, 2H); 2.95–2.83 (m,4H); 1.95 (m, 1H); 1.74 (s, 2H); 1.62 (m, 2H); 0.88 (s, 9H). <sup>13</sup>C NMR (100 MHz, CD<sub>3</sub>OD) δ (ppm): 165.41, 137.46, 135.50, 131.46, 126.36, 55.69, 53.11, 41.94, 39.42, 30.07, 29.44, 21.71; HRMS calc’d for C<sub>19</sub>H<sub>26</sub>Cl<sub>2</sub>N<sub>2</sub>O, 369.1500 [M+H]; found 369.1501.</p></div></div><div id="ml218.s14"><h2 id="_ml218_s14_">3. Results</h2><p>The Vanderbilt Screening/Chemistry Center completed the primary screen using fluorescence-based assay provided by the PI Dr. Xie. AfaSci Lab and JHU have been undertaking secondary screening using electrophysiologic techniques. The primary screen using the Ca<sub>v</sub>3.2 channel expressed in HEK293 cells (provided by the project Consultant Dr. Perez-Reyes, University of Virginia) was performed in a 384-well format using an FDSS 6000 kinetic imaging plate reader (Hamamatsu). The 2008 collection of MLSCN library containing 110,720 compounds was screened at 10μM in a single measurement. Data quality was controlled by the known T channel inhibitor <b>7</b> as the positive control and the 0.1% DMSO vehicle as the negative control. The assay quality control used a screening window coefficient “Z-factor”. The Z′ values were between 0.6–0.8, indicating excellent assay quality. Primary data analysis was performed by comparing the activity of any test compound with all the other test compounds on the same plate, taking the ratio max in the kinetic time window of 12–40 sec with 0–1 sec as the baseline. Primary hits were defined as any outliers that were different from all the others based on a <i>z</i>-score threshold of 3. That is, after hits were picked using z-score, those z-scores were calculated again on the remaining compounds. Total 4,246 initial hits were identified from these 2 iterations.</p><div id="ml218.s15"><h3>3.1. Dose Response Curves for Probe</h3><p><a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a> inhibition of Ca<sub>v</sub>3 channels was evaluated in electrophysiological and fluorescent assays (<a class="figpopup" href="/books/NBK143195/figure/ml218.f1/?report=objectonly" target="object" rid-figpopup="figml218f1" rid-ob="figobml218f1">Figure 1</a>). Currents through Ca<sub>v</sub>3 channels were recorded using the IonWorks Quattro (patch EP). <a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a> is a potent and well-behaved inhibitor of Ca<sub>v</sub>3 channels.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml218f1" co-legend-rid="figlgndml218f1"><a href="/books/NBK143195/figure/ml218.f1/?report=objectonly" target="object" title="Figure 1" class="img_link icnblk_img figpopup" rid-figpopup="figml218f1" rid-ob="figobml218f1"><img class="small-thumb" src="/books/NBK143195/bin/ml218f1.gif" src-large="/books/NBK143195/bin/ml218f1.jpg" alt="Figure 1. Dose-response curves for ML218." /></a><div class="icnblk_cntnt" id="figlgndml218f1"><h4 id="ml218.f1"><a href="/books/NBK143195/figure/ml218.f1/?report=objectonly" target="object" rid-ob="figobml218f1">Figure 1</a></h4><p class="float-caption no_bottom_margin">Dose-response curves for ML218. A) Structure of ML218; B) Raw fluorescence-based HTS assay traces; C) CRC from fluorescence-based assay (IC<sub>50</sub> = 150 nM); D) Inhibition time-course of ML218. Ba<sup>2+</sup> currents were elicited by pulse depolarized to −20mV <a href="/books/NBK143195/figure/ml218.f1/?report=objectonly" target="object" rid-ob="figobml218f1">(more...)</a></p></div></div></div><div id="ml218.s16"><h3>3.2. Cellular Activity</h3><p>All assays (fluorescence and EP) for Ca<sub>v</sub>3 and other ion channels described in this report are cell-based assays. The activity of <a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a> in these assays indicates that the probe compound exhibits adequate cell permeability to support further studies with native cells or tissues. No acute toxicity was observed in the cell based assays. <a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a> was also evaluated in our tier 1 DMPK profiling assays as well as in <i>in vivo</i> PK and brain level studies in rats. No acute toxicity was observed in any cell based assay or <i>in vivo</i>. No toxicity was observed in a haloperiodol-induced catalepsy model up to 30 mg/kg p.o.