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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="Inhibitors of FAP-fluorogen interaction as a multiplex assay tool compound for receptor internalization assays" /><meta name="citation_publisher" content="National Center for Biotechnology Information (US)" /><meta name="citation_date" content="2013/03/22" /><meta name="citation_author" content="Yang Wu" /><meta name="citation_author" content="Philip H. Tapia" /><meta name="citation_author" content="Kristine Gouveia" /><meta name="citation_author" content="Larry A. Sklar" /><meta name="citation_author" content="Gregory W Fisher" /><meta name="citation_author" content="Alan S. Waggoner" /><meta name="citation_author" content="Jonathan W. Jarvik" /><meta name="citation_author" content="Alison R. 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Waggoner" /><meta name="DC.Contributor" content="Jonathan W. Jarvik" /><meta name="DC.Contributor" content="Alison R. Gregro" /><meta name="DC.Contributor" content="Mike Poslusney" /><meta name="DC.Contributor" content="Craig W. Lindsley" /><meta name="DC.Contributor" content="Shaun R. Stauffer" /><meta name="DC.Date" content="2013/03/22" /><meta name="DC.Identifier" content="https://www.ncbi.nlm.nih.gov/books/NBK148495/" /><meta name="description" content="A novel assay using fluorogen activating peptide (FAP) technology for G protein-coupled receptor (GPCR) activation and internalization was applied to the human β2AR. This technology avoids microscopy and antibody-based detection methods. A major goal for the project was to identify G-protein independent/β2AR ligands or β2AR biased ligands that induce β2AR internalization. Analysis of the most potent hits in the primary project revealed that they interfered with fluorogen activation by the FAP rather than interacting with the receptor itself. These molecules were pursued further because they had the potential to enable improved assay protocols to monitor receptor trafficking and receptor location in real time. A highly potent compound (ML342, CID 2953239) was declared as a Molecular Libraries Probe Center Network (MLPCN) probe molecule." /><meta name="og:title" content="Inhibitors of FAP-fluorogen interaction as a multiplex assay tool compound for receptor internalization assays" /><meta name="og:type" content="book" /><meta name="og:description" content="A novel assay using fluorogen activating peptide (FAP) technology for G protein-coupled receptor (GPCR) activation and internalization was applied to the human β2AR. This technology avoids microscopy and antibody-based detection methods. A major goal for the project was to identify G-protein independent/β2AR ligands or β2AR biased ligands that induce β2AR internalization. Analysis of the most potent hits in the primary project revealed that they interfered with fluorogen activation by the FAP rather than interacting with the receptor itself. These molecules were pursued further because they had the potential to enable improved assay protocols to monitor receptor trafficking and receptor location in real time. 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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="#__NBK148495_dtls__">Show details</a><div style="display:none" class="ui-widget" id="__NBK148495_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/ml344/" title="Previous page in this title">< Prev</a><a class="active page_link next" href="/books/n/mlprobe/ml341/" 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="_NBK148495_"><span class="title" itemprop="name">Inhibitors of FAP-fluorogen interaction as a multiplex assay tool compound for receptor internalization assays</span></h1><p class="contrib-group"><span itemprop="author">Yang Wu</span>, <span itemprop="author">Philip H. Tapia</span>, <span itemprop="author">Kristine Gouveia</span>, <span itemprop="author">Larry A. Sklar</span>, <span itemprop="author">Gregory W Fisher</span>, <span itemprop="author">Alan S. Waggoner</span>, <span itemprop="author">Jonathan W. Jarvik</span>, <span itemprop="author">Alison R. Gregro</span>, <span itemprop="author">Mike Poslusney</span>, <span itemprop="author">Craig W. Lindsley</span>, and <span itemprop="author">Shaun R. Stauffer</span>.</p><a data-jig="ncbitoggler" href="#__NBK148495_ai__" style="border:0;text-decoration:none">Author Information and Affiliations</a><div style="display:none" class="ui-widget" id="__NBK148495_ai__"><p class="contrib-group"><h4>Authors</h4><span itemprop="author">Yang Wu</span>,<sup>1</sup> <span itemprop="author">Philip H. Tapia</span>,<sup>1</sup> <span itemprop="author">Kristine Gouveia</span>,<sup>1</sup> <span itemprop="author">Larry A. Sklar</span>,<sup>1</sup> <span itemprop="author">Gregory W Fisher</span>,<sup>2</sup> <span itemprop="author">Alan S. Waggoner</span>,<sup>2</sup> <span itemprop="author">Jonathan W. Jarvik</span>,<sup>2</sup> <span itemprop="author">Alison R. Gregro</span>,<sup>3</sup> <span itemprop="author">Mike Poslusney</span>,<sup>3</sup> <span itemprop="author">Craig W. Lindsley</span>,<sup>3</sup> and <span itemprop="author">Shaun R. Stauffer</span><sup>3</sup><sup>,*</sup>.</p><h4>Affiliations</h4><div class="affiliation"><sup>1</sup>
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University of New Mexico Center for Molecular Discovery</div><div class="affiliation"><sup>2</sup>
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Carnegie Mellon University Technology Center for Networks and Pathway</div><div class="affiliation"><sup>3</sup>
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Vanderbilt Specialized Chemistry Center, Vanderbilt University Medical Center</div><div class="affiliation">
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<sup>*</sup>
