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<script type="text/javascript" src="/corehtml/pmc/jatsreader/ptpmc_3.22/js/jr.boots.min.js"> </script><title>High throughput screening for SMA - 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="High throughput screening for SMA">
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<meta name="citation_publisher" content="National Center for Biotechnology Information (US)">
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<meta name="citation_date" content="2010/09/02">
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<meta name="citation_author" content="Steve Titus">
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<meta name="citation_author" content="Juan Marugan">
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<meta name="citation_author" content="Noel Southall">
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<meta name="citation_author" content="Jim Inglese">
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<meta name="citation_author" content="Chris Austin">
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<meta name="citation_author" content="Wei Zheng">
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<meta name="citation_author" content="Elliot Androphy">
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<meta name="citation_pmid" content="21433373">
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<meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK47357/">
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<meta name="DC.Title" content="High throughput screening for SMA">
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<meta name="DC.Type" content="Text">
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<meta name="DC.Publisher" content="National Center for Biotechnology Information (US)">
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<meta name="DC.Contributor" content="Steve Titus">
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<meta name="DC.Contributor" content="Juan Marugan">
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<meta name="DC.Contributor" content="Noel Southall">
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<meta name="DC.Contributor" content="Jim Inglese">
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<meta name="DC.Contributor" content="Chris Austin">
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<meta name="DC.Contributor" content="Wei Zheng">
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<meta name="DC.Contributor" content="Elliot Androphy">
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<meta name="DC.Date" content="2010/09/02">
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<meta name="description" content="Spinal muscular atrophy (SMA) is caused by insufficient levels of the survival motor neuron protein SMN. The SMN locus on chromosome 5q13 contains two inverted copies of SMN, called SMN1 and SMN2. In the SMA disease state, various mutations in the SMN1 locus render that protein nonfunctional and induce the disease state. A therapy to either increase the amount of SMN2 product made, or to increase the inclusion of exon 7, has been proposed for the treatment of SMA. Previous probes have worked through non-specific mechanisms of activation (HDAC inhibition, RNA decapping), and effect an increase in protein abundance in both SMN2 and SMN1 reporter cell lines. The present probe ML104 (CID-6404603) appears to operate through a unique mechanism of action, showing an increase in SMN2 protein production in fibroblast line derived from an SMA patient, without having an effect on SMN1 protein production in the analogous cell line. The compound may alter splicing of SMN2 protein, but it has not been evaluated for use in vivo, and mode of action studies are ongoing.">
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<meta name="og:title" content="High throughput screening for SMA">
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<meta name="og:description" content="Spinal muscular atrophy (SMA) is caused by insufficient levels of the survival motor neuron protein SMN. The SMN locus on chromosome 5q13 contains two inverted copies of SMN, called SMN1 and SMN2. In the SMA disease state, various mutations in the SMN1 locus render that protein nonfunctional and induce the disease state. A therapy to either increase the amount of SMN2 product made, or to increase the inclusion of exon 7, has been proposed for the treatment of SMA. Previous probes have worked through non-specific mechanisms of activation (HDAC inhibition, RNA decapping), and effect an increase in protein abundance in both SMN2 and SMN1 reporter cell lines. The present probe ML104 (CID-6404603) appears to operate through a unique mechanism of action, showing an increase in SMN2 protein production in fibroblast line derived from an SMA patient, without having an effect on SMN1 protein production in the analogous cell line. The compound may alter splicing of SMN2 protein, but it has not been evaluated for use in vivo, and mode of action studies are ongoing.">
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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="_NBK47357_"><span class="title" itemprop="name">High throughput screening for SMA</span></h1><p class="contribs">Titus S, Marugan J, Southall N, et al.</p><p class="fm-aai"><a href="#_NBK47357_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>Spinal muscular atrophy (SMA) is caused by insufficient levels of the survival motor neuron protein SMN. The SMN locus on chromosome 5q13 contains two inverted copies of SMN, called SMN1 and SMN2. In the SMA disease state, various mutations in the SMN1 locus render that protein nonfunctional and induce the disease state. A therapy to either increase the amount of SMN2 product made, or to increase the inclusion of exon 7, has been proposed for the treatment of SMA. Previous probes have worked through non-specific mechanisms of activation (HDAC inhibition, RNA decapping), and effect an increase in protein abundance in both SMN2 and SMN1 reporter cell lines. The present probe ML104 (CID-6404603) appears to operate through a unique mechanism of action, showing an increase in SMN2 protein production in fibroblast line derived from an SMA patient, without having an effect on SMN1 protein production in the analogous cell line. The compound may alter splicing of SMN2 protein, but it has not been evaluated for use in vivo, and mode of action studies are ongoing.</p></div><div class="h2"></div><p><b>Assigned Assay Grant #:</b> 1 R03 MH084179-01</p><p><b>Screening Center Name & PI:</b> NIH Chemical Genomics Center,
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Christopher Austin</p><p><b>Chemistry Center Name & PI:</b> NIH Chemical Genomics Center,
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Christopher Austin</p><p><b>Assay Submitter & Institution:</b> Elliot Androphy, University of
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Massachusetts Medical School</p><p><b>PubChem Summary Bioassay Identifier (AID):</b>
