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<script type="text/javascript" src="/corehtml/pmc/jatsreader/ptpmc_3.22/js/jr.boots.min.js"> </script><title>Identification of a small molecule that selectively activates alpha-synuclein translational expression - 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="Identification of a small molecule that selectively activates alpha-synuclein translational expression">
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<meta name="citation_publisher" content="National Center for Biotechnology Information (US)">
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<meta name="citation_date" content="2011/02/15">
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<meta name="citation_author" content="Nathan T Ross">
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<meta name="citation_author" content="Shailesh R Metkar">
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<meta name="citation_author" content="Hanh Le">
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<meta name="citation_author" content="Jason Burbank">
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<meta name="citation_author" content="Catherine Cahill">
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<meta name="citation_author" content="Andrew Germain">
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<meta name="citation_author" content="Lawrence MacPherson">
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<meta name="citation_author" content="Joshua Bittker">
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<meta name="citation_author" content="Michelle Palmer">
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<meta name="citation_author" content="Jack Rogers">
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<meta name="citation_author" content="Stuart L Schreiber">
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<meta name="citation_pmid" content="21634084">
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<meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK55072/">
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<meta name="DC.Title" content="Identification of a small molecule that selectively activates alpha-synuclein translational expression">
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<meta name="DC.Publisher" content="National Center for Biotechnology Information (US)">
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<meta name="DC.Contributor" content="Nathan T Ross">
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<meta name="DC.Contributor" content="Shailesh R Metkar">
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<meta name="DC.Contributor" content="Hanh Le">
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<meta name="DC.Contributor" content="Jason Burbank">
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<meta name="DC.Contributor" content="Catherine Cahill">
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<meta name="DC.Contributor" content="Andrew Germain">
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<meta name="DC.Contributor" content="Lawrence MacPherson">
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<meta name="DC.Contributor" content="Joshua Bittker">
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<meta name="DC.Contributor" content="Michelle Palmer">
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<meta name="DC.Contributor" content="Jack Rogers">
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<meta name="DC.Contributor" content="Stuart L Schreiber">
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<meta name="DC.Date" content="2011/02/15">
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<meta name="description" content="Alpha-synuclein is a protein implicated in the pathogenesis of neurodegenerative alpha-synucleinopathies including Parkinson’s disease (PD), the most prevalent movement disorder in humans. Evidence suggests that misregulation of alpha-synuclein translation plays a clear role in the pathology of this disease. We report the outcome of a high-throughput chemical library screen to identify novel, nontoxic, and selective small-molecule activators of alpha-synuclein expression, which will aid understanding of the role of alpha-synuclein in PD and other neurodegenerative diseases. Of the 303,224-screened compounds, 22 hits were identified as activators of alpha-synuclein expression and subsequently validated to confirm their activation activity and selectivity. One of these compounds (ML163) displayed greater than 100-fold selective activation of alpha-synuclein expression compared with a related system and is inactive in a control system at the highest tested dose. Our results indicate that ML163 has a drug-like structure with no obvious chemical liabilities, excellent solubility, and stability in aqueous conditions. This new probe should be very useful in future cell-based investigations and in vivo studies of alpha-synuclein expression.">
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<meta name="og:description" content="Alpha-synuclein is a protein implicated in the pathogenesis of neurodegenerative alpha-synucleinopathies including Parkinson’s disease (PD), the most prevalent movement disorder in humans. Evidence suggests that misregulation of alpha-synuclein translation plays a clear role in the pathology of this disease. We report the outcome of a high-throughput chemical library screen to identify novel, nontoxic, and selective small-molecule activators of alpha-synuclein expression, which will aid understanding of the role of alpha-synuclein in PD and other neurodegenerative diseases. Of the 303,224-screened compounds, 22 hits were identified as activators of alpha-synuclein expression and subsequently validated to confirm their activation activity and selectivity. One of these compounds (ML163) displayed greater than 100-fold selective activation of alpha-synuclein expression compared with a related system and is inactive in a control system at the highest tested dose. Our results indicate that ML163 has a drug-like structure with no obvious chemical liabilities, excellent solubility, and stability in aqueous conditions. This new probe should be very useful in future cell-based investigations and in vivo studies of alpha-synuclein expression.">
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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="_NBK55072_"><span class="title" itemprop="name">Identification of a small molecule that selectively activates alpha-synuclein translational expression</span></h1><p class="contribs">Ross NT, Metkar SR, Le H, et al.</p><p class="fm-aai"><a href="#_NBK55072_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>Alpha-synuclein is a protein implicated in the pathogenesis of neurodegenerative alpha-synucleinopathies including Parkinson’s disease (PD), the most prevalent movement disorder in humans. Evidence suggests that misregulation of alpha-synuclein translation plays a clear role in the pathology of this disease. We report the outcome of a high-throughput chemical library screen to identify novel, nontoxic, and selective small-molecule activators of alpha-synuclein expression, which will aid understanding of the role of alpha-synuclein in PD and other neurodegenerative diseases. Of the 303,224-screened compounds, 22 hits were identified as activators of alpha-synuclein expression and subsequently validated to confirm their activation activity and selectivity. One of these compounds (<a href="/pcsubstance/?term=ML163[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML163</a>) displayed greater than 100-fold selective activation of alpha-synuclein expression compared with a related system and is inactive in a control system at the highest tested dose. Our results indicate that <a href="/pcsubstance/?term=ML163[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML163</a> has a drug-like structure with no obvious chemical liabilities, excellent solubility, and stability in aqueous conditions. This new probe should be very useful in future cell-based investigations and <i>in vivo</i> studies of alpha-synuclein expression.</p></div><div class="h2"></div><p><b>Assigned Assay Grant #:</b> 1 R21 NS059434-01</p><p><b>Screening Center Name & PI:</b> Broad Institute Probe Development Center, Stuart Schreiber, PhD</p><p><b>Chemistry Center Name & PI:</b> Broad Institute Probe Development Center, Stuart Schreiber, PhD</p><p><b>Assay Submitter & Institution:</b> Jack Rogers, PhD. Massachusetts General Hospital</p><p><b>PubChem Summary Bioassay Identifier (AID):</b>
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<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1829" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1829</a></p><div id="ml163.s1"><h2 id="_ml163_s1_">Probe Structure & Characteristics</h2><div id="ml163.fu1" class="figure"><div class="graphic"><img src="/books/NBK55072/bin/ml163fu1.jpg" alt="Image ml163fu1" /></div></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml163tu1"><a href="/books/NBK55072/table/ml163.tu1/?report=objectonly" target="object" title="Table" class="img_link icnblk_img figpopup" rid-figpopup="figml163tu1" rid-ob="figobml163tu1"><img class="small-thumb" src="/books/NBK55072/table/ml163.tu1/?report=thumb" src-large="/books/NBK55072/table/ml163.tu1/?report=previmg" alt="Image " /></a><div class="icnblk_cntnt"><h4 id="ml163.tu1"><a href="/books/NBK55072/table/ml163.tu1/?report=objectonly" target="object" rid-ob="figobml163tu1">Table</a></h4></div></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml163tu2"><a href="/books/NBK55072/table/ml163.tu2/?report=objectonly" target="object" title="Table" class="img_link icnblk_img figpopup" rid-figpopup="figml163tu2" rid-ob="figobml163tu2"><img class="small-thumb" src="/books/NBK55072/table/ml163.tu2/?report=thumb" src-large="/books/NBK55072/table/ml163.tu2/?report=previmg" alt="Image " /></a><div class="icnblk_cntnt"><h4 id="ml163.tu2"><a href="/books/NBK55072/table/ml163.tu2/?report=objectonly" target="object" rid-ob="figobml163tu2">Table</a></h4></div></div></div><div id="ml163.s2"><h2 id="_ml163_s2_">Recommendations for scientific use of the probe</h2><p>The goal of this project is to identify novel small molecule probes that activate alpha-synuclein translational expression in dopaminergic neurons by targeting the 5′-untranslated region (5′UTR) stem-loop of alpha-synuclein as a novel probe for alpha-synuclein translational expression. The 5′UTR stem-loop of alpha-synuclein mRNA can interact with Iron Regulatory Protein-1 (IRP1), which upon interaction causes an increase in alpha-synuclein mRNA translation. Probes that can successfully increase alpha-synuclein expression levels as measured in a luciferase reporter assay will be further tested for specificity in cells lacking the alpha-synuclein 5′UTR stem-loop (H4-C) to confirm that the probes are acting through the intended target. Probe selectivity will be tested in cells containing the prion protein (PRP) 5′UTR mRNA stem-loop, which is also fused to a luciferase reporter. Finally, probe candidates will be verified using Western blot analysis in untransfected H4 neuroglioblastoma cells.