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<span class="before-email-separator"></span><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="ude.tlibrednav@reffuats.nuahs" class="oemail">ude.tlibrednav@reffuats.nuahs</a></div></div><p class="small">Received: <span itemprop="datePublished">December 14, 2012</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 novel assay using fluorogen activating peptide (FAP) technology for G protein-coupled receptor (GPCR) activation and internalization was applied to the human β2AR. This technology avoids microscopy and antibody-based detection methods. A major goal for the project was to identify G-protein independent/β2AR ligands or β2AR biased ligands that induce β2AR internalization. Analysis of the most potent hits in the primary project revealed that they interfered with fluorogen activation by the FAP rather than interacting with the receptor itself. These molecules were pursued further because they had the potential to enable improved assay protocols to monitor receptor trafficking and receptor location in real time. A highly potent compound (<a href="/pcsubstance/?term=ML342[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML342</a>, CID 2953239) was declared as a Molecular Libraries Probe Center Network (MLPCN) probe molecule.</p></div><div class="h2"></div><p><b>Assigned Assay Grant #: R03</b> DA031668-01</p><p><b>Screening Center Name & PI:</b> University of New Mexico Center for Molecular Discovery, Larry A. Sklar</p><p><b>Chemistry Center Name & PI:</b> Vanderbilt Specialized Chemistry Center, Craig Lindsley</p><p><b>Assay Submitter & Institution:</b> Jonathan Jarvik, Carnegie Mellon University</p><p><b>PubChem Summary Bioassay Identifier (AID):</b>
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<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651701" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">651701</a></p><div id="ml342.s1"><h2 id="_ml342_s1_">Probe Structure & Characteristics</h2><div id="ml342.fu1" class="figure bk_fig"><div class="graphic"><img src="/books/NBK148495/bin/ml342fu1.jpg" alt="ML342." /></div><h3><span class="title">ML342</span></h3></div><div id="ml342.tu1" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK148495/table/ml342.tu1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml342.tu1_lrgtbl__"><table><thead><tr><th id="hd_h_ml342.tu1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CID/ML#</th><th id="hd_h_ml342.tu1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Target Name</th><th id="hd_h_ml342.tu1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">EC<sub>50</sub> (nM) [SID, AID]</th><th id="hd_h_ml342.tu1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Antitargets Name(s)</th><th id="hd_h_ml342.tu1_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Activity [SID, AIDs]</th><th id="hd_h_ml342.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Fold Selective</th><th id="hd_h_ml342.tu1_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Cytotoxicity</th><th id="hd_h_ml342.tu1_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Reversibility</th><th id="hd_h_ml342.tu1_1_1_1_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Soluble Fluorogen Binding</th></tr></thead><tbody><tr><td headers="hd_h_ml342.tu1_1_1_1_1" rowspan="2" colspan="1" style="text-align:center;vertical-align:middle;">CID 2953239/<a href="/pcsubstance/?term=ML342[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML342</a></td><td headers="hd_h_ml342.tu1_1_1_1_2" rowspan="2" colspan="1" style="text-align:center;vertical-align:middle;">FAP-tag</td><td headers="hd_h_ml342.tu1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2.24±0.51 nM</td><td headers="hd_h_ml342.tu1_1_1_1_4" rowspan="2" colspan="1" style="text-align:center;vertical-align:middle;">MG13</td><td headers="hd_h_ml342.tu1_1_1_1_5" rowspan="2" colspan="1" style="text-align:center;vertical-align:middle;">Inactive<br /><br /><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651698" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651698</a><br /><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651873" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651873</a></td><td headers="hd_h_ml342.tu1_1_1_1_6" rowspan="2" colspan="1" style="text-align:center;vertical-align:middle;">>800</td><td headers="hd_h_ml342.tu1_1_1_1_7" rowspan="2" colspan="1" style="text-align:center;vertical-align:middle;">non-cytotoxic at 100 μM<br /><br /><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651694" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651694</a></td><td headers="hd_h_ml342.tu1_1_1_1_8" rowspan="2" colspan="1" style="text-align:center;vertical-align:middle;">Reversible<br /><br /><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651936" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651936</a></td><td headers="hd_h_ml342.tu1_1_1_1_9" rowspan="2" colspan="1" style="text-align:center;vertical-align:middle;">Active<br /><br /><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651938" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651938</a></td></tr><tr><td headers="hd_h_ml342.tu1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/125240931" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 125240931</a><br /><a href="https://pubchem.ncbi.nlm.nih.gov/substance/136348701" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 136348701</a><br /><br /><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/588775" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 588775</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651872" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651872</a></td></tr></tbody></table></div></div></div><div id="ml342.s2"><h2 id="_ml342_s2_">1. Recommendations for Scientific Use of the Probe</h2><p>This probe (<a href="/pcsubstance/?term=ML342[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML342</a>, CID 2953239, <a href="https://pubchem.ncbi.nlm.nih.gov/substance/125240931" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 125240931</a>) is a potent disruptor of the activation of thiazole-orange fluorogens TO1-2p by FAP AM2.2 and is selective as compared to the MG13 FAP-tag that activates fluorogen malachite green.