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<script type="text/javascript" src="/corehtml/pmc/jatsreader/ptpmc_3.22/js/jr.boots.min.js"> </script><title>Selective GPR35 Antagonists - Probes 1 & 2 - Probe Reports from the NIH Molecular Libraries Program - NCBI Bookshelf</title>
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<meta name="citation_inbook_title" content="Probe Reports from the NIH Molecular Libraries Program [Internet]">
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<meta name="citation_title" content="Selective GPR35 Antagonists - Probes 1 & 2">
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<meta name="citation_publisher" content="National Center for Biotechnology Information (US)">
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<meta name="citation_date" content="2010/10/04">
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<meta name="citation_author" content="Susanne Heynen-Genel">
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<meta name="citation_author" content="Russell Dahl">
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<meta name="citation_author" content="Shenghua Shi">
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<meta name="citation_author" content="Michelle Sauer">
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<meta name="citation_author" content="Santosh Hariharan">
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<meta name="citation_author" content="Eduard Sergienko">
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<meta name="citation_author" content="Shakeela Dad">
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<meta name="citation_author" content="Thomas DY Chung">
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<meta name="citation_author" content="Derek Stonich">
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<meta name="citation_author" content="Ying Su">
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<meta name="citation_author" content="Marc Caron">
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<meta name="citation_author" content="Pingwei Zhao">
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<meta name="citation_author" content="Mary E Abood">
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<meta name="citation_author" content="Lawrence S Barak">
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<meta name="citation_pmid" content="21433393">
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<meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK50703/">
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<meta name="DC.Title" content="Selective GPR35 Antagonists - Probes 1 & 2">
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<meta name="DC.Contributor" content="Susanne Heynen-Genel">
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<meta name="DC.Contributor" content="Russell Dahl">
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<meta name="DC.Contributor" content="Shenghua Shi">
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<meta name="DC.Contributor" content="Michelle Sauer">
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<meta name="DC.Contributor" content="Santosh Hariharan">
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<meta name="DC.Contributor" content="Eduard Sergienko">
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<meta name="DC.Contributor" content="Shakeela Dad">
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<meta name="DC.Contributor" content="Thomas DY Chung">
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<meta name="DC.Contributor" content="Derek Stonich">
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<meta name="DC.Contributor" content="Ying Su">
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<meta name="DC.Contributor" content="Marc Caron">
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<meta name="DC.Contributor" content="Pingwei Zhao">
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<meta name="DC.Contributor" content="Mary E Abood">
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<meta name="DC.Contributor" content="Lawrence S Barak">
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<meta name="DC.Date" content="2010/10/04">
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<meta name="description" content="Although many known receptors that regulate addiction have been pharmacologically and biochemically well characterized, some orphan receptors with homology to known receptors of abuse (i.e. GPR35) remain uncharacterized. GPR35 is a G-protein coupled receptor, first identified in 1998 after a screen of a human genomic library. More recent RT-PCR studies have now confirmed the presence of GPR35 in dorsal root ganglion, the cerebellum and brain, as well as GPR35b, which was cloned from a human whole brain cDNA library. Thus, GPR35 regulation appears to have profound physiological and pathophysiological implications. In line with the specific aim of this project, the identified probes ML145 (CID-2286812) and ML144 (CID-1542103) represent selective antagonists for GPR35, but not for the related GPR55 orphan receptor, supporting the hypothesis that they do not produce non-specific interference with signaling directly at or downstream of the β-arrestin signaling pathway. These probes will serve as novel tools to delineate the biochemistry of GPR35 as potential therapeutics to selectively target pathways underlying pain and to enhance our understanding of the molecular basis of addiction.">
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<meta name="og:description" content="Although many known receptors that regulate addiction have been pharmacologically and biochemically well characterized, some orphan receptors with homology to known receptors of abuse (i.e. GPR35) remain uncharacterized. GPR35 is a G-protein coupled receptor, first identified in 1998 after a screen of a human genomic library. More recent RT-PCR studies have now confirmed the presence of GPR35 in dorsal root ganglion, the cerebellum and brain, as well as GPR35b, which was cloned from a human whole brain cDNA library. Thus, GPR35 regulation appears to have profound physiological and pathophysiological implications. In line with the specific aim of this project, the identified probes ML145 (CID-2286812) and ML144 (CID-1542103) represent selective antagonists for GPR35, but not for the related GPR55 orphan receptor, supporting the hypothesis that they do not produce non-specific interference with signaling directly at or downstream of the β-arrestin signaling pathway. These probes will serve as novel tools to delineate the biochemistry of GPR35 as potential therapeutics to selectively target pathways underlying pain and to enhance our understanding of the molecular basis of addiction.">
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id="jr-fip-info-p"><a id="jr-fip-prev" class="wsprkl btn" title="Jump to previuos match">◀</a><button id="jr-fip-matches">no matches yet</button><a id="jr-fip-next" class="wsprkl btn" title="Jump to next match">▶</a></nav></nav></div><div id="jr-epub-interstitial" class="hidden"></div><div id="jr-content"><article data-type="main"><div class="main-content lit-style" itemscope="itemscope" itemtype="http://schema.org/CreativeWork"><div class="meta-content fm-sec"><div class="fm-sec"><h1 id="_NBK50703_"><span class="title" itemprop="name">Selective GPR35 Antagonists - Probes 1 & 2</span></h1><p class="contribs">Heynen-Genel S, Dahl R, Shi S, et al.</p><p class="fm-aai"><a href="#_NBK50703_pubdet_">Publication Details</a></p></div></div><div class="jig-ncbiinpagenav body-content whole_rhythm" data-jigconfig="allHeadingLevels: ['h2'],smoothScroll: false" itemprop="text"><div id="_abs_rndgid_" itemprop="description"><p>Although many known receptors that regulate addiction have been pharmacologically and biochemically well characterized, some orphan receptors with homology to known receptors of abuse (i.e. GPR35) remain uncharacterized. GPR35 is a G-protein coupled receptor, first identified in 1998 after a screen of a human genomic library. More recent RT-PCR studies have now confirmed the presence of GPR35 in dorsal root ganglion, the cerebellum and brain, as well as GPR35b, which was cloned from a human whole brain cDNA library. Thus, GPR35 regulation appears to have profound physiological and pathophysiological implications. In line with the specific aim of this project, the identified probes ML145 (CID-2286812) and ML144 (CID-1542103) represent selective antagonists for GPR35, but not for the related GPR55 orphan receptor, supporting the hypothesis that they do not produce non-specific interference with signaling directly at or downstream of the β-arrestin signaling pathway. These probes will serve as novel tools to delineate the biochemistry of GPR35 as potential therapeutics to selectively target pathways underlying pain and to enhance our understanding of the molecular basis of addiction.</p></div><div class="h2"></div><p><b>Probe project:</b> Selective GPR35 Antagonists</p><p><b>Assigned Assay Grant #:</b> 1 X01 MH085708-01</p><p><b>Screening Center Name & PI:</b> Sanford-Burnham Center for Chemical Genomics (<i>NIH PubChem & MLPCN designation</i>) & John C. Reed</p><p><b>Chemistry Center Name & PI:</b> Burnham Center for Chemical Genomics & John C. Reed</p><p><b>Assay Submitter & Institution:</b> Lawrence S. Barak, Duke University Medical Center Collaborating PI: (Mary E. Abood, Temple University, PA)</p><p><b>PubChem Summary Bioassay Identifier (AID):</b>
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<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2079" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-2079</a></p><div id="ml145.s2"><h2 id="_ml145_s2_">Probe(s) Structure & Characteristics</h2><p><i>This Center Probe Report describes two selective antagonists for GPR35 an orphan GPCR receptor that represent two novel scaffolds or chemical series: (1) CID2286812 and(2) CID1542103</i></p><div id="ml145.fu1" class="figure"><div class="graphic"><img src="/books/NBK50703/bin/ml145fu1.jpg" alt="Image ml145fu1" /></div></div><div id="ml145.fu2" class="figure"><div class="graphic"><img src="/books/NBK50703/bin/ml145fu2.jpg" alt="Image ml145fu2" /></div></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml145tu1"><a href="/books/NBK50703/table/ml145.tu1/?report=objectonly" target="object" title="Table" class="img_link icnblk_img figpopup" rid-figpopup="figml145tu1" rid-ob="figobml145tu1"><img class="small-thumb" src="/books/NBK50703/table/ml145.tu1/?report=thumb" src-large="/books/NBK50703/table/ml145.tu1/?report=previmg" alt="Image " /></a><div class="icnblk_cntnt"><h4 id="ml145.tu1"><a href="/books/NBK50703/table/ml145.tu1/?report=objectonly" target="object" rid-ob="figobml145tu1">Table</a></h4></div></div></div><div id="ml145.s3"><h2 id="_ml145_s3_">Recommendations for the scientific use of this probe</h2><p>Many receptors regulating addiction are pharmacologically and biochemically well characterized, but some orphan receptors like GPR35 with homology to known receptors of abuse remain uncharacterized. The aim of this research is to identify small molecule antagonists of human GPR35. The identification of small molecules capable of selectively inhibiting or activating orphans will provide tools for elucidating novel molecular pathways underlying addictive behaviors. These novel compounds will then be utilized to elucidate a number of things such as characterize GPR35 biology in vitro, GPR35 in animal models of pain and enhance the understanding of the molecular basis of addiction.</p></div><div id="ml145.s4"><h2 id="_ml145_s4_">1. Scientific Rationale for Project</h2><p>Drug addiction continues to remain a major public health concern in the United States. Addictive behavior results from changes in central nervous system signaling pathways that are modified after exposure to drugs of abuse. In particular, compounds such as cannabinoids and opiates that influence mood and pain perception are commonly associated with addictive behaviors. Many receptors regulating addiction are pharmacologically and biochemically well characterized, but some orphan receptors like GPR35 with homology to known receptors of abuse remain almost totally uncharacterized. GPR35 is a G-protein coupled receptor that was first identified in 1998 after a screen of a human genomic library (<a class="bibr" href="#ml145.r1" rid="ml145.r1">1</a>). GPR35 is homologous to P2Y purinergic receptors and GPR23, whose ligand is lysophosphatidic acid. GPR35 shares a 30 percent identity with the putative cannabinoid receptor GPR55 (<a class="bibr" href="#ml145.r2" rid="ml145.r2 ml145.r3 ml145.r4 ml145.r5">2–5</a>). The ability of GPR55 to recognize cannabinoids was first described in a yeast expression system, where the CB1 antagonists AM251 and SR141716A (rimonabant) acted as agonists (<a class="bibr" href="#ml145.r6" rid="ml145.r6">6</a>). Preliminary studies of GPR35 by mRNA expression showed it expressed predominantly in the immune and gastrointestinal systems (<a class="bibr" href="#ml145.r1" rid="ml145.r1">1</a>). However, recent RT-PCR studies have confirmed the presence of GPR35 in dorsal root ganglion, the cerebellum and brain, and GPR35b was cloned from a human whole brain cDNA library (<a class="bibr" href="#ml145.r2" rid="ml145.r2">2</a>, <a class="bibr" href="#ml145.r5" rid="ml145.r5">5</a>, <a class="bibr" href="#ml145.r7" rid="ml145.r7">7</a>). Variable Gi/o protein activation by GPR35 that was pertussis toxin sensitive was subsequently observed in rat sympathetic neurons (<a class="bibr" href="#ml145.r2" rid="ml145.r2">2</a>).</p><p>There are approximately fifteen papers characterizing GPR35 in the PubMed listed peer reviewed literature. An N-terminal splice variant of GPR35, GPR35b, was identified from a genetic screen of gastric carcinomas (<a class="bibr" href="#ml145.r8" rid="ml145.r8">8</a>), leading to speculation that GPR-35 regulates cell growth. The observation that the <i>a</i> isoform possessed a stronger transforming activity than the <i>b</i> also led the authors to postulate that GPR-35a possesses constitutive activity (<a class="bibr" href="#ml145.r8" rid="ml145.r8">8</a>). While GPR35 has been implicated in the formation of gastric cancers (<a class="bibr" href="#ml145.r8" rid="ml145.r8">8</a>), conversely, deletion of GPR35 may be responsible for a mental retardation syndrome associated with deletions on 2q37.3 (<a class="bibr" href="#ml145.r9" rid="ml145.r9">9</a>).</p><p>GPR35 regulation appears to have profound physiological and pathophysiological implications so that defining compounds that regulate GPR35 will be important. The specific aim of this grant is to identify small molecule antagonists of human GPR35. These novel compounds will be utilized to characterize GPR35 biology in vitro and GPR35 in animal models of pain. Thus, this proposal will provide tools for delineating the biochemistry of GPR35, potentially provide compounds for targeted therapeutics of pathways underlying pain, and enhance our understanding of the molecular basis of addiction.</p></div><div id="ml145.s5"><h2 id="_ml145_s5_">2. Project Description</h2><div id="ml145.s6"><h3>a. Original goal for probe characteristics</h3><p>The goal of the HTS was to identify novel and specific inhibitors of GPR35. To date, no antagonists for GPR35 are known and the goal of this project was to identify small molecules that had an IC50 of 5 µM or less in the primary GPR35 β-arrestin HCS assay, with at least 10-fold antagonist selectivity against the related receptor GPR55.</p></div><div id="ml145.s7"><h3>b. Information for each Assay Implemented and Screening Run</h3><div id="ml145.s8"><h4>i. PubChem Bioassay Name(s), AID(s), Assay-Type (Primary, DR, Counterscreen, Secondary)</h4><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml145t1"><a href="/books/NBK50703/table/ml145.t1/?report=objectonly" target="object" title="Table 1" class="img_link icnblk_img figpopup" rid-figpopup="figml145t1" rid-ob="figobml145t1"><img class="small-thumb" src="/books/NBK50703/table/ml145.t1/?report=thumb" src-large="/books/NBK50703/table/ml145.t1/?report=previmg" alt="Table 1. Assays for GPR35." /></a><div class="icnblk_cntnt"><h4 id="ml145.t1"><a href="/books/NBK50703/table/ml145.t1/?report=objectonly" target="object" rid-ob="figobml145t1">Table 1</a></h4><p class="float-caption no_bottom_margin">Assays for GPR35. </p></div></div></div><div id="ml145.s9"><h4>ii. Assay Rationale & Description</h4><div id="ml145.s10"><h5>Primary Screen</h5><p>This image-based high-content screen (HCS) is based on fluorescence redistribution of a GFP-β-arrestin complex from homogeneous distribution in the cytoplasm via the plasma membrane to intracellular pits and vesicles (assay technology marketed as Transfluor® assay by Molecular Devices). Upon activation by ligand binding, GPCRs undergo deactivation or “desensitization” by binding of the β-arrestin protein to the activated receptor. The GPCR-β-arrestin complex internalizes, the ligand is removed and the receptor is recycled back to the cell membrane. Localization of the fluorescently labeled β-arrestin can be monitored by image analysis (<a class="bibr" href="#ml145.r10" rid="ml145.r10">10</a>). The primary screen assay is designed to identify compounds inhibiting GPR35 signaling induced by the GPR35 agonist Zaprinast at approx. EC80 concentration (<a class="bibr" href="#ml145.r5" rid="ml145.r5">5</a>).</p><p>The primary screening protocol is described below.</p><div id="ml145.fu3" class="figure bk_fig"><div class="graphic"><img src="/books/NBK50703/bin/ml145fu3.jpg" alt="GPR35 Example Images of Positive and Negative Controls." /></div><h3><span class="title">GPR35 Example Images of Positive and Negative Controls</span></h3><div class="caption"><p>Effect of 10 µM agonist