</p><p>The probe (CID 1090779/<a href="/pcsubstance/?term=ML163[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML163</a>) described in this report selectively increases alpha-synuclein expression in transfected H4 neuroglioblastoma cells and also increases protein levels in native H4 cells without affecting actin levels. The probe has an EC<sub>50</sub> of 2 μM in the H4 primary screening cell line (H4-2a) and was found to be over 20-fold selective versus the H4-C cell line and over 95-fold versus the H4-PRP counterscreen cell lines. Additionally, a dose-response increase of alpha-synuclein levels was observed in H4 cells by Western blotting.</p><p>This probe is a first-in-class compound; there are no other published examples of compounds that act via this mechanism. In the future, this probe will be used to examine the effect of increasing alpha-synuclein levels in neurons and mouse models of Parkinson’s disease (PD). The probe may also serve as a positive control in future cell-based and animal-based experiments.</p></div><div id="ml163.s3"><h2 id="_ml163_s3_">1. Introduction</h2><div id="ml163.s4"><h3>Scientific Rationale</h3><p>Alpha-synuclein is an approximately 15 KDa protein implicated in the pathogenesis of neurodegenerative alpha-synucleinopathies (<a class="bibr" href="#ml163.r1" rid="ml163.r1">1</a>), including Parkinson’s disease (PD), the most prevalent movement disorder in humans. In these disorders, alpha-synuclein undergoes a conformational change and subsequent oligomerization, which causes a toxic gain of function, neurodegeneration, and deposition of alpha-synuclein aggregates in the form of Lewy bodies. Increased alpha-synuclein levels caused by gene duplication causes familial PD (<a class="bibr" href="#ml163.r2" rid="ml163.r2">2</a>), and GATA transcription factors (<a class="bibr" href="#ml163.r3" rid="ml163.r3">3</a>) directly regulate the PD-linked alpha-synuclein gene. In this regard, regulation of alpha-synuclein translation is physiologically relevant to Lewy body dementia brains, which exhibit lowered alpha-synuclein mRNA but higher insoluble protein. This suggests misregulation of alpha-synuclein translation in addition to protein clearance by chaperones (<a class="bibr" href="#ml163.r3" rid="ml163.r3">3</a>, <a class="bibr" href="#ml163.r4" rid="ml163.r4">4</a>). Consequently, our therapeutic strategy is to limit alpha-synuclein translation.</p><p>Alpha-synuclein translation is governed, at least in part, by an iron-responsive element (IRE) in the 5′ untranslated region (5′-UTR) stem-loop of alpha-synuclein mRNA. Iron-responsive elements (IREs) are RNA stem loops in the UTRs of ferritin and transferring-receptor mRNAs that are critical to iron homeostasis (<a class="bibr" href="#ml163.r5" rid="ml163.r5">5</a>, <a class="bibr" href="#ml163.r6" rid="ml163.r6">6</a>). However, the protein binding profile of the alpha-synuclein IRE is different from the canonical IREs of iron homeostasis. These findings reflect the sensitivity of this RNA sequence as a probe target. The IRE of alpha-synuclein is known to interact with iron regulatory protein-1 (IRP-1), which causes an increase in alpha-synuclein translation. There are currently no small molecules which activate the expression of alpha-synuclein by this mechanism (see <a href="#ml163.s24">Section 4.1</a>, <a class="figpopup" href="/books/NBK55072/table/ml163.t2/?report=objectonly" target="object" rid-figpopup="figml163t2" rid-ob="figobml163t2">Table 2</a>).</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml163t2"><a href="/books/NBK55072/table/ml163.t2/?report=objectonly" target="object" title="Table 2" class="img_link icnblk_img figpopup" rid-figpopup="figml163t2" rid-ob="figobml163t2"><img class="small-thumb" src="/books/NBK55072/table/ml163.t2/?report=thumb" src-large="/books/NBK55072/table/ml163.t2/?report=previmg" alt="Table 2. Search Strings and Databases Employed in the Prior Art Search." /></a><div class="icnblk_cntnt"><h4 id="ml163.t2"><a href="/books/NBK55072/table/ml163.t2/?report=objectonly" target="object" rid-ob="figobml163t2">Table 2</a></h4><p class="float-caption no_bottom_margin">Search Strings and Databases Employed in the Prior Art Search. </p></div></div><p>Through this screening campaign, we have discovered a probe that specifically activates this RNA-protein interaction and increases alpha-synuclein translation. This probe, N-naphthalen-1-yl-5-pyridin-4-yl-1,3,4-thiadiazol-2 amine, inhibits IRE driven alpha-synuclein translation with an EC<sub>50</sub> of 2 μM and is over 20-fold selective over cells lacking an IRE (H4-C) and over 95-fold selective over cells with an IRE from Scrapies prion protein (H4-PRP).</p></div></div><div id="ml163.s5"><h2 id="_ml163_s5_">2. Materials and Methods</h2><div id="ml163.s6"><h3>Materials and Reagents</h3><p>Dulbeccos modified essential medium (DMEM, catalog no. 12-614Q); FBS (catalog no.14-503E), L-glutamine (catalog no. 17-605E), penicillin/streptomycin (catalog no. 17-602E); phenol red free media (phenol red-free DMEM with 4.5 g/L D-glucose (catalog no. 12-917F) were purchased from Lonza (Portsmouth, NH). Geneticin (catalog no. 10131-027)) and alpha-synuclein ELISA kit KHB0061 were acquired from Invitrogen. Trypsin/EDTA (catalog no. 25-053-Cl) was purchased from Cellgro, and strophanthidine (catalog no. S6626-250MG, Lot 038K1036) was purchased from Sigma. Steady-Glo (catalog no. E2250) was purchased from Promega (Madison, WI). For the Western Blot secondary screen, penicillin/streptomycin was acquired from Bio-Whittaker (Walkerville, MD), mouse monoclonal anti-alpha-synuclein was purchased from BD Transduction Laboratories, and anti-beta-actin was acquired from Chemicon. 384-well plates (catalog no. 3570) were purchased from Corning and Falcon TC flasks (catalog no. 353112) were purchased from Becton Dickison (Waltham, MA).</p></div><div id="ml163.s7"><h3>2.1. Assays</h3><p>A summary listing of completed assays and corresponding PubChem AID numbers is provided in <a href="#ml163.app1">Appendix A</a> (<a class="figpopup" href="/books/NBK55072/table/ml163.t3/?report=objectonly" target="object" rid-figpopup="figml163t3" rid-ob="figobml163t3">Table A1</a>). Refer to <a href="#ml163.app2">Appendix B</a> for the detailed assay protocols.</p><div id="ml163.s8"><h4>2.1.1. Primary Screen for ASYN 5′-UTR IRE-driven Luciferase Expression in H4 Cells</h4><p>Stably transfected H4 neuroglioblastoma cells were grown to confluency in 35 mL complete DMEM with 4.5 g/L D-glucose supplemented with 10% FBS, 200 μM L-glutamine, 100 μM penicillin/streptomycin, and 200 μg/mL geneticin in a T175 TC flask in a TC incubator (37°C, 95% humidity, 5% CO<sub>2</sub>) (doubling time = 24 h). Cells were harvested by washing the monolayer quickly with 5 mL trypsin/EDTA (1X), aspirating, then adding 5 mL trypsin/EDTA and incubating for 5 min at 37°C, 95% humidity, 5% CO<sub>2</sub>. Then, 5 mL of complete media was added and mixed by pipetting up and down. Then, 10 mL of cell solution was aspirated and added to a conical tube; cells were pelleted for 3 min at 1000 rpm in a swinging bucket centrifuge. The media was aspirated, and the cell pellet was resuspended in 10 mL fresh complete media. Cells were then counted and expanded by plating 1×10<sup>6</sup> cells in 35 mL complete media per T175 flask (20×T175 flasks per 200 assay plate run) and allowing 72 h for cells to grow to confluency. Cells were harvested as above, but were resuspended in phenol-red free complete media (phenol red-free DMEM with 4.5 g/L D-glucose supplemented with 10% FBS, 200 μM L-glutamine, 100 μM penstrep, and 200 μg/mL geneticin, and then were diluted to 60,000 cells/mL in phenol-red free complete media prewarmed to 37°C in 1-liter sterile plastic bottles. Cells were plated into white TC treated 384-well plates (batch size = 200–220 assay plates) at 3,000 cells/well in 50 μL phenol-red free complete media using a Thermo MultiDrop Combi liquid dispenser and a sterilized dispensing cassette and stir bar, in a TC hood. Assay plates were loaded into 22 slot holders which were then placed into an online Liconic (STX 2201C) incubator set to 37°C, 95 % humidity, 5% CO<sub>2</sub>, and were incubated overnight.</p><p>Screening was performed using an open system (HiRes Biosolutions). Each run was initiated in CBIP (Broad Chemical Biology Informatics Platform) and scheduled with Cellario software (HiRes Biosolutions). Staubli arms moved plates from different instruments on the robotic system. Replicate assay plates were delidded and then pinned with 100 nL of 3.75 mM compound (final concentration = 7.5 μM) each with a 100 nL pin head using a MicroPin pin tool (HiRes Biosolutions). The positive control used in this assay was strophanthidine (Sigma, Cat. S6626-250MG, Lot038K1036), which was used in dose (12-point, 2-fold dilution, from 20 μM final [compound]), and was incorporated at the beginning and end of each run. Plates with compound were then relidded and returned to the Liconic incubator (STX 2201C) and incubated for 48 h at 37°C, 95% humidity, 5% CO<sub>2</sub>. Plates were then individually moved to a room temperature Liconic Carousel (LPX 220) and were allowed to temperature equilibrate for 30 min. Plates were then delidded and moved to a MultiDrop Combi liquid dispenser where 30 μL of 0.5X Steady-Glo was added (Steady-Glo was maintained at 4°C and warmed to room temperature using a Combi cassette with a 6-foot input line submerged in a room temperature water bath). Plates were then relidded, returned to the Liconic Carousel, and incubated at room temperature for 30 min. Plates were then delidded and moved to an Envision plate reader (Perkin Elmer); luminescence values were collected using with 100 ms read time per well. Plates were then relidded and discarded.</p></div><div id="ml163.s9"><h4>2.1.2. Primary ReTest in H4 Cells</h4><p>As described in the primary screen in H4 cells but the retest samples were cherry picked from the central library by Discovery Partners Inc.</p></div><div id="ml163.s10"><h4>2.1.3. Secondary Assay in H4-C Cells</h4><p>As described in the primary retest in H4 cells but using the H4-C cell line.</p></div><div id="ml163.s11"><h4>2.1.4. Secondary Assay in H4-PRP Cells</h4><p>As described in the primary retest in H4 cells but using the H4-PRP cell line.</p></div><div id="ml163.s12"><h4>2.1.5. Western Blot Secondary Screen in H4 Cells</h4><p>Human H4 neuroglioblastoma cells were cultured in DMEM supplemented with 10% FBS and penicillin/streptomycin. Cells were exposed to increasing concentrations of compound (0, 0.1, 1, 10, 100 μM) for 48 hours. Cytoplasmic protein lysates were prepared by homogenizing the cells in midRIPA buffer (25 mM Tris pH 7.4, 1% NP40, 0.5% sodium deoxycholate, 15 mM NaCl, protease inhibitors, RNase inhibitor, and 10 mM DTT). Western blotting for alpha-synuclein was performed using mouse monoclonal anti-alpha-synuclein, and anti-beta-actin. The blots were developed using chemiluminescence (Pierce), visualized with a PhosphoImager (BioRad, Hercules, CA), and the bands were quantified using QuantityOne software (BioRad).