</p><p>FAP tags can be genetically fused to proteins of interest. These fusion proteins are not fluorescent except when exposed to fluorogen, whereupon the fluorogen binds to the FAP and its fluorescence immediately increases by as much as 20,000-fold rendering the FAP fusion protein highly fluorescent. Fluorogens can be made membrane impermeant, thus allowing detection of only those receptors expressed on the cell surface. Researchers in both basic biology and in high throughput screening can benefit greatly from this new technology (<a class="bk_pop" href="#ml342.r5">5</a>,<a class="bk_pop" href="#ml342.r6">6</a>). There is currently no inhibitor of the single chain human antibody (scFv) AM2.2 FAP-tag that blocks the binding of its fluorogen, thiazole orange. Probes that specifically inhibit or reverse fluorogen activation by a FAP will have a number of immediate applications.</p><ol><li class="half_rhythm"><div><i>Study of receptor proteins</i> – By adding probe, one may selectively eliminate the fluorescence signal from FAP-tagged receptor molecules at the cell surface without affecting internalized signal. This enables more accurate quantitation of receptor (GPCR) internalization post-stimulation. We have demonstrated the utility of such an approach using trypsin treatment to remove the surface signal. Trypsin indiscriminately digests all surface proteins however, which can produce a variety of off-target effects. Use of the new probe in place of trypsin is expected to yield fewer false positives than the current trypsin-based protocols.</div></li><li class="half_rhythm"><div><i>Pulse-chase experiments</i> – Enablement of multi-color pulse-chase experiments in which signal from one FAP but not another is specifically eliminated by the addition of the probe. By splitting the sample and adding probe over time, it would be possible to assess the level of extracellular and intracellular protein.</div></li><li class="half_rhythm"><div><i>High throughput screening</i> - The proposed probe can be used as a baseline control for high throughput assays. This is particularly useful for assays where there is no known inhibitor of the target protein. In addition, the new probe would be used to make more precise measurements of the fraction of fluorescent signal that is due to background fluorescence. This will be done by measuring the fluorescence of cells that have been treated with fluorogen in the presence of excess eraser compound and subtracting this background value from the values collected from cells treated with fluorogen only. This more accurate determination of the net pre-stimulation and post-stimulation signal should lead to higher Z′ factors, which should permit the identification of weak (but genuine) agonists and antagonists in the library which would otherwise be missed.</div></li><li class="half_rhythm"><div><i>In vivo</i> - The FAP-tags are likely to find utility <i>in vivo</i>. The total FAP signal would represent the amount of labeled protein. Through additional SAR and targeted design it may be possible to develop new impermeable probes to distinguish the amount of protein available to the extracellular from the intracellular environment.</div></li></ol></div><div id="ml342.s3"><h2 id="_ml342_s3_">2. Materials and Methods</h2><div id="ml342.s4"><h3>2.1. Assays</h3><div id="ml342.s5"><h4>HTS for Beta-2AR agonists (UNMCMD)</h4><p>This is the primary assay to measure fluorescence signal decrease induced by sample compounds blocking the interaction between fluorogen TO1-2p and AM2.2.</p><ol><li class="half_rhythm"><div>Spin down AM2.2-beta2AR cells, discard supernatant, and resuspend in fresh RPMI1640 full medium. Final cell density will be 5×10<sup>6</sup> cells/mL.</div></li><li class="half_rhythm"><div>Add 5 μL serum free RPMI to the assay plate except for columns 11 and 23 by Microflo.</div></li><li class="half_rhythm"><div>Add 5 μL of freshly prepared 32 μM ISO in RPMI full media to Column 11 and 23 of all the plates as PCntrls by Microflo.</div></li><li class="half_rhythm"><div>Add 100 nL of library compounds between 3 nM and 100 μM (final concentration) to assay plates by FX.</div></li><li class="half_rhythm"><div>Add 3 μL of cells to Columns 1 – 11, 13–23 of the assay plates by Microflo.</div></li><li class="half_rhythm"><div>Shake the plates and put them in 37°C incubator for 90 mins.</div></li><li class="half_rhythm"><div>Add 3 μL 650nM TO1-2p to assay plates by Microflo or Nanoquot to assay plates and read by high-throughput flow cytometers immediately.</div></li></ol><div id="ml342.s6"><h5>Calculation</h5><p>If EC50 is reported and EC50 < 10 then PUBCHEM_ACTIVITY_SCORE = 10</p><p>Otherwise PUBCHEM_ACTIVITY_SCORE = 100</p><p>IF PUBCHEM_ACTIVITY_SCORE <= 10 then PUBCHEM_ACTIVITY_OUTCOME = 2 (or active)</p><p>IF PUBCHEM_ACTIVITY_SCORE >10 then PUBCHEM_ACTIVITY_OUTCOME = 1 (or inactive)</p></div></div><div id="ml342.s7"><h4>High throughput counter screen assay with AM2.2-β2AR/MG13-CCR5 cells (UNMCMD)</h4><p>This is a counter screen for the beta-2AR screens. This assay measures binding of fluorogen MG-2p to FAP MG13-tagged mouse CCR5 in addition to the binding of TO1-2p to FAP AM2.2-tagged β2AR in the presence of test compounds to assess if the compound interferes the binding between fluorogen MG-2p and FAP MG13.