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Zaprinast (left panel) compared to DMSO control (right panel)</p></div></div><div id="ml145.s11"><h5>Primary Assay Materials</h5><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml145t2"><a href="/books/NBK50703/table/ml145.t2/?report=objectonly" target="object" title="Table 2" class="img_link icnblk_img figpopup" rid-figpopup="figml145t2" rid-ob="figobml145t2"><img class="small-thumb" src="/books/NBK50703/table/ml145.t2/?report=thumb" src-large="/books/NBK50703/table/ml145.t2/?report=previmg" alt="Table 2. Critical Reagents used for the uHTS experiments." /></a><div class="icnblk_cntnt"><h4 id="ml145.t2"><a href="/books/NBK50703/table/ml145.t2/?report=objectonly" target="object" rid-ob="figobml145t2">Table 2</a></h4><p class="float-caption no_bottom_margin">Critical Reagents used for the uHTS experiments. </p></div></div></div><div id="ml145.s12"><h5>Primary Screen Protocol</h5><p>A. Plate Preparation:</p><ol><li class="half_rhythm"><div>45ul of cell suspension (133,000 cells/ml in culture medium) was dispensed in each well of the assay plates using a Wellmate bulk dispenser.</div></li><li class="half_rhythm"><div>Plates were incubated overnight or approximately 20 hours at 37 degree C and 5% CO2.</div></li><li class="half_rhythm"><div>Serum was removed by media aspiration and replaced with 45ul serum-free MEM prior to addition of compounds.</div></li><li class="half_rhythm"><div>Compound addition was done on a Biomek FX with 384-head dispenser (Beckman):</div><ol class="lower-alpha"><li class="half_rhythm"><div>5ul of 100µM compound solution was added to columns 3 through 24 of the assay plates for a final assay compound concentration of 10µM and 0.5% DMSO.</div></li><li class="half_rhythm"><div>5ul of 5% DMSO was added to columns 1 and 2 to balance the volume and DMSO concentration across the plate.</div></li><li class="half_rhythm"><div>5ul of positive control (2% DMSO) working solution was added to column 1.</div></li></ol></li><li class="half_rhythm"><div>Plates were incubated for 15 minutes at room temperature.</div></li><li class="half_rhythm"><div>Agonist addition was done on a Biomek FX with 384-head dispenser (Beckman). 5ul of agonist (100µM Zaprinast) working solution was added to columns 2–24. (This also serves as the negative control in column 2.)</div></li><li class="half_rhythm"><div>Plates were incubated for 45 minutes at 37 degrees C and 5% CO2.</div></li><li class="half_rhythm"><div>Media was aspirated leaving 20ul liquid in each well using a Titertek plate washer.</div></li><li class="half_rhythm"><div>40ul of fixative working solution was added to each well using a Wellmate bulk dispenser (Matrix) for a final concentration of 4% PFA.</div></li><li class="half_rhythm"><div>Plates were incubated for 40 minutes at room temperature.</div></li><li class="half_rhythm"><div>Fixative was aspirated and plates were washed twice with 50ul PBS leaving 20ul liquid in each well using a Titertek plate washer.</div></li><li class="half_rhythm"><div>40ul of DAPI working solution was added using a Wellmate bulk dispenser for a final DAPI concentration of 100ng/ml. Aluminum plate seals were applied to each plate.</div></li></ol><p>B. Image Acquisition and Analysis:</p><ol><li class="half_rhythm"><div>Image acquisition was performed on an Opera QEHS (Perkin Elmer) with 45 plate capacity loader/stacker and the following settings:</div><dl class="temp-labeled-list"><dl class="bkr_refwrap"><dt>-</dt><dd><p class="no_top_margin">40x 0.6 NA air objective</p></dd></dl><dl class="bkr_refwrap"><dt>-</dt><dd><p class="no_top_margin">Acquisition camera set to 2-by-2 binning for an image size of 688 by 512 pixels</p></dd></dl><dl class="bkr_refwrap"><dt>-</dt><dd><p class="no_top_margin">2 channels acquired sequentially: Exp1Cam1 = B-arrestin GFP using 488 nm laser excitation</p><p>and 540/70 nm 4mission filters, Exp2Cam2 = DAPI (nuclei) using 365 nm Xenon lamp excitation and 450/50 nm emission filters</p></dd></dl><dl class="bkr_refwrap"><dt>-</dt><dd><p class="no_top_margin">3 fields per well</p></dd></dl></dl></li><li class="half_rhythm"><div>Image analysis was performed using the Acapella™ Spot Detection Algorithm with the following analysis settings:</div><div>
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<div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml145tu2"><a href="/books/NBK50703/table/ml145.tu2/?report=objectonly" target="object" title="Table" class="img_link icnblk_img figpopup" rid-figpopup="figml145tu2" rid-ob="figobml145tu2"><img class="small-thumb" src="/books/NBK50703/table/ml145.tu2/?report=thumb" src-large="/books/NBK50703/table/ml145.tu2/?report=previmg" alt="Image " /></a><div class="icnblk_cntnt"><h4 id="ml145.tu2"><a href="/books/NBK50703/table/ml145.tu2/?report=objectonly" target="object" rid-ob="figobml145tu2">Table</a></h4></div></div></div></li><li class="half_rhythm"><div>Metrics calculated from…</div><div>NUCLEI IMAGES: Cell Count (“Number of Cells Analyzed”), Nuclei Area (“Area of the Nucleus”), Integrated Intensity of the Nuclei (“Total Integrated Intensity of the Nucleus”), Average Intensity of the Nuclei (“Average Intensity of the Nucleus”)</div><div>GFP IMAGES: Integrated Intensity of the Cytoplasm (“Total Cytoplasm Intensity”), Integrated Intensity of the Detected Spots (“Total Spot Intensity”), Ratio of the Integrated Spot to Integrated Cytoplasm Intensities (“Ratio of Spot Intensity to Cytoplasmintensity”), Number of Spots per Cell (“Average Spots Per Cell”)</div></li></ol><p>The primary screen was performed at a compound concentration of 10 µM in 384-well format. The average Z’ for the screen was 0.65 and Z’ values ranged from 0.44 to 0.82 using the ratio of the GFP intensity of the spots over the GFP intensity of the cytoplasm (“Ratio of Spot Intensity to Cytoplasmintensity”) as the primary assay read-out.</p></div></div><div id="ml145.s13"><h5>Confirmation Assays</h5><p>Initial hit confirmation of compound solutions resupplied by the MLSMR was done at a single compound concentration (10 µM) in duplicates using the primary screen assay to confirm activity of the hit compounds. Compounds with confirmed activity at 10 µM were tested from stock solutions resupplied by the MLSMR in 7-point dose responses (0.5 to 32 µM) to evaluate potency. Potent compounds (IC50 <5 µM) were clustered into scaffolds and 10-point dose responses (0.06 to 32 µM) were performed for dry powder compounds selected from hits and their commercially available analogs.</p></div><div id="ml145.s14"><h5>Counterscreen/Selectivity Assays</h5><p>To eliminate artifacts introduced by the β-arrestin-GFP assay technology, an image-based high-content assay using the same assay technology was performed against the putative cannabinoid receptor GPR55 in antagonist mode. In addition to eliminating false positives caused by assay artifacts, this assay also evaluates selectivity of the GPR35 hit compounds against the GPR55 receptor. This is of additional interest since GPR35 and GPR55 share ~30% identity (<a class="bibr" href="#ml145.r2" rid="ml145.r2 ml145.r3 ml145.r4 ml145.r5">2–5</a>). In addition, GFP intensity of the cells was quantified to identify compounds causing cellular fluorescence resulting in a decrease in number of detected spots and thus false positive results.</p></div><div id="ml145.s15"><h5>Secondary Probe Characterization Assays</h5><p>The identified GPR35 antagonist probes are characterized further by two assays performed in the assay provider’s and his collaborator’s labs. This checks for assay technology artifacts using the same assay technology on another receptor, the vasopressin receptor, which has been well characterized and is not related to GPR35. Any compounds that were also active in the vasopressin antagonist assay would be likely interfering with the GFP-tagged β-arrestin and thus identified as false positives. The second assay evaluates ERK1/2 activity downstream in the GPR35 signaling pathway.</p></div></div><div id="ml145.s16"><h4>iii. Summary of Results</h4><p>The GPR35 antagonist primary screen of 291,994 compounds resulted in 549 compounds that were considered as hits using the hit criteria of >50% activity as compared to cells without agonist addition, >30 cells in the imaged area of the well, and a total GFP intensity of <10,000,000 relative units. The upper limit for the total GFP intensity was added as hit criterion to eliminate cell-permeable autofluorescent compounds interfering with detection of spot formation.</p><p>Stock solutions resupplied by the MLSMR of 490 compounds were tested for hit confirmation. Single point confirmations at 10 µM concentration were conducted in duplicate on these compounds. 102 of these compounds confirmed using the same hit criteria as for the primary screening campaign. Further testing of these compounds using a seven-point dose response (0.5 to 32 µM concentration range) identified 33 compounds with an IC50 of less than 1 µM and 57 compounds with an IC50 between 1 and 10 µM.</p><p>The hits were clustered into scaffolds by using a maximum-common-substructure-based algorithm. Analyzing the assay data in terms of scaffolds therefore, yielded 22 hits from 8 scaffolds and 38 of their commercially available analogs were selected for dry powder purchase. Testing of these dry powder compounds in 10-point dose responses using the primary screen assay and the GPR55 antagonist counterscreen/selectivity assay resulted in 26 compounds with IC50 < 5 µM and GPR55 antagonist selectivity of IC50(GPR55) > 10x IC50(GPR35) spanning 5 scaffolds (<a class="figpopup" href="/books/NBK50703/figure/ml145.f1/?report=objectonly" target="object" rid-figpopup="figml145f1" rid-ob="figobml145f1">Figure 1</a>).</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml145f1" co-legend-rid="figlgndml145f1"><a href="/books/NBK50703/figure/ml145.f1/?report=objectonly" target="object" title="Figure 1" class="img_link icnblk_img figpopup" rid-figpopup="figml145f1" rid-ob="figobml145f1"><img class="small-thumb" src="/books/NBK50703/bin/ml145f1.gif" src-large="/books/NBK50703/bin/ml145f1.jpg" alt="Figure 1. The GPR35 Antagonist Probe Flowchart summarizes the compound triage and decision tree for advancement of the compounds." /></a><div class="icnblk_cntnt" id="figlgndml145f1"><h4 id="ml145.f1"><a href="/books/NBK50703/figure/ml145.f1/?report=objectonly" target="object" rid-ob="figobml145f1">Figure 1</a></h4><p class="float-caption no_bottom_margin">The GPR35 Antagonist Probe Flowchart summarizes the compound triage and decision tree for advancement of the compounds. </p></div></div><p>The critical path for the development of the probes is shown below:</p><div id="ml145.fu4" class="figure"><div class="graphic"><img src="/books/NBK50703/bin/ml145fu4.jpg" alt="Image ml145fu4" /></div></div></div></div><div id="ml145.s17"><h3>c. Probe Optimization</h3><div id="ml145.s18"><h4>i. Description of SAR & chemistry strategy (including structure and data) that led to the probe</h4><p>As shown in <a class="figpopup" href="/books/NBK50703/table/ml145.t3/?report=objectonly" target="object" rid-figpopup="figml145t3" rid-ob="figobml145t3">Table 3</a>, the probe compound, CID2286812 (<a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544499" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-87544499</a>) and 5 close analogs all show potent GPR35 antagonism, are selective over GPR55. This class of selective GPR35 antagonists consists of a thioxothiazolidinone with an aromatic or styryl portion consisting of substituent R1 and a 3-carbon spacer connected to a pendant amidobenzoic acid moiety having substitutions R2 and R3. The styryl substituent at R1 provides the most potent and selective inhibitors, as is seen in compounds CID2286812 and CID2286888. While differentially substituted aromatic groups at R1 still provide potent antagonists, the overall profile of the styryl-substituted examples is superior. Substitutions of the amidobenzoic acid portion, R2 and R3, show tolerance for the <i>ortho</i> and <i>meta</i> positions of this ring. The most potent compounds have the carboxylic acid moiety in the <i>para</i> position, with either hydroxyl or H at the meta position. These examples constitute clear SAR of those features required for selective GPR35 antagonism and provide multiple compounds exceeding the set probe criteria.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml145t3"><a href="/books/NBK50703/table/ml145.t3/?report=objectonly" target="object" title="Table 3" class="img_link icnblk_img figpopup" rid-figpopup="figml145t3" rid-ob="figobml145t3"><img class="small-thumb" src="/books/NBK50703/table/ml145.t3/?report=thumb" src-large="/books/NBK50703/table/ml145.t3/?report=previmg" alt="Table 3. Probe #1: SAR of selective GPR35 antagonists, including probe CID2286812." /></a><div class="icnblk_cntnt"><h4 id="ml145.t3"><a href="/books/NBK50703/table/ml145.t3/?report=objectonly" target="object" rid-ob="figobml145t3">Table 3</a></h4><p class="float-caption no_bottom_margin">Probe #1: SAR of selective GPR35 antagonists, including probe CID2286812. </p></div></div><p>The SAR of the second probe CID1542103 and related analogs can be seen in <a class="figpopup" href="/books/NBK50703/table/ml145.t4/?report=objectonly" target="object" rid-figpopup="figml145t4" rid-ob="figobml145t4">Table 4</a>. This probe series consists of a pyrazolo-pyrimidine core containing a substituted piperazine moiety (<a class="figpopup" href="/books/NBK50703/table/ml145.t4/?report=objectonly" target="object" rid-figpopup="figml145t4" rid-ob="figobml145t4">Table 4</a>, R<sub>1</sub>) on the pyrimidine ring and a pendant aromatic group (R<sub>2</sub>) connected to the pyrazole portion. A variety of aryl groups are tolerated at the R1 position, providing selective GPR35 antagonists in the single-digit micromolar range for GPR35 IC<sub>50</sub> and no GPR55 inhibitory activity. Briefly, at R1, <i>ortho</i> substituted groups provide the most potent derivatives as seen with the probe CID1542103 and the close analogue CID1502520. Removal of all substitution from the aryl ring at R<sub>1</sub> shows a decrease in GPR35 antagonism, as in CID8056691 and CID8056641. Finally, para substitution here leads to the weakest antagonists, CID661907 and CID2474060. At R<sub>2</sub>, para substitution is favored, with chloro and methyl substituents providing superior antagonists. It can be noted from CID2474060 that substitution at positions other than the para position result in a loss of GPR35 antagonist activity.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml145t4"><a href="/books/NBK50703/table/ml145.t4/?report=objectonly" target="object" title="Table 4" class="img_link icnblk_img figpopup" rid-figpopup="figml145t4" rid-ob="figobml145t4"><img class="small-thumb" src="/books/NBK50703/table/ml145.t4/?report=thumb" src-large="/books/NBK50703/table/ml145.t4/?report=previmg" alt="Table 4. Probe #2: SAR of selective GPR35 antagonists, including probe CID1542103." /></a><div class="icnblk_cntnt"><h4 id="ml145.t4"><a href="/books/NBK50703/table/ml145.t4/?report=objectonly" target="object" rid-ob="figobml145t4">Table 4</a></h4><p class="float-caption no_bottom_margin">Probe #2: SAR of selective GPR35 antagonists, including probe CID1542103. </p></div></div></div></div></div><div id="ml145.s19"><h2 id="_ml145_s19_">3. Probe(s)</h2><div id="ml145.s20"><h3>a. Chemical name of probe compound (s) (Chemical IUPAC)</h3><p><i>Probe 1 (series 1):</i> 2-hydroxy-4-[4-[(5Z)-5-[(E)-2-methyl-3-phenylprop-2 enylidene]-4-oxo-2- sulfanylidene-1,3-thiazolidin-3-yl]butanoylamino] benzoic acid <b>[<a href="/pcsubstance/?term=ML145[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML145</a>]</b></p><p><i>Probe 2 (series 2):</i> 1-[(4-chlorophenyl)methyl]-4-[4-(2-methylphenyl)piperazin-1-yl]pyrazolo[3,4-d]pyrimidine <b>[<a href="/pcsubstance/?term=ML144[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML144</a>]</b></p></div><div id="ml145.s21"><h3>b. Probe chemical structure(s) including stereochemistry</h3><div id="ml145.fu5" class="figure"><div class="graphic"><img src="/books/NBK50703/bin/ml145fu10.jpg" alt="Image ml145fu10" /></div></div><div id="ml145.fu6" class="figure"><div class="graphic"><img src="/books/NBK50703/bin/ml145fu11.jpg" alt="Image ml145fu11" /></div></div></div><div id="ml145.s22"><h3>c. Structural Verification Information of probe SID</h3><p>The probe SIDs are for Probe #1 <a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544499" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-87544499</a> (corresponding to CID2286812) and Probe #2 <a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544496" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-87544496</a> (corresponding to CID1542103) please see below at the spectra.</p><p><i>(1)</i>
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<i>PubChem <a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544499" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-87544499</a></i>:</p><div id="ml145.s23"><h4>NMR Purity</h4><p>>95% (<sup>1</sup>H-NMR): <sup>1</sup>H NMR (400 MHz, DMSO-<i>d</i><sub>6</sub>) δ ppm:</p><div id="ml145.fu7" class="figure"><div class="graphic"><img src="/books/NBK50703/bin/ml145fu12.jpg" alt="Image ml145fu12" /></div></div></div><div id="ml145.s24"><h4>Purity = 98% (HPLC-MS)</h4><div id="ml145.fu8" class="figure"><div class="graphic"><img src="/books/NBK50703/bin/ml145fu13.jpg" alt="Image ml145fu13" /></div></div><div id="ml145.fu9" class="figure"><div class="graphic"><img src="/books/NBK50703/bin/ml145fu14.jpg" alt="Image ml145fu14" /></div></div></div><div id="ml145.s25"><h4>Mass Spec</h4><p><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544499" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-87544499</a>: ESI-MS <i>m/z</i> calcd for C<sub>24</sub>H<sub>22</sub>N<sub>2</sub>O<sub>5</sub>S<sub>2</sub> [M+H]<sup>+</sup>: 483, found: 483.</p><p><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544496" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-87544496</a>: ESI-MS <i>m/z</i> calcd for C<sub>23</sub>H<sub>23</sub>ClN<sub>6</sub> [M+H]<sup>+</sup>: 419, found: 419.</p></div></div><div id="ml145.s26"><h3>d. PubChem CID(s) (corresponding to the SID)</h3><p>For Probe #1: PubChem CID-2286812 (corresponding to the <a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544499" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-87544499</a>).</p><p>For Probe #2: PubChem CID-1542103 (corresponding to the <a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544496" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-87544496</a>).</p></div><div id="ml145.s27"><h3>e. Availability from a vendor</h3><ol><li class="half_rhythm"><div><i>Scaffold 1</i>, CID2286812 is commercially available from ChemBridge CAT # 6791281</div></li><li class="half_rhythm"><div><i>Scaffold 2</i>, CID1542103 is commercially available ChemBridge: Cat#7652281</div></li></ol></div><div id="ml145.s28"><h3>f. MLS#'s of probe molecule and five related samples that were submitted to the SMR collection</h3><p>See <a class="figpopup" href="/books/NBK50703/table/ml145.t5/?report=objectonly" target="object" rid-figpopup="figml145t5" rid-ob="figobml145t5">Tables 5A</a> & <a class="figpopup" href="/books/NBK50703/table/ml145.t6/?report=objectonly" target="object" rid-figpopup="figml145t6" rid-ob="figobml145t6">5B</a>.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml145t5"><a href="/books/NBK50703/table/ml145.t5/?report=objectonly" target="object" title="Table 5A" class="img_link icnblk_img figpopup" rid-figpopup="figml145t5" rid-ob="figobml145t5"><img class="small-thumb" src="/books/NBK50703/table/ml145.t5/?report=thumb" src-large="/books/NBK50703/table/ml145.t5/?report=previmg" alt="Table 5A. Submission information on Probe 1: CID2286812 and analogs." /></a><div class="icnblk_cntnt"><h4 id="ml145.t5"><a href="/books/NBK50703/table/ml145.t5/?report=objectonly" target="object" rid-ob="figobml145t5">Table 5A</a></h4><p class="float-caption no_bottom_margin">Submission information on Probe 1: CID2286812 and analogs. </p></div></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml145t6"><a href="/books/NBK50703/table/ml145.t6/?report=objectonly" target="object" title="Table 5B" class="img_link icnblk_img figpopup" rid-figpopup="figml145t6" rid-ob="figobml145t6"><img class="small-thumb" src="/books/NBK50703/table/ml145.t6/?report=thumb" src-large="/books/NBK50703/table/ml145.t6/?report=previmg" alt="Table 5B. Submission information on Probe#2: CID1542103 and analogs." /></a><div class="icnblk_cntnt"><h4 id="ml145.t6"><a href="/books/NBK50703/table/ml145.t6/?report=objectonly" target="object" rid-ob="figobml145t6">Table 5B</a></h4><p class="float-caption no_bottom_margin">Submission information on Probe#2: CID1542103 and analogs. </p></div></div></div><div id="ml145.s29"><h3>g. Mode of action for biological activity of probe</h3><p>Probes identified in this project are acting at the beginning of the GPR35 signaling pathway.