</p></div><div id="ml218.s17"><h3>3.3. Profiling Assays</h3><p>Beyond the basic CRCs in <a class="figpopup" href="/books/NBK143195/figure/ml218.f1/?report=objectonly" target="object" rid-figpopup="figml218f1" rid-ob="figobml218f1">Figure 1</a>, we assessed <a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a> activity on the native T-type currents for selectivity over other ion channels in rat DRG neurons (small diameter 10–20μm) in which the Ca<sub>v</sub>3.2 channel is prominently expressed. Using pharmacological tools to isolate the current of interest, <a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a> at 1μM produced approximately 70% inhibition of the T-type Ca<sup>2+</sup> current without affecting the tetrodotoxin (TTX)-sensitive voltage-gated Na<sup>+</sup> current and K<sup>+</sup> currents (mainly delay rectifier, <a class="figpopup" href="/books/NBK143195/figure/ml218.f2/?report=objectonly" target="object" rid-figpopup="figml218f2" rid-ob="figobml218f2">Figure 2</a>). Although the potency of <a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a> in blocking the native T-type current is less potent compared to its inhibition on the recombinant Ca<sub>v</sub>3.2 channel expressed in HEK cells, it shows great selectivity over other major membrane ion channels in native neurons. The effects of <a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a> on high-voltage-activated Ca<sup>2+</sup> channels (e.g., L- and N- type) in the DRG neurons will be investigated in an extended probe characterization project.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml218f2" co-legend-rid="figlgndml218f2"><a href="/books/NBK143195/figure/ml218.f2/?report=objectonly" target="object" title="Figure 2" class="img_link icnblk_img figpopup" rid-figpopup="figml218f2" rid-ob="figobml218f2"><img class="small-thumb" src="/books/NBK143195/bin/ml218f2.gif" src-large="/books/NBK143195/bin/ml218f2.jpg" alt="Figure 2. ML218 selectively blocks T-type Ca2+ currents without affecting voltage-gated Na+ and K+ currents in DRG neurons." /></a><div class="icnblk_cntnt" id="figlgndml218f2"><h4 id="ml218.f2"><a href="/books/NBK143195/figure/ml218.f2/?report=objectonly" target="object" rid-ob="figobml218f2">Figure 2</a></h4><p class="float-caption no_bottom_margin">ML218 selectively blocks T-type Ca<sup>2+</sup> currents without affecting voltage-gated Na<sup>+</sup> and K<sup>+</sup> currents in DRG neurons. <i>A.</i>
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ML218 (1μM) reversibly blocks the T-type Ca<sup>2+</sup> currents elicited by depolarizing steps to −30mV from a Vh of −100mV <a href="/books/NBK143195/figure/ml218.f2/?report=objectonly" target="object" rid-ob="figobml218f2">(more...)</a></p></div></div><p><a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a> was evaluated in the parental HEK fluorescent assay which contains native Ca<sup>2+</sup> channels, presumably both L- and N-Type Ca<sup>2+</sup> channels. <a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a> had no affect on the parental line and was therefore thought to be selective versus L- and N-calcium channels (later confirmed at Ricerca panel, where <a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a> showed no significant activity in radioligand binding at L- and N-type Ca<sup>2+</sup> channels, 49%@10 μM and 17%@10 μM, respectively). As mentioned above, the effects of <a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a> on high-voltage-activated Ca<sup>2+</sup> channels (e.g., L- and N- type) in the DRG neurons via EP will be investigated in a later extended probe characterization effort. To more fully characterize this novel Ca<sub>v</sub>3 inhibitor probe molecule, <a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a> was tested on Ricerca’s (formerly MDS Pharma’s) Lead Profiling Screen (binding assay panel of 68 GPCRs, ion channels and transporters screened at 10 μM), and was found to only significantly bind two targets (sodium channel site 2 and sigma 1) of the 68 assays conducted (no inhibition of radio ligand binding > 50% at 10 μM). Interestingly, the Merck T-type inhibitors also showed similar activity in binding assays at sodium channel site 2 and sigma 1, but neither Merck T-type inhibitors were active in functional sodium channel EP assays. In radioligand binding assays at Ricerca,<a class="bk_pop" href="#ml218.r43">43</a>