</p><ol><li class="half_rhythm"><div>Spin down AM2.2-β2AR/MG13-mCCR5 dual expressing cells, discard supernatant, and resuspend in fresh RPMI1640 full medium. Final cell density will be 5×10<sup>6</sup> cells/mL.</div></li><li class="half_rhythm"><div>Add 5 μL serum free RPMI to Columns 2–24 of the assay plate by Nanoquot.</div></li><li class="half_rhythm"><div>Add 5 μL of freshly prepared Rantes in RPMI full media to Column 1 of all the plates as PCntrls by Microflow.</div></li><li class="half_rhythm"><div>Add 100 nL of library compounds to assay plates by FX or NX.</div></li><li class="half_rhythm"><div>Add 3 μL of cells to Columns 1 – 22 of the assay plates by Nanoquot.</div></li><li class="half_rhythm"><div>Shake the plates and put them in 37°C incubator for 90 mins.</div></li><li class="half_rhythm"><div>Add 3 μL 650nM TO1-2p/210 nM MG-2p mixture to assay plates by Microflow or Nanoquot to assay plates and read by high-throughput flow cytometers immediately.</div></li></ol><div id="ml342.s8"><h5>Calculations</h5><p>Median Channel fluorescence is calculated from flow cytometric data by HyperView (IntelliCyt, Albuquerque, NM). These values for the entire concentration range of a test compound are fitted by Prism(R) software (GraphPad Software, Inc., San Diego, CA) using nonlinear least-squares regression in a sigmoidal dose response model with variable slope, also known as the four parameter logistic equation. Curve fit statistics are used to determine the following parameters of the model: EC50, microM - concentration of added test compound competitor that inhibited fluorescent ligand binding by 50 percent; LOGEC50 - the logarithm of EC50; TOP - the response value at the top plateau; BOTTOM - the response value at the bottom plateau; HILLSLOPE - the slope factor, or the Hill coefficient; STD_LOGEC50, STD_TOP, STD_BOTTOM, STD_HILLSLOPE - standard errors of LOGEC50, TOP, BOTTOM, and HILLSLOPE ; EC50_95CI_LOW, EC50_95CI_HIGH - the low and high boundaries of the 95% confidence interval of the EC50 estimate, RSQR - the correlation coefficient (r squared) indicative of goodness-of-fit.</p><p>Compounds with percent viability at the highest concentration is less than 50% are labeled active and the PubChem_Score is calculated based on EC50 of cytotoxicity by the following equation:</p><div class="pmc_disp_formula whole_rhythm clearfix" id="ml342.eq1"><div class="inline_block pmc_inline_block pmc_va_middle pmc_hide_overflow twelve_col">PubChem Score = 100 * (1 − EC50/30 μM)</div><div class="inline_block pmc_inline_block pmc_va_middle pmc_hide_overflow last bk_equ_label "><div><span class="nowrap"></span></div></div></div></div></div><div id="ml342.s9"><h4>Fluorogen binding competition assay using AM2.2-β2AR cells and AM2.2-GPR32 cells (UNMCMD)</h4><p>This is a counter screen for the beta-2AR screens. This assay measures binding of fluorogen to FAP-tagged GPR32 in the presence of test compounds to assess whether the compound interferes with FAP binding.</p><ol><li class="half_rhythm"><div>Spin down AM2.2-GPR32 cells, discard supernatant, and resuspend in fresh RPMI1640 full medium. Final cell density will be 5×10^6 cells/mL.</div></li><li class="half_rhythm"><div>Add 5 μL serum free RPMI to Columns 2–24 of the assay plate by Nanoquot.</div></li><li class="half_rhythm"><div>Add 5 μL of RPMI full media to Column 1 of all the plates, i.e., there is no Positive control.</div></li><li class="half_rhythm"><div>Add 100 nL of library compounds to assay plates by FX or NX.</div></li><li class="half_rhythm"><div>Add 3 μL of cells to Columns 1 – 22 of the assay plates by Nanoquot.</div></li><li class="half_rhythm"><div>Shake the plates and put them in 37 °C incubator for 90 mins.</div></li><li class="half_rhythm"><div>Add 3 μL 650 nM TO1-2pto assay plates by Microflo or Nanoquot to assay plates and read by high-throughput flow cytometers immediately.</div></li></ol><div id="ml342.s10"><h5>Calculations</h5><p>Median Channel fluorescence is calculated from flow cytometric data by HyperView (IntelliCyt, Albuquerque, NM). These values for the entire concentration range of a test compound are fitted by Prism(R) software (GraphPad Software, Inc., San Diego, CA) using nonlinear least-squares regression in a sigmoidal dose response model with variable slope, also known as the four parameter logistic equation. Curve fit statistics are used to determine the following parameters of the model: EC50, microM - concentration of added test compound competitor that inhibited fluorescent ligand binding by 50 percent; LOGEC50 - the logarithm of EC50; TOP - the response value at the top plateau; BOTTOM - the response value at the bottom plateau; HILLSLOPE - the slope factor, or the Hill coefficient; STD_LOGEC50, STD_TOP, STD_BOTTOM, STD_HILLSLOPE - standard errors of LOGEC50, TOP, BOTTOM, and HILLSLOPE ; EC50_95CI_LOW, EC50_95CI_HIGH - the low and high boundaries of the 95% confidence interval of the EC50 estimate, RSQR - the correlation coefficient (r squared) indicative of goodness-of-fit.</p><p>Compounds with EC50 less than 10 μM are labeled active and the PubChem_Score is calculated based on EC50 by the following equation:</p><div class="pmc_disp_formula whole_rhythm clearfix" id="ml342.eq2"><div class="inline_block pmc_inline_block pmc_va_middle pmc_hide_overflow twelve_col">PubChem Score = 100 * (1 − EC50/10 μM)</div><div class="inline_block pmc_inline_block pmc_va_middle pmc_hide_overflow last bk_equ_label "><div><span class="nowrap"></span></div></div></div></div></div><div id="ml342.s11"><h4>Fluorogen/soluble FAP binding competition assay (CMU)</h4><p>Competition binding assays are performed by adding differing amounts of compounds to mixtures of soluble AM2.2 (FAP-tag) and TO1-2p (fluorogen), and the fluorescent signal measured by spectrofluorometry.