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Ligand binding causes phosphorylation of the GPCR, which in turn causes translocation of the
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β-arrestin to the membrane, where it binds to the GPCR. The β-arrestin-GPCR complex
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internalizes into clathrin-coated pits within the cell, where it dissociates and the receptor
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recycles back to the membrane. Since the assay read-out quantifies formation of
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β-arrestin-GPCR pits, the identified antagonists interfere with the pit formation or any
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process upstream. The selectivity of these two probes for GPR35 (as described in our <a class="figpopup" href="/books/NBK50703/figure/ml145.fu4/?report=objectonly" target="object" rid-figpopup="figml145fu4" rid-ob="figobml145fu4">Critical Path
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Flowchart</a> above), but not for the related GPR55 orphan receptor in a cognate β-arrestin HCS
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assay supports that they are not non-specifically interfering with signaling directly at or
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downstream of the β-arrestin signaling pathway. In follow-up studies by Dr. Barak
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(<i>see</i>
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<a href="#ml145.s37">sec. 5</a> below), these probes also did not, as expected, agonize nor antagonize β-arrestin mediated signaling of the completely unrelated vasopressin receptor, again supporting their GPR35 specificity. Additional secondary assays by Dr. Abood demonstrated that the probes did inhibit Erk1/2 phosphorylation. This confirms that our imaging assay based results translate to the authentic downstream biological response.</p></div><div id="ml145.s30"><h3>h. Detailed synthetic pathway for synthesizing probe#1</h3><div id="ml145.fu10" class="figure"><div class="graphic"><img src="/books/NBK50703/bin/ml145fu15.jpg" alt="Image ml145fu15" /></div></div></div><div id="ml145.s31"><h3>i. Detailed synthetic pathway for synthesizing probe#2</h3><div id="ml145.fu11" class="figure"><div class="graphic"><img src="/books/NBK50703/bin/ml145fu16.jpg" alt="Image ml145fu16" /></div></div></div><div id="ml145.s32"><h3>j. Summary of probe properties (solubility, absorbance/fluorescence, reactivity,
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toxicity, etc.)</h3><p><i>In Vitro</i> Pharmacology Profiles of Probes CID2286812 [<b><a href="/pcsubstance/?term=ML145[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML145</a></b>] and CID1542103 [<b><a href="/pcsubstance/?term=ML144[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML144</a></b>] <b>(</b><i>See</i>
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<a class="figpopup" href="/books/NBK50703/table/ml145.t7/?report=objectonly" target="object" rid-figpopup="figml145t7" rid-ob="figobml145t7">Table 6</a>
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<i>below</i><b>).</b> Both nominated probes, CID2286812 [<b><a href="/pcsubstance/?term=ML145[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML145</a></b>] and CID1542103 [<b><a href="/pcsubstance/?term=ML144[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML144</a></b>] were evaluated in a detailed <i>in vitro</i> pharmacology screen.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml145t7"><a href="/books/NBK50703/table/ml145.t7/?report=objectonly" target="object" title="Table 6" class="img_link icnblk_img figpopup" rid-figpopup="figml145t7" rid-ob="figobml145t7"><img class="small-thumb" src="/books/NBK50703/table/ml145.t7/?report=thumb" src-large="/books/NBK50703/table/ml145.t7/?report=previmg" alt="Table 6. Summary of in vitro ADMET/PK Properties of the GPR35 Antagonist Probes." /></a><div class="icnblk_cntnt"><h4 id="ml145.t7"><a href="/books/NBK50703/table/ml145.t7/?report=objectonly" target="object" rid-ob="figobml145t7">Table 6</a></h4><p class="float-caption no_bottom_margin">Summary of <i>in vitro</i> ADMET/PK Properties of the GPR35 Antagonist Probes. </p></div></div><p>Neither probe molecule had appreciable solubility at pH 5.0. CID2286812 [<b><a href="/pcsubstance/?term=ML145[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML145</a></b>] had good solubility at the higher pHs (pH 6.2 & pH 7.4), however, the solubility of CID1542103 [<b><a href="/pcsubstance/?term=ML144[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML144</a></b>] remained poor at the higher pHs.</p><p>The PAMPA (<b>P</b>arallel <b>A</b>rtificial <b>M</b>embrane <b>P</b>ermeability <b>A</b>ssay) assay is used as an <i>in vitro</i> model of passive, transcellular permeability. An artificial membrane immobilized on a filter is placed between a donor and acceptor compartment. At the start of the test, drug is introduced in the donor compartment. Following the permeation period, the concentration of drug in the donor and acceptor compartments is measured using UV spectroscopy. In this assay CID2286812 [<b><a href="/pcsubstance/?term=ML145[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML145</a></b>] has a bell-shaped permeability with good permeability at pH 6.2, while permeability decreased to moderate/poor levels at pH 5 and pH 7.4. In contrast, CID1542103 [<b><a href="/pcsubstance/?term=ML144[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML144</a>]</b> has moderate permeability at pH 5.0, but undetectable or poor permeability at pH 6.2 and 7.4.</p><p>Plasma Protein Binding is a measure of a drug’s efficiency to bind to the proteins within blood plasma. The less bound a drug is, the more efficiently it can traverse cell membranes or diffuse. Highly plasma protein bound drugs are confined to the vascular space, thereby having a relatively low volume of distribution. In contrast, drugs that remain largely unbound in plasma are generally available for distribution to other organs and tissues. Both CID2286812 [<b><a href="/pcsubstance/?term=ML145[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML145</a></b>] and CID1542103 [<b><a href="/pcsubstance/?term=ML144[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML144</a></b>] are highly bound (98–99.9%) to both human and mouse plasma.</p><p>Plasma Stability is a measure of the stability of small molecules and peptides in plasma and is an important parameter, which strongly can influence the <i>in vivo</i> efficacy of a test compound. Drug candidates are exposed in plasma to enzymatic processes (proteinases, esterases), and they can undergo intramolecular rearrangement or bind irreversibly (covalently) to proteins. Interestingly, CID2286812 [<b><a href="/pcsubstance/?term=ML145[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML145</a></b>] shows some instability in human plasma (77%), but more stable to mouse plasma (94%). In contrast, CID1542103 [<b><a href="/pcsubstance/?term=ML144[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML144</a></b>] shows excellent stability (90 & 100%) in both human and mouse plasma.</p><p>The microsomal stability assay is commonly used to rank compounds according to their metabolic stability. This assay addresses the pharmacologic question of how long the parent compound will remain circulating in plasma within the body. CID2286812 [<b><a href="/pcsubstance/?term=ML145[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML145</a></b>] shows poor stability (2.4% & 7.0%) in both human and mouse liver homogenates. By comparison CID1542103 [<b><a href="/pcsubstance/?term=ML144[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML144</a></b>] shows modest hepatic microsome stability (57% & 31%) in both human and mouse liver homogenates.</p><p>CID2286812 [<b><a href="/pcsubstance/?term=ML145[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML145</a></b>] shows no toxicity (>50 µM) toward human hepatocyctes, whereas CID1542103 [<b><a href="/pcsubstance/?term=ML144[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML144</a></b>] yields some moderate toxicity in our assay.</p><p>A recent retrospective study (February 4, 2010) describes a number of substructural features that
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identifies compounds as “frequent hitters” or promiscuous in a number of biochemical
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high-throughput screens (<a class="bibr" href="#ml145.r11" rid="ml145.r11">11</a>). The authors recommended
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that Pan Assay Interference Compounds (PAINS) be excluded from bioassay libraries, as they have been
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shown to be generally interfering of HTS assays through general chemical reactivities, chelation,
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aggregation, spectroscopic overlaps and have also been cited as “<i>cul de
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sac</i>” compounds that waste resources. One of the substructures of concern, the
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rhodanine element (see <a class="figpopup" href="/books/NBK50703/figure/ml145.fu12/?report=objectonly" target="object" rid-figpopup="figml145fu12" rid-ob="figobml145fu12">structure</a>), is present in our first probe molecule, CID2286812, therefore raises some concerns for the potential promiscuosity for this probe. However, this probe has been shown to be strongly potent yet selective against the related GPR55 receptor in the same assay format, so general PAINS reactivity does not appear to be present. Furthermore during the initial scaffold triage, two close analogs of this probe molecule were not active in any of the 70–72 assays in PubChem in which they were tested. From this report, we surmise that rhodanine reactivity is dependent upon having an unsubstituted ring nitrogen or substituents that are in aromatic conjugation with it. In our probe, CID 2286812, this nitrogen has an aliphatic chain substituent that yields a tertiary amine that renders this rhodanine less reactive.</p><div id="ml145.fu12" class="figure"><div class="graphic"><img src="/books/NBK50703/bin/ml145fu17.jpg" alt="Image ml145fu17" /></div></div><p>The 2nd probe, CID1542103, does not have these concerning features, and indeed was found to score in only 11 of 354 assays when it was tested in PubChem. While this probe is 100-fold less potent than the 1<sup>st</sup> probe and not as dramatically <i>pro forma</i> selective against GPR55 compared to CID2286812, it still does meet the probe criteria set by the team and the assay provider. Furthermore, based on its narrow spectrum of cross-reactivity as assessed by its very low hit rate in over 350 PubChem assays, it is expected to be a useful and selective tool compound for studying GPR35.</p></div><div id="ml145.s33"><h3>k. Probe properties</h3><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml145t8"><a href="/books/NBK50703/table/ml145.t8/?report=objectonly" target="object" title="Table 7" class="img_link icnblk_img figpopup" rid-figpopup="figml145t8" rid-ob="figobml145t8"><img class="small-thumb" src="/books/NBK50703/table/ml145.t8/?report=thumb" src-large="/books/NBK50703/table/ml145.t8/?report=previmg" alt="Table 7. Properties computed from Structure." /></a><div class="icnblk_cntnt"><h4 id="ml145.t8"><a href="/books/NBK50703/table/ml145.t8/?report=objectonly" target="object" rid-ob="figobml145t8">Table 7</a></h4><p class="float-caption no_bottom_margin">Properties computed from Structure. </p></div></div></div></div><div id="ml145.s34"><h2 id="_ml145_s34_">4. Comparative data showing probe specificity for target in biologically relevant assays</h2><p>There is no current state of art probes except from this probe report. Further follow-up work has been conducted in the assay provider’s laboratories (Dr. Barak and collaborator Dr. Abood) as outlined in the CPDP as post Probe Nomination characterization. The three additional downstream assays are as described below and the SAR data summarized for the 1<sup>st</sup> probe series (<a class="figpopup" href="/books/NBK50703/table/ml145.t9/?report=objectonly" target="object" rid-figpopup="figml145t9" rid-ob="figobml145t9">Table 8A</a>) and 2<sup>nd</sup> probe series below (<a class="figpopup" href="/books/NBK50703/table/ml145.t10/?report=objectonly" target="object" rid-figpopup="figml145t10" rid-ob="figobml145t10">Table 8B</a>).</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml145t9"><a href="/books/NBK50703/table/ml145.t9/?report=objectonly" target="object" title="Table 8A" class="img_link icnblk_img figpopup" rid-figpopup="figml145t9" rid-ob="figobml145t9"><img class="small-thumb" src="/books/NBK50703/table/ml145.t9/?report=thumb" src-large="/books/NBK50703/table/ml145.t9/?report=previmg" alt="Table 8A. Comparative Downstream and Selectivity assays by Assay Provider for CID2286812." /></a><div class="icnblk_cntnt"><h4 id="ml145.t9"><a href="/books/NBK50703/table/ml145.t9/?report=objectonly" target="object" rid-ob="figobml145t9">Table 8A</a></h4><p class="float-caption no_bottom_margin">Comparative Downstream and Selectivity assays by Assay Provider for CID2286812. </p></div></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml145t10"><a href="/books/NBK50703/table/ml145.t10/?report=objectonly" target="object" title="Table 8B" class="img_link icnblk_img figpopup" rid-figpopup="figml145t10" rid-ob="figobml145t10"><img class="small-thumb" src="/books/NBK50703/table/ml145.t10/?report=thumb" src-large="/books/NBK50703/table/ml145.t10/?report=previmg" alt="Table 8B. Comparative Downstream and Selectivity assays by Assay Provider for CID1542103." /></a><div class="icnblk_cntnt"><h4 id="ml145.t10"><a href="/books/NBK50703/table/ml145.t10/?report=objectonly" target="object" rid-ob="figobml145t10">Table 8B</a></h4><p class="float-caption no_bottom_margin">Comparative Downstream and Selectivity assays by Assay Provider for CID1542103. </p></div></div><div id="ml145.s35"><h3>Assay for Agonists (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463201" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-463201</a>) and Antagonists (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463202" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-463202</a>) of Vasopressin Receptor Type IIa [GI: 208973254; Gene: 554]</h3><p>This assay was originally proposed as the primary selectivity assay, but as our Center was
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already performing the cognate GPR55 assays for a different project, and since it was an “in