</p></div></div><div id="ml163.s13"><h3>2.2. Probe Chemical Characterization</h3><p>The probe <b>1</b> (CID 1090779/<a href="/pcsubstance/?term=ML163[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML163</a>) was purchased from ASINEX (ID BAS 07778946), however it could be synthesized starting from known hydrazide <b>2</b> in one step as shown in <a class="figpopup" href="/books/NBK55072/figure/ml163.f5/?report=objectonly" target="object" rid-figpopup="figml163f5" rid-ob="figobml163f5">Scheme 1</a>, based on literature precedent (<a class="bibr" href="#ml163.r7" rid="ml163.r7">7</a>). Treatment of <b>2</b> with 1-isothiocyanatonapthalene <b>3</b> would form the thiosemicarbazide <b>4</b>. Subsequent treatment with concentrated sulfuric acid at room temperature would result in closure to the probe compound (CID 1090779, CID 17410558, <a href="/pcsubstance/?term=ML163[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML163</a>). The probe was determined to have a solubility of <1 μM in PBS at room temperature. The probe and five analogs were submitted to the SMR collection (MLS002825686, MLS002725688, MLS002725689, MLS002729069, MLS002825679).</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml163f5" co-legend-rid="figlgndml163f5"><a href="/books/NBK55072/figure/ml163.f5/?report=objectonly" target="object" title="Scheme 1" class="img_link icnblk_img figpopup" rid-figpopup="figml163f5" rid-ob="figobml163f5"><img class="small-thumb" src="/books/NBK55072/bin/ml163f5.gif" src-large="/books/NBK55072/bin/ml163f5.jpg" alt="Scheme 1. Synthesis of Probe." /></a><div class="icnblk_cntnt" id="figlgndml163f5"><h4 id="ml163.f5"><a href="/books/NBK55072/figure/ml163.f5/?report=objectonly" target="object" rid-ob="figobml163f5">Scheme 1</a></h4><p class="float-caption no_bottom_margin">Synthesis of Probe. </p></div></div><p>The <sup>1</sup>H NMR spectra and LC-MS chromatograms of the probe (CID 1090779/<a href="/pcsubstance/?term=ML163[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML163</a>) and analogs are provided in <a href="#ml163.app3">Appendix C</a>.</p></div><div id="ml163.s14"><h3>2.3. Probe Preparation</h3><p>Not applicable. The probe (CID 1090779/<a href="/pcsubstance/?term=ML163[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML163</a>) was purchased from ASINEX (ID BAS 07778946).</p></div></div><div id="ml163.s15"><h2 id="_ml163_s15_">3. Results</h2><div id="ml163.s16"><h3>Probe Attributes</h3><ul><li class="half_rhythm"><div>EC<sub>50</sub> ≤ 10 μM in the alpha-synuclein IRE containing H4 neuroglioblastoma primary screening cell line (H4-2a).</div></li><li class="half_rhythm"><div>Greater than 10-fold selectivity as defined by: EC<sub>50</sub> ratios of the primary screening cell line (H4-2a) and both the non-IRE containing H4 cell line (H4-C) and the prion protein IRE containing H4 cell line (H4-PRP).</div></li><li class="half_rhythm"><div>Compound has a dose-response activation in native non-transfected H4 cells as observed by Western blot.</div></li></ul></div><div id="ml163.s17"><h3>3.1. Summary of Screening Results</h3><p>A high-throughput screen of 303,811 compounds (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1814" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1814</a>) was performed in duplicate in the alpha-synuclein IRE containing the H4 neuroglioblastoma primary screening cell line (H4-2a). Using a screening “hit” cutoff of ≥ 62% activation at a screening concentration of 7.5 μM, 21,690 compounds were identified as activators of IRE-driven luciferase expression (<a class="figpopup" href="/books/NBK55072/figure/ml163.f1/?report=objectonly" target="object" rid-figpopup="figml163f1" rid-ob="figobml163f1">Figure 1</a>). These active compounds were then clustered based upon chemical structure, which resulted in 2,600 clusters. The most active compound from each cluster was then cherry picked. Out of these 2,600 compounds, 2,336 were available and these were retested in eight-point dose (20 to 0.16 μM) in the primary screening cell line (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2003" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 2003</a>). Compounds that had EC<sub>50</sub> values ≤ 20 μM were considered to be successful primary retests (1272/2336, 54% retest rate).</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml163f1" co-legend-rid="figlgndml163f1"><a href="/books/NBK55072/figure/ml163.f1/?report=objectonly" target="object" title="Figure 1" class="img_link icnblk_img figpopup" rid-figpopup="figml163f1" rid-ob="figobml163f1"><img class="small-thumb" src="/books/NBK55072/bin/ml163f1.gif" src-large="/books/NBK55072/bin/ml163f1.jpg" alt="Figure 1. Critical Path for Probe Development." /></a><div class="icnblk_cntnt" id="figlgndml163f1"><h4 id="ml163.f1"><a href="/books/NBK55072/figure/ml163.f1/?report=objectonly" target="object" rid-ob="figobml163f1">Figure 1</a></h4><p class="float-caption no_bottom_margin">Critical Path for Probe Development. </p></div></div><p>Compounds were also tested in two counterscreens, the first of which was an H4 neuroglioblastoma cell line transfected with the same plasmid as the primary screening line, except the alpha-synuclein IRE stem-loop was removed from the 5′UTR (H4-C). The purpose of this counterscreen was to determine if active compounds were either cytotoxic, inhibiting luciferase activity, or causing global reductions in transcription/translation. Compounds were tested in dose, and hit criteria were the same as the primary retest. Of these, 1720 compounds were active (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2002" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 2002</a>). The second counterscreen was with H4 neuroglioblastoma cells transfected with a construct containing the prion protein IRE in the 5′UTR stem-loop upstream of luciferase (H4-PRP). The purpose of this counterscreen was to determine if compounds active in the primary retest were specific for the alpha-synuclein IRE stem-loop or if they were capable of binding a closely related IRE stem-loop. Compounds were tested in the same means as in the previous confirmatory screens using the same activity criteria. Of these, 1353 compounds were active (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1999" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1999</a>).</p><p>Compounds with at least 10-fold selectivity for the primary H4-2a screening line over the H4-C cell line and 10-fold selectivity for H4-2a over the H4-PRP cell line were prioritized for further development. Of these 22 compounds 13 were obtained as dry powders and were then retested in the primary and counterscreen assays (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2442" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 2442</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2453" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 2453</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2446" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 2446</a>). Three compounds met the selectivity criteria using dry powder samples. These compounds were then tested in non-transfected H4 neuroglioblastoma cells. Western blot analysis of alpha-synuclein protein levels in H4 cells showed a dose dependent increase corresponding to increasing doses of compound CID 1090779/<a href="/pcsubstance/?term=ML163[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML163</a> (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2627" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 2627</a>). This compound became probe series 1.</p></div><div id="ml163.s18"><h3>3.2. Dose Response Curves for Probe</h3><p>The probe (CID 1090779/<a href="/pcsubstance/?term=ML163[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML163</a>) met the defined probe criteria and has acceptable solubility and stability in aqueous conditions. The activity curves of the probe in the aforementioned cell lines and alpha-synuclein Western blot are depicted in <a class="figpopup" href="/books/NBK55072/figure/ml163.f2/?report=objectonly" target="object" rid-figpopup="figml163f2" rid-ob="figobml163f2">Figure 2</a> and <a class="figpopup" href="/books/NBK55072/figure/ml163.f3/?report=objectonly" target="object" rid-figpopup="figml163f3" rid-ob="figobml163f3">Figure 3</a>, respectively.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml163f2" co-legend-rid="figlgndml163f2"><a href="/books/NBK55072/figure/ml163.f2/?report=objectonly" target="object" title="Figure 2" class="img_link icnblk_img figpopup" rid-figpopup="figml163f2" rid-ob="figobml163f2"><img class="small-thumb" src="/books/NBK55072/bin/ml163f2.gif" src-large="/books/NBK55072/bin/ml163f2.jpg" alt="Figure 2. Concentration-dependent Activities of the Probe (CID 1090779/ML163) in Target and Counterscreen Assays." /></a><div class="icnblk_cntnt" id="figlgndml163f2"><h4 id="ml163.f2"><a href="/books/NBK55072/figure/ml163.f2/?report=objectonly" target="object" rid-ob="figobml163f2">Figure 2</a></h4><p class="float-caption no_bottom_margin">Concentration-dependent Activities of the Probe (CID 1090779/ML163) in Target and Counterscreen Assays. Activity curves for probe (CID 1090779/ML163): the alpha-synuclein IRE containing H4 neuroglioblastoma primary screening cell line (<i>A</i>); the H4 neuroglioblastoma <a href="/books/NBK55072/figure/ml163.f2/?report=objectonly" target="object" rid-ob="figobml163f2">(more...)</a></p></div></div><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml163f3" co-legend-rid="figlgndml163f3"><a href="/books/NBK55072/figure/ml163.f3/?report=objectonly" target="object" title="Figure 3" class="img_link icnblk_img figpopup" rid-figpopup="figml163f3" rid-ob="figobml163f3"><img class="small-thumb" src="/books/NBK55072/bin/ml163f3.gif" src-large="/books/NBK55072/bin/ml163f3.jpg" alt="Figure 3. Western Blot Results From Probe (CID 1090779/ML163) in H4 Cells (AID 2627)." /></a><div class="icnblk_cntnt" id="figlgndml163f3"><h4 id="ml163.f3"><a href="/books/NBK55072/figure/ml163.f3/?report=objectonly" target="object" rid-ob="figobml163f3">Figure 3</a></h4><p class="float-caption no_bottom_margin">Western Blot Results From Probe (CID 1090779/ML163) in H4 Cells (AID 2627). </p></div></div></div><div id="ml163.s19"><h3>3.3. Scaffold/Moiety Chemical Liabilities</h3><p>A search of PubChem for the probe compound (CID 1090779) revealed that the probe has been tested in 413 BioAssays and was confirmed as active in only four other assays. Therefore, the compound is not promiscuous. The compound has a drug-like structure with no obvious chemical liabilities that would be a concern.</p></div><div id="ml163.s20"><h3>3.4. SAR Tables</h3><p>The structures and the activities of the probe and analogs in the primary assay and the suite of secondary assays are summarized in <a class="figpopup" href="/books/NBK55072/table/ml163.t1/?report=objectonly" target="object" rid-figpopup="figml163t1" rid-ob="figobml163t1">Table 1</a>.