</p></div><div id="ml342.s12"><h4>Compound cytotoxicity in U937 cells (UNMCMD)</h4><p>This assay is used to determine whether a compound is causing a decrease in signal by killing the cell instead of actually inhibiting beta2AR internalization.</p><p>CellTiter-Glo, a luminescent cell viability assay kit from Promega (Madison, WI), will be used according to the manufacturer’s instruction. Briefly, the cell cultures of AM2.2-beta2AR cells are seeded in complete medium at twelve different cell densities in 96-well white polypropylene opaque plates (50 μL/well per 384 well) (Corning, Corning, NY). At the time of passage, 90 μL of cell suspension (10^5 cells/mL) are added into the plates. After stabilization for 2h, test compounds are added to the wells at 10 μL per well to a final concentration range of 380 nM to 100 μM. Vehicle control wells contain 0.01% DMSO alone. Following treatment, cells are incubated at 37°C and 5% CO2 for 18 hours. At the end of the respective time point, 1x CellTiter-Glo is added to each well (10 μL/well per 384 well). Plates are read after 30 minutes. Luminescence intensity (LI) is collected using a Wallac 1420 plate reader (PerkinElmer, Norwalk, CT).</p><div id="ml342.s13"><h5>Calculations</h5><p>Background luminescence were subtracted from all readings and then luminescence values for the entire concentration range of a test compound were fitted by Prism(R) software (GraphPad Software, Inc., San Diego, CA) using nonlinear least-squares regression in a sigmoidal dose response model with variable slope, also known as the four parameter logistic equation. Curve fit statistics were used to determine the following parameters of the model: EC50, μM - concentration of added test compound competitor that inhibited fluorescent ligand binding by 50 percent; LOGEC50 - the logarithm of EC50; TOP - the response value at the top plateau; BOTTOM - the response value at the bottom plateau; HILLSLOPE - the slope factor, or the Hill coefficient; STD_LOGEC50, STD_TOP, STD_BOTTOM, STD_HILLSLOPE - standard errors of LOGEC50, TOP, BOTTOM, and HILLSLOPE ; EC50_95CI_LOW, EC50_95CI_HIGH - the low and high boundaries of the 95% confidence interval of the EC50 estimate, RSQR - the correlation coefficient (r squared) indicative of goodness-of-fit.</p><p>Compounds with percent viability at the highest concentration is less than 50% are labeled active and the PubChem_Score is calculated based on EC50 of cytotoxicity by the following equation:</p><div class="pmc_disp_formula whole_rhythm clearfix" id="ml342.eq3"><div class="inline_block pmc_inline_block pmc_va_middle pmc_hide_overflow twelve_col">PubChem Score = 100 * (1 − EC50/50 μM)</div><div class="inline_block pmc_inline_block pmc_va_middle pmc_hide_overflow last bk_equ_label "><div><span class="nowrap"></span></div></div></div></div></div><div id="ml342.s14"><h4>Compound reversibility (UNMCMD)</h4><p>This is a secondary assay to determine whether the binding between sample compound and the FAP is covalent. Binding between non-covalent compounds and the FAP is reversible.</p><ol><li class="half_rhythm"><div>Spin down AM2.2-GPR32 cells and resuspend in serum free RPMI media and yielded a final cell density of 5×10<sup>5</sup>/mL.</div></li><li class="half_rhythm"><div>Add 99 μL of cells to two sets of assay tubes followed by 1 μL of 100 × compound in DMSO or 1 μL of DMSO only for NCntrls to both sets of tubes.</div></li><li class="half_rhythm"><div>Mix well by mild vortexing and keep it at 37°C incubator for 90 mins.</div></li><li class="half_rhythm"><div>Spin down cells from one set of the assay samples, discard supernatant, wash the cells in 500 μL of serum free RPMI, then spin and discard supernatant. Resuspend the cells in 100 μL RPMI.</div></li><li class="half_rhythm"><div>Add 150 nM TO1-2p to the cells and read by Accuri flow cytometer immediately.</div></li></ol></div></div><div id="ml342.s15"><h3>2.2. Probe Chemical Characterization</h3><p>Synthetic procedure and spectral data for <a href="/pcsubstance/?term=ML342[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML342</a> (CID 2953239, <a href="https://pubchem.ncbi.nlm.nih.gov/substance/125240931" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 125240931</a>, <i>N</i>,4-dimethyl-<i>N</i>-(2-oxo-2-(4-(pyridin-2-yl)piperazin-1-yl)ethyl)benzenesulfonamide:</p><p>Probe compound <a href="/pcsubstance/?term=ML342[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML342</a> (CID 2953239) was prepared according to <a class="figpopup" href="/books/NBK148495/figure/ml342.f3/?report=objectonly" target="object" rid-figpopup="figml342f3" rid-ob="figobml342f3">Scheme 1</a> and provided the following characterization data: LCMS (>99% 215 nm, 254 nm), R<sub>t</sub> = 0.65 min; <i>m/z</i> (M+H)<sup>+</sup> = 389. <sup>1</sup>H NMR (400 MHz, CDCl<sub>3</sub>) δ 8.1–8.2 (m, 1H), 7.9–8.0 (m, 1H), 7.7 (d, <i>J</i>=8.2 Hz, 2H), 7.4 (d, <i>J</i>=8 Hz, 2H), 7.1 (d, <i>J</i>=9.2 Hz, 1H), 7.0 (t, <i>J</i>=6.5 Hz, 1H), 3.8–4.0 (m, 10H), 2.8 (s, 3H), 2.5 (s, 3H); <sup>13</sup>C NMR 166.2, 152.3, 144.1, 144.1, 138.1, 133.0, 129.9, 128.9, 127.5, 113.3, 111.8, 52.2, 45.9, 45.7, 43.8, 40.8, 35.7, 21.4; HRMS (ESI) <i>m/z</i> 389.1646 ([M+H]<sup>+</sup>, 100%) calcd for C<sub>19</sub>H<sub>25</sub>N<sub>4</sub>O<sub>3</sub>S, 389.1647.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml342f3" co-legend-rid="figlgndml342f3"><a href="/books/NBK148495/figure/ml342.f3/?report=objectonly" target="object" title="Scheme 1" class="img_link icnblk_img figpopup" rid-figpopup="figml342f3" rid-ob="figobml342f3"><img class="small-thumb" src="/books/NBK148495/bin/ml342f3.gif" src-large="/books/NBK148495/bin/ml342f3.jpg" alt="Scheme 1. Synthesis of ML342." /></a><div class="icnblk_cntnt" id="figlgndml342f3"><h4 id="ml342.f3"><a href="/books/NBK148495/figure/ml342.f3/?report=objectonly" target="object" rid-ob="figobml342f3">Scheme 1</a></h4><p class="float-caption no_bottom_margin">Synthesis of ML342. </p></div></div><p><b>Solubility.