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<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1474" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1474</a></p><div id="ml104.s2"><h2 id="_ml104_s2_">Probe Structure & Characteristics</h2><div id="ml104.fu1" class="figure"><div class="graphic"><img src="/books/NBK47357/bin/ml104fu1.jpg" alt="Image ml104fu1" /></div></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml104tu1"><a href="/books/NBK47357/table/ml104.tu1/?report=objectonly" target="object" title="Table" class="img_link icnblk_img figpopup" rid-figpopup="figml104tu1" rid-ob="figobml104tu1"><img class="small-thumb" src="/books/NBK47357/table/ml104.tu1/?report=thumb" src-large="/books/NBK47357/table/ml104.tu1/?report=previmg" alt="Image " /></a><div class="icnblk_cntnt"><h4 id="ml104.tu1"><a href="/books/NBK47357/table/ml104.tu1/?report=objectonly" target="object" rid-ob="figobml104tu1">Table</a></h4></div></div></div><div id="ml104.s3"><h2 id="_ml104_s3_">Recommendations for the scientific use of this probe</h2><p>This probe should be useful for modulating the expression of SMN2 protein in
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cell-based models of SMN protein expression. This probe has not been evaluated for
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use <i>in vivo</i>, though it has been shown to increase SMN2 protein
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production in fibroblasts derived from SMA patients. Compound may alter splicing of
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SMN2 protein, but mode of action studies are ongoing. The goal of the project was to
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identify a compound 10-fold selective for SMN2 versus SMN1 expression in
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reporter-based assays which increase SMN2 protein abundance in primary SMA derived
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fibroblasts, or immortalized SMA derived fibroblasts. CID 6404603 satisfies these
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criteria, increasing protein expression in fibroblasts derived from SMA patients at
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a compound concentration of 1 μM, and was the most potent member of the
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series in the primary screening assay.</p></div><div id="ml104.s4"><h2 id="_ml104_s4_">1. Scientific Rationale for Project</h2><p>Spinal muscular atrophy (SMA) is caused by insufficient levels of the survival motor
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neuron protein SMN. The SMN locus on chromosome 5q13 contains two inverted copies of
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SMN, called SMN1 and SMN2, which are 99% identical at the amino acid
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level. At the splicing level, SMN1 mainly produces one splice variant
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(90%) containing exons 1–8; this variant is identified as
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SMN protein, which is the fully functional protein. Protein from SMN2 expression
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excludes exon 7 90% of the time. Skipping of exon 7 produces
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non-functional SMN protein product. In the SMA disease state, various mutations in
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the SMN1 locus render that protein nonfunctional and induce the disease state. The
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10% of SMN2 translation that yields SMN protein is not sufficient to
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overcome the deficiency produced by the loss of the SMN1 expression product. A
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therapy to either increase the amount of SMN2 product made, or to increase the
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inclusion of exon 7, has been proposed for the treatment of SMA.</p><p>We have designed a luciferase reporter gene assay by combining the promoter and
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splicing-based cassettes in tandem with the major portion of the native SMN2 cDNA. A
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screen of this assay with a compound library can identify compounds that increase
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SMN2 levels by three mechanisms: modulating alternative splicing of SMN exon 7,
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increasing transcription from the SMN2 promoter, or stabilizing the SMN protein.
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Earlier screening assays for SMA only targeted compounds that either stimulate the
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SMN2 promoter or decrease exon 7 skipping.</p></div><div id="ml104.s5"><h2 id="_ml104_s5_">2. Project Description</h2><p>208,516 compounds were screened in the primary assay during the course of this
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screen. Some active compounds were chosen for confirmation according to the scheme
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shown in <a class="figpopup" href="/books/NBK47357/figure/ml104.f1/?report=objectonly" target="object" rid-figpopup="figml104f1" rid-ob="figobml104f1">figure 1</a>.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml104f1" co-legend-rid="figlgndml104f1"><a href="/books/NBK47357/figure/ml104.f1/?report=objectonly" target="object" title="Figure 1" class="img_link icnblk_img figpopup" rid-figpopup="figml104f1" rid-ob="figobml104f1"><img class="small-thumb" src="/books/NBK47357/bin/ml104f1.gif" src-large="/books/NBK47357/bin/ml104f1.jpg" alt="Figure 1. Overview of screening process." /></a><div class="icnblk_cntnt" id="figlgndml104f1"><h4 id="ml104.f1"><a href="/books/NBK47357/figure/ml104.f1/?report=objectonly" target="object" rid-ob="figobml104f1">Figure 1</a></h4><p class="float-caption no_bottom_margin">Overview of screening process. </p></div></div><div id="ml104.s6"><h3>a. Information for each Assay Implemented and Screening Run</h3><div id="ml104.s7"><h4>i. qHTS Assay for modulators of SMN2 splicing [<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1458" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1458</a>; Primary
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DR]</h4><div id="ml104.s8"><h5>Assay Description</h5><p>Cells stably expressing the luciferase reporter gene fused to exon 8 were
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used as the primary screening cell line (<a class="figpopup" href="/books/NBK47357/figure/ml104.f2/?report=objectonly" target="object" rid-figpopup="figml104f2" rid-ob="figobml104f2">figure 2</a>). When exon 7 is included in the mRNA
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transcript, full length protein (fused to luciferase) is generated.