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family” target, we agreed substitute GPR55 as the selectivity filter in the <a class="figpopup" href="/books/NBK50703/figure/ml145.fu4/?report=objectonly" target="object" rid-figpopup="figml145fu4" rid-ob="figobml145fu4">Critical Path Flowchart</a> (<i>above on p. 7</i>). These assays were performed by the assay provider and characterize the selectivity of compounds against the unrelated vasopressin receptor. It is a confirmatory assay for the final probes and analogs that are already known to be selective against GPR55. This imaging assay utilizes a cell line permanently expressing a beta-arrestin GFP biosensor and human vasopressin receptor type IIa. Upon agonist-mediated GPCR activation, the arrestin-GFP redistributes from the cytosolic compartment to the plasma membrane to coated pits. This arrestin-GFP redistribution is measured as increased local concentrations of fluorescent arrestins. As can be seen in <a class="figpopup" href="/books/NBK50703/table/ml145.t9/?report=objectonly" target="object" rid-figpopup="figml145t9" rid-ob="figobml145t9">Tables 8A</a> and <a class="figpopup" href="/books/NBK50703/table/ml145.t10/?report=objectonly" target="object" rid-figpopup="figml145t10" rid-ob="figobml145t10">8B</a>, both probes and analogs do not agonize (<1% activation) nor antagonize (< 37% inhibition) the vasopressin receptor. We note the high variance in the vasopressin antagonist assays.</p></div><div id="ml145.s36"><h3>Assay for ERK1/2 Activity (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463217" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-463217</a>) in GPR35-Overexpressing U2OS Cells [GI: 33695097; Gene: 2859]</h3><p>Assay performed by the assay provider characterizes the downstream ERK phosphorylation activity of probe compounds. This is an “in-cell” Western assay utilizes a cell line permanently expressing a beta-arrestin GFP biosensor and human GPR35 receptor. Upon agonist-mediated GPCR activation, ERK1/2 phosphorylation occurs as measured by pERK1/2 antibodies. The ERK1/2 assay is generally much less sensitive than the beta-arrestin GPCR assays and the assay protocol does not allow for compound pre-incubation before agonist addition, which may result in much higher IC50 values (10 – 40-fold) for the pERK-ICW potencies as compared the GPR35 assay. This can be seen in <a class="figpopup" href="/books/NBK50703/table/ml145.t9/?report=objectonly" target="object" rid-figpopup="figml145t9" rid-ob="figobml145t9">Tables 8A</a> where the nanomolar GPR35 probe and analogs yield micromolar potency in ERK phosphorylation. For the less potent probe and analogs in <a class="figpopup" href="/books/NBK50703/table/ml145.t10/?report=objectonly" target="object" rid-figpopup="figml145t10" rid-ob="figobml145t10">Table 8B</a>, the rightward shift in potency in ERK limits the obtainment of a good IC50 due to solubility limits. We note that while there are some rank order differences in the apparent pERK IC50’s versus the GPR35 IC50s, possibly due different solubility or ADME properties, the overall conclusion is that both probe scaffold classes do have measureable activity in the downstream assays on the authentic signaling pathway.</p></div></div><div id="ml145.s37"><h2 id="_ml145_s37_">5. References</h2><dl class="temp-labeled-list"><dl class="bkr_refwrap"><dt>1.</dt><dd><div class="bk_ref" id="ml145.r1">O’Dowd BF, Nguyen T, Marchese A, Cheng R, Lynch KR, Heng HH, Kolakowski LF Jr, George SR. Discovery of three novel G-protein-coupled receptor genes. <span><span class="ref-journal">Genomics. </span>1998;<span class="ref-vol">47</span>:310–3.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/9479505" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 9479505</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>2.</dt><dd><div class="bk_ref" id="ml145.r2">Guo J, Williams DJ, Puhl HL 3rd, Ikeda SR. Inhibition of N-type calcium channels by activation of GPR35, an orphan receptor, heterologously expressed in rat sympathetic neurons. <span><span class="ref-journal">J Pharmacol Exp Ther. </span>2008;<span class="ref-vol">324</span>:342–51.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/17940199" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17940199</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>3.</dt><dd><div class="bk_ref" id="ml145.r3">Johns DG, Behm DJ, Walker DJ, Ao Z, Shapland EM, Daniels DA, Riddick M, Dowell S, Staton PC, Green P, Shabon U, Bao W, Aiyar N, Yue TL, Brown AJ, Morrison AD, Douglas SA. The novel endocannabinoid receptor GPR55 is activated by atypical cannabinoids but does not mediate their vasodilator effects. <span><span class="ref-journal">Br J Pharmacol. </span>2007;<span class="ref-vol">152</span>:825–31.</span> [<a href="/pmc/articles/PMC2190033/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2190033</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/17704827" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17704827</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>4.</dt><dd><div class="bk_ref" id="ml145.r4">Ryberg E, Larsson N, Sjogren S, Hjorth S, Hermansson NO, Leonova J, Elebring T, Nilsson K, Drmota T, Greasley PJ. The orphan receptor GPR55 is a novel cannabinoid receptor. <span><span class="ref-journal">Br J Pharmacol. </span>2007;<span class="ref-vol">152</span>:1092–101.</span> [<a href="/pmc/articles/PMC2095107/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2095107</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/17876302" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17876302</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>5.</dt><dd><div class="bk_ref" id="ml145.r5">Taniguchi Y, Tonai-Kachi H, Shinjo K. Zaprinast, a well-known cyclic guanosine monophosphate-specific phosphodiesterase inhibitor, is an agonist for GPR35. <span><span class="ref-journal">FEBS Lett. </span>2006;<span class="ref-vol">580</span>:5003–8.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/16934253" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 16934253</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>6.</dt><dd><div class="bk_ref" id="ml145.r6">Brown AJ. Novel cannabinoid receptors. <span><span class="ref-journal">Br J Pharmacol. </span>2007;<span class="ref-vol">152</span>:567–75.</span> [<a href="/pmc/articles/PMC2190013/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2190013</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/17906678" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17906678</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>7.</dt><dd><div class="bk_ref" id="ml145.r7">Ohshiro H, Tonai-Kachi H, Ichikawa K. GPR35 is a functional receptor in rat dorsal root ganglion neurons. <span><span class="ref-journal">Biochem Biophys Res Commun. </span>2008;<span class="ref-vol">365</span>:344–8.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/17996730" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17996730</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>8.</dt><dd><div class="bk_ref" id="ml145.r8">Okumura S, Baba H, Kumada T, Nanmoku K, Nakajima H, Nakane Y, Hioki K, Ikenaka K. Cloning of a G-protein-coupled receptor that shows an activity to transform NIH3T3 cells and is expressed in gastric cancer cells. <span><span class="ref-journal">Cancer Sci. </span>2004;<span class="ref-vol">95</span>:131–5.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/14965362" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 14965362</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>9.</dt><dd><div class="bk_ref" id="ml145.r9">Shrimpton AE, Braddock BR, Thomson LL, Stein CK, Hoo JJ. Molecular delineation of deletions on 2q37.3 in three cases with an Albright hereditary osteodystrophy-like phenotype. <span><span class="ref-journal">Clin Genet. </span>2004;<span class="ref-vol">66</span>:537–44.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/15521982" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 15521982</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>10.</dt><dd><div class="bk_ref" id="ml145.r10">Barak LS, Ferguson SS, Zhang J, Martenson C, Meyer T, Caron MG. Internal trafficking and surface mobility of a functionally intact beta2-adrenergic receptor-green fluorescent protein conjugate. <span><span class="ref-journal">Mol Pharmacol. </span>1997;<span class="ref-vol">51</span>:177–84.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/9203621" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 9203621</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>11.</dt><dd><div class="bk_ref" id="ml145.r11">Baell BJ, Holloway AG. New Substructure Filters for Removal of Pan Assay Interference Compounds (PAINS) from Screening Libraries and for Their Exclusion in Bioassays. <span><span class="ref-journal">J Med Chem. </span>2010 2010 Feb 4;</span> [Epub ahead of print] [PubMed - as supplied by publisher] [<a href="https://pubmed.ncbi.nlm.nih.gov/20131845" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 20131845</span></a>]</div></dd></dl></dl></div><div style="display:none"><div style="display:none" id="figml145fu4"><img alt="Image ml145fu4" src-large="/books/NBK50703/bin/ml145fu4.jpg" /></div><div style="display:none" id="figml145fu12"><img alt="Image ml145fu17" src-large="/books/NBK50703/bin/ml145fu17.jpg" /></div></div><div id="bk_toc_contnr"></div></div></div><div class="fm-sec"><h2 id="_NBK50703_pubdet_">Publication Details</h2><h3>Author Information and Affiliations</h3><p class="contrib-group"><h4>Authors</h4><span itemprop="author">Susanne Heynen-Genel</span>, <span itemprop="author">Russell Dahl</span>, <span itemprop="author">Shenghua Shi</span>, <span itemprop="author">Michelle Sauer</span>, <span itemprop="author">Santosh Hariharan</span>, <span itemprop="author">Eduard Sergienko</span>, <span itemprop="author">Shakeela Dad</span>, <span itemprop="author">Thomas DY Chung</span>, <span itemprop="author">Derek Stonich</span>, <span itemprop="author">Ying Su</span>, <span itemprop="author">Marc Caron</span>, <span itemprop="author">Pingwei Zhao</span>, <span itemprop="author">Mary E Abood</span>, and <span itemprop="author">Lawrence S Barak</span>.</p><h3>Publication History</h3><p class="small">Received: <span itemprop="datePublished">February 28, 2010</span>; Last Update: <span itemprop="dateModified">October 4, 2010</span>.</p><h3>Copyright</h3><div><div class="half_rhythm"><a href="/books/about/copyright/">Copyright Notice</a></div></div><h3>Publisher</h3><p>National Center for Biotechnology Information (US), Bethesda (MD)</p><h3>NLM Citation</h3><p>Heynen-Genel S, Dahl R, Shi S, et al. Selective GPR35 Antagonists - Probes 1 & 2. 2010 Feb 28 [Updated 2010 Oct 4]. In: Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-. <span class="bk_cite_avail"></span></p></div><div class="small-screen-prev"><a href="/books/n/mlprobe/ml146/?report=reader"><svg xmlns="http://www.w3.org/2000/svg" viewBox="0 0 100 100" preserveAspectRatio="none"><path d="M75,30 c-80,60 -80,0 0,60 c-30,-60 -30,0 0,-60"></path><text x="20" y="28" textLength="60" style="font-size:25px">Prev</text></svg></a></div><div class="small-screen-next"><a href="/books/n/mlprobe/ml143/?report=reader"><svg xmlns="http://www.w3.org/2000/svg" viewBox="0 0 100 100" preserveAspectRatio="none"><path d="M25,30c80,60 80,0 0,60 c30,-60 30,0 0,-60"></path><text x="20" y="28" textLength="60" style="font-size:25px">Next</text></svg></a></div></article><article data-type="fig" id="figobml145fu1"><div id="ml145.fu1" class="figure"><div class="graphic"><img data-src="/books/NBK50703/bin/ml145fu1.jpg" alt="Image ml145fu1" /></div></div></article><article data-type="fig" id="figobml145fu2"><div id="ml145.fu2" class="figure"><div class="graphic"><img data-src="/books/NBK50703/bin/ml145fu2.jpg" alt="Image ml145fu2" /></div></div></article><article data-type="table-wrap" id="figobml145tu1"><div id="ml145.tu1" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK50703/table/ml145.tu1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml145.tu1_lrgtbl__"><table><thead><tr><th id="hd_h_ml145.tu1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CID</th><th id="hd_h_ml145.tu1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Target Name</th><th id="hd_h_ml145.tu1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">IC<sub>50</sub>/EC<sub>50</sub> (nM) [SID, AID]</th><th id="hd_h_ml145.tu1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Anti-target Name(s)</th><th id="hd_h_ml145.tu1_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">IC<sub>50</sub>/EC<sub>50</sub> (μM) [SID, AID]</th><th id="hd_h_ml145.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Selectivity</th><th id="hd_h_ml145.tu1_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Secondary Assay(s) Name: IC<sub>50</sub>/EC<sub>50</sub> (nM) [SID, AID]</th></tr></thead><tbody><tr><td headers="hd_h_ml145.tu1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CID2286812 <i>(scaffold1)</i><br /><br /><b><a href="/pcsubstance/?term=ML145[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML145</a></b></td><td headers="hd_h_ml145.tu1_1_1_1_2" rowspan="2" colspan="1" style="text-align:center;vertical-align:top;">GPR35 Antagonist <i>Orphan GPCR receptor</i></td><td headers="hd_h_ml145.tu1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">20.1 nM <a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544499" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-87544499</a>
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<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2480" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-2480</a></td><td headers="hd_h_ml145.tu1_1_1_1_4" rowspan="2" colspan="1" style="text-align:center;vertical-align:top;">GPR55 Antagonist <i>Orphan GPCR receptor</i></td><td headers="hd_h_ml145.tu1_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">~ 21.7 µM <a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544499" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-87544499</a>
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<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2397" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-2397</a></td><td headers="hd_h_ml145.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">~1,080-fold vs. GPR55 Antagonist</td><td headers="hd_h_ml145.tu1_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">N/A</td></tr><tr><td headers="hd_h_ml145.tu1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CID1542103 <i>(scaffold2)</i><br /><br /><b><a href="/pcsubstance/?term=ML144[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML144</a></b></td><td headers="hd_h_ml145.tu1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">2220 nM <a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544496" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-87544496</a>
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<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2480" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-2480</a></td><td headers="hd_h_ml145.tu1_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">>32 µM <a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544496" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-87544496</a>
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<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2397" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-2397</a></td><td headers="hd_h_ml145.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">>15-fold vs. GPR55 Antagonist</td><td headers="hd_h_ml145.tu1_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">N/A</td></tr></tbody></table></div></div></article><article data-type="table-wrap" id="figobml145t1"><div id="ml145.t1" class="table"><h3><span class="label">Table 1</span><span class="title">Assays for GPR35</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK50703/table/ml145.t1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml145.t1_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml145.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">PubChem BioAssay Name</th><th id="hd_h_ml145.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">AID</th><th id="hd_h_ml145.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Probe Type</th><th id="hd_h_ml145.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Assay Type</th><th id="hd_h_ml145.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Assay Format</th><th id="hd_h_ml145.t1_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Assay Detection (well format)</th></tr></thead><tbody><tr><td headers="hd_h_ml145.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Summary of Image-based HTS for Selective Antagonists of GPR35</td><td headers="hd_h_ml145.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2079" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2079</a></td><td headers="hd_h_ml145.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">N/A</td><td headers="hd_h_ml145.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Summary</td><td headers="hd_h_ml145.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">N/A</td><td headers="hd_h_ml145.t1_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">N/A</td></tr><tr><td headers="hd_h_ml145.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Image-Based HTS for Selective Antagonists of GPR35 [Primary]</td><td headers="hd_h_ml145.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2058" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2058</a></td><td headers="hd_h_ml145.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Inhibitors</td><td headers="hd_h_ml145.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Primary</td><td headers="hd_h_ml145.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Cell-based</td><td headers="hd_h_ml145.t1_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Imaging method (384)</td></tr><tr><td headers="hd_h_ml145.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">HCS GPR35 Antagonist SAR-primary assay used as secondary</td><td headers="hd_h_ml145.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2480" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2480</a></td><td headers="hd_h_ml145.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Inhibitors</td><td headers="hd_h_ml145.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SAR</td><td headers="hd_h_ml145.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Cell-based</td><td headers="hd_h_ml145.t1_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Imaging method (384)</td></tr><tr><td headers="hd_h_ml145.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">HCS GPR55 antagonist – Counterscreen - SAR</td><td headers="hd_h_ml145.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2397" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2397</a></td><td headers="hd_h_ml145.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Inhibitors</td><td headers="hd_h_ml145.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SAR</td><td headers="hd_h_ml145.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Cell-based</td><td headers="hd_h_ml145.t1_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Imaging method (384)</td></tr></tbody></table></div></div></article><article data-type="fig" id="figobml145fu3"><div id="ml145.fu3" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK50703/bin/ml145fu3.jpg" alt="GPR35 Example Images of Positive and Negative Controls." /></div><h3><span class="title">GPR35 Example Images of Positive and Negative Controls</span></h3><div class="caption"><p>Effect of 10 µM agonist