</b> In-house solubility assay in PBS at pH 7.4 was determined for <a href="/pcsubstance/?term=ML342[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML342</a> to be 95.1±2.7 μM or 38 μg/mL based upon triplicate testing. <a href="/pcsubstance/?term=ML342[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML342</a> shows excellent solubility up to 10 mM DMSO. Overall <a href="/pcsubstance/?term=ML342[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML342</a> is in the moderate to highly soluble range for an <i>in vitro</i> tool compound at neutral pH.</p><p><b>Stability.</b> Stability was determined for <a href="/pcsubstance/?term=ML342[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML342</a> in PBS buffer at room temperature with time course evaluation over 48h. After 1.5 hour, the percent of parent compound remaining was >95%. After 24 and 48h, there is <10% apparent loss of compound. Thus <a href="/pcsubstance/?term=ML342[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML342</a> appears to have excellent stability after prolonged exposure to PBS buffer.</p><p><b>Compounds added to the SMR collection (MLS#s):</b> MLS004645659 (<a href="/pcsubstance/?term=ML342[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML342</a>, CID 2953239, 24.5 mg); MLS004645660 (CID 16323094, 8.3 mg); MLS004645661 (CID 3164412, 9.7 mg); MLS004645662 (CID 16365258, 7.7 mg); MLS004645663 (CID 17477879; 9.1 mg); MLS004645664 (CID 1001608, 5.4 mg)</p></div><div id="ml342.s16"><h3>2.3. Probe Preparation</h3><div id="ml342.fu2" class="figure"><div class="graphic"><img src="/books/NBK148495/bin/ml342fu2.jpg" alt="Image ml342fu2" /></div></div><div id="ml342.s17"><h4>Step 1. Preparation of 2-bromo-1-(4-(pyridin-2-yl)piperazin-1-yl)ethanone (1)</h4><p>To a round bottom flask equipped with a magnetic stir bar was added bromoacetyl bromide (201 mg, 1 mmol), followed by dichloromethane (10 mL). This mixture was cooled in an ice bath, and 1-(pyridin-2-yl)piperazine (326 mg, 2 mmol) was added dropwise as a solution in 5 mL dichloromethane. The mixture was allowed to stir at 0 °C for 30 min., then allowed to warm to ambient temperature and stirred for an additional 60 min. The mixture was diluted with a saturated aqueous ammonium chloride solution and extracted with diethyl ether. The combined organic layers were dried and evaporated under reduced pressure to afford 2-bromo-1-(4-(pyridin-2-yl)piperazin-1-yl)ethanone (<b>1</b>) as a clear oil, which solidified on standing. Bromide <b>1</b> was used in the next step without further purification.</p><div id="ml342.fu3" class="figure"><div class="graphic"><img src="/books/NBK148495/bin/ml342fu3.jpg" alt="Image ml342fu3" /></div></div></div><div id="ml342.s18"><h4>Step 2. Preparation of 2-(methylamino)-1-(4-(pyridin-2-yl)piperazin-1-yl)ethanone (2)</h4><p>A solution of 2-bromo-1-(4-(pyridin-2-yl)piperazin-1-yl)ethanone (300 mg, 1 mmol) in dichloromethane (10 mL) was added slowly to a solution of methylamine (1M in THF, large excess). This mixture was allowed to stir at ambient temperature for 10 minutes, then diluted with water and extracted with dichloromethane. The combined organic layers were dried and evaporated to give 205 mg of title compound <b>2</b> as an oil which was used without further purification (89%): LCMS (>99% 215 nm, 254 nm) <i>m/z</i> (M+H)<sup>+</sup> = 235.</p></div><div id="ml342.s19"><h4>Step 3. Amide coupling to prepare ML342</h4><p>2-(methylamino)-1-(4-(pyridin-2-yl)piperazin-1-yl)ethanone (246 mg, 1 mmol) was added to a round bottom flask and dissolved in dichloromethane (20 mL). N,N-Diisopropylethylamine (0.35 mL, 2 mmol) was added, followed by 4-methylbenzene-1-sulfonyl chloride (380 mg, 2 mmol). Mixture allowed to stir at room temperature for 30 minutes, then washed with water. Organic layers collected, dried, evaporated and purified by HPLC to afford N,4-dimethyl-N-(2-oxo-2-(4-(pyridin-2-yl)piperazin-1-yl)ethyl)benzenesulfonamide as a colorless oil (see final full characterization data above).</p></div></div></div><div id="ml342.s20"><h2 id="_ml342_s20_">3. Results</h2><div id="ml342.s21"><h3>3.1. Dose Response Curves for Probe</h3><div id="ml342.f1" class="figure bk_fig"><div class="graphic"><img src="/books/NBK148495/bin/ml342f1.jpg" alt="Figure 1. Concentration Response Curve for Probe ML342 (CID 2953239) in AM2.2-β2AR/TO1-2p (fluorogen) assay." /></div><h3><span class="label">Figure 1</span><span class="title">Concentration Response Curve for Probe ML342 (CID 2953239) in AM2.2-β2AR/TO1-2p (fluorogen) assay</span></h3></div><div id="ml342.f2" class="figure bk_fig"><div class="graphic"><img src="/books/NBK148495/bin/ml342f2.jpg" alt="Figure 2. Concentration Response Curve for Probe ML342 (CID 2953239) in MG13-CCR5 counterscreen binding assay." /></div><h3><span class="label">Figure 2</span><span class="title">Concentration Response Curve for Probe ML342 (CID 2953239) in MG13-CCR5 counterscreen binding assay</span></h3></div></div><div id="ml342.s22"><h3>3.2. Cellular Activity</h3><p>This probe is directed at an extracellular target. We plan to pursue cellular permeability in future studies.</p></div><div id="ml342.s23"><h3>3.3. Profiling Assays</h3><p>Due to the goal and unique nature of this probe development project, which is currently focused on <i>in vitro</i> studies, ancillary pharmacology was not assessed at this time for <a href="/pcsubstance/?term=ML342[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML342</a>. Should future efforts lead to the identification of more advanced and highly selective small molecule inhibitors of FAP-tagged cell surface proteins, applications could be envisioned which might require such an assessment of biological activity.