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Compounds that either increase the inclusion of exon 7 or increase the
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overall level of transcript produced will generate increased levels of
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luciferase. The assay measures the luminescent signal produced by
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luciferase, and compounds with increases in luminescent signaling will
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be flagged as potential actives. The luciferase enzyme requires ATP,
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luciferin, and oxygen as substrates for enzymatic activity. All of the
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required components are present in Promega’s One Glo
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Luicferase detection kit. The luminescent signal is read on the Viewlux
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CCD camera-based plate reader, which has a very sensitive, cooled CCD
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camera that captures data from the entire plate in one read. The
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reagents required for the cell propagation and the assay are listed in
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<a class="figpopup" href="/books/NBK47357/table/ml104.t1/?report=objectonly" target="object" rid-figpopup="figml104t1" rid-ob="figobml104t1">table 2</a>.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml104f2" co-legend-rid="figlgndml104f2"><a href="/books/NBK47357/figure/ml104.f2/?report=objectonly" target="object" title="Figure 2" class="img_link icnblk_img figpopup" rid-figpopup="figml104f2" rid-ob="figobml104f2"><img class="small-thumb" src="/books/NBK47357/bin/ml104f2.gif" src-large="/books/NBK47357/bin/ml104f2.jpg" alt="Figure 2. Schematic of SMN2 reporter cell line." /></a><div class="icnblk_cntnt" id="figlgndml104f2"><h4 id="ml104.f2"><a href="/books/NBK47357/figure/ml104.f2/?report=objectonly" target="object" rid-ob="figobml104f2">Figure 2</a></h4><p class="float-caption no_bottom_margin">Schematic of SMN2 reporter cell line. </p></div></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml104t1"><a href="/books/NBK47357/table/ml104.t1/?report=objectonly" target="object" title="Table 2" class="img_link icnblk_img figpopup" rid-figpopup="figml104t1" rid-ob="figobml104t1"><img class="small-thumb" src="/books/NBK47357/table/ml104.t1/?report=thumb" src-large="/books/NBK47357/table/ml104.t1/?report=previmg" alt="Table 2. Reagents and resources for the SMN2 Luciferase screen." /></a><div class="icnblk_cntnt"><h4 id="ml104.t1"><a href="/books/NBK47357/table/ml104.t1/?report=objectonly" target="object" rid-ob="figobml104t1">Table 2</a></h4><p class="float-caption no_bottom_margin">Reagents and resources for the SMN2 Luciferase screen. </p></div></div></div><div id="ml104.s9"><h5>Assay Protocol</h5><p>The protocol is presented in <a class="figpopup" href="/books/NBK47357/table/ml104.t2/?report=objectonly" target="object" rid-figpopup="figml104t2" rid-ob="figobml104t2">table
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3</a>. Freshly trypsinized cells were resuspended in phenol red
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free DMEM containing 10% fetal bovine serum, 1x
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penicillin/streptomycin, and 1x sodium pyruvate at a density of 2000
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cells/well in 5ul of media. The assay plates used were white solid
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bottom TC-treated 1536 well plates. Cells were incubated at 37 C for 10
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to 12 hours to allow recovery. After incubation, cells were treated with
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control (sodium butyrate) or library compounds. Cells were then
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incubated at 37 C for 30 to 36 hours prior to being assayed. Luciferase
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activity was detected by adding 3 ul of OneGlo detection reagent,
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incubating for 5 minutes at room temperature and measuring luminescent
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signal in the Viewlux (using a 60 second integration time, high speed
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capture with 2x pixel binning).</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml104t2"><a href="/books/NBK47357/table/ml104.t2/?report=objectonly" target="object" title="Table 3" class="img_link icnblk_img figpopup" rid-figpopup="figml104t2" rid-ob="figobml104t2"><img class="small-thumb" src="/books/NBK47357/table/ml104.t2/?report=thumb" src-large="/books/NBK47357/table/ml104.t2/?report=previmg" alt="Table 3. Assay protocol for the SMN2 luciferase cell line in 1536 well format." /></a><div class="icnblk_cntnt"><h4 id="ml104.t2"><a href="/books/NBK47357/table/ml104.t2/?report=objectonly" target="object" rid-ob="figobml104t2">Table 3</a></h4><p class="float-caption no_bottom_margin">Assay protocol for the SMN2 luciferase cell line in 1536 well
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format. </p></div></div></div><div id="ml104.s10"><h5>Summary of Results</h5><p>NCGC tested 1229 1536-well plates in the primary screen. The average
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Z’ for the screen was 0.47 +/− 0.12,
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excluding 8 plates which failed visual QC. Results are reported for
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210,836 samples. 6,128 compounds gave significant
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concentration-responses in the primary screen. Compounds were
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deprioritized for confirmation if they were suspected to interfere with
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the readout of the assay platform, or cellular viability using internal
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NCGC SAR data from other related assays.</p></div></div><div id="ml104.s11"><h4>ii. Confirmation Concentration-Response Assay for SMN2 activators
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[<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1740" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1740</a>; Confirmatory DR]</h4><div id="ml104.s12"><h5>Assay Description</h5><p>To confirm activity in the original assay, select samples active in the
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primary screen were obtained in DMSO solution from the MLSMR and/or as
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powders from compound vendors.</p></div><div id="ml104.s13"><h5>Assay Protocol</h5><p>The assay protocol and reagents are identical to: qHTS Assay for
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Enhancers of SMN2 Splice Variant Expression [<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1458" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1458</a>;
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Primary]</p></div><div id="ml104.s14"><h5>Summary of Results</h5><p>Prioritized compounds had a high confirmation rate, but the most potent
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compounds from the MLSMR were only in the micromolar range using the
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HTRF assay format.</p></div></div><div id="ml104.s15"><h4>iii. Counterscreen in SMN1 cell line for SMN1 activators.
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[<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1739" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1739</a>; Confirmatory DR]</h4><div id="ml104.s16"><h5>Assay Description</h5><p>The SMN1 splicing pattern typically includes exon 7. A cell line
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harboring the SMN1 gene fused to luciferase was used as a counterscreen.