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Zaprinast (left panel) compared to DMSO control (right panel)</p></div></div></article><article data-type="table-wrap" id="figobml145t2"><div id="ml145.t2" class="table"><h3><span class="label">Table 2</span><span class="title">Critical Reagents used for the uHTS experiments</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK50703/table/ml145.t2/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml145.t2_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml145.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Reagent</th><th id="hd_h_ml145.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Vendor</th></tr></thead><tbody><tr><td headers="hd_h_ml145.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">U2OS (Human Osteosarcoma) cell line stably expressing GFP-tagged β-arrestin and over-expressing the GPR35 receptor</td><td headers="hd_h_ml145.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Cells from AP, scaled-up by BCCG</td></tr><tr><td headers="hd_h_ml145.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Zaprinast - GPR35 Agonist</td><td headers="hd_h_ml145.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">ALEXIS Biochemicals (now Enzo Life Sciences)</td></tr><tr><td headers="hd_h_ml145.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Paraformaldehyde - Fixative</td><td headers="hd_h_ml145.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">ACROS Organics</td></tr><tr><td headers="hd_h_ml145.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">DAPI – Nuclei Stain</td><td headers="hd_h_ml145.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Invitrogen</td></tr></tbody></table></div></div></article><article data-type="table-wrap" id="figobml145tu2"><div id="ml145.tu2" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK50703/table/ml145.tu2/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml145.tu2_lrgtbl__"><table><tbody><tr><td colspan="2" rowspan="1" style="text-align:left;vertical-align:top;">NUCLEI DETECTION</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">- Threshold Adjustment:</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">4</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">- Nuclear Splitting Adjustment:</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">10</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">- Individual Threshold Adjustment</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.05</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">- Minimum Nuclear Area:</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">200</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">- Minimum Nuclear Contrast:</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">CYTOPLASM DETECTION</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">- Cytoplasm Individual Threshold Adjustment:</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0</td></tr><tr><td colspan="2" rowspan="1" style="text-align:left;vertical-align:top;">SPOT DETECTION</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">- Spot Minimum Distance</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">3</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">- Spot Peak Radius</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">- Spot Reference Radius</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">3</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">- Spot Minimum Contrast</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.26</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">- Spot Minimum to Cell Intensity</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0.5</td></tr></tbody></table></div></div></article><article data-type="fig" id="figobml145f1"><div id="ml145.f1" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK50703/bin/ml145f1.jpg" alt="Figure 1. The GPR35 Antagonist Probe Flowchart summarizes the compound triage and decision tree for advancement of the compounds." /></div><h3><span class="label">Figure 1</span><span class="title">The GPR35 Antagonist Probe Flowchart summarizes the compound triage and decision tree for advancement of the compounds</span></h3></div></article><article data-type="fig" id="figobml145fu4"><div id="ml145.fu4" class="figure"><div class="graphic"><img data-src="/books/NBK50703/bin/ml145fu4.jpg" alt="Image ml145fu4" /></div></div></article><article data-type="table-wrap" id="figobml145t3"><div id="ml145.t3" class="table"><h3><span class="label">Table 3</span><span class="title">Probe #1: SAR of selective GPR35 antagonists, including probe CID2286812</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK50703/table/ml145.t3/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml145.t3_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml145.t3_1_1_1_1" colspan="2" rowspan="1" style="text-align:center;vertical-align:top;"><div class="graphic"><img src="/books/NBK50703/bin/ml145fu5.jpg" alt="Image ml145fu5.jpg" /></div></th><th id="hd_h_ml145.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></th><th id="hd_h_ml145.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></th><th id="hd_h_ml145.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></th><th id="hd_h_ml145.t3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></th><th id="hd_h_ml145.t3_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"></th></tr><tr><th headers="hd_h_ml145.t3_1_1_1_1" id="hd_h_ml145.t3_1_1_2_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;">R1</th><th headers="hd_h_ml145.t3_1_1_1_1" id="hd_h_ml145.t3_1_1_2_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;">R2, R3</th><th headers="hd_h_ml145.t3_1_1_1_2" id="hd_h_ml145.t3_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">PubChem CID</th><th headers="hd_h_ml145.t3_1_1_1_3" id="hd_h_ml145.t3_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">PubChem SID</th><th headers="hd_h_ml145.t3_1_1_1_4" id="hd_h_ml145.t3_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Burnham ID</th><th headers="hd_h_ml145.t3_1_1_1_5" id="hd_h_ml145.t3_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">GPR35 IC<sub>50</sub> (µM)</th><th headers="hd_h_ml145.t3_1_1_1_6" id="hd_h_ml145.t3_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">GPR55 IC<sub>50</sub> (µM)</th></tr></thead><tbody><tr><td headers="hd_h_ml145.t3_1_1_1_1 hd_h_ml145.t3_1_1_2_1" rowspan="1" colspan="1" style="background-color:#00FFFF;text-align:left;vertical-align:middle;"><div class="graphic"><img src="/books/NBK50703/bin/ml145fu6.jpg" alt="Image ml145fu6.jpg" /></div></td><td headers="hd_h_ml145.t3_1_1_1_1 hd_h_ml145.t3_1_1_2_2" rowspan="1" colspan="1" style="background-color:#00FFFF;text-align:left;vertical-align:middle;"><b>OH, COOH</b></td><td headers="hd_h_ml145.t3_1_1_1_2 hd_h_ml145.t3_1_1_2_3" rowspan="1" colspan="1" style="background-color:#00FFFF;text-align:center;vertical-align:middle;">2286812</td><td headers="hd_h_ml145.t3_1_1_1_3 hd_h_ml145.t3_1_1_2_4" rowspan="1" colspan="1" style="background-color:#00FFFF;text-align:center;vertical-align:middle;">87544499</td><td headers="hd_h_ml145.t3_1_1_1_4 hd_h_ml145.t3_1_1_2_5" rowspan="1" colspan="1" style="background-color:#00FFFF;text-align:center;vertical-align:middle;">MLS-0205231</td><td headers="hd_h_ml145.t3_1_1_1_5 hd_h_ml145.t3_1_1_2_6" rowspan="1" colspan="1" style="background-color:#00FFFF;text-align:center;vertical-align:middle;"><0.06/<0.06/0.02</td><td headers="hd_h_ml145.t3_1_1_1_6 hd_h_ml145.t3_1_1_2_7" rowspan="1" colspan="1" style="background-color:#00FFFF;text-align:center;vertical-align:middle;">>32/11.3</td></tr><tr><td headers="hd_h_ml145.t3_1_1_1_1 hd_h_ml145.t3_1_1_2_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;"><div class="graphic"><img src="/books/NBK50703/bin/ml145fu7.jpg" alt="Image ml145fu7.jpg" /></div></td><td headers="hd_h_ml145.t3_1_1_1_1 hd_h_ml145.t3_1_1_2_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;"><b>H, COOH</b></td><td headers="hd_h_ml145.t3_1_1_1_2 hd_h_ml145.t3_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2286888</td><td headers="hd_h_ml145.t3_1_1_1_3 hd_h_ml145.t3_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">87544500</td><td headers="hd_h_ml145.t3_1_1_1_4 hd_h_ml145.t3_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS-0216981</td><td headers="hd_h_ml145.t3_1_1_1_5 hd_h_ml145.t3_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><0.06/<0.06/0.10</td><td headers="hd_h_ml145.t3_1_1_1_6 hd_h_ml145.t3_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>32/17.9</td></tr><tr><td headers="hd_h_ml145.t3_1_1_1_1 hd_h_ml145.t3_1_1_2_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;"><b>4-Et-Ph</b></td><td headers="hd_h_ml145.t3_1_1_1_1 hd_h_ml145.t3_1_1_2_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;"><b>H, COOH</b></td><td headers="hd_h_ml145.t3_1_1_1_2 hd_h_ml145.t3_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1985027</td><td headers="hd_h_ml145.t3_1_1_1_3 hd_h_ml145.t3_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">87544525</td><td headers="hd_h_ml145.t3_1_1_1_4 hd_h_ml145.t3_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS-0370945</td><td headers="hd_h_ml145.t3_1_1_1_5 hd_h_ml145.t3_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><0.06/<0.06/0.07</td><td headers="hd_h_ml145.t3_1_1_1_6 hd_h_ml145.t3_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">24.5/10.1</td></tr><tr><td headers="hd_h_ml145.t3_1_1_1_1 hd_h_ml145.t3_1_1_2_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;"><b>3,4- diOMe- Ph</b></td><td headers="hd_h_ml145.t3_1_1_1_1 hd_h_ml145.t3_1_1_2_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;"><b>OH, COOH</b></td><td headers="hd_h_ml145.t3_1_1_1_2 hd_h_ml145.t3_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">6202912</td><td headers="hd_h_ml145.t3_1_1_1_3 hd_h_ml145.t3_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">87544534</td><td headers="hd_h_ml145.t3_1_1_1_4 hd_h_ml145.t3_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS-0435437</td><td headers="hd_h_ml145.t3_1_1_1_5 hd_h_ml145.t3_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.12/0.14/0.18 Ave 0.15</td><td headers="hd_h_ml145.t3_1_1_1_6 hd_h_ml145.t3_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>32/>32</td></tr><tr><td headers="hd_h_ml145.t3_1_1_1_1 hd_h_ml145.t3_1_1_2_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;"><div class="graphic"><img src="/books/NBK50703/bin/ml145fu8.jpg" alt="Image ml145fu8.jpg" /></div></td><td headers="hd_h_ml145.t3_1_1_1_1 hd_h_ml145.t3_1_1_2_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;"><b>OH, COOH</b></td><td headers="hd_h_ml145.t3_1_1_1_2 hd_h_ml145.t3_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">6203183</td><td headers="hd_h_ml145.t3_1_1_1_3 hd_h_ml145.t3_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">87544535</td><td headers="hd_h_ml145.t3_1_1_1_4 hd_h_ml145.t3_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS-0435438</td><td headers="hd_h_ml145.t3_1_1_1_5 hd_h_ml145.t3_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.32/0.28 Ave 0.30</td><td headers="hd_h_ml145.t3_1_1_1_6 hd_h_ml145.t3_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>32/>32</td></tr><tr><td headers="hd_h_ml145.t3_1_1_1_1 hd_h_ml145.t3_1_1_2_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><b>4-Me-Ph</b></td><td headers="hd_h_ml145.t3_1_1_1_1 hd_h_ml145.t3_1_1_2_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;"><b>COOH, H</b></td><td headers="hd_h_ml145.t3_1_1_1_2 hd_h_ml145.t3_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1346721</td><td headers="hd_h_ml145.t3_1_1_1_3 hd_h_ml145.t3_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">87544505</td><td headers="hd_h_ml145.t3_1_1_1_4 hd_h_ml145.t3_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS-0374311</td><td headers="hd_h_ml145.t3_1_1_1_5 hd_h_ml145.t3_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.23/0.25/Ave 0.24</td><td headers="hd_h_ml145.t3_1_1_1_6 hd_h_ml145.t3_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>32/>32</td></tr></tbody></table></div></div></article><article data-type="table-wrap" id="figobml145t4"><div id="ml145.t4" class="table"><h3><span class="label">Table 4</span><span class="title">Probe #2: SAR of selective GPR35 antagonists, including probe CID1542103</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK50703/table/ml145.t4/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml145.t4_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml145.t4_1_1_1_1" colspan="2" rowspan="1" style="text-align:center;vertical-align:middle;"><div class="graphic"><img src="/books/NBK50703/bin/ml145fu9.jpg" alt="Image ml145fu9.jpg" /></div></th><th id="hd_h_ml145.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></th><th id="hd_h_ml145.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></th><th id="hd_h_ml145.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></th><th id="hd_h_ml145.t4_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></th><th id="hd_h_ml145.t4_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"></th></tr><tr><th headers="hd_h_ml145.t4_1_1_1_1" id="hd_h_ml145.t4_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">R1</th><th headers="hd_h_ml145.t4_1_1_1_1" id="hd_h_ml145.t4_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">R2</th><th headers="hd_h_ml145.t4_1_1_1_2" id="hd_h_ml145.t4_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">PubChem CID</th><th headers="hd_h_ml145.t4_1_1_1_3" id="hd_h_ml145.t4_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">PubChem SID</th><th headers="hd_h_ml145.t4_1_1_1_4" id="hd_h_ml145.t4_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Burnham ID</th><th headers="hd_h_ml145.t4_1_1_1_5" id="hd_h_ml145.t4_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">GPR35 IC<sub>50</sub> (µM)</th><th headers="hd_h_ml145.t4_1_1_1_6" id="hd_h_ml145.t4_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">GPR55 IC<sub>50</sub> (µM)</th></tr></thead><tbody><tr><td headers="hd_h_ml145.t4_1_1_1_1 hd_h_ml145.t4_1_1_2_1" rowspan="1" colspan="1" style="background-color:#00FFFF;text-align:center;vertical-align:middle;">2-Me-Ph</td><td headers="hd_h_ml145.t4_1_1_1_1 hd_h_ml145.t4_1_1_2_2" rowspan="1" colspan="1" style="background-color:#00FFFF;text-align:center;vertical-align:middle;">4-Cl-Ph</td><td headers="hd_h_ml145.t4_1_1_1_2 hd_h_ml145.t4_1_1_2_3" rowspan="1" colspan="1" style="background-color:#00FFFF;text-align:center;vertical-align:middle;">1542103</td><td headers="hd_h_ml145.t4_1_1_1_3 hd_h_ml145.t4_1_1_2_4" rowspan="1" colspan="1" style="background-color:#00FFFF;text-align:center;vertical-align:middle;">87544496</td><td headers="hd_h_ml145.t4_1_1_1_4 hd_h_ml145.t4_1_1_2_5" rowspan="1" colspan="1" style="background-color:#00FFFF;text-align:center;vertical-align:middle;">MLS-0013034</td><td headers="hd_h_ml145.t4_1_1_1_5 hd_h_ml145.t4_1_1_2_6" rowspan="1" colspan="1" style="background-color:#00FFFF;text-align:center;vertical-align:middle;">1.65/2.78/Ave 2.22</td><td headers="hd_h_ml145.t4_1_1_1_6 hd_h_ml145.t4_1_1_2_7" rowspan="1" colspan="1" style="background-color:#00FFFF;text-align:center;vertical-align:middle;">>32/>32</td></tr><tr><td headers="hd_h_ml145.t4_1_1_1_1 hd_h_ml145.t4_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2-Me-Ph</td><td headers="hd_h_ml145.t4_1_1_1_1 hd_h_ml145.t4_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">4-Me-Ph</td><td headers="hd_h_ml145.t4_1_1_1_2 hd_h_ml145.t4_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1502520</td><td headers="hd_h_ml145.t4_1_1_1_3 hd_h_ml145.t4_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">87544497</td><td headers="hd_h_ml145.t4_1_1_1_4 hd_h_ml145.t4_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS-0037657</td><td headers="hd_h_ml145.t4_1_1_1_5 hd_h_ml145.t4_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2.23/3.05/Ave 2.65</td><td headers="hd_h_ml145.t4_1_1_1_6 hd_h_ml145.t4_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>32/>32</td></tr><tr><td headers="hd_h_ml145.t4_1_1_1_1 hd_h_ml145.t4_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Ph</td><td headers="hd_h_ml145.t4_1_1_1_1 hd_h_ml145.t4_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">4-Cl-Ph</td><td headers="hd_h_ml145.t4_1_1_1_2 hd_h_ml145.t4_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">8056691</td><td headers="hd_h_ml145.t4_1_1_1_3 hd_h_ml145.t4_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">87544526</td><td headers="hd_h_ml145.t4_1_1_1_4 hd_h_ml145.t4_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS-0435432</td><td headers="hd_h_ml145.t4_1_1_1_5 