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<a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a> had no significant inhibition of L- and N-type calcium channels (17–49% inhibition @10 μM, respectively), K<sub>ATP</sub> potassium channel (4% inhibition @10 μM) or hERG (48% inhibition @10 μM). Thus, from the Ricerca selectivity profile and the selectivity versus closely related ion channels in which <a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a> affords greater than 60-fold selectivity for block of related ion channels and other distantly related potassium channels.</p><div id="ml218.fu4" class="figure"><div class="graphic"><img src="/books/NBK143195/bin/ml218fu4.jpg" alt="Image ml218fu4" /></div></div><p><b>DMPK Profiling.</b>
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<a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a> was evaluated in our tier 1 <i>in vitro</i> DMPK panel. In plasma protein binding studies (equilibrium dialysis), <a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a> possessed good free fraction in both rat (<i>f<sub>u</sub></i> = 9.1%) and human (<i>f<sub>u</sub></i> = 3.3%). The CYP profile across four major CYP450s was also good: 3A4 (IC<sub>50</sub> >30 μM), 2C9 (IC<sub>50</sub> >30 μM), 1A2 (IC<sub>50</sub> = 10.8 μM), 2D6 (IC<sub>50</sub> = 1.7 μM). Intrinsic clearance experiments in liver microsomes indicated that <a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a> was highly cleared in rat (rat CL<sub>INT</sub> = 115 ml/min/kg), but low to moderate clearance in human (human CL<sub>INT</sub> = 12.7 ml/min/kg). <i>In vivo</i> rat PK mirrored the <i>in vitro</i> work, with a CL of 56 ml/min/kg; however, the t<sub>1/2</sub> of <a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a> was 6.8 hours with a mean residence time of ~7 hours. In a 10 mg/kg po plasma/brain study at a 1 hour time point, the plasma concentration of <a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a> was 145 ng/mL and the brain concentration of <a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a> was 1018 ng/mL. Importantly, <a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a> was found to be highly brain penetrant with a Brain<sub>AUC</sub>/Plasma<sub>AUC</sub> of ~7.4, making <a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a> an exciting tool compound for CNS studies. The profile of <a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a> is virtually identical to Merck’s, which showed poor rat PK, but excellent dog, rhesus and predicted human PK. Thus, <a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a> (CID 45115620) is highly selective and can be used to dissect the role of Ca T-type inhibition <i>in vitro</i> and potentially <i>in vivo</i>.</p><p><b><i>In Vivo</i></b>
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<b>Efficacy in a Preclinical Model of Parkinson’s disease.</b> Parkinson’s disease (PD) is therapeutic indication for T-Type Ca<sup>2+</sup> channel inhibitors, and Merck’s displayed robust efficacy in a preclinical cataleptic PD model. As we have this same model, haloperidol-induced catalepsy, running for our other PD programs, we evaluated Merck’s and <a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a> in this model, wherein reversal of the cataleptic state is consistent with an anti-Parkinsonian phenotype.<sup><a class="bk_pop" href="#ml218.r24">24</a>,<a class="bk_pop" href="#ml218.r42">42</a></sup> Here, we dosed i.p. in dose response and observed robust efficacy comparable to an A2A standard.<sup><a class="bk_pop" href="#ml218.r24">24</a>,<a class="bk_pop" href="#ml218.r42">42</a></sup> Excitingly, <a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a> showed similar efficacy upon oral dosing, with a 30 mg/kg dose (1.8 μM plasma (160 nM free) with 17.7 μM brain) essentially equivalent to a 56.6 mg/kg dose of the A2A standard (<a class="figpopup" href="/books/NBK143195/figure/ml218.f3/?report=objectonly" target="object" rid-figpopup="figml218f3" rid-ob="figobml218f3">Figure 3</a>). Thus, <a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a> is comparable to Merck’s (both <i>in vitro</i> and <i>in vivo</i>), and a valuable probe to study T-Type Ca<sup>2+</sup> channel inhibition <i>in vivo</i> without IP restrictions.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml218f3" co-legend-rid="figlgndml218f3"><a href="/books/NBK143195/figure/ml218.f3/?report=objectonly" target="object" title="Figure 3" class="img_link icnblk_img figpopup" rid-figpopup="figml218f3" rid-ob="figobml218f3"><img class="small-thumb" src="/books/NBK143195/bin/ml218f3.gif" src-large="/books/NBK143195/bin/ml218f3.jpg" alt="Figure 3. ML218 reverses haloperidol-induced catalepsy in a dose-dependent manner upon oral dosing." /></a><div class="icnblk_cntnt" id="figlgndml218f3"><h4 id="ml218.f3"><a href="/books/NBK143195/figure/ml218.f3/?report=objectonly" target="object" rid-ob="figobml218f3">Figure 3</a></h4><p class="float-caption no_bottom_margin">ML218 reverses haloperidol-induced catalepsy in a dose-dependent manner upon oral dosing. A) Merck’s <i>7</i> dose-dependent reversal of haloperidol-induced catalepsy upon i.p. dosing. B) ML218 dose-dependent reversal of haloperidol-induced catalepsy <a href="/books/NBK143195/figure/ml218.f3/?report=objectonly" target="object" rid-ob="figobml218f3">(more...)