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml163t1"><a href="/books/NBK55072/table/ml163.t1/?report=objectonly" target="object" title="Table 1" class="img_link icnblk_img figpopup" rid-figpopup="figml163t1" rid-ob="figobml163t1"><img class="small-thumb" src="/books/NBK55072/table/ml163.t1/?report=thumb" src-large="/books/NBK55072/table/ml163.t1/?report=previmg" alt="Table 1. SAR Analysis and Properties of the Alpha-synuclein Probe and Analogs." /></a><div class="icnblk_cntnt"><h4 id="ml163.t1"><a href="/books/NBK55072/table/ml163.t1/?report=objectonly" target="object" rid-ob="figobml163t1">Table 1</a></h4><p class="float-caption no_bottom_margin">SAR Analysis and Properties of the Alpha-synuclein Probe and Analogs. </p></div></div><p>Analogs to the probe were generated and tested in the H4-2a primary screening cell line in dose format. Screening results for the primary assay are shown in <a class="figpopup" href="/books/NBK55072/figure/ml163.f4/?report=objectonly" target="object" rid-figpopup="figml163f4" rid-ob="figobml163f4">Figure 4</a> and <a class="figpopup" href="/books/NBK55072/table/ml163.t2/?report=objectonly" target="object" rid-figpopup="figml163t2" rid-ob="figobml163t2">Table 2</a>. The highest level of activation was measured with compound 1 (CID 1090779/<a href="/pcsubstance/?term=ML163[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML163</a>), which has been designated as the probe for this series. Replacement of the napthalene with other aromatic groups results in a decrease in activity (CID 44826047). Several other aromatic groups were tested with various substituents and showed a range of activity (CID 1257201, CID 1864426, and CID 1294885). This probe will serve quite well in further cell-based investigations, but additional probes will be sought with good potency and improved physical properties.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml163f4" co-legend-rid="figlgndml163f4"><a href="/books/NBK55072/figure/ml163.f4/?report=objectonly" target="object" title="Figure 4" class="img_link icnblk_img figpopup" rid-figpopup="figml163f4" rid-ob="figobml163f4"><img class="small-thumb" src="/books/NBK55072/bin/ml163f4.gif" src-large="/books/NBK55072/bin/ml163f4.jpg" alt="Figure 4. Concentration-dependent Activity of Alpha-synuclein Analogs." /></a><div class="icnblk_cntnt" id="figlgndml163f4"><h4 id="ml163.f4"><a href="/books/NBK55072/figure/ml163.f4/?report=objectonly" target="object" rid-ob="figobml163f4">Figure 4</a></h4><p class="float-caption no_bottom_margin">Concentration-dependent Activity of Alpha-synuclein Analogs. Dose-response curves of five probe analogs: CID 1090779/ML163 (<i>A</i>), CID 44826047(<i>B</i>), CID 1257201(<i>C</i>), CID 1864426(<i>D</i>), CID 1294885(<i>E</i>). </p></div></div></div><div id="ml163.s21"><h3>3.5. Cellular Activity</h3><p>No specific assays were conducted to measure cellular toxicity or permeability of the probe or analogs. Since, however, the probe and analogs cause an increased readout of the reporter enzyme luciferase in the screening cell line, these compounds do not appear to be toxic at the concentrations measured over the 48-hour incubation time of the assays.</p></div><div id="ml163.s22"><h3>3.6. Profiling Assays</h3><p>No profiling assays were conducted.</p></div></div><div id="ml163.s23"><h2 id="_ml163_s23_">4. Discussion</h2><div id="ml163.s24"><h3>4.1. Comparison to Existing Art and How the New Probe is an Improvement</h3><p>The literature searches described in <a class="figpopup" href="/books/NBK55072/table/ml163.t2/?report=objectonly" target="object" rid-figpopup="figml163t2" rid-ob="figobml163t2">Table 2</a> were unable to find any small molecules that activated the 5′UTR of alpha synuclein mRNA.</p></div><div id="ml163.s25"><h3>4.2. Mechanism of Action Studies</h3><p>No mechanism of action studies were conducted.</p></div><div id="ml163.s26"><h3>4.3. Planned Future Studies</h3><p>The probe and analogs will be tested in primary neuronal cells to confirm their activity. Furthermore, they will be used in mouse models of Parkinson’s disease</p></div></div><div id="ml163.reflist"><h2 id="_ml163_reflist_">5. References</h2><dl class="temp-labeled-list"><dl class="bkr_refwrap"><dt>1.</dt><dd><div class="bk_ref" id="ml163.r1">Uéda K, Fukushima H, Masliah E, Xia Y, Iwai A, Yoshimoto M, Otero DA, Kondo J, Ihara Y, Saitoh T. Molecular cloning of cDNA encoding an unrecognized component of amyloid in Alzheimer disease. <span><span class="ref-journal">Proc Natl Acad Sci USA. </span>1993 Dec 1;<span class="ref-vol">90</span>:12282–12286.</span> [<a href="/pmc/articles/PMC47966/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC47966</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/8248242" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 8248242</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>2.</dt><dd><div class="bk_ref" id="ml163.r2">Singleton A, Gwinn-Hardy K. Parkinson’s disease and dementia with Lewy bodies: a difference in dose. <span><span class="ref-journal">Lancet. </span>2004 Sep 25-Oct 1;<span class="ref-vol">364</span>(9440):1105–1107.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/15451205" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 15451205</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>3.</dt><dd><div class="bk_ref" id="ml163.r3">Scherzer CR, Grass JA, Liao Z, Pepivani I, Zheng B, Eklund AC, Ney PA, Ng J, McGoldrick M, Mollenhauer B, et al. GATA transcription factors directly regulate the Parkinson’s disease-linked gene alpha-synuclein. <span><span class="ref-journal">Proc Natl Acad Sci USA. </span>2008 Aug 5;<span class="ref-vol">105</span>(31):10907–10912.</span> Epub 2008 Jul 31. [<a href="/pmc/articles/PMC2504800/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2504800</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/18669654" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18669654</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>4.</dt><dd><div class="bk_ref" id="ml163.r4">Cantuti-Castelvetri I, Klucken J, Ingelsson M, Ramasamy K, McLean PJ, Frosch MP, Hyman BT, Standaert DG. Alpha-synuclein and chaperones in dementia with Lewy bodies. <span><span class="ref-journal">J Neuropathol Exp Neurol. </span>2005 Dec;<span class="ref-vol">64</span>(12):1058–1066.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/16319716" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 16319716</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>5.</dt><dd><div class="bk_ref" id="ml163.r5">Thomson AM, Rogers JT, Leedman PJ. Iron-regulatory proteins, iron-responsive elements and ferritin mRNA translation. <span><span class="ref-journal">Int J Biochem Cell Biol. </span>1999 Oct;<span class="ref-vol">31</span>(10):1139–1152.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/10582343" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 10582343</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>6.</dt><dd><div class="bk_ref" id="ml163.r6">Thomson AM, Cahill CM, Cho HH, Kassachau KD, Epis MR, Bridges KR, Leedman PJ, Rogers JT. The acute box cis-element in human heavy ferritin mRNA 5′-untranslated region is a unique translation enhancer that binds poly(C)-binding proteins. <span><span class="ref-journal">J Biol Chem. </span>2005 Aug 26;<span class="ref-vol">280</span>(34):30032–30045.</span> Epub 2005 Jun 20. [<a href="https://pubmed.ncbi.nlm.nih.gov/15967798" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 15967798</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>7.</dt><dd><div class="bk_ref" id="ml163.r7">Oruç EE, Rollas S, Kandemirli F, Shvets N, Dimoglo AS. 1,3,4-thiadiazole derivatives: Synthesis, structure elucidation, and structure-antituberculosis activity relationship investigation. <span><span class="ref-journal">J Med Chem. </span>2004 Dec 30;<span class="ref-vol">47</span>(27):6760–6767.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/15615525" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 15615525</span></a>]</div></dd></dl></dl></div><div id="ml163.appgroup1"><h2 id="_ml163_appgroup1_">6. Appendices</h2><div id="ml163.app1"><h3>Appendix A. Compound Characterization</h3><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml163t3"><a href="/books/NBK55072/table/ml163.t3/?report=objectonly" target="object" title="Table A1" class="img_link icnblk_img figpopup" rid-figpopup="figml163t3" rid-ob="figobml163t3"><img class="small-thumb" src="/books/NBK55072/table/ml163.t3/?report=thumb" src-large="/books/NBK55072/table/ml163.t3/?report=previmg" alt="Table A1. Summary of Completed Assays and AIDs." /></a><div class="icnblk_cntnt"><h4 id="ml163.t3"><a href="/books/NBK55072/table/ml163.t3/?report=objectonly" target="object" rid-ob="figobml163t3">Table A1</a></h4><p class="float-caption no_bottom_margin">Summary of Completed Assays and AIDs. </p></div></div></div><div id="ml163.app2"><h3>Appendix B. Detailed Assay Protocols</h3><div id="ml163.s31"><h4>Primary Screen in H4 Cells</h4><ol><li class="half_rhythm"><div>Grow stably transfected H4 neuroglioblastoma cells to confluency in 35 mL complete media (DMEM with 4.5 g/L D-glucose [Lonza, 12-614Q] supplemented with 10% FBS [Lonza, 14-503E], 200 μM L-glutamine [Lonza, 17-605E], 100 μM penstrep [Lonza, 17-602E] and 200 μg/mL geneticin [Invitrogen, 10131-027]) in a T175 TC flask (BD Falcon, 353112).</div></li><li class="half_rhythm"><div>Incubate in a TC incubator (37°C, 95% humidity, 5 % CO<sub>2</sub>) (doubling time = 24 h).</div></li><li class="half_rhythm"><div>Harvest cells by washing the monolayer quickly with 5 mL trypsin/EDTA (1X, Cellgro, 25-053-Cl). Aspirate, then add 5 mL trypsin/EDTA.</div></li><li class="half_rhythm"><div>Incubate for 5 min (37°C, 95% humidity, 5% CO<sub>2)</sub>.</div></li><li class="half_rhythm"><div>Add 5 mL of complete media and mix by pipetting up and down. Aspirate 10 mL of cell solution and add to a conical tube.</div></li><li class="half_rhythm"><div>Pellet the cells for 3 min at 1000 rpm in a swinging bucket centrifuge.</div></li><li class="half_rhythm"><div>Aspirate the media and resuspend the cell pellet in 10 mL fresh complete media.</div></li><li class="half_rhythm"><div>Count the cells and expand by plating 1×10<sup>6</sup> cells in 35 mL complete media per T175 flask (20×T175 flasks per 200 assay plate run). Allowing 72 h for cells to grow to confluency.</div></li><li class="half_rhythm"><div>Harvest the cells as above, but resuspend cells in phenol-red free complete media (phenol red-free DMEM with 4.5 g/L D-glucose [Lonza, 12-917F] supplemented with 10% FBS [Lonza, 14-503E], 200 μM L-glutamine [Lonza, 17-605E], 100 μM penstrep [Lonza, 17-602E], and 200 μg/mL geneticin [Invitrogen, 10131-027]).</div></li><li class="half_rhythm"><div>Dilute resuspended cells to 60,000 cells/mL in phenol-red free complete media prewarmed to 37°C in 1-L sterile plastic bottles.