</p></div></div><div id="ml342.s24"><h2 id="_ml342_s24_">4. Discussion</h2><div id="ml342.s25"><h3>4.1. Comparison to Existing Art and How the New Probe is an Improvement</h3><p>In the course of identifying candidate β2AR agonists, we identified a number of compounds that act at the level of the FAP reporter and not at the level of the receptor to which it is fused. In the context of screen development, as described above, these compounds have provided a new and unpredicted opportunity. To date, no non-fluorescent analogs of fluorogen thiazole orange and its derivatives have been discovered or reported in literature. Thus <a href="/pcsubstance/?term=ML342[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML342</a> represents the first probe molecule of this type.</p></div></div><div id="ml342.s26"><h2 id="_ml342_s26_">5. References</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="ml342.r1">Szent-Gyorgyi C, Schmidt BF, Creeger Y, Fisher GW, Zakel KL, Adler S, Fitzpatrick JA, Woolford CA, Yan Q, Vasilev KV, Berget PB, Bruchez MP, Jarvik JW, Waggoner A. Fluorogen-activating single-chain antibodies for imaging cell surface proteins. <span><span class="ref-journal">Nature biotechnology. </span>2008;<span class="ref-vol">26</span>:235–240.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/18157118" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18157118</span></a>]</div></dd><dt>2.</dt><dd><div class="bk_ref" id="ml342.r2">Holleran J, Brown D, Fuhrman MH, Adler SA, Fisher GW, Jarvik JW. Fluorogen-activating proteins as biosensors of cell-surface proteins in living cells. <span><span class="ref-journal">Cytometry. Part A : the journal of the International Society for Analytical Cytology. </span>2010;<span class="ref-vol">77</span>:776–782.</span> [<a href="/pmc/articles/PMC2945705/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2945705</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/20653017" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 20653017</span></a>]</div></dd><dt>3.</dt><dd><div class="bk_ref" id="ml342.r3">Saunders MJ, Szent-Gyorgyi C, Fisher GW, Jarvik JW, Bruchez MP, Waggoner AS. Fluorogen activating proteins in flow cytometry for the study of surface molecules and receptors. <span><span class="ref-journal">Methods. </span>2012;<span class="ref-vol">57</span>:308–317.</span> [<a href="/pmc/articles/PMC3432715/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3432715</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/22366230" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 22366230</span></a>]</div></dd><dt>4.</dt><dd><div class="bk_ref" id="ml342.r4">Keseru GM, Makara GM. The influence of lead discovery strategies on the properties of drug candidates. <span><span class="ref-journal">Nat Rev Drug Discov. </span>2009;<span class="ref-vol">8</span>:203–212.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/19247303" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19247303</span></a>]</div></dd><dt>5.</dt><dd><div class="bk_ref" id="ml342.r5">Wu Y, Tapia PH, Fisher GW, Simons PC, Strouse JJ, Foutz T, Waggoner AS, Jarvik J, Sklar LA. Discovery of regulators of receptor internalization with high-throughput flow cytometry. <span><span class="ref-journal">Mol Pharm. </span>2012;<span class="ref-vol">82</span>:645–657.</span> [<a href="/pmc/articles/PMC3463215/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3463215</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/22767611" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 22767611</span></a>]</div></dd><dt>6.</dt><dd><div class="bk_ref" id="ml342.r6">Wu Y, Tapia P, Fisher GW, Waggoner AS, Jarvik J, Sklar LA. High-Throughput Flow Cytometry Compatible Biosensor Based on Fluorogen Activating Protein Technology. <span><span class="ref-journal">Cytometry. </span></span> in Press. [<a href="/pmc/articles/PMC3621705/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3621705</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/23303704" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 23303704</span></a>]</div></dd></dl></div><div id="bk_toc_contnr"></div></div></div>
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<div xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance"></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Views</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="PDF_download" id="Shutter"></a></div><div class="portlet_content"><ul xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="simple-list"><li><a href="/books/NBK148495/?report=reader">PubReader</a></li><li><a href="/books/NBK148495/?report=printable">Print View</a></li><li><a data-jig="ncbidialog" href="#_ncbi_dlg_citbx_NBK148495" data-jigconfig="width:400,modal:true">Cite this Page</a><div id="_ncbi_dlg_citbx_NBK148495" style="display:none" title="Cite this Page"><div class="bk_tt">Wu Y, Tapia PH, Gouveia K, et al. Inhibitors of FAP-fluorogen interaction as a multiplex assay tool compound for receptor internalization assays. 