</a></p></div></div></div></div><div id="ml218.s18"><h2 id="_ml218_s18_">4. Discussion</h2><div id="ml218.s19"><h3>4.1. Comparison to Existing Art and How the New Probe is an Improvement</h3><p>A number of structural classes of compounds have been described in the scientific and patent literature that act as Ca<sup>2+</sup> T-Type channel inhibitors (<i>vide supra</i>). At least 20 structural classes of compounds that inhibit Ca<sup>2+</sup> T-Type channel have been described in the scientific and/or patent literature. The known compounds either lack the required selectivity for the Ca<sub>v</sub>3 channels, or are covered by patents.<sup><a class="bk_pop" href="#ml218.r11">11</a>–<a class="bk_pop" href="#ml218.r26">26</a>,<a class="bk_pop" href="#ml218.r40">40</a></sup> This was known prior to running the HTS screen, and NIH desired that we develop a Ca<sup>2+</sup> T-Type inhibitor free of IP issues so that the biomedical community could possess a potent and selective probe without concern over use restrictions. <a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a> is a potent inhibitor (Ca<sub>v</sub>3.2, IC<sub>50</sub> = 150 nM in Tl<sup>+</sup> flux; Ca<sub>v</sub>3.2 IC<sub>50</sub> = 310 nM and Ca<sub>v</sub>3.3 IC<sub>50</sub> = 270 nM in patch clamp electrophysiology) and selective against the other members of the calcium family of ion channels (L- and N-type calcium channels) as well as multiple sodium and potassium channels. Lastly, <a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a> was tested at Ricerca’s (formerly MDS Pharma’s) Lead Profiling Screen (binding assay panel of 68 GPCRs, ion channels and transporters screened at 10 μM) and was found to significantly bind to only 2 of the 68 targets; importantly, <a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a> was found to be selective against hERG and many other ion channels. <a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a> possess excellent DMPK properties, acceptable <i>in vivo</i> rat PK, excellent brain levels and was efficacious in a haloperidol-induced catalepsy assay, a preclinical Parkinsonian model. Thus, <a href="/pcsubstance/?term=ML218[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML218</a> is a powerful new probe to study Ca<sup>2+</sup> T-Type function <i>in vitro</i> and <i>in vivo</i>.</p></div></div><div id="ml218.s20"><h2 id="_ml218_s20_">5. References</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="ml218.r1">Catterall WA. <span><span class="ref-journal">Neurosci
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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/NBK143195/?report=reader">PubReader</a></li><li><a href="/books/NBK143195/?report=printable">Print View</a></li><li><a data-jig="ncbidialog" href="#_ncbi_dlg_citbx_NBK143195" data-jigconfig="width:400,modal:true">Cite this Page</a><div id="_ncbi_dlg_citbx_NBK143195" style="display:none" title="Cite this Page"><div class="bk_tt">Xie X, Brogan JT, Schulte ML, et al. Scaffold Hopping Affords a Highly Selective in vitro and in vivo T-Type Calcium Inhibitor Probe Free From IP Issues. 2011 Mar 31 [Updated 2013 Mar 22]. 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></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="#ml218.rp" ref="log$=inpage&link_id=inpage">Resulting Publications</a></li><li><a href="#ml218.s1" ref="log$=inpage&link_id=inpage">Probe Structure & Characteristics</a></li><li><a href="#ml218.s2" ref="log$=inpage&link_id=inpage">Recommendations for Scientific Use of the Probe</a></li><li><a href="#ml218.s3" ref="log$=inpage&link_id=inpage">Materials and Methods</a></li><li><a href="#ml218.s14" ref="log$=inpage&link_id=inpage">Results</a></li><li><a href="#ml218.s18" ref="log$=inpage&link_id=inpage">Discussion</a></li><li><a href="#ml218.s20" ref="log$=inpage&link_id=inpage">References</a></li></ul></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Related information</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="discovery_db_links" id="Shutter"></a></div><div