</div></li><li class="half_rhythm"><div>Plate cells into white TC-treated 384-well plates (Corning, 3570; batch size = 200–220 assay plates) at 3,000 cells/well in 50-μL phenol-red free complete media using a Thermo MultiDrop Combi liquid dispenser, a sterilized dispensing cassette, stir bar in a TC hood.</div></li><li class="half_rhythm"><div>Load assay plates into 22 slot holder and place into an online Liconic (STX 2201C) incubator set to 37°C, 95% humidity, 5% CO<sub>2.</sub> Incubate plates overnight.</div></li><li class="half_rhythm"><div>Perform screening using an open system (HiRes Biosolutions). Initiate each run in Broad Chemical Biology Informatics Platform (CBIP) and schedule with Cellario software (HiRes Biosolutions). Staubli arms moved plates from different instruments on the robotic system.</div></li><li class="half_rhythm"><div>Delid replicate assay plates and pin with 100 nL of 3.75 μM compound (final concentration = 7.5 μM) each with a 100 nL pin head using a MicroPin pin tool (HiRes Biosolutions).</div></li><li class="half_rhythm"><div>Use strophanthidine (Sigma, Cat. No. S6626-250MG, Lot No.038K1036) as the positive control in dose (12-point, 2-fold dilution, from 20 μM final [compound]) at the beginning and end of each run. Relid plates with compound and return to the Liconic incubator (STX 2201C).</div></li><li class="half_rhythm"><div>Incubate plates for 48h at 37°C, 95% humidity, 5% CO<sub>2</sub>. Move plates individually to a room temperature Liconic Carousel (LPX 220) and allow to temperature equilibrate for 30 min.</div></li><li class="half_rhythm"><div>Delid plates and move to a MultiDrop Combi liquid dispenser and add 30 μL of 0.5X Steady-Glo (Promega, E2250). Maintain Steady-Glo at 4°C and warm to room temperature using a Combi cassette with a 6-foot input line submerged in a room temperature water bath.</div></li><li class="half_rhythm"><div>Relid plates and return to the Liconic Carousel. Incubate at room temperature for 30 min.</div></li><li class="half_rhythm"><div>Delid plates and move to an Envision plate reader (Perkin Elmer). Collect luminescence values using with 100 ms read time per well. Relid the plates and discard.</div></li></ol></div><div id="ml163.s32"><h4>Western Blot Secondary Screen in H4 Cells</h4><ol><li class="half_rhythm"><div>Culture human H4 neuroglioblastoma cells in Dulbeccos modified essential medium (Invitrogen) supplemented with 10% FBS (Invitrogen) and penicillin/streptomycin (Bio-Whittaker, Walkerville, MD).</div></li><li class="half_rhythm"><div>Expose cells to increasing concentrations of compound (0, 0.1, 1, 10, 100 μM) for 48 hours.</div></li><li class="half_rhythm"><div>Prepare cytoplasmic protein lysates by homogenizing the cells in midRIPA buffer (25 mM Tris pH 7.4, 1% NP40, 0.5% sodium deoxycholate, 15 mM NaCl, protease inhibitors, RNase inhibitor and 10 mM DTT).</div></li><li class="half_rhythm"><div>Perform Western blotting for alpha-synuclein using mouse monoclonal anti-alpha-synuclein (BD Transduction Laboratories), and anti-beta-actin (Chemicon). Develop the blots using chemiluminescence (PIERCE) and visualize with a PhosphoImager (BioRad, Hercules, CA). Quantify the bands using QuantityOne software (BioRad).</div></li></ol></div></div><div id="ml163.app3"><h3>Appendix C. NMR and LC Data of Probe and Analogs</h3><div id="ml163.fu2" class="figure bk_fig"><div class="graphic"><img src="/books/NBK55072/bin/ml163fu8.jpg" alt="1H NMR Spectra of Probe (CID 1090779/ML163)." /></div><h3><span class="title"><sup>1</sup>H NMR Spectra of Probe (CID 1090779/<a href="/pcsubstance/?term=ML163[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML163</a>)</span></h3></div><div id="ml163.fu3" class="figure bk_fig"><div class="graphic"><img src="/books/NBK55072/bin/ml163fu9.jpg" alt="LC/MS Chromatogram of Probe (CID 1090779/ML163)." /></div><h3><span class="title">LC/MS Chromatogram of Probe (CID 1090779/<a href="/pcsubstance/?term=ML163[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML163</a>)</span></h3></div><div id="ml163.fu4" class="figure bk_fig"><div class="graphic"><img src="/books/NBK55072/bin/ml163fu10.jpg" alt="1H NMR Spectra of Analog CID 44826047." /></div><h3><span class="title"><sup>1</sup>H NMR Spectra of Analog CID 44826047</span></h3></div><div id="ml163.fu5" class="figure bk_fig"><div class="graphic"><img src="/books/NBK55072/bin/ml163fu11.jpg" alt="LC/MS Chromatogram of Analog CID 44826047." /></div><h3><span class="title">LC/MS Chromatogram of Analog CID 44826047</span></h3></div><div id="ml163.fu6" class="figure bk_fig"><div class="graphic"><img src="/books/NBK55072/bin/ml163fu12.jpg" alt="1H NMR Spectra of Analog CID 1257201." /></div><h3><span class="title"><sup>1</sup>H NMR Spectra of Analog CID 1257201</span></h3></div><div id="ml163.fu7" class="figure bk_fig"><div class="graphic"><img src="/books/NBK55072/bin/ml163fu13.jpg" alt="LC/MS Chromatogram of Analog CID 1257201." /></div><h3><span class="title">LC/MS Chromatogram of Analog CID 1257201</span></h3></div><div id="ml163.fu8" class="figure bk_fig"><div class="graphic"><img src="/books/NBK55072/bin/ml163fu14.jpg" alt="1H NMR Spectra of Analog CID 1864426." /></div><h3><span class="title"><sup>1</sup>H NMR Spectra of Analog CID 1864426</span></h3></div><div id="ml163.fu9" class="figure bk_fig"><div class="graphic"><img src="/books/NBK55072/bin/ml163fu15.jpg" alt="LC/MS Chromatogram of Analog CID 1864426." /></div><h3><span class="title">LC/MS Chromatogram of Analog CID 1864426</span></h3></div><div id="ml163.fu10" class="figure bk_fig"><div class="graphic"><img src="/books/NBK55072/bin/ml163fu16.jpg" alt="1H NMR Spectra of Analog CID 1294885." /></div><h3><span class="title"><sup>1</sup>H NMR Spectra of Analog CID 1294885</span></h3></div><div id="ml163.fu11" class="figure bk_fig"><div class="graphic"><img src="/books/NBK55072/bin/ml163fu17.jpg" alt="LC/MS Chromatogram of Analog CID 1294885." /></div><h3><span class="title">LC/MS Chromatogram of Analog CID 1294885</span></h3></div></div><div id="ml163.app4"><h3>Appendix D. Compounds Submitted to BioFocus</h3><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml163t4"><a href="/books/NBK55072/table/ml163.t4/?report=objectonly" target="object" title="Table A2" class="img_link icnblk_img figpopup" rid-figpopup="figml163t4" rid-ob="figobml163t4"><img class="small-thumb" src="/books/NBK55072/table/ml163.t4/?report=thumb" src-large="/books/NBK55072/table/ml163.t4/?report=previmg" alt="Table A2. Probe and Analog Information." /></a><div class="icnblk_cntnt"><h4 id="ml163.t4"><a href="/books/NBK55072/table/ml163.t4/?report=objectonly" target="object" rid-ob="figobml163t4">Table A2</a></h4><p class="float-caption no_bottom_margin">Probe and Analog Information. </p></div></div></div></div><div id="bk_toc_contnr"></div></div></div><div class="fm-sec"><h2 id="_NBK55072_pubdet_">Publication Details</h2><h3>Author Information and Affiliations</h3><p class="contrib-group"><h4>Authors</h4><span itemprop="author">Nathan T Ross</span>,<sup>1</sup> <span itemprop="author">Shailesh R Metkar</span>,<sup>1</sup> <span itemprop="author">Hanh Le</span>,<sup>1</sup> <span itemprop="author">Jason Burbank</span>,<sup>1</sup> <span itemprop="author">Catherine Cahill</span>,<sup>2</sup> <span itemprop="author">Andrew Germain</span>,<sup>1</sup> <span itemprop="author">Lawrence MacPherson</span>,<sup>1</sup> <span itemprop="author">Joshua Bittker</span>,<sup>1</sup> <span itemprop="author">Michelle Palmer</span>,<sup>1</sup> <span itemprop="author">Jack Rogers</span>,<sup>2</sup> and <span itemprop="author">Stuart L Schreiber</span><sup>1,3</sup>.</p><h4>Affiliations</h4><div class="affiliation"><sup>1</sup>
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The Broad Institute Probe Development Center, Cambridge, MA</div><div class="affiliation"><sup>2</sup>
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Massachussetts General Hospital, Charlestown, MA</div><div class="affiliation"><sup>3</sup>
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Howard Hughes Medical Institute, Chemistry & Chemical Biology, Harvard University, Cambridge, MA</div><h3>Publication History</h3><p class="small">Received: <span itemprop="datePublished">February 4, 2011</span>; Last Update: <span itemprop="dateModified">February 15, 2011</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>Ross NT, Metkar SR, Le H, et al. Identification of a small molecule that selectively activates alpha-synuclein translational expression. 2011 Feb 4 [Updated 2011 Feb 15]. 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/ml164/?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/ml162/?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="figobml163fu1"><div id="ml163.fu1" class="figure"><div class="graphic"><img data-src="/books/NBK55072/bin/ml163fu1.jpg" alt="Image ml163fu1" /></div></div></article><article data-type="table-wrap" id="figobml163tu1"><div id="ml163.tu1" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK55072/table/ml163.tu1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml163.tu1_lrgtbl__"><table><tbody><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">IUPAC Chemical Name</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><i>N</i>-naphthalen-1-yl-5-pyridin-4-yl-1,3,4-thiadiazol-2-amine</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">PubChem CID/ML#</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CID 1090779/<a href="/pcsubstance/?term=ML163[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML163</a></td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Molecular Weight</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">304.37</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Molecular Formula</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">C17H12N4S</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">XlogP</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">4.1</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">H-Bond Donor</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">1</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">H-Bond Acceptor</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">4</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Rotatable Bond Count</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">3</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Exact Mass</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">304.08</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Topological Polar Surface Area</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">78.9</td></tr></tbody></table></div></div></article><article data-type="table-wrap" id="figobml163tu2"><div id="ml163.tu2" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK55072/table/ml163.tu2/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml163.tu2_lrgtbl__"><table><thead><tr><th id="hd_h_ml163.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CID/ML#</th><th id="hd_h_ml163.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Target Name</th><th id="hd_h_ml163.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">EC<sub>50</sub> (μM) [SID, AID]</th><th id="hd_h_ml163.