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Follow-up compounds exhibiting increased signal in the SMN1 cell line
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were flagged as non specific activators (i.e. transcription or protein
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stability).</p></div><div id="ml104.s17"><h5>Assay Protocol</h5><p>The assay protocol and reagents are identical to: qHTS Assay for
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Enhancers of SMN2 Splice Variant Expression [<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1458" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1458</a>;
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Confirmatory DR]</p></div><div id="ml104.s18"><h5>Summary of Results</h5><p>About half of the compounds from the MLSMR <i>inhibited</i>
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SMN1 protein production in the micromolar range using the HTRF assay
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format.</p></div></div><div id="ml104.s19"><h4>iv. Counterscreen of active compounds against purified luciferase enzyme
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[<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1733" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1733</a>; Confirmatory DR]</h4><div id="ml104.s20"><h5>Assay Description</h5><p>Luciferase inhibitors have been shown to stabilize the structure (and
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also the half life) of the enzyme. Compounds which act as luciferase
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inhibitors could show up in the SMN2 assay as false positives due to
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this phenomenon. Purified luciferase enzyme was used in a counter-screen
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of the active compounds.</p></div><div id="ml104.s21"><h5>Assay Protocol</h5><p>The assay protocol and reagents are listed in <a class="figpopup" href="/books/NBK47357/table/ml104.t3/?report=objectonly" target="object" rid-figpopup="figml104t3" rid-ob="figobml104t3">table 4</a> and <a class="figpopup" href="/books/NBK47357/table/ml104.t4/?report=objectonly" target="object" rid-figpopup="figml104t4" rid-ob="figobml104t4">5</a>, respectively. A white solid bottom 1536
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well plate was used in the assay. The enzyme mixture consisted of 50 mM
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Tris-acetate pH 7.6, 10 mM Mg acetate 0.05% BSA,
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0.01% Tween and 5 nM firefly luciferase. Each well received
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2.5 ul of enzyme and 23 nl of follow-up compound, and was incubated for
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5 minutes at room temperature. After incubation, 5 ul of detection
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reagent (OneGlo) was added to each well and luminescent enzyme activity
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was recorded on the ViewLux (using medium sensitivity, 1x pixel binning
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and 1 second of integration). The control compound used was AMP at 100
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nM and dose responses thereof.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml104t3"><a href="/books/NBK47357/table/ml104.t3/?report=objectonly" target="object" title="Table 4" class="img_link icnblk_img figpopup" rid-figpopup="figml104t3" rid-ob="figobml104t3"><img class="small-thumb" src="/books/NBK47357/table/ml104.t3/?report=thumb" src-large="/books/NBK47357/table/ml104.t3/?report=previmg" alt="Table 4. Purified Luciferase Enzyme Assay Protocol." /></a><div class="icnblk_cntnt"><h4 id="ml104.t3"><a href="/books/NBK47357/table/ml104.t3/?report=objectonly" target="object" rid-ob="figobml104t3">Table 4</a></h4><p class="float-caption no_bottom_margin">Purified Luciferase Enzyme Assay Protocol. </p></div></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml104t4"><a href="/books/NBK47357/table/ml104.t4/?report=objectonly" target="object" title="Table 5" class="img_link icnblk_img figpopup" rid-figpopup="figml104t4" rid-ob="figobml104t4"><img class="small-thumb" src="/books/NBK47357/table/ml104.t4/?report=thumb" src-large="/books/NBK47357/table/ml104.t4/?report=previmg" alt="Table 5. Reagents for purified luciferase counterscreen." /></a><div class="icnblk_cntnt"><h4 id="ml104.t4"><a href="/books/NBK47357/table/ml104.t4/?report=objectonly" target="object" rid-ob="figobml104t4">Table 5</a></h4><p class="float-caption no_bottom_margin">Reagents for purified luciferase counterscreen. </p></div></div></div></div><div id="ml104.s22"><h4>v. SMN2 protein expression in fibroblasts derived from an SMA
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patient</h4><div id="ml104.s23"><h5>Assay Description</h5><p>Ideally, compounds modulating SMN2 protein production in the luciferase
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reporter line would also modulate SMN2 protein production in fibroblasts
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derived from patients. Protein production is quantified via a Western
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blot.</p></div><div id="ml104.s24"><h5>Assay Protocol</h5><p>Human fibroblast cell lines from a 3-year-old type I SMA patient
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(GM03813; Coriell Cell Repository) with 2 copies of the SMN2 gene were
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maintained as previously described (<a class="bibr" href="#ml104.r1" rid="ml104.r1">1</a>). For drug treatments, cells were either plated in media
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alone or with TSA (BIOMOL International), which was dissolved in DMSO
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immediately before each use. The cells were then harvested for either
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RNA or protein isolation and quantification as previously described
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(<a class="bibr" href="#ml104.r1" rid="ml104.r1">1</a>).</p></div><div id="ml104.s25"><h5>Summary of Results</h5><p>The probe compound enhanced SMN2 protein production in fibroblast line
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derived from an SMA patient.</p></div></div></div><div id="ml104.s26"><h3>b. Probe Optimization</h3><div id="ml104.s27"><h4>i. SAR & chemistry strategy</h4><p>Initial hit from screen was confirmed. Powder sample was ordered from
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supplier and Quality Controlled, and reconfirmed in screening assay.