class="portlet_content"><ul><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pmc&DbFrom=books&Cmd=Link&LinkName=books_pmc_refs&IdsFromResult=3034046" ref="log$=recordlinks">PMC</a><div class="brieflinkpop offscreen_noflow">PubMed Central citations</div></li><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pcsubstance&DbFrom=books&Cmd=Link&LinkName=books_pcsubstance&IdsFromResult=3034046" ref="log$=recordlinks">PubChem Substance</a><div class="brieflinkpop offscreen_noflow">Related PubChem Substances</div></li><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pubmed&DbFrom=books&Cmd=Link&LinkName=books_pubmed_refs&IdsFromResult=3034046" ref="log$=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/22368764" ref="ordinalpos=1&linkpos=1&log$=relatedarticles&logdbfrom=pubmed">The Discovery and Characterization of ML218: A Novel, Centrally Active T-Type Calcium Channel Inhibitor with Robust Effects in STN Neurons and in a Rodent Model of Parkinson's Disease.</a><span class="source">[ACS Chem Neurosci. 2011]</span><div class="brieflinkpop offscreen_noflow">The Discovery and Characterization of ML218: A Novel, Centrally Active T-Type Calcium Channel Inhibitor with Robust Effects in STN Neurons and in a Rodent Model of Parkinson's Disease.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Xiang Z, Thompson AD, Brogan JT, Schulte ML, Melancon BJ, Mi D, Lewis LM, Zou B, Yang L, Morrison R, et al. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">ACS Chem Neurosci. 2011 Dec 21; 2(12):730-742. </em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/23762925" ref="ordinalpos=1&linkpos=2&log$=relatedreviews&logdbfrom=pubmed"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> Development of a Selective, Allosteric PLD1/2 Inhibitor in a Novel Scaffold.</a><span class="source">[Probe Reports from the NIH Mol...]</span><div class="brieflinkpop offscreen_noflow"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> Development of a Selective, Allosteric PLD1/2 Inhibitor in a Novel Scaffold.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Scott SA, O’Reilly MC, Daniels JS, Morrison R, Ptak R, Dawson ES, Tower N, Engers JL, Engers DW, Oguin T, et al. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Probe Reports from the NIH Molecular Libraries Program. 2010</em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/23762931" ref="ordinalpos=1&linkpos=3&log$=relatedreviews&logdbfrom=pubmed"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> Development of a Selective, Allosteric PLD2 Inhibitor.</a><span class="source">[Probe Reports from the NIH Mol...]</span><div class="brieflinkpop offscreen_noflow"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> Development of a Selective, Allosteric PLD2 Inhibitor.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Scott SA, O’Reilly MC, Daniels JS, Morrison R, Ptak R, Dawson ES, Tower N, Engers JL, Engers DW, Oguin T, et al. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Probe Reports from the NIH Molecular Libraries Program. 2010</em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/25834904" ref="ordinalpos=1&linkpos=4&log$=relatedreviews&logdbfrom=pubmed"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> A Next generation PLD2 inhibitor with improved physiochemical properties and DMPK profile for translational in vivo.</a><span class="source">[Probe Reports from the NIH Mol...]</span><div class="brieflinkpop offscreen_noflow"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> A Next generation PLD2 inhibitor with improved physiochemical properties and DMPK profile for translational in vivo.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">O'Reilly MC, Scott SA, Daniels JS, Morrison R, Engers JL, Oguin T, Thomas P, Brown HA, Lindsley CW. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Probe Reports from the NIH Molecular Libraries Program. 2010</em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/23613468" ref="ordinalpos=1&linkpos=5&log$=relatedarticles&logdbfrom=pubmed">Protein kinase A regulation of T-type Ca2+ channels in rat cerebral arterial smooth muscle.</a><span class="source">[J Cell Sci. 2013]</span><div class="brieflinkpop offscreen_noflow">Protein kinase A regulation of T-type Ca2+ channels in rat cerebral arterial smooth muscle.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Harraz OF, Welsh DG. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">J Cell Sci. 2013 Jul 1; 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