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Anti-target Name(s)</th><th id="hd_h_ml163.tu2_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_ml163.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Fold Selective</th><th id="hd_h_ml163.tu2_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> (μM) [SID, AID]</th></tr></thead><tbody><tr><td headers="hd_h_ml163.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">1090779/163</td><td headers="hd_h_ml163.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">α-synuclein<br />5′-UTR</td><td headers="hd_h_ml163.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">2 [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/87218802" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 87218802</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2442" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 2442</a>]</td><td headers="hd_h_ml163.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Prion 5′-UTR (H4-PRP)</td><td headers="hd_h_ml163.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">>200 [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/17410558" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 17410558</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2446" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 2446</a>]</td><td headers="hd_h_ml163.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">>100</td><td headers="hd_h_ml163.tu2_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">H4-C: 40 [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/17410558" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 17410558</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2453" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 2453</a>]</td></tr></tbody></table></div></div></article><article data-type="table-wrap" id="figobml163t2"><div id="ml163.t2" class="table"><h3><span class="label">Table 2</span><span class="title">Search Strings and Databases Employed in the Prior Art Search</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK55072/table/ml163.t2/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml163.t2_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml163.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Search String</th><th id="hd_h_ml163.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Database</th><th id="hd_h_ml163.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">No. of References Found</th></tr></thead><tbody><tr><td headers="hd_h_ml163.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">“alpha synuclein”</td><td headers="hd_h_ml163.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">SciFinder</td><td headers="hd_h_ml163.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">7627</td></tr><tr><td headers="hd_h_ml163.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">“alpha synuclein + translation”</td><td headers="hd_h_ml163.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">SciFinder</td><td headers="hd_h_ml163.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">1054</td></tr><tr><td headers="hd_h_ml163.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">“alpha synuclein + expression activation”</td><td headers="hd_h_ml163.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">SciFinder</td><td headers="hd_h_ml163.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">304</td></tr><tr><td headers="hd_h_ml163.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">“alpha synuclein + 5′UTR”</td><td headers="hd_h_ml163.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">SciFinder</td><td headers="hd_h_ml163.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">8</td></tr><tr><td headers="hd_h_ml163.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">“alpha synuclein + 5′UTR activation”</td><td headers="hd_h_ml163.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">SciFinder</td><td headers="hd_h_ml163.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0</td></tr><tr><td headers="hd_h_ml163.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">“alpha synuclein + translation activation”</td><td headers="hd_h_ml163.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Reaxys</td><td headers="hd_h_ml163.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0</td></tr><tr><td headers="hd_h_ml163.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">“alpha synuclein + expression activation”</td><td headers="hd_h_ml163.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Reaxys</td><td headers="hd_h_ml163.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0</td></tr><tr><td headers="hd_h_ml163.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">“alpha synuclein + 5′UTR”</td><td headers="hd_h_ml163.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Reaxys</td><td headers="hd_h_ml163.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">0</td></tr></tbody></table></div></div></article><article data-type="fig" id="figobml163f5"><div id="ml163.f5" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK55072/bin/ml163f5.jpg" alt="Scheme 1. Synthesis of Probe." /></div><h3><span class="label">Scheme 1</span><span class="title">Synthesis of Probe</span></h3></div></article><article data-type="fig" id="figobml163f1"><div id="ml163.f1" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK55072/bin/ml163f1.jpg" alt="Figure 1. Critical Path for Probe Development." /></div><h3><span class="label">Figure 1</span><span class="title">Critical Path for Probe Development</span></h3></div></article><article data-type="fig" id="figobml163f2"><div id="ml163.f2" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK55072/bin/ml163f2.jpg" alt="Figure 2. Concentration-dependent Activities of the Probe (CID 1090779/ML163) in Target and Counterscreen Assays." /></div><h3><span class="label">Figure 2</span><span class="title">Concentration-dependent Activities of the Probe (CID 1090779/<a href="/pcsubstance/?term=ML163[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML163</a>) in Target and Counterscreen Assays</span></h3><div class="caption"><p>Activity curves for probe (CID 1090779/<a href="/pcsubstance/?term=ML163[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML163</a>): the alpha-synuclein IRE containing H4 neuroglioblastoma primary screening cell line (<i>A</i>); the H4 neuroglioblastoma cell line with the IRE stem-loop removed from the 5′UTR of alpha-synuclein (<i>B</i>); the H4 neuroglioblastoma cells containing the prion protein IRE containing in the 5′UTR upstream of luciferase (<i>C</i>).</p></div></div></article><article data-type="fig" id="figobml163f3"><div id="ml163.f3" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK55072/bin/ml163f3.jpg" alt="Figure 3. Western Blot Results From Probe (CID 1090779/ML163) in H4 Cells (AID 2627)." /></div><h3><span class="label">Figure 3</span><span class="title">Western Blot Results From Probe (CID 1090779/<a href="/pcsubstance/?term=ML163[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML163</a>) in H4 Cells (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2627" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 2627</a>)</span></h3></div></article><article data-type="table-wrap" id="figobml163t1"><div id="ml163.t1" class="table"><h3><span class="label">Table 1</span><span class="title">SAR Analysis and Properties of the Alpha-synuclein Probe and Analogs</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK55072/table/ml163.t1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml163.t1_lrgtbl__"><table class="no_margin"><thead><tr><th id="hd_h_ml163.t1_1_1_1_1" colspan="5" rowspan="1" style="text-align:center;vertical-align:middle;">SAR Analysis for Alpha-synuclein</th><th id="hd_h_ml163.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><div class="graphic"><img src="/books/NBK55072/bin/ml163fu2.jpg" alt="Image ml163fu2.jpg" /></div></th><th id="hd_h_ml163.t1_1_1_1_3" colspan="4" rowspan="1" style="text-align:center;vertical-align:middle;">Potency (μM) mean ± S.E.M. (n=replicates)</th><th id="hd_h_ml163.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Target to Antitarget Fold Selectivity</th></tr><tr><th headers="hd_h_ml163.t1_1_1_1_1" id="hd_h_ml163.t1_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Entry</th><th headers="hd_h_ml163.t1_1_1_1_1" id="hd_h_ml163.t1_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CID</th><th headers="hd_h_ml163.t1_1_1_1_1" id="hd_h_ml163.t1_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SID</th><th headers="hd_h_ml163.t1_1_1_1_1" id="hd_h_ml163.t1_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Broad No.</th><th headers="hd_h_ml163.t1_1_1_1_1" id="hd_h_ml163.t1_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><sup>*</sup></th><th headers="hd_h_ml163.t1_1_1_1_2" id="hd_h_ml163.t1_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">R</th><th headers="hd_h_ml163.t1_1_1_1_3" id="hd_h_ml163.t1_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">n</th><th headers="hd_h_ml163.t1_1_1_1_3" id="hd_h_ml163.t1_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Target</th><th headers="hd_h_ml163.t1_1_1_1_3" id="hd_h_ml163.t1_1_1_2_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">n</th><th headers="hd_h_ml163.t1_1_1_1_3" id="hd_h_ml163.t1_1_1_2_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Control</th><th headers="hd_h_ml163.t1_1_1_1_4" id="hd_h_ml163.t1_1_1_2_11" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Prion</th></tr></thead><tbody><tr><td headers="hd_h_ml163.t1_1_1_1_1 hd_h_ml163.t1_1_1_2_1" rowspan="2" colspan="1" style="text-align:center;vertical-align:middle;">1</td><td headers="hd_h_ml163.t1_1_1_1_1 hd_h_ml163.t1_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1090779</td><td headers="hd_h_ml163.t1_1_1_1_1 hd_h_ml163.t1_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">87218802</td><td headers="hd_h_ml163.t1_1_1_1_1 hd_h_ml163.t1_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BRD-K32644160</td><td headers="hd_h_ml163.t1_1_1_1_1 hd_h_ml163.t1_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">P</td><td headers="hd_h_ml163.t1_1_1_1_2 hd_h_ml163.t1_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><div class="graphic"><img src="/books/NBK55072/bin/ml163fu3.jpg" alt="Image ml163fu3.jpg" /></div></td><td headers="hd_h_ml163.t1_1_1_1_3 hd_h_ml163.t1_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1</td><td headers="hd_h_ml163.t1_1_1_1_3 hd_h_ml163.t1_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2</td><td headers="hd_h_ml163.t1_1_1_1_3 hd_h_ml163.t1_1_1_2_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1</td><td headers="hd_h_ml163.t1_1_1_1_3 hd_h_ml163.t1_1_1_2_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">40</td><td headers="hd_h_ml163.t1_1_1_1_4 hd_h_ml163.t1_1_1_2_11" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>200</td></tr><tr><td headers="hd_h_ml163.t1_1_1_1_1 hd_h_ml163.t1_1_1_2_2 hd_h_ml163.t1_1_1_2_3 hd_h_ml163.t1_1_1_2_4 hd_h_ml163.t1_1_1_2_5 hd_h_ml163.t1_1_1_1_2 hd_h_ml163.t1_1_1_2_6" colspan="5" rowspan="1" style="text-align:center;vertical-align:middle;">Solubility: <1 μM</td><td headers="hd_h_ml163.t1_1_1_1_3 hd_h_ml163.t1_1_1_2_7 hd_h_ml163.t1_1_1_2_8 hd_h_ml163.t1_1_1_2_9 hd_h_ml163.t1_1_1_2_10 hd_h_ml163.t1_1_1_1_4 hd_h_ml163.t1_1_1_2_11" colspan="5" rowspan="1" style="text-align:left;vertical-align:middle;">Purity: >95%</td></tr><tr><td headers="hd_h_ml163.t1_1_1_1_1 hd_h_ml163.t1_1_1_2_1" rowspan="2" colspan="1" style="text-align:center;vertical-align:middle;">2</td><td headers="hd_h_ml163.t1_1_1_1_1 hd_h_ml163.t1_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">44826047</td><td headers="hd_h_ml163.t1_1_1_1_1 hd_h_ml163.t1_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">89856174</td><td headers="hd_h_ml163.t1_1_1_1_1 hd_h_ml163.t1_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BRD-K44893543</td><td headers="hd_h_ml163.t1_1_1_1_1 hd_h_ml163.t1_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">P</td><td headers="hd_h_ml163.t1_1_1_1_2 hd_h_ml163.t1_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><div class="graphic"><img src="/books/NBK55072/bin/ml163fu4.jpg" alt="Image ml163fu4.jpg" /></div></td><td headers="hd_h_ml163.t1_1_1_1_3 hd_h_ml163.t1_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1</td><td headers="hd_h_ml163.t1_1_1_1_3 hd_h_ml163.t1_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">4</td><td headers="hd_h_ml163.t1_1_1_1_3 hd_h_ml163.t1_1_1_2_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1</td><td headers="hd_h_ml163.t1_1_1_1_3 hd_h_ml163.t1_1_1_2_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">ND</td><td headers="hd_h_ml163.t1_1_1_1_4 hd_h_ml163.t1_1_1_2_11" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">ND</td></tr><tr><td headers="hd_h_ml163.t1_1_1_1_1 hd_h_ml163.t1_1_1_2_2 hd_h_ml163.t1_1_1_2_3 hd_h_ml163.t1_1_1_2_4 hd_h_ml163.t1_1_1_2_5 hd_h_ml163.t1_1_1_1_2 hd_h_ml163.t1_1_1_2_6" colspan="5" rowspan="1" style="text-align:center;vertical-align:middle;">Solubility: 2.0 μM</td><td headers="hd_h_ml163.t1_1_1_1_3 hd_h_ml163.t1_1_1_2_7 hd_h_ml163.t1_1_1_2_8 hd_h_ml163.t1_1_1_2_9 hd_h_ml163.t1_1_1_2_10 hd_h_ml163.t1_1_1_1_4 hd_h_ml163.t1_1_1_2_11" colspan="5" rowspan="1" style="text-align:left;vertical-align:middle;">Purity: 97%</td></tr><tr><td headers="hd_h_ml163.t1_1_1_1_1 hd_h_ml163.t1_1_1_2_1" rowspan="2" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml163.t1_1_1_1_1 hd_h_ml163.t1_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1257201</td><td headers="hd_h_ml163.t1_1_1_1_1 hd_h_ml163.t1_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">87457166</td><td headers="hd_h_ml163.t1_1_1_1_1 hd_h_ml163.t1_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BRD-K53011428</td><td headers="hd_h_ml163.t1_1_1_1_1 hd_h_ml163.t1_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">P</td><td headers="hd_h_ml163.t1_1_1_1_2 hd_h_ml163.t1_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><div class="graphic"><img src="/books/NBK55072/bin/ml163fu5.jpg" alt="Image ml163fu5.jpg" /></div></td><td headers="hd_h_ml163.t1_1_1_1_3 hd_h_ml163.t1_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1</td><td headers="hd_h_ml163.t1_1_1_1_3 hd_h_ml163.t1_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">10</td><td headers="hd_h_ml163.t1_1_1_1_3 hd_h_ml163.t1_1_1_2_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1</td><td headers="hd_h_ml163.t1_1_1_1_3 hd_h_ml163.t1_1_1_2_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">ND</td><td headers="hd_h_ml163.t1_1_1_1_4 hd_h_ml163.t1_1_1_2_11" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">ND</td></tr><tr><td headers="hd_h_ml163.t1_1_1_1_1 hd_h_ml163.t1_1_1_2_2 hd_h_ml163.t1_1_1_2_3 hd_h_ml163.t1_1_1_2_4 hd_h_ml163.t1_1_1_2_5 hd_h_ml163.t1_1_1_1_2 hd_h_ml163.t1_1_1_2_6" colspan="5" rowspan="1" style="text-align:center;vertical-align:middle;">Solubility: <1 μM</td><td headers="hd_h_ml163.t1_1_1_1_3 hd_h_ml163.t1_1_1_2_7 hd_h_ml163.t1_1_1_2_8 hd_h_ml163.t1_1_1_2_9 hd_h_ml163.t1_1_1_2_10 hd_h_ml163.t1_1_1_1_4 hd_h_ml163.t1_1_1_2_11" colspan="5" rowspan="1" style="text-align:left;vertical-align:middle;">Purity: >99%</td></tr><tr><td headers="hd_h_ml163.t1_1_1_1_1 hd_h_ml163.t1_1_1_2_1" rowspan="2" colspan="1" style="text-align:center;vertical-align:middle;">4</td><td headers="hd_h_ml163.t1_1_1_1_1 hd_h_ml163.t1_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1864426</td><td headers="hd_h_ml163.t1_1_1_1_1 hd_h_ml163.t1_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">87457167</td><td headers="hd_h_ml163.t1_1_1_1_1 hd_h_ml163.t1_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BRD-K07530279</td><td headers="hd_h_ml163.t1_1_1_1_1 hd_h_ml163.t1_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">P</td><td headers="hd_h_ml163.t1_1_1_1_2 hd_h_ml163.t1_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><div class="graphic"><img src="/books/NBK55072/bin/ml163fu6.jpg" alt="Image ml163fu6.jpg" /></div></td><td headers="hd_h_ml163.t1_1_1_1_3 hd_h_ml163.t1_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1</td><td headers="hd_h_ml163.t1_1_1_1_3 hd_h_ml163.t1_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">70</td><td headers="hd_h_ml163.t1_1_1_1_3 hd_h_ml163.t1_1_1_2_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1</td><td headers="hd_h_ml163.t1_1_1_1_3 hd_h_ml163.t1_1_1_2_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">ND</td><td headers="hd_h_ml163.t1_1_1_1_4 hd_h_ml163.t1_1_1_2_11" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">ND</td></tr><tr><td headers="hd_h_ml163.t1_1_1_1_1 hd_h_ml163.t1_1_1_2_2 hd_h_ml163.t1_1_1_2_3 hd_h_ml163.t1_1_1_2_4 hd_h_ml163.t1_1_1_2_5 hd_h_ml163.t1_1_1_1_2 hd_h_ml163.t1_1_1_2_6" colspan="5" rowspan="1" style="text-align:center;vertical-align:middle;">Solubility: <1 μM</td><td headers="hd_h_ml163.t1_1_1_1_3 hd_h_ml163.t1_1_1_2_7 hd_h_ml163.t1_1_1_2_8 hd_h_ml163.t1_1_1_2_9 hd_h_ml163.t1_1_1_2_10 hd_h_ml163.t1_1_1_1_4 hd_h_ml163.t1_1_1_2_11" colspan="5" rowspan="1" style="text-align:left;vertical-align:middle;">Purity: 93%</td></tr><tr><td headers="hd_h_ml163.t1_1_1_1_1 hd_h_ml163.t1_1_1_2_1" rowspan="2" colspan="1" style="text-align:center;vertical-align:middle;">5</td><td headers="hd_h_ml163.t1_1_1_1_1 hd_h_ml163.t1_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1294885</td><td headers="hd_h_ml163.t1_1_1_1_1 hd_h_ml163.t1_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">87457165</td><td headers="hd_h_ml163.t1_1_1_1_1 hd_h_ml163.t1_1_1_2_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BRD-K06020002</td><td headers="hd_h_ml163.t1_1_1_1_1 hd_h_ml163.t1_1_1_2_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">P</td><td headers="hd_h_ml163.t1_1_1_1_2 hd_h_ml163.t1_1_1_2_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><div class="graphic"><img src="/books/NBK55072/bin/ml163fu7.jpg" alt="Image ml163fu7.jpg" /></div></td><td headers="hd_h_ml163.t1_1_1_1_3 hd_h_ml163.t1_1_1_2_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1</td><td headers="hd_h_ml163.t1_1_1_1_3 hd_h_ml163.t1_1_1_2_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>200</td><td headers="hd_h_ml163.t1_1_1_1_3 hd_h_ml163.t1_1_1_2_9" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1</td><td headers="hd_h_ml163.t1_1_1_1_3 hd_h_ml163.t1_1_1_2_10" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">ND</td><td headers="hd_h_ml163.t1_1_1_1_4 hd_h_ml163.t1_1_1_2_11" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">ND</td></tr><tr><td headers="hd_h_ml163.t1_1_1_1_1 hd_h_ml163.t1_1_1_2_2 hd_h_ml163.t1_1_1_2_3 hd_h_ml163.t1_1_1_2_4 hd_h_ml163.t1_1_1_2_5 hd_h_ml163.t1_1_1_1_2 hd_h_ml163.t1_1_1_2_6" colspan="5" rowspan="1" style="text-align:center;vertical-align:middle;">Solubility: <1 μM</td><td headers="hd_h_ml163.t1_1_1_1_3 hd_h_ml163.t1_1_1_2_7 hd_h_ml163.t1_1_1_2_8 hd_h_ml163.t1_1_1_2_9 hd_h_ml163.t1_1_1_2_10 hd_h_ml163.t1_1_1_1_4 hd_h_ml163.t1_1_1_2_11" colspan="5" rowspan="1" style="text-align:left;vertical-align:middle;">Purity: 98%</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="ml163.tfn1"><p class="no_margin">P=Purchased</p></div></dd></dl></dl></div></div></div></article><article data-type="fig" id="figobml163f4"><div id="ml163.f4" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK55072/bin/ml163f4.jpg" alt="Figure 4. Concentration-dependent Activity of Alpha-synuclein Analogs." /></div><h3><span class="label">Figure 4</span><span class="title">Concentration-dependent Activity of Alpha-synuclein Analogs</span></h3><div class="caption"><p>Dose-response curves of five probe analogs: CID 1090779/<a href="/pcsubstance/?term=ML163[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML163</a> (<i>A</i>), CID 44826047(<i>B</i>), CID 1257201(<i>C</i>), CID 1864426(<i>D</i>), CID 1294885(<i>E</i>).</p></div></div></article><article data-type="table-wrap" id="figobml163t3"><div id="ml163.t3" class="table"><h3><span class="label">Table A1</span><span class="title">Summary of Completed Assays and AIDs</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK55072/table/ml163.t3/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml163.t3_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml163.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">PubChem AID No.</th><th id="hd_h_ml163.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Type</th><th id="hd_h_ml163.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Target</th><th id="hd_h_ml163.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Concentration Range (μM)</th><th id="hd_h_ml163.t3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Samples Tested</th></tr></thead><tbody><tr><td headers="hd_h_ml163.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1829" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1829</a></td><td headers="hd_h_ml163.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Summary</td><td headers="hd_h_ml163.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">NA</td><td headers="hd_h_ml163.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">NA</td><td headers="hd_h_ml163.t3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">NA</td></tr><tr><td headers="hd_h_ml163.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1814" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1814</a></td><td headers="hd_h_ml163.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Primary</td><td headers="hd_h_ml163.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">ASYN 5′UTR</td><td headers="hd_h_ml163.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">7.5</td><td headers="hd_h_ml163.t3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">303811</td></tr><tr><td headers="hd_h_ml163.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2003" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2003</a></td><td headers="hd_h_ml163.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Confirmatory</td><td headers="hd_h_ml163.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">ASYN 5′UTR</td><td headers="hd_h_ml163.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">20 - 0.16</td><td headers="hd_h_ml163.t3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2336</td></tr><tr><td headers="hd_h_ml163.