hd_h_ml145.t4_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">5.76/8.57/Ave 7.17</td><td headers="hd_h_ml145.t4_1_1_1_6 hd_h_ml145.t4_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">31.6/>26.9</td></tr><tr><td headers="hd_h_ml145.t4_1_1_1_1 hd_h_ml145.t4_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Ph</td><td headers="hd_h_ml145.t4_1_1_1_1 hd_h_ml145.t4_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Ph</td><td headers="hd_h_ml145.t4_1_1_1_2 hd_h_ml145.t4_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">8056641</td><td headers="hd_h_ml145.t4_1_1_1_3 hd_h_ml145.t4_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">87544527</td><td headers="hd_h_ml145.t4_1_1_1_4 hd_h_ml145.t4_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS-0435433</td><td headers="hd_h_ml145.t4_1_1_1_5 hd_h_ml145.t4_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">12.8/21.2/Ave 17.0</td><td headers="hd_h_ml145.t4_1_1_1_6 hd_h_ml145.t4_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>32/29.1</td></tr><tr><td headers="hd_h_ml145.t4_1_1_1_1 hd_h_ml145.t4_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">4-Me-Ph</td><td headers="hd_h_ml145.t4_1_1_1_1 hd_h_ml145.t4_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">4-Me-Ph</td><td headers="hd_h_ml145.t4_1_1_1_2 hd_h_ml145.t4_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">661907</td><td headers="hd_h_ml145.t4_1_1_1_3 hd_h_ml145.t4_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">87544523</td><td headers="hd_h_ml145.t4_1_1_1_4 hd_h_ml145.t4_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS-0002494</td><td headers="hd_h_ml145.t4_1_1_1_5 hd_h_ml145.t4_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">23.0/12.4/Ave 17.7</td><td headers="hd_h_ml145.t4_1_1_1_6 hd_h_ml145.t4_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>32/>32</td></tr><tr><td headers="hd_h_ml145.t4_1_1_1_1 hd_h_ml145.t4_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">4-F-Ph</td><td headers="hd_h_ml145.t4_1_1_1_1 hd_h_ml145.t4_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2,4-di-Cl - Ph</td><td headers="hd_h_ml145.t4_1_1_1_2 hd_h_ml145.t4_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2474060</td><td headers="hd_h_ml145.t4_1_1_1_3 hd_h_ml145.t4_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">87544537</td><td headers="hd_h_ml145.t4_1_1_1_4 hd_h_ml145.t4_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS-0435440</td><td headers="hd_h_ml145.t4_1_1_1_5 hd_h_ml145.t4_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>32/>32/Ave >32</td><td headers="hd_h_ml145.t4_1_1_1_6 hd_h_ml145.t4_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>32/>32</td></tr></tbody></table></div></div></article><article data-type="fig" id="figobml145fu5"><div id="ml145.fu5" class="figure"><div class="graphic"><img data-src="/books/NBK50703/bin/ml145fu10.jpg" alt="Image ml145fu10" /></div></div></article><article data-type="fig" id="figobml145fu6"><div id="ml145.fu6" class="figure"><div class="graphic"><img data-src="/books/NBK50703/bin/ml145fu11.jpg" alt="Image ml145fu11" /></div></div></article><article data-type="fig" id="figobml145fu7"><div id="ml145.fu7" class="figure"><div class="graphic"><img data-src="/books/NBK50703/bin/ml145fu12.jpg" alt="Image ml145fu12" /></div></div></article><article data-type="fig" id="figobml145fu8"><div id="ml145.fu8" class="figure"><div class="graphic"><img data-src="/books/NBK50703/bin/ml145fu13.jpg" alt="Image ml145fu13" /></div></div></article><article data-type="fig" id="figobml145fu9"><div id="ml145.fu9" class="figure"><div class="graphic"><img data-src="/books/NBK50703/bin/ml145fu14.jpg" alt="Image ml145fu14" /></div></div></article><article data-type="table-wrap" id="figobml145t5"><div id="ml145.t5" class="table"><h3><span class="label">Table 5A</span><span class="title">Submission information on Probe 1: CID2286812 and analogs</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK50703/table/ml145.t5/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml145.t5_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml145.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Probe/Analog</th><th id="hd_h_ml145.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS#<br /><br />(DPI)</th><th id="hd_h_ml145.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS-(BCCG#)</th><th id="hd_h_ml145.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CID</th><th id="hd_h_ml145.t5_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SID</th><th id="hd_h_ml145.t5_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Source (vendor/BCCG syn)</th><th id="hd_h_ml145.t5_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Amt (mg)</th><th id="hd_h_ml145.t5_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Date ordered/submitted</th></tr></thead><tbody><tr><td headers="hd_h_ml145.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Probe<br /><b><a href="/pcsubstance/?term=ML145[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML145</a></b></td><td headers="hd_h_ml145.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS002699443</td><td headers="hd_h_ml145.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0205231</td><td headers="hd_h_ml145.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2286812</td><td headers="hd_h_ml145.t5_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544499" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87544499</a></td><td headers="hd_h_ml145.t5_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">ChemBridge</td><td headers="hd_h_ml145.t5_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">50</td><td headers="hd_h_ml145.t5_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1/28/10</td></tr><tr><td headers="hd_h_ml145.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Analog 1</td><td headers="hd_h_ml145.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">MLS002699444</td><td headers="hd_h_ml145.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">0216981</td><td headers="hd_h_ml145.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">2286888</td><td headers="hd_h_ml145.t5_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544500" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87544500</a></td><td headers="hd_h_ml145.t5_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">ChemBridge</td><td headers="hd_h_ml145.t5_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">20</td><td headers="hd_h_ml145.t5_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">2/17/10</td></tr><tr><td headers="hd_h_ml145.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Analog 2</td><td headers="hd_h_ml145.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">MLS002699445</td><td headers="hd_h_ml145.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">0370945</td><td headers="hd_h_ml145.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">1985027</td><td headers="hd_h_ml145.t5_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544525" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87544525</a></td><td headers="hd_h_ml145.t5_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">InterBioScreen</td><td headers="hd_h_ml145.t5_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">20</td><td headers="hd_h_ml145.t5_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">2/17/10</td></tr><tr><td headers="hd_h_ml145.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Analog 3</td><td headers="hd_h_ml145.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">MLS002699446</td><td headers="hd_h_ml145.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">0435437</td><td headers="hd_h_ml145.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">6202912</td><td headers="hd_h_ml145.t5_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544534" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87544534</a></td><td headers="hd_h_ml145.t5_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Life Chemicals</td><td headers="hd_h_ml145.t5_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">20</td><td headers="hd_h_ml145.t5_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">2/17/10</td></tr><tr><td headers="hd_h_ml145.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Analog 4</td><td headers="hd_h_ml145.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">MLS002699447</td><td headers="hd_h_ml145.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">0435438</td><td headers="hd_h_ml145.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">6203183</td><td headers="hd_h_ml145.t5_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544535" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87544535</a></td><td headers="hd_h_ml145.t5_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Life Chemicals</td><td headers="hd_h_ml145.t5_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">20</td><td headers="hd_h_ml145.t5_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">2/17/10</td></tr><tr><td headers="hd_h_ml145.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Analog 5</td><td headers="hd_h_ml145.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS002699448</td><td headers="hd_h_ml145.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">0374311</td><td headers="hd_h_ml145.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">1346721</td><td headers="hd_h_ml145.t5_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544505" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87544505</a></td><td headers="hd_h_ml145.t5_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">ChemBridge</td><td headers="hd_h_ml145.t5_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">20</td><td headers="hd_h_ml145.t5_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">2/17/10</td></tr></tbody></table></div></div></article><article data-type="table-wrap" id="figobml145t6"><div id="ml145.t6" class="table"><h3><span class="label">Table 5B</span><span class="title">Submission information on Probe#2: CID1542103 and analogs</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK50703/table/ml145.t6/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml145.t6_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml145.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Probe/Analog</th><th id="hd_h_ml145.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS# (DPI)</th><th id="hd_h_ml145.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS-(BCCG#)</th><th id="hd_h_ml145.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CID</th><th id="hd_h_ml145.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SID</th><th id="hd_h_ml145.t6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Source (vendor/BCCG syn)</th><th id="hd_h_ml145.t6_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Amt (mg)</th><th id="hd_h_ml145.t6_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Date ordered/submitted</th></tr></thead><tbody><tr><td headers="hd_h_ml145.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Probe<br /><b><a href="/pcsubstance/?term=ML144[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML144</a></b></td><td headers="hd_h_ml145.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLS002699449</td><td headers="hd_h_ml145.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0013034</td><td headers="hd_h_ml145.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">1542103</td><td headers="hd_h_ml145.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544496" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87544496</a></td><td headers="hd_h_ml145.t6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">ChemBridge</td><td headers="hd_h_ml145.t6_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">50</td><td headers="hd_h_ml145.t6_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1/28/10</td></tr><tr><td headers="hd_h_ml145.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Analog 1</td><td headers="hd_h_ml145.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS002699450</td><td headers="hd_h_ml145.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0037657</td><td headers="hd_h_ml145.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1502520</td><td headers="hd_h_ml145.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544497" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87544497</a></td><td headers="hd_h_ml145.t6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">ChemBridge</td><td headers="hd_h_ml145.t6_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">20</td><td headers="hd_h_ml145.t6_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2/17/10</td></tr><tr><td headers="hd_h_ml145.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Analog 2</td><td headers="hd_h_ml145.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS002699451</td><td headers="hd_h_ml145.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0435432</td><td headers="hd_h_ml145.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">8056691</td><td headers="hd_h_ml145.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544526" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87544526</a></td><td headers="hd_h_ml145.t6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">InterBioScreen</td><td headers="hd_h_ml145.t6_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">20</td><td headers="hd_h_ml145.t6_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2/17/10</td></tr><tr><td headers="hd_h_ml145.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Analog 3</td><td headers="hd_h_ml145.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS002699452</td><td headers="hd_h_ml145.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0435433</td><td headers="hd_h_ml145.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">8056641</td><td headers="hd_h_ml145.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544527" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87544527</a></td><td headers="hd_h_ml145.t6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">InterBioScreen</td><td headers="hd_h_ml145.t6_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">20</td><td headers="hd_h_ml145.t6_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2/17/10</td></tr><tr><td headers="hd_h_ml145.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Analog 4</td><td headers="hd_h_ml145.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS002699453</td><td headers="hd_h_ml145.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0002494</td><td headers="hd_h_ml145.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">661907</td><td headers="hd_h_ml145.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544523" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87544523</a></td><td headers="hd_h_ml145.t6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">InterBioScreen</td><td headers="hd_h_ml145.t6_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">20</td><td headers="hd_h_ml145.t6_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2/17/10</td></tr><tr><td headers="hd_h_ml145.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Analog 5</td><td headers="hd_h_ml145.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS002699454</td><td headers="hd_h_ml145.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0435440</td><td headers="hd_h_ml145.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2474060</td><td headers="hd_h_ml145.