2012 Dec 14 [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="#ml342.s1" ref="log$=inpage&link_id=inpage">Probe Structure & Characteristics</a></li><li><a href="#ml342.s2" ref="log$=inpage&link_id=inpage">Recommendations for Scientific Use of the Probe</a></li><li><a href="#ml342.s3" ref="log$=inpage&link_id=inpage">Materials and Methods</a></li><li><a href="#ml342.s20" ref="log$=inpage&link_id=inpage">Results</a></li><li><a href="#ml342.s24" ref="log$=inpage&link_id=inpage">Discussion</a></li><li><a href="#ml342.s26" ref="log$=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=3040604" ref="log$=recordlinks">PMC</a><div class="brieflinkpop offscreen_noflow">PubMed Central citations</div></li><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pcassay&DbFrom=books&Cmd=Link&LinkName=books_pcassay_probe&IdsFromResult=3040604" ref="log$=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=3040604" ref="log$=recordlinks">PubChem Substance</a><div class="brieflinkpop offscreen_noflow">Related PubChem Substances</div></li><li class="brieflinkpopper"><a class="brieflinkpopperctrl" href="/books/?Db=pubmed&DbFrom=books&Cmd=Link&LinkName=books_pubmed_refs&IdsFromResult=3040604" ref="log$=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/26442911" ref="ordinalpos=1&linkpos=1&log$=relatedarticles&logdbfrom=pubmed">Discovery of Small-Molecule Nonfluorescent Inhibitors of Fluorogen-Fluorogen Activating Protein Binding Pair.</a><span class="source">[J Biomol Screen. 2016]</span><div class="brieflinkpop offscreen_noflow">Discovery of Small-Molecule Nonfluorescent Inhibitors of Fluorogen-Fluorogen Activating Protein Binding Pair.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Wu Y, Stauffer SR, Stanfield RL, Tapia PH, Ursu O, Fisher GW, Szent-Gyorgyi C, Evangelisti A, Waller A, Strouse JJ, et al. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">J Biomol Screen. 2016 Jan; 21(1):74-87. Epub 2015 Oct 6.</em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/24510772" ref="ordinalpos=1&linkpos=2&log$=relatedarticles&logdbfrom=pubmed">Real-time detection of protein trafficking with high-throughput flow cytometry (HTFC) and fluorogen-activating protein (FAP) base biosensor.</a><span class="source">[Curr Protoc Cytom. 2014]</span><div class="brieflinkpop offscreen_noflow">Real-time detection of protein trafficking with high-throughput flow cytometry (HTFC) and fluorogen-activating protein (FAP) base biosensor.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Wu Y, Tapia PH, Jarvik J, Waggoner AS, Sklar LA. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Curr Protoc Cytom. 2014 Jan 2; 67:9.43.1-9.43.11. Epub 2014 Jan 2.</em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/23303704" ref="ordinalpos=1&linkpos=3&log$=relatedarticles&logdbfrom=pubmed">High-throughput flow cytometry compatible biosensor based on fluorogen activating protein technology.</a><span class="source">[Cytometry A. 2013]</span><div class="brieflinkpop offscreen_noflow">High-throughput flow cytometry compatible biosensor based on fluorogen activating protein technology.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Wu Y, Tapia PH, Fisher GW, Waggoner AS, Jarvik J, Sklar LA. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Cytometry A. 2013 Feb; 83(2):220-6. Epub 2013 Jan 9.</em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/29881673" ref="ordinalpos=1&linkpos=4&log$=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> Fluorogen-activating proteins: beyond classical fluorescent proteins.</a><span class="source">[Acta Pharm Sin B. 2018]</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> Fluorogen-activating proteins: beyond classical fluorescent proteins.<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Xu S, Hu HY. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Acta Pharm Sin B. 2018 May; 8(3):339-348. Epub 2018 Mar 24.</em></div></div></li><li class="brieflinkpopper two_line"><a class="brieflinkpopperctrl" href="/pubmed/23762929" ref="ordinalpos=1&linkpos=5&log$=relatedreviews&logdbfrom=pubmed"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> Identification of a Selective Allosteric Agonist of mGlu(5).</a><span class="source">[Probe Reports from the NIH Mol...]</span><div class="brieflinkpop offscreen_noflow"><span xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="invert">Review</span> Identification of a Selective Allosteric Agonist of mGlu(5).<div class="brieflinkpopdesc"><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="author">Manka J, Zhou Y, Chun A, Dawson ES, Vinson PN, Niswender CM, Noetzel MJ, Rook JM, Bridges TM, Daniels JS, et al. </em><em xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="cit">Probe Reports from the NIH Molecular Libraries Program. 2010</em></div></div></li></ul><a class="seemore" href="/sites/entrez?db=pubmed&cmd=link&linkname=pubmed_pubmed_reviews&uid=23833798" ref="ordinalpos=1&log$=relatedreviews_seeall&logdbfrom=pubmed">See reviews...</a><a class="seemore" href="/sites/entrez?db=pubmed&cmd=link&linkname=pubmed_pubmed&uid=23833798" ref="ordinalpos=1&log$=relatedarticles_seeall&logdbfrom=pubmed">See all...</a></div></div><div class="portlet"><div class="portlet_head"><div class="portlet_title"><h3><span>Recent Activity</span></h3></div><a name="Shutter" sid="1" href="#" class="portlet_shutter" title="Show/hide content" remembercollapsed="true" pgsec_name="recent_activity" id="Shutter"></a></div><div class="portlet_content"><div xmlns:np="http://ncbi.gov/portal/XSLT/namespace" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" id="HTDisplay" class=""><div class="action"><a href="javascript:historyDisplayState('ClearHT')">Clear</a><a href="javascript:historyDisplayState('HTOff')" class="HTOn">Turn Off</a><a href="javascript:historyDisplayState('HTOn')" class="HTOff">Turn On</a></div><ul id="activity"><li class="ra_rcd ralinkpopper two_line"><a class="htb ralinkpopperctrl" ref="log$=activity&linkpos=1" href="/portal/utils/pageresolver.fcgi?recordid=67d66b50cde49f3df7da1a1e">Inhibitors of FAP-fluorogen interaction as a multiplex assay tool compound for r...</a><div class="ralinkpop offscreen_noflow">Inhibitors of FAP-fluorogen interaction as a multiplex assay tool compound for receptor internalization assays - 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