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2002" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2002</a></td><td headers="hd_h_ml163.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Counterscreen</td><td headers="hd_h_ml163.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">H4-C</td><td headers="hd_h_ml163.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">20 - 0.16</td><td headers="hd_h_ml163.t3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2336</td></tr><tr><td headers="hd_h_ml163.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1999" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1999</a></td><td headers="hd_h_ml163.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Confirmatory-powder</td><td headers="hd_h_ml163.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">H4-PRP</td><td headers="hd_h_ml163.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">20 - 0.16</td><td headers="hd_h_ml163.t3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2336</td></tr><tr><td headers="hd_h_ml163.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2442" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2442</a></td><td headers="hd_h_ml163.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Confirmatory-powder</td><td headers="hd_h_ml163.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">ASYN 5′UTR</td><td headers="hd_h_ml163.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">20 - 0.16</td><td headers="hd_h_ml163.t3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">13</td></tr><tr><td headers="hd_h_ml163.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2453" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2453</a></td><td headers="hd_h_ml163.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Counterscreen-powder</td><td headers="hd_h_ml163.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">H4-C</td><td headers="hd_h_ml163.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">20 - 0.16</td><td headers="hd_h_ml163.t3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">13</td></tr><tr><td headers="hd_h_ml163.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2446" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2446</a></td><td headers="hd_h_ml163.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Counterscreen-powder</td><td headers="hd_h_ml163.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">H4-PRP</td><td headers="hd_h_ml163.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">20 - 0.16</td><td headers="hd_h_ml163.t3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">13</td></tr><tr><td headers="hd_h_ml163.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2627" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2627</a></td><td headers="hd_h_ml163.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Western Blot</td><td headers="hd_h_ml163.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">ASYN</td><td headers="hd_h_ml163.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">100 - 0.1</td><td headers="hd_h_ml163.t3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1</td></tr><tr><td headers="hd_h_ml163.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/2670" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">2670</a></td><td headers="hd_h_ml163.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Analogs</td><td headers="hd_h_ml163.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">ASYN 5′UTR</td><td headers="hd_h_ml163.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">20 - 0.16</td><td headers="hd_h_ml163.t3_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">4</td></tr></tbody></table></div></div></article><article data-type="fig" id="figobml163fu2"><div id="ml163.fu2" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK55072/bin/ml163fu8.jpg" alt="1H NMR Spectra of Probe (CID 1090779/ML163)." /></div><h3><span class="title"><sup>1</sup>H NMR Spectra of Probe (CID 1090779/<a href="/pcsubstance/?term=ML163[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML163</a>)</span></h3></div></article><article data-type="fig" id="figobml163fu3"><div id="ml163.fu3" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK55072/bin/ml163fu9.jpg" alt="LC/MS Chromatogram of Probe (CID 1090779/ML163)." /></div><h3><span class="title">LC/MS Chromatogram of Probe (CID 1090779/<a href="/pcsubstance/?term=ML163[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML163</a>)</span></h3></div></article><article data-type="fig" id="figobml163fu4"><div id="ml163.fu4" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK55072/bin/ml163fu10.jpg" alt="1H NMR Spectra of Analog CID 44826047." /></div><h3><span class="title"><sup>1</sup>H NMR Spectra of Analog CID 44826047</span></h3></div></article><article data-type="fig" id="figobml163fu5"><div id="ml163.fu5" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK55072/bin/ml163fu11.jpg" alt="LC/MS Chromatogram of Analog CID 44826047." /></div><h3><span class="title">LC/MS Chromatogram of Analog CID 44826047</span></h3></div></article><article data-type="fig" id="figobml163fu6"><div id="ml163.fu6" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK55072/bin/ml163fu12.jpg" alt="1H NMR Spectra of Analog CID 1257201." /></div><h3><span class="title"><sup>1</sup>H NMR Spectra of Analog CID 1257201</span></h3></div></article><article data-type="fig" id="figobml163fu7"><div id="ml163.fu7" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK55072/bin/ml163fu13.jpg" alt="LC/MS Chromatogram of Analog CID 1257201." /></div><h3><span class="title">LC/MS Chromatogram of Analog CID 1257201</span></h3></div></article><article data-type="fig" id="figobml163fu8"><div id="ml163.fu8" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK55072/bin/ml163fu14.jpg" alt="1H NMR Spectra of Analog CID 1864426." /></div><h3><span class="title"><sup>1</sup>H NMR Spectra of Analog CID 1864426</span></h3></div></article><article data-type="fig" id="figobml163fu9"><div id="ml163.fu9" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK55072/bin/ml163fu15.jpg" alt="LC/MS Chromatogram of Analog CID 1864426." /></div><h3><span class="title">LC/MS Chromatogram of Analog CID 1864426</span></h3></div></article><article data-type="fig" id="figobml163fu10"><div id="ml163.fu10" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK55072/bin/ml163fu16.jpg" alt="1H NMR Spectra of Analog CID 1294885." /></div><h3><span class="title"><sup>1</sup>H NMR Spectra of Analog CID 1294885</span></h3></div></article><article data-type="fig" id="figobml163fu11"><div id="ml163.fu11" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK55072/bin/ml163fu17.jpg" alt="LC/MS Chromatogram of Analog CID 1294885." /></div><h3><span class="title">LC/MS Chromatogram of Analog CID 1294885</span></h3></div></article><article data-type="table-wrap" id="figobml163t4"><div id="ml163.t4" class="table"><h3><span class="label">Table A2</span><span class="title">Probe and Analog Information</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK55072/table/ml163.t4/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml163.t4_lrgtbl__"><table class="no_margin"><thead><tr><th id="hd_h_ml163.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">BRD</th><th id="hd_h_ml163.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">SID</th><th id="hd_h_ml163.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CID</th><th id="hd_h_ml163.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">P/A</th><th id="hd_h_ml163.t4_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">MLSID</th><th id="hd_h_ml163.t4_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">ML</th></tr></thead><tbody><tr><td headers="hd_h_ml163.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BRD-K32644160</td><td headers="hd_h_ml163.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/99495808" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">99495808</a></td><td headers="hd_h_ml163.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1090779</td><td headers="hd_h_ml163.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">P</td><td headers="hd_h_ml163.t4_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS002725686</td><td headers="hd_h_ml163.t4_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="/pcsubstance/?term=ML163[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">163</a></td></tr><tr><td headers="hd_h_ml163.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BRD-K44893543</td><td headers="hd_h_ml163.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/89856174" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">89856174</a></td><td headers="hd_h_ml163.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">44826047</td><td headers="hd_h_ml163.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">A</td><td headers="hd_h_ml163.t4_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS002725688</td><td headers="hd_h_ml163.t4_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">NA</td></tr><tr><td headers="hd_h_ml163.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BRD-K53011428</td><td headers="hd_h_ml163.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87457166" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87457166</a></td><td headers="hd_h_ml163.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1257201</td><td headers="hd_h_ml163.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">A</td><td headers="hd_h_ml163.t4_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS002725689</td><td headers="hd_h_ml163.t4_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">NA</td></tr><tr><td headers="hd_h_ml163.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BRD-K07530279</td><td headers="hd_h_ml163.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87457167" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87457167</a></td><td headers="hd_h_ml163.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1864426</td><td headers="hd_h_ml163.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">A</td><td headers="hd_h_ml163.t4_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS002729069</td><td headers="hd_h_ml163.t4_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">NA</td></tr><tr><td headers="hd_h_ml163.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BRD-K06020002</td><td headers="hd_h_ml163.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/87457165" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">87457165</a></td><td headers="hd_h_ml163.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1294885</td><td headers="hd_h_ml163.t4_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">A</td><td headers="hd_h_ml163.t4_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">MLS002825679</td><td headers="hd_h_ml163.t4_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">NA</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="ml163.tfn2"><p class="no_margin">P = probe, A = analog</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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