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87544537" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87544537</a></td><td headers="hd_h_ml145.t6_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Enamine</td><td headers="hd_h_ml145.t6_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">20</td><td headers="hd_h_ml145.t6_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2/17/10</td></tr></tbody></table></div></div></article><article data-type="fig" id="figobml145fu10"><div id="ml145.fu10" class="figure"><div class="graphic"><img data-src="/books/NBK50703/bin/ml145fu15.jpg" alt="Image ml145fu15" /></div></div></article><article data-type="fig" id="figobml145fu11"><div id="ml145.fu11" class="figure"><div class="graphic"><img data-src="/books/NBK50703/bin/ml145fu16.jpg" alt="Image ml145fu16" /></div></div></article><article data-type="table-wrap" id="figobml145t7"><div id="ml145.t7" class="table"><h3><span class="label">Table 6</span><span class="title">Summary of <i>in vitro</i> ADMET/PK Properties of the GPR35 Antagonist Probes</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK50703/table/ml145.t7/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml145.t7_lrgtbl__"><table class="no_margin"><thead><tr><th id="hd_h_ml145.t7_1_1_1_1" rowspan="2" colspan="1" headers="hd_h_ml145.t7_1_1_1_1" style="text-align:center;vertical-align:top;">Probe CID<br />Probe ML#<br />BCCG MLS-#</th><th id="hd_h_ml145.t7_1_1_1_2" rowspan="2" colspan="1" headers="hd_h_ml145.t7_1_1_1_2" style="text-align:center;vertical-align:top;">Aqueous Solubility<br />(μg/mL)<sup>a</sup> (@ pH)</th><th id="hd_h_ml145.t7_1_1_1_3" rowspan="2" colspan="1" headers="hd_h_ml145.t7_1_1_1_3" style="text-align:center;vertical-align:top;">PAMPA Pe<br />(×10<sup>−6</sup> cm/s)<sup>b</sup> (@ pH)</th><th id="hd_h_ml145.t7_1_1_1_4" colspan="2" rowspan="1" style="text-align:center;vertical-align:top;">Plasma Protein Binding (% Bound)</th><th id="hd_h_ml145.t7_1_1_1_5" rowspan="2" colspan="1" headers="hd_h_ml145.t7_1_1_1_5" style="text-align:center;vertical-align:top;">Plasma Stability<sup>c</sup><br />Human Mouse/</th><th id="hd_h_ml145.t7_1_1_1_6" rowspan="2" colspan="1" headers="hd_h_ml145.t7_1_1_1_6" style="text-align:center;vertical-align:top;">Hepatic Microsome Stability<sup>d</sup><br />Human/Mouse</th><th id="hd_h_ml145.t7_1_1_1_7" rowspan="2" colspan="1" headers="hd_h_ml145.t7_1_1_1_7" style="text-align:center;vertical-align:top;">Hepatic Toxicity<sup>e</sup><br />LC50 (μM)</th></tr><tr><th headers="hd_h_ml145.t7_1_1_1_4" id="hd_h_ml145.t7_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Human<br />1μM/10μM</th><th headers="hd_h_ml145.t7_1_1_1_4" id="hd_h_ml145.t7_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Mouse<br />1μM/10μM</th></tr></thead><tbody><tr><td headers="hd_h_ml145.t7_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CID2286812</td><td headers="hd_h_ml145.t7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.76 (5.0)</td><td headers="hd_h_ml145.t7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">55 (5.0)</td><td headers="hd_h_ml145.t7_1_1_1_4 hd_h_ml145.t7_1_1_2_1" rowspan="3" colspan="1" style="text-align:center;vertical-align:middle;">98.17/98.37</td><td headers="hd_h_ml145.t7_1_1_1_4 hd_h_ml145.t7_1_1_2_2" rowspan="3" colspan="1" style="text-align:center;vertical-align:middle;">98.65/97.31</td><td headers="hd_h_ml145.t7_1_1_1_5" rowspan="3" colspan="1" style="text-align:center;vertical-align:middle;">77.58/94.44</td><td headers="hd_h_ml145.t7_1_1_1_6" rowspan="3" colspan="1" style="text-align:center;vertical-align:middle;">2.41/7.01</td><td headers="hd_h_ml145.t7_1_1_1_7" rowspan="3" colspan="1" style="text-align:center;vertical-align:middle;">>50</td></tr><tr><td headers="hd_h_ml145.t7_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b><a href="/pcsubstance/?term=ML145[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML145</a></b></td><td headers="hd_h_ml145.t7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">31.4 (6.2)</td><td headers="hd_h_ml145.t7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">244 (6.2)</td></tr><tr><td headers="hd_h_ml145.t7_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS-0205231</td><td headers="hd_h_ml145.t7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">44.4 (7.4)</td><td headers="hd_h_ml145.t7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">11 (7.4)</td></tr><tr><td headers="hd_h_ml145.t7_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CID1542103</td><td headers="hd_h_ml145.t7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.02 (5.0)</td><td headers="hd_h_ml145.t7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">40 (5.0)</td><td headers="hd_h_ml145.t7_1_1_1_4 hd_h_ml145.t7_1_1_2_1" rowspan="3" colspan="1" style="text-align:center;vertical-align:middle;">99.92/99.90</td><td headers="hd_h_ml145.t7_1_1_1_4 hd_h_ml145.t7_1_1_2_2" rowspan="3" colspan="1" style="text-align:center;vertical-align:middle;">99.16/99.13</td><td headers="hd_h_ml145.t7_1_1_1_5" rowspan="3" colspan="1" style="text-align:center;vertical-align:middle;">90.00/100.37</td><td headers="hd_h_ml145.t7_1_1_1_6" rowspan="3" colspan="1" style="text-align:center;vertical-align:middle;">56.23/30.58</td><td headers="hd_h_ml145.t7_1_1_1_7" rowspan="3" colspan="1" style="text-align:center;vertical-align:middle;">13.8</td></tr><tr><td headers="hd_h_ml145.t7_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><b><a href="/pcsubstance/?term=ML144[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML144</a></b></td><td headers="hd_h_ml145.t7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.07 (6.2)</td><td headers="hd_h_ml145.t7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">und* (6.2)</td></tr><tr><td headers="hd_h_ml145.t7_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS-0013034</td><td headers="hd_h_ml145.t7_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><0.01 (7.4)</td><td headers="hd_h_ml145.t7_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0 (7.4)</td></tr></tbody></table></div><div class="tblwrap-foot"><div><dl class="temp-labeled-list small"><dl class="bkr_refwrap"><dt>a</dt><dd><div id="ml145.tfn1"><p class="no_margin">in aqueous buffer, pH’s 5.0/6.2/7.4</p></div></dd></dl><dl class="bkr_refwrap"><dt>b</dt><dd><div id="ml145.tfn2"><p class="no_margin">in aqueous buffer; Donor compartment pH’s 5.0/6.2/7.4; Acceptor compartment pH 7.4</p></div></dd></dl><dl class="bkr_refwrap"><dt>c</dt><dd><div id="ml145.tfn3"><p class="no_margin">% remaining at 3 hr</p></div></dd></dl><dl class="bkr_refwrap"><dt>d</dt><dd><div id="ml145.tfn4"><p class="no_margin">% remaining at 1 hr</p></div></dd></dl><dl class="bkr_refwrap"><dt>e</dt><dd><div id="ml145.tfn5"><p class="no_margin">towards Fa2N-4 immortalized human hepatocytes</p></div></dd></dl><dl class="bkr_refwrap"><dt>*</dt><dd><div id="ml145.tfn6"><p class="no_margin">undeterminable</p></div></dd></dl></dl></div></div></div></article><article data-type="fig" id="figobml145fu12"><div id="ml145.fu12" class="figure"><div class="graphic"><img data-src="/books/NBK50703/bin/ml145fu17.jpg" alt="Image ml145fu17" /></div></div></article><article data-type="table-wrap" id="figobml145t8"><div id="ml145.t8" class="table"><h3><span class="label">Table 7</span><span class="title">Properties computed from Structure</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK50703/table/ml145.t8/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml145.t8_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml145.t8_1_1_1_1" rowspan="3" colspan="1" headers="hd_h_ml145.t8_1_1_1_1" style="text-align:center;vertical-align:middle;">Calculated Property</th><th id="hd_h_ml145.t8_1_1_1_2" colspan="2" rowspan="1" style="text-align:center;vertical-align:middle;">Probe Identity</th></tr><tr><th headers="hd_h_ml145.t8_1_1_1_2" id="hd_h_ml145.t8_1_1_2_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">CID2286812 <i>(scaffold 1)</i></th><th headers="hd_h_ml145.t8_1_1_1_2" id="hd_h_ml145.t8_1_1_2_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">CID1542103 <i>(Scaffold 2)</i></th></tr><tr><th headers="hd_h_ml145.t8_1_1_1_2 hd_h_ml145.t8_1_1_2_1" id="hd_h_ml145.t8_1_1_3_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">MLS-0205231</th><th headers="hd_h_ml145.t8_1_1_1_2 hd_h_ml145.t8_1_1_2_2" id="hd_h_ml145.t8_1_1_3_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">MLS-0013034</th></tr></thead><tbody><tr><td headers="hd_h_ml145.t8_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Molecular Weight [g/mol]</td><td headers="hd_h_ml145.t8_1_1_1_2 hd_h_ml145.t8_1_1_2_1 hd_h_ml145.t8_1_1_3_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">482.57188</td><td headers="hd_h_ml145.t8_1_1_1_2 hd_h_ml145.t8_1_1_2_2 hd_h_ml145.t8_1_1_3_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">418.92192</td></tr><tr><td headers="hd_h_ml145.t8_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Molecular Formula</td><td headers="hd_h_ml145.t8_1_1_1_2 hd_h_ml145.t8_1_1_2_1 hd_h_ml145.t8_1_1_3_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">C<sub>24</sub>H<sub>22</sub>N<sub>2</sub>O<sub>5</sub>S<sub>2</sub></td><td headers="hd_h_ml145.t8_1_1_1_2 hd_h_ml145.t8_1_1_2_2 hd_h_ml145.t8_1_1_3_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">C<sub>23</sub>H<sub>23</sub>ClN<sub>6</sub></td></tr><tr><td headers="hd_h_ml145.t8_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">XLogP3-AA</td><td headers="hd_h_ml145.t8_1_1_1_2 hd_h_ml145.t8_1_1_2_1 hd_h_ml145.t8_1_1_3_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">5.1</td><td headers="hd_h_ml145.t8_1_1_1_2 hd_h_ml145.t8_1_1_2_2 hd_h_ml145.t8_1_1_3_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">4.8</td></tr><tr><td headers="hd_h_ml145.t8_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">H-Bond Donor</td><td headers="hd_h_ml145.t8_1_1_1_2 hd_h_ml145.t8_1_1_2_1 hd_h_ml145.t8_1_1_3_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">3</td><td headers="hd_h_ml145.t8_1_1_1_2 hd_h_ml145.t8_1_1_2_2 hd_h_ml145.t8_1_1_3_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0</td></tr><tr><td headers="hd_h_ml145.t8_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">H-Bond Acceptor</td><td headers="hd_h_ml145.t8_1_1_1_2 hd_h_ml145.t8_1_1_2_1 hd_h_ml145.t8_1_1_3_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">5</td><td headers="hd_h_ml145.t8_1_1_1_2 hd_h_ml145.t8_1_1_2_2 hd_h_ml145.t8_1_1_3_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">5</td></tr><tr><td headers="hd_h_ml145.t8_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Rotatable Bond Count</td><td headers="hd_h_ml145.t8_1_1_1_2 hd_h_ml145.t8_1_1_2_1 hd_h_ml145.t8_1_1_3_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">8</td><td headers="hd_h_ml145.t8_1_1_1_2 hd_h_ml145.t8_1_1_2_2 hd_h_ml145.t8_1_1_3_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">4</td></tr><tr><td headers="hd_h_ml145.t8_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Tautomer Count</td><td headers="hd_h_ml145.t8_1_1_1_2 hd_h_ml145.t8_1_1_2_1 hd_h_ml145.t8_1_1_3_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">17</td><td headers="hd_h_ml145.t8_1_1_1_2 hd_h_ml145.t8_1_1_2_2 hd_h_ml145.t8_1_1_3_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">-</td></tr><tr><td headers="hd_h_ml145.t8_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Exact Mass</td><td headers="hd_h_ml145.t8_1_1_1_2 hd_h_ml145.t8_1_1_2_1 hd_h_ml145.t8_1_1_3_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">482.097013</td><td headers="hd_h_ml145.t8_1_1_1_2 hd_h_ml145.t8_1_1_2_2 hd_h_ml145.t8_1_1_3_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">418.167272</td></tr><tr><td headers="hd_h_ml145.t8_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">MonoIsotopic Mass</td><td headers="hd_h_ml145.t8_1_1_1_2 hd_h_ml145.t8_1_1_2_1 hd_h_ml145.t8_1_1_3_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">482.097013</td><td headers="hd_h_ml145.t8_1_1_1_2 hd_h_ml145.t8_1_1_2_2 hd_h_ml145.t8_1_1_3_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">418.167272</td></tr><tr><td headers="hd_h_ml145.t8_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Topological Polar Surface Area</td><td headers="hd_h_ml145.t8_1_1_1_2 hd_h_ml145.t8_1_1_2_1 hd_h_ml145.t8_1_1_3_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">164</td><td headers="hd_h_ml145.t8_1_1_1_2 hd_h_ml145.t8_1_1_2_2 hd_h_ml145.t8_1_1_3_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">50.1</td></tr><tr><td headers="hd_h_ml145.t8_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Heavy Atom Count</td><td headers="hd_h_ml145.t8_1_1_1_2 hd_h_ml145.t8_1_1_2_1 hd_h_ml145.t8_1_1_3_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">33</td><td headers="hd_h_ml145.t8_1_1_1_2 hd_h_ml145.t8_1_1_2_2 hd_h_ml145.t8_1_1_3_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">30</td></tr><tr><td headers="hd_h_ml145.t8_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Formal Charge</td><td headers="hd_h_ml145.t8_1_1_1_2 hd_h_ml145.t8_1_1_2_1 hd_h_ml145.t8_1_1_3_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0</td><td headers="hd_h_ml145.t8_1_1_1_2 hd_h_ml145.t8_1_1_2_2 hd_h_ml145.t8_1_1_3_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0</td></tr><tr><td headers="hd_h_ml145.t8_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Complexity</td><td headers="hd_h_ml145.t8_1_1_1_2 hd_h_ml145.t8_1_1_2_1 hd_h_ml145.t8_1_1_3_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">835</td><td headers="hd_h_ml145.t8_1_1_1_2 hd_h_ml145.t8_1_1_2_2 hd_h_ml145.t8_1_1_3_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">548</td></tr><tr><td headers="hd_h_ml145.t8_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Isotope Atom Count</td><td headers="hd_h_ml145.t8_1_1_1_2 hd_h_ml145.t8_1_1_2_1 hd_h_ml145.t8_1_1_3_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0</td><td headers="hd_h_ml145.t8_1_1_1_2 hd_h_ml145.t8_1_1_2_2 hd_h_ml145.t8_1_1_3_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0</td></tr><tr><td headers="hd_h_ml145.t8_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Defined Atom StereoCenter Count</td><td headers="hd_h_ml145.t8_1_1_1_2 hd_h_ml145.t8_1_1_2_1 hd_h_ml145.t8_1_1_3_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0</td><td headers="hd_h_ml145.t8_1_1_1_2 hd_h_ml145.t8_1_1_2_2 hd_h_ml145.t8_1_1_3_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0</td></tr><tr><td headers="hd_h_ml145.t8_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Undefined Atom StereoCenter Count</td><td headers="hd_h_ml145.t8_1_1_1_2 hd_h_ml145.t8_1_1_2_1 hd_h_ml145.t8_1_1_3_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0</td><td headers="hd_h_ml145.t8_1_1_1_2 hd_h_ml145.t8_1_1_2_2 hd_h_ml145.t8_1_1_3_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0</td></tr><tr><td headers="hd_h_ml145.t8_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Defined Bond StereoCenter Count</td><td headers="hd_h_ml145.t8_1_1_1_2 hd_h_ml145.t8_1_1_2_1 hd_h_ml145.t8_1_1_3_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">2</td><td headers="hd_h_ml145.t8_1_1_1_2 hd_h_ml145.t8_1_1_2_2 hd_h_ml145.t8_1_1_3_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0</td></tr><tr><td headers="hd_h_ml145.t8_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Undefined Bond StereoCenter Count</td><td headers="hd_h_ml145.t8_1_1_1_2 hd_h_ml145.t8_1_1_2_1 hd_h_ml145.t8_1_1_3_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0</td><td headers="hd_h_ml145.t8_1_1_1_2 hd_h_ml145.t8_1_1_2_2 hd_h_ml145.t8_1_1_3_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0</td></tr><tr><td headers="hd_h_ml145.t8_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Covalently-Bonded Unit Count</td><td headers="hd_h_ml145.t8_1_1_1_2 hd_h_ml145.t8_1_1_2_1 hd_h_ml145.t8_1_1_3_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">1</td><td headers="hd_h_ml145.t8_1_1_1_2 hd_h_ml145.t8_1_1_2_2 hd_h_ml145.t8_1_1_3_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">1</td></tr></tbody></table></div></div></article><article data-type="table-wrap" id="figobml145t9"><div id="ml145.t9" class="table"><h3><span class="label">Table 8A</span><span class="title">Comparative Downstream and Selectivity assays by Assay Provider for CID2286812</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK50703/table/ml145.t9/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml145.t9_lrgtbl__"><table class="no_margin"><thead><tr><th id="hd_h_ml145.t9_1_1_1_1" colspan="5" rowspan="1" style="text-align:center;vertical-align:middle;"><div class="graphic"><img src="/books/NBK50703/bin/ml145fu18.jpg" alt="Image ml145fu18.jpg" /></div></th><th id="hd_h_ml145.t9_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">GPR35<br /><i><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2480" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-2480</a></i></th><th id="hd_h_ml145.t9_1_1_1_3" colspan="2" rowspan="1" style="text-align:center;vertical-align:middle;">pERK-ICW<br /><i><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463217" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-463217</a></i></th><th id="hd_h_ml145.t9_1_1_1_4" rowspan="2" colspan="1" headers="hd_h_ml145.t9_1_1_1_4" style="text-align:center;vertical-align:middle;">Vasopressin IIa Agonist<br /><i><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463201" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-463201</a></i><br /><br />%Activation @ 10 µM</th><th id="hd_h_ml145.t9_1_1_1_5" rowspan="2" colspan="1" headers="hd_h_ml145.t9_1_1_1_5" style="text-align:center;vertical-align:middle;">Vaopressin IIa Antagonist<br /><i><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463202" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-463202</a></i><br /><br />%Inhibition @ 10 µM</th></tr><tr><th headers="hd_h_ml145.t9_1_1_1_1" id="hd_h_ml145.t9_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CID</th><th headers="hd_h_ml145.t9_1_1_1_1" id="hd_h_ml145.t9_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SID</th><th headers="hd_h_ml145.t9_1_1_1_1" id="hd_h_ml145.t9_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">R1</th><th headers="hd_h_ml145.t9_1_1_1_1" id="hd_h_ml145.t9_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">R2</th><th headers="hd_h_ml145.t9_1_1_1_1" id="hd_h_ml145.t9_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">R3</th><th headers="hd_h_ml145.t9_1_1_1_2" id="hd_h_ml145.t9_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">IC50 (µM)</th><th headers="hd_h_ml145.t9_1_1_1_3" id="hd_h_ml145.t9_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">IC50 (µM)</th><th