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Structurally similar compounds were ordered and nearly all were inactive,
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indicating a very restrictive SAR.</p></div></div></div><div id="ml104.s28"><h2 id="_ml104_s28_">3. Probe</h2><div id="ml104.s29"><h3>a. Chemical name</h3><p>5-methoxy-3,6-di(phenyl)-1,2,4-triazine
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<b>[<a href="/pcsubstance/?term=ML104[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML104</a>]</b></p></div><div id="ml104.s30"><h3>b. Probe chemical structure</h3><div id="ml104.fu2" class="figure"><div class="graphic"><img src="/books/NBK47357/bin/ml104fu2.jpg" alt="Image ml104fu2" /></div></div></div><div id="ml104.s31"><h3>c. Structural Verification Information of probe</h3><p><a href="https://pubchem.ncbi.nlm.nih.gov/substance/24819285" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-24819285</a></p><p>Structural verification and initial purity quantification was performed by
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|
<sup>1</sup>H NMR analysis using a Varian spectrometer dissolving the sample
|
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in deuterated DMSO.</p><p><sup>1</sup>H δ (ppm): 8.50 (m, 2H), 8.05 (m, 2H), 7.60 (m, 6H), 4.19
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(s, 3H).</p><p>In addition, further analysis was carried out by LC/MS using an Agilent system in
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the following conditions:</p><ul class="simple-list"><li class="half_rhythm"><div>Column: 3 × 75 mm Luna C18, 3 micron</div></li><li class="half_rhythm"><div>Run time: 4.5 minutes</div></li><li class="half_rhythm"><div>Gradient: 4% to 100% over 2.8 minutes</div></li><li class="half_rhythm"><div>Mobile phase: acetonitrile (0.025% TFA), water
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(0.05% TFA)</div></li><li class="half_rhythm"><div>Flow rate: 0.8 to 1.0 mL/min</div></li><li class="half_rhythm"><div>Temperature: 50 C</div></li><li class="half_rhythm"><div>UV Wavelength: 220 nm, 254 nm</div></li></ul><p>The retention time of MLS-000763654 (CID-6404603, <a href="https://pubchem.ncbi.nlm.nih.gov/substance/24819285" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-24819285</a>) in those
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conditions was of 3.84 minutes. The purity of the Mass spectra was recorded in
|
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the positive ionization mode using an electrospray (API-ES) ionizing source with
|
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nitrogen as drying gas. Both NMR and LC/MS analysis showed purity greater than
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99% for those batches of MLS000763654 use in the biological
|
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evaluation.</p></div><div id="ml104.s32"><h3>d. PubChem CID (corresponding to the SID)</h3><p>CID-6404603</p></div><div id="ml104.s33"><h3>e. Availability from a vendor</h3><p>Ambinter </p></div><div id="ml104.s34"><h3>f. Mode of action for biological activity of probe</h3><p>Compound may alter splicing of SMN2 protein, but mode of action studies are
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ongoing.</p></div><div id="ml104.s35"><h3>g. Detailed synthetic pathway for making probe</h3><p>There several published methods for the synthesis of
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3,6-disubtitued-1,2,4-triazines and analogues (<a class="bibr" href="#ml104.r2" rid="ml104.r2 ml104.r3 ml104.r4">2–4</a>).</p><p><a class="figpopup" href="/books/NBK47357/figure/ml104.f3/?report=objectonly" target="object" rid-figpopup="figml104f3" rid-ob="figobml104f3">Scheme 1</a> shows a general method for
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|
the synthesis of this probe. Thus, commercially available benzimidohydrazide and
|
|
2-oxo-2-phenylacetic acid were reacted in the presence of phosphorus
|
|
pentachloride using toluene as solvent. The intermediate
|
|
5-chloro-3,6-diphenyl-1,2,4-triazine reacted with methanol in the present of a
|
|
suitable base such us Butyl Lithium to yield
|
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5-methoxy-3,6-diphenyl-1,2,4-triazine.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml104f3" co-legend-rid="figlgndml104f3"><a href="/books/NBK47357/figure/ml104.f3/?report=objectonly" target="object" title="Scheme 1" class="img_link icnblk_img figpopup" rid-figpopup="figml104f3" rid-ob="figobml104f3"><img class="small-thumb" src="/books/NBK47357/bin/ml104f3.gif" src-large="/books/NBK47357/bin/ml104f3.jpg" alt="Scheme 1. Synthesis of 5-methoxy-3,6-diphenyl-1,2,4-triazine." /></a><div class="icnblk_cntnt" id="figlgndml104f3"><h4 id="ml104.f3"><a href="/books/NBK47357/figure/ml104.f3/?report=objectonly" target="object" rid-ob="figobml104f3">Scheme 1</a></h4><p class="float-caption no_bottom_margin">Synthesis of 5-methoxy-3,6-diphenyl-1,2,4-triazine. </p></div></div></div><div id="ml104.s36"><h3>h. Summary of probe properties</h3><p>MLS-000763654 and other compounds in this series easily dissolve in organic
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solvents such us MeOH, Cl2CH2, acetone or DMSO. As pure compound, MLS-000763654
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is a white powder at room temperature, chemically stable and with no apparent
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reactivity with air.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml104tu2"><a href="/books/NBK47357/table/ml104.tu2/?report=objectonly" target="object" title="Table" class="img_link icnblk_img figpopup" rid-figpopup="figml104tu2" rid-ob="figobml104tu2"><img class="small-thumb" src="/books/NBK47357/table/ml104.tu2/?report=thumb" src-large="/books/NBK47357/table/ml104.tu2/?report=previmg" alt="Image " /></a><div class="icnblk_cntnt"><h4 id="ml104.tu2"><a href="/books/NBK47357/table/ml104.tu2/?report=objectonly" target="object" rid-ob="figobml104tu2">Table</a></h4></div></div></div></div><div id="ml104.s37"><h2 id="_ml104_s37_">4. Appendices</h2><div id="ml104.s38"><h3>a. Activity of probe and structurally-related compounds</h3><p>MLS-000763654 is uniquely active within the set of compounds tested. See <a class="figpopup" href="/books/NBK47357/table/ml104.t5/?report=objectonly" target="object" rid-figpopup="figml104t5" rid-ob="figobml104t5">Table 6</a></p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml104t5"><a href="/books/NBK47357/table/ml104.t5/?report=objectonly" target="object" title="Table 6" class="img_link icnblk_img figpopup" rid-figpopup="figml104t5" rid-ob="figobml104t5"><img class="small-thumb" src="/books/NBK47357/table/ml104.t5/?report=thumb" src-large="/books/NBK47357/table/ml104.t5/?report=previmg" alt="Table 6. Activity of MLS-000763654 and related compounds in reporter assays of SMN2 and SMN1 expression." /></a><div class="icnblk_cntnt"><h4 id="ml104.t5"><a href="/books/NBK47357/table/ml104.t5/?report=objectonly" target="object" rid-ob="figobml104t5">Table 6</a></h4><p class="float-caption no_bottom_margin">Activity of MLS-000763654 and related compounds in reporter assays of
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SMN2 and SMN1 expression. </p></div></div></div><div id="ml104.s39"><h3>b. Comparative data showing probe specificity for target</h3><p>Previous probes have worked through non-specific mechanisms of activation (HDAC
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inhibition, RNA decapping), and effect an increase in protein abundance in both
|
|
SMN2 and SMN1 reporter cell lines. The present probe appears to operate through
|
|
a unique mechanism of action, showing an increase in SMN2 protein production
|
|
without having an effect on SMN1 protein production in the analogous cell
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|
line.</p><p>Activity of the probe molecule in the luciferase assay was initially of concern
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|
(<a class="figpopup" href="/books/NBK47357/figure/ml104.f4/?report=objectonly" target="object" rid-figpopup="figml104f4" rid-ob="figobml104f4">figure 3</a>), but the probe
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molecule exhibits similar activity in a luciferase-free assay – the
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patient fibroblasts shown below (<a class="figpopup" href="/books/NBK47357/figure/ml104.f5/?report=objectonly" target="object" rid-figpopup="figml104f5" rid-ob="figobml104f5">figure
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4</a>). In addition, it is worth noting the luciferase inhibition SAR
|
|
also does not track with SMN2 activity in the reporter cell line.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml104f4" co-legend-rid="figlgndml104f4"><a href="/books/NBK47357/figure/ml104.f4/?report=objectonly" target="object" title="Figure 3" class="img_link icnblk_img figpopup" rid-figpopup="figml104f4" rid-ob="figobml104f4"><img class="small-thumb" src="/books/NBK47357/bin/ml104f4.gif" src-large="/books/NBK47357/bin/ml104f4.jpg" alt="Figure 3. Activity of the probe molecule in SMN2, SMN1 reporter cell lines and in an in vitro assay against purified luciferase enzyme." /></a><div class="icnblk_cntnt" id="figlgndml104f4"><h4 id="ml104.f4"><a href="/books/NBK47357/figure/ml104.f4/?report=objectonly" target="object" rid-ob="figobml104f4">Figure 3</a></h4><p class="float-caption no_bottom_margin">Activity of the probe molecule in SMN2, SMN1 reporter cell lines and
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|
in an <i>in vitro</i> assay against purified luciferase
|
|
enzyme. </p></div></div><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml104f5" co-legend-rid="figlgndml104f5"><a href="/books/NBK47357/figure/ml104.f5/?report=objectonly" target="object" title="Figure 4" class="img_link icnblk_img figpopup" rid-figpopup="figml104f5" rid-ob="figobml104f5"><img class="small-thumb" src="/books/NBK47357/bin/ml104f5.gif" src-large="/books/NBK47357/bin/ml104f5.jpg" alt="Figure 4. SMN protein abundance in patient fibroblasts after incubation with probe molecule." /></a><div class="icnblk_cntnt" id="figlgndml104f5"><h4 id="ml104.f5"><a href="/books/NBK47357/figure/ml104.f5/?report=objectonly" target="object" rid-ob="figobml104f5">Figure 4</a></h4><p class="float-caption no_bottom_margin">SMN protein abundance in patient fibroblasts after incubation with
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probe molecule. Sodium butyrate was used as a positive control; it has been observed to
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increase SMN protein roughly 10-fold. The probe molecule effected an
|
|
approximately 2-fold increase <a href="/books/NBK47357/figure/ml104.f5/?report=objectonly" target="object" rid-ob="figobml104f5">(more...)</a></p></div></div><p>Data for this project is available from PubChem as shown in <a class="figpopup" href="/books/NBK47357/table/ml104.t6/?report=objectonly" target="object" rid-figpopup="figml104t6" rid-ob="figobml104t6">Table 7</a>.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml104t6"><a href="/books/NBK47357/table/ml104.t6/?report=objectonly" target="object" title="Table 7" class="img_link icnblk_img figpopup" rid-figpopup="figml104t6" rid-ob="figobml104t6"><img class="small-thumb" src="/books/NBK47357/table/ml104.t6/?report=thumb" src-large="/books/NBK47357/table/ml104.t6/?report=previmg" alt="Table 7. Summary of data in PubChem." /></a><div class="icnblk_cntnt"><h4 id="ml104.t6"><a href="/books/NBK47357/table/ml104.t6/?report=objectonly" target="object" rid-ob="figobml104t6">Table 7</a></h4><p class="float-caption no_bottom_margin">Summary of data in PubChem. </p></div></div></div></div><div id="ml104.bib"><h2 id="_ml104_bib_">5. Bibliography</h2><dl class="temp-labeled-list"><dl class="bkr_refwrap"><dt>1.</dt><dd><div class="bk_ref" id="ml104.r1">Lunn MR, et al. Indoprofen upregulates the survival motor neuron protein
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through a cyclooxygenase-independent mechanism. <span><span class="ref-journal">Chem Biol. </span>2004;<span class="ref-vol">11</span>(11):1489–1493.</span> [<a href="/pmc/articles/PMC3160629/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3160629</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/15555999" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 15555999</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>2.</dt><dd><div class="bk_ref" id="ml104.r2">Kozhevnikov DN, et al. Transformations of 1,2,4-Triazines in Reactions with
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Nucleophiles: V. SNH and ipso-Substitution in the Synthesis and
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Transformations of 5-Cyano-1,2,4-triazines. <span><span class="ref-journal">Russian Journal of Organic Chemistry (Translation of Zhurnal
|
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Organicheskoi Khimii). </span>2002;<span class="ref-vol">38</span>(5):744–750.</span></div></dd></dl><dl class="bkr_refwrap"><dt>3.</dt><dd><div class="bk_ref" id="ml104.r3">Neunhoeffer Hans, et al. The chemistry of 1,2,4-triazines. XIV. Synthesis and
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reactions of 5-chloro-1,2,4-triazines. <span><span class="ref-journal">Liebigs Annalen der Chemie. </span>1990;(7):631–40.</span></div></dd></dl><dl class="bkr_refwrap"><dt>4.</dt><dd><div class="bk_ref" id="ml104.r4">Konno Shoetsu, et al. as-Triazine derivatives. VIII. Synthesis of 5-substituted
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1,2,4-triazines. <span><span class="ref-journal">Heterocycles. </span>1987;<span class="ref-vol">26</span>(12):3259–64.</span></div></dd></dl></dl></div><div id="bk_toc_contnr"></div></div></div><div class="fm-sec"><h2 id="_NBK47357_pubdet_">Publication Details</h2><h3>Author Information and Affiliations</h3><p class="contrib-group"><h4>Authors</h4><span itemprop="author">Steve Titus</span>, <span itemprop="author">Juan Marugan</span>, <span itemprop="author">Noel Southall</span>, <span itemprop="author">Jim Inglese</span>, <span itemprop="author">Chris Austin</span>, and <span itemprop="author">Wei Zheng</span>.<sup>1</sup> <span itemprop="author">Elliot Androphy</span>.<sup>2</sup></p><h4>Affiliations</h4><div class="affiliation"><sup>1</sup>
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NIH Chemical Genomics Center, 9800 Medical Center Dr.,
|
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Building B, Bethesda, MD, 20892-3370</div><div class="affiliation"><sup>2</sup>
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University of Massachusetts Medical School</div><h3>Publication History</h3><p class="small">Received: <span itemprop="datePublished">May 18, 2009</span>; Last Update: <span itemprop="dateModified">September 2, 2010</span>.</p><h3>Copyright</h3><div><div class="half_rhythm"><a href="/books/about/copyright/">Copyright Notice</a></div></div><h3>Publisher</h3><p>National Center for Biotechnology Information (US), Bethesda (MD)</p><h3>NLM Citation</h3><p>Titus S, Marugan J, Southall N, et al. High throughput screening for SMA. 2009 May 18 [Updated 2010 Sep 2]. In: Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-. <span class="bk_cite_avail"></span></p></div><div class="small-screen-prev"><a href="/books/n/mlprobe/ml106/?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/ml103/?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="figobml104fu1"><div id="ml104.fu1" class="figure"><div class="graphic"><img data-src="/books/NBK47357/bin/ml104fu1.jpg" alt="Image ml104fu1" /></div></div></article><article data-type="table-wrap" id="figobml104tu1"><div id="ml104.tu1" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK47357/table/ml104.tu1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml104.tu1_lrgtbl__"><table><thead><tr><th id="hd_h_ml104.tu1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">CID/ML</th><th id="hd_h_ml104.tu1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Target Name</th><th id="hd_h_ml104.tu1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">IC50/EC 50 (nM) [SID,
|
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AID]</th><th id="hd_h_ml104.tu1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Anti-target Name(s)</th><th id="hd_h_ml104.tu1_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">IC50/EC50 (μM)
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[SID, AID]</th><th id="hd_h_ml104.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Selectivity</th><th id="hd_h_ml104.tu1_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">Secondary Assay(s) Name: IC50/EC50 (nM)
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[SID, AID]</th></tr></thead><tbody><tr><td headers="hd_h_ml104.tu1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">CID-6404603/<a href="/pcsubstance/?term=ML104[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML104</a></td><td headers="hd_h_ml104.tu1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">SMN2 protein expression</td><td headers="hd_h_ml104.tu1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">2512 nM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/24819285" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-24819285</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1740" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1740</a>]</td><td headers="hd_h_ml104.tu1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">SMN1 protein expression</td><td headers="hd_h_ml104.tu1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">>50 µM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/24819285" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-24819285</a>,
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<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1739" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1739</a>]</td><td headers="hd_h_ml104.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">>50-fold</td><td headers="hd_h_ml104.tu1_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Western blot of SMN protein in patient
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fibroblasts: 1000 nM [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/24819285" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID-24819285</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1474" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID-1474</a>]</td></tr></tbody></table></div></div></article><article data-type="fig" id="figobml104f1"><div id="ml104.f1" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK47357/bin/ml104f1.jpg" alt="Figure 1. Overview of screening process." /></div><h3><span class="label">Figure 1</span><span class="title">Overview of screening process</span></h3></div></article><article data-type="fig" id="figobml104f2"><div id="ml104.f2" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK47357/bin/ml104f2.jpg" alt="Figure 2. Schematic of SMN2 reporter cell line." /></div><h3><span class="label">Figure 2</span><span class="title">Schematic of SMN2 reporter cell line</span></h3></div></article><article data-type="table-wrap" id="figobml104t1"><div id="ml104.t1" class="table"><h3><span class="label">Table 2</span><span class="title">Reagents and resources for the SMN2 Luciferase screen</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK47357/table/ml104.t1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml104.t1_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml104.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;">Reagents</th><th id="hd_h_ml104.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;">Resources</th><th id="hd_h_ml104.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;">Catalog Number</th></tr></thead><tbody><tr><td headers="hd_h_ml104.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">OneGlo Luicferase assay
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system</td><td headers="hd_h_ml104.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Promega, Madison WI</td><td headers="hd_h_ml104.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">E6130</td></tr><tr><td headers="hd_h_ml104.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">DMEM Media (phenol red
|
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free)</td><td headers="hd_h_ml104.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"></td><td headers="hd_h_ml104.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">21063–045</td></tr><tr><td headers="hd_h_ml104.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">DMEM Media</td><td headers="hd_h_ml104.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Invitrogen</td><td headers="hd_h_ml104.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">11765</td></tr><tr><td headers="hd_h_ml104.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Sodium Pyruvate</td><td headers="hd_h_ml104.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Invitrogen</td><td headers="hd_h_ml104.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">11360–070</td></tr><tr><td headers="hd_h_ml104.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Fetal Bovine Serum</td><td headers="hd_h_ml104.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Invitrogen</td><td headers="hd_h_ml104.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">10091</td></tr><tr><td headers="hd_h_ml104.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Penicillin-Streptomycin</td><td headers="hd_h_ml104.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Invitrogen</td><td headers="hd_h_ml104.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">15140</td></tr><tr><td headers="hd_h_ml104.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Hygromycin B</td><td headers="hd_h_ml104.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Invitrogen</td><td headers="hd_h_ml104.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">10687–010</td></tr></tbody></table></div></div></article><article data-type="table-wrap" id="figobml104t2"><div id="ml104.t2" class="table"><h3><span class="label">Table 3</span><span class="title">Assay protocol for the SMN2 luciferase cell line in 1536 well
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format</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK47357/table/ml104.t2/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml104.t2_lrgtbl__"><table class="no_margin"><thead><tr><th id="hd_h_ml104.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Sequence</th><th id="hd_h_ml104.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Parameter</th><th id="hd_h_ml104.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Value</th><th id="hd_h_ml104.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Description</th></tr></thead><tbody><tr><td headers="hd_h_ml104.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">
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<b>1</b>
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</td><td headers="hd_h_ml104.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
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<b>Cells</b>
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</td><td headers="hd_h_ml104.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
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<b>5μL</b>
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</td><td headers="hd_h_ml104.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
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<b>2000 cells/well 1536 TC treated White solid bottom
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plate</b>
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</td></tr><tr><td headers="hd_h_ml104.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">
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<b>2</b>
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</td><td headers="hd_h_ml104.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
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<b>Time</b>
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</td><td headers="hd_h_ml104.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
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<b>10–12 hours</b>
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</td><td headers="hd_h_ml104.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
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<b>37°C 5% CO2</b>
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</td></tr><tr><td headers="hd_h_ml104.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">3</td><td headers="hd_h_ml104.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Compound</td><td headers="hd_h_ml104.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">23 <b>nl</b></td><td headers="hd_h_ml104.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Libraries see plate map</td></tr><tr><td headers="hd_h_ml104.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">
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<b>4</b>
|
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</td><td headers="hd_h_ml104.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
|
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<b>Control Compound</b>
|
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</td><td headers="hd_h_ml104.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
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<b>23 nL</b>
|
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</td><td headers="hd_h_ml104.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
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<b>Sodium butyrate</b>
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<sup>A</sup>
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</td></tr><tr><td headers="hd_h_ml104.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">5</td><td headers="hd_h_ml104.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Time</td><td headers="hd_h_ml104.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">30–36 hours</td><td headers="hd_h_ml104.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">37°C
|
|
5% CO2</td></tr><tr><td headers="hd_h_ml104.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">
|
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<b>6</b>
|
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</td><td headers="hd_h_ml104.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
|
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<b>Reagent</b>
|
|
</td><td headers="hd_h_ml104.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
|
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<b>3 ul</b>
|
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</td><td headers="hd_h_ml104.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
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<b>One Glo</b>
|
|
</td></tr><tr><td headers="hd_h_ml104.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">
|
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<b>7</b>
|
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</td><td headers="hd_h_ml104.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
|
|
<b>Time</b>
|
|
</td><td headers="hd_h_ml104.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
|
|
<b>5–15 minutes</b>
|
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</td><td headers="hd_h_ml104.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
|
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<b>Room temp</b>
|
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</td></tr><tr><td headers="hd_h_ml104.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">
|
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<b>8</b>
|
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</td><td headers="hd_h_ml104.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
|
|
<b>Detector</b>
|
|
</td><td headers="hd_h_ml104.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
|
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<b>See below</b>
|
|
<sup>B</sup>
|
|
</td><td headers="hd_h_ml104.t2_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
|
|
<b>Envision</b>
|
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</td></tr></tbody></table></div><div class="tblwrap-foot"><div><dl class="temp-labeled-list small"><dl class="bkr_refwrap"><dt>A</dt><dd><div id="ml104.tfn1"><p class="no_margin">Sodium butyrate is at 4.5 mM (final) concentration and 1:1
|
|
dilns.</p></div></dd></dl><dl class="bkr_refwrap"><dt>B</dt><dd><div id="ml104.tfn2"><p class="no_margin">Viewlux settings; luminescent read, 60 second integration,
|
|
high speed 2× binning</p></div></dd></dl><dl class="bkr_refwrap"><dt></dt><dd><div id="ml104.tfn3"><p class="no_margin">Passaging media contained DMEM w/glutamax
|
|
(+phenol red) 10% FBS,
|
|
1× pen/strep, 200 ug/ml hygro, 1×
|
|
sodium pyruvate. Assay media contained DMEM w/glutamax
|
|
(−phenol red) 10% FBS