headers="hd_h_ml145.t9_1_1_1_3" id="hd_h_ml145.t9_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">n</th></tr></thead><tbody><tr><td headers="hd_h_ml145.t9_1_1_1_1 hd_h_ml145.t9_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2286812<sup>†</sup></td><td headers="hd_h_ml145.t9_1_1_1_1 hd_h_ml145.t9_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/87544499" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87544499</a></td><td headers="hd_h_ml145.t9_1_1_1_1 hd_h_ml145.t9_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><div class="graphic"><img src="/books/NBK50703/bin/ml145fu19.jpg" alt="Image ml145fu19.jpg" /></div></td><td headers="hd_h_ml145.t9_1_1_1_1 hd_h_ml145.t9_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">OH</td><td headers="hd_h_ml145.t9_1_1_1_1 hd_h_ml145.t9_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">COOH</td><td headers="hd_h_ml145.t9_1_1_1_2 hd_h_ml145.t9_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.061</td><td headers="hd_h_ml145.t9_1_1_1_3 hd_h_ml145.t9_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2.4±0.08</td><td headers="hd_h_ml145.t9_1_1_1_3 hd_h_ml145.t9_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">9</td><td headers="hd_h_ml145.t9_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.4/0.1; Avg. 0.21</td><td headers="hd_h_ml145.t9_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">31.9/28.1; Avg. 30.0</td></tr><tr><td headers="hd_h_ml145.t9_1_1_1_1 hd_h_ml145.t9_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2286888<sup>*</sup></td><td headers="hd_h_ml145.t9_1_1_1_1 hd_h_ml145.t9_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/87544500" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87544500</a></td><td headers="hd_h_ml145.t9_1_1_1_1 hd_h_ml145.t9_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><div class="graphic"><img src="/books/NBK50703/bin/ml145fu20.jpg" alt="Image ml145fu20.jpg" /></div></td><td headers="hd_h_ml145.t9_1_1_1_1 hd_h_ml145.t9_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">H</td><td headers="hd_h_ml145.t9_1_1_1_1 hd_h_ml145.t9_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">COOH</td><td headers="hd_h_ml145.t9_1_1_1_2 hd_h_ml145.t9_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.088</td><td headers="hd_h_ml145.t9_1_1_1_3 hd_h_ml145.t9_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1.6±0.10</td><td headers="hd_h_ml145.t9_1_1_1_3 hd_h_ml145.t9_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">6</td><td headers="hd_h_ml145.t9_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">−0.1/−0.2; Avg. −0.11</td><td headers="hd_h_ml145.t9_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">40.3/−3.4; Avg. 18.5</td></tr><tr><td headers="hd_h_ml145.t9_1_1_1_1 hd_h_ml145.t9_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1985027<sup>*</sup></td><td headers="hd_h_ml145.t9_1_1_1_1 hd_h_ml145.t9_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/87544525" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87544525</a></td><td headers="hd_h_ml145.t9_1_1_1_1 hd_h_ml145.t9_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">4-Et-Ph</td><td headers="hd_h_ml145.t9_1_1_1_1 hd_h_ml145.t9_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">H</td><td headers="hd_h_ml145.t9_1_1_1_1 hd_h_ml145.t9_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">COOH</td><td headers="hd_h_ml145.t9_1_1_1_2 hd_h_ml145.t9_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.081</td><td headers="hd_h_ml145.t9_1_1_1_3 hd_h_ml145.t9_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.64±0.27</td><td headers="hd_h_ml145.t9_1_1_1_3 hd_h_ml145.t9_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">6</td><td headers="hd_h_ml145.t9_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">−0.4/−0.6; Avg. −0.5</td><td headers="hd_h_ml145.t9_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">31.5/−45.1; Avg. −6.8</td></tr><tr><td headers="hd_h_ml145.t9_1_1_1_1 hd_h_ml145.t9_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1346721<sup>†</sup></td><td headers="hd_h_ml145.t9_1_1_1_1 hd_h_ml145.t9_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/87544505" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87544505</a></td><td headers="hd_h_ml145.t9_1_1_1_1 hd_h_ml145.t9_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">4-Me-Ph</td><td headers="hd_h_ml145.t9_1_1_1_1 hd_h_ml145.t9_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">COOH</td><td headers="hd_h_ml145.t9_1_1_1_1 hd_h_ml145.t9_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">H</td><td headers="hd_h_ml145.t9_1_1_1_2 hd_h_ml145.t9_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.35</td><td headers="hd_h_ml145.t9_1_1_1_3 hd_h_ml145.t9_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1.4±0.33</td><td headers="hd_h_ml145.t9_1_1_1_3 hd_h_ml145.t9_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">6</td><td headers="hd_h_ml145.t9_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">−0.3/0.2; Avg. −0.08</td><td headers="hd_h_ml145.t9_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">22.3/−5.9; Avg. 8.2</td></tr><tr><td headers="hd_h_ml145.t9_1_1_1_1 hd_h_ml145.t9_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">6202912<sup>†</sup></td><td headers="hd_h_ml145.t9_1_1_1_1 hd_h_ml145.t9_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/87544534" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87544534</a></td><td headers="hd_h_ml145.t9_1_1_1_1 hd_h_ml145.t9_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3,4-di-OMe-Ph</td><td headers="hd_h_ml145.t9_1_1_1_1 hd_h_ml145.t9_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">OH</td><td headers="hd_h_ml145.t9_1_1_1_1 hd_h_ml145.t9_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">COOH</td><td headers="hd_h_ml145.t9_1_1_1_2 hd_h_ml145.t9_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.17</td><td headers="hd_h_ml145.t9_1_1_1_3 hd_h_ml145.t9_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.52±0.20</td><td headers="hd_h_ml145.t9_1_1_1_3 hd_h_ml145.t9_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">9</td><td headers="hd_h_ml145.t9_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">−0.3/−0.2; Avg. −0.24</td><td headers="hd_h_ml145.t9_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">32.4/13.5; 22.9</td></tr><tr><td headers="hd_h_ml145.t9_1_1_1_1 hd_h_ml145.t9_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">6203183<sup>†</sup></td><td headers="hd_h_ml145.t9_1_1_1_1 hd_h_ml145.t9_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/87544535" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87544535</a></td><td headers="hd_h_ml145.t9_1_1_1_1 hd_h_ml145.t9_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><div class="graphic"><img src="/books/NBK50703/bin/ml145fu21.jpg" alt="Image ml145fu21.jpg" /></div></td><td headers="hd_h_ml145.t9_1_1_1_1 hd_h_ml145.t9_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">OH</td><td headers="hd_h_ml145.t9_1_1_1_1 hd_h_ml145.t9_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">COOH</td><td headers="hd_h_ml145.t9_1_1_1_2 hd_h_ml145.t9_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.34</td><td headers="hd_h_ml145.t9_1_1_1_3 hd_h_ml145.t9_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2.1±0.27</td><td headers="hd_h_ml145.t9_1_1_1_3 hd_h_ml145.t9_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">5</td><td headers="hd_h_ml145.t9_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">−0.2/0.6; Avg. 0.21</td><td headers="hd_h_ml145.t9_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">42.8/14.0; Avg. 28.4</td></tr></tbody></table></div><div class="tblwrap-foot"><div><dl class="temp-labeled-list small"><dl class="bkr_refwrap"><dt>†</dt><dd><div id="ml145.tfn7"><p class="no_margin">Not fully dissolved at 10 mM Nominal in DMSO</p></div></dd></dl><dl class="bkr_refwrap"><dt>*</dt><dd><div id="ml145.tfn8"><p class="no_margin">Clear solution at 10 mM Nominal in DMSO</p></div></dd></dl></dl></div></div></div></article><article data-type="table-wrap" id="figobml145t10"><div id="ml145.t10" class="table"><h3><span class="label">Table 8B</span><span class="title">Comparative Downstream and Selectivity assays by Assay Provider for CID1542103</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK50703/table/ml145.t10/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml145.t10_lrgtbl__"><table class="no_margin"><thead><tr><th id="hd_h_ml145.t10_1_1_1_1" colspan="4" rowspan="1" style="text-align:center;vertical-align:bottom;"><div class="graphic"><img src="/books/NBK50703/bin/ml145fu22.jpg" alt="Image ml145fu22.jpg" /></div></th><th id="hd_h_ml145.t10_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">GPR35<br /><i><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2480" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-2480</a></i></th><th id="hd_h_ml145.t10_1_1_1_3" colspan="2" rowspan="1" style="text-align:center;vertical-align:bottom;">pERK-ICW<br /><i><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463217" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-463217</a></i></th><th id="hd_h_ml145.t10_1_1_1_4" rowspan="2" colspan="1" headers="hd_h_ml145.t10_1_1_1_4" style="text-align:center;vertical-align:bottom;">Vasopressin IIa Agonist<br /><i><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463201" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-463201</a></i><br /><br />%Activation @ 10 µM</th><th id="hd_h_ml145.t10_1_1_1_5" rowspan="2" colspan="1" headers="hd_h_ml145.t10_1_1_1_5" style="text-align:center;vertical-align:bottom;">Vaopressin IIa Antagonist<br /><i><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/463202" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-463202</a></i><br /><br />%Inhibition @ 10 µM</th></tr><tr><th headers="hd_h_ml145.t10_1_1_1_1" id="hd_h_ml145.t10_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">CID</th><th headers="hd_h_ml145.t10_1_1_1_1" id="hd_h_ml145.t10_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">SID</th><th headers="hd_h_ml145.t10_1_1_1_1" id="hd_h_ml145.t10_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">R1</th><th headers="hd_h_ml145.t10_1_1_1_1" id="hd_h_ml145.t10_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">R2</th><th headers="hd_h_ml145.t10_1_1_1_2" id="hd_h_ml145.t10_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">IC50 (µM)</th><th headers="hd_h_ml145.t10_1_1_1_3" id="hd_h_ml145.t10_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">IC50(µM)</th><th headers="hd_h_ml145.t10_1_1_1_3" id="hd_h_ml145.t10_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">n</th></tr></thead><tbody><tr><td headers="hd_h_ml145.t10_1_1_1_1 hd_h_ml145.t10_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1542103<sup>*</sup></td><td headers="hd_h_ml145.t10_1_1_1_1 hd_h_ml145.t10_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/87544496" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87544496</a></td><td headers="hd_h_ml145.t10_1_1_1_1 hd_h_ml145.t10_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2-Me</td><td headers="hd_h_ml145.t10_1_1_1_1 hd_h_ml145.t10_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">4-Cl</td><td headers="hd_h_ml145.t10_1_1_1_2 hd_h_ml145.t10_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">4.72</td><td headers="hd_h_ml145.t10_1_1_1_3 hd_h_ml145.t10_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>32</td><td headers="hd_h_ml145.t10_1_1_1_3 hd_h_ml145.t10_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">9</td><td headers="hd_h_ml145.t10_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.0/−0.3; Avg. −0.16</td><td headers="hd_h_ml145.t10_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">13.6/1.6; Avg. 7.6</td></tr><tr><td headers="hd_h_ml145.t10_1_1_1_1 hd_h_ml145.t10_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1502520<sup>*</sup></td><td headers="hd_h_ml145.t10_1_1_1_1 hd_h_ml145.t10_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/87544497" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87544497</a></td><td headers="hd_h_ml145.t10_1_1_1_1 hd_h_ml145.t10_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2-Me</td><td headers="hd_h_ml145.t10_1_1_1_1 hd_h_ml145.t10_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">4-Me</td><td headers="hd_h_ml145.t10_1_1_1_2 hd_h_ml145.t10_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">5.01</td><td headers="hd_h_ml145.t10_1_1_1_3 hd_h_ml145.t10_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">17.7 ±0.10</td><td headers="hd_h_ml145.t10_1_1_1_3 hd_h_ml145.t10_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">9</td><td headers="hd_h_ml145.t10_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">−0.4/0.2; Avg. −0.08</td><td headers="hd_h_ml145.t10_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">40.4/33.3; Avg. 36.9</td></tr><tr><td headers="hd_h_ml145.t10_1_1_1_1 hd_h_ml145.t10_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">8056691<sup>*</sup></td><td headers="hd_h_ml145.t10_1_1_1_1 hd_h_ml145.t10_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/87544526" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87544526</a></td><td headers="hd_h_ml145.t10_1_1_1_1 hd_h_ml145.t10_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">H</td><td headers="hd_h_ml145.t10_1_1_1_1 hd_h_ml145.t10_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">4-Cl</td><td headers="hd_h_ml145.t10_1_1_1_2 hd_h_ml145.t10_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">9.76</td><td headers="hd_h_ml145.t10_1_1_1_3 hd_h_ml145.t10_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>32</td><td headers="hd_h_ml145.t10_1_1_1_3 hd_h_ml145.t10_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">5</td><td headers="hd_h_ml145.t10_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.3/−0.4; Avg. −0.08</td><td headers="hd_h_ml145.t10_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">−42.7/49.5; Avg. 3.4</td></tr><tr><td headers="hd_h_ml145.t10_1_1_1_1 hd_h_ml145.t10_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">8056641</td><td headers="hd_h_ml145.t10_1_1_1_1 hd_h_ml145.t10_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/87544527" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87544527</a></td><td headers="hd_h_ml145.t10_1_1_1_1 hd_h_ml145.t10_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">H</td><td headers="hd_h_ml145.t10_1_1_1_1 hd_h_ml145.t10_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">H</td><td headers="hd_h_ml145.t10_1_1_1_2 hd_h_ml145.t10_1_1_2_5 hd_h_ml145.t10_1_1_1_3 hd_h_ml145.t10_1_1_2_6 hd_h_ml145.t10_1_1_2_7 hd_h_ml145.t10_1_1_1_4 hd_h_ml145.t10_1_1_1_5" colspan="5" rowspan="1" style="text-align:center;vertical-align:top;">Not potent in GPR35 Primary assay so not tested for the downstream assays.</td></tr><tr><td headers="hd_h_ml145.t10_1_1_1_1 hd_h_ml145.t10_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">661907</td><td headers="hd_h_ml145.t10_1_1_1_1 hd_h_ml145.t10_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/87544523" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87544523</a></td><td headers="hd_h_ml145.t10_1_1_1_1 hd_h_ml145.t10_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">4-Me</td><td headers="hd_h_ml145.t10_1_1_1_1 hd_h_ml145.t10_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">4-Me</td><td headers="hd_h_ml145.t10_1_1_1_2 hd_h_ml145.t10_1_1_2_5 hd_h_ml145.t10_1_1_1_3 hd_h_ml145.t10_1_1_2_6 hd_h_ml145.t10_1_1_2_7 hd_h_ml145.t10_1_1_1_4 hd_h_ml145.t10_1_1_1_5" colspan="5" rowspan="1" style="text-align:center;vertical-align:top;">Not potent in GPR35 Primary assay so not tested for the downstream assays.</td></tr><tr><td headers="hd_h_ml145.t10_1_1_1_1 hd_h_ml145.t10_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2474060</td><td headers="hd_h_ml145.t10_1_1_1_1 hd_h_ml145.t10_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/87544537" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87544537</a></td><td headers="hd_h_ml145.t10_1_1_1_1 hd_h_ml145.t10_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">4-F</td><td headers="hd_h_ml145.t10_1_1_1_1 hd_h_ml145.t10_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2,4-Cl</td><td headers="hd_h_ml145.t10_1_1_1_2 hd_h_ml145.t10_1_1_2_5 hd_h_ml145.t10_1_1_1_3 hd_h_ml145.t10_1_1_2_6 hd_h_ml145.t10_1_1_2_7 hd_h_ml145.t10_1_1_1_4 hd_h_ml145.t10_1_1_1_5" colspan="5" rowspan="1" style="text-align:center;vertical-align:top;">Not potent in GPR35 Primary assay so not tested for the downstream assays.</td></tr></tbody></table></div><div class="tblwrap-foot"><div><dl class="temp-labeled-list small"><dl class="bkr_refwrap"><dt>*</dt><dd><div id="ml145.tfn9"><p class="no_margin">Clear solution at 10 mM nominal in DMSO</p></div></dd></dl></dl></div></div></div></article></div><div id="jr-scripts"><script src="/corehtml/pmc/jatsreader/ptpmc_3.22/js/libs.min.js"> </script><script src="/corehtml/pmc/jatsreader/ptpmc_3.22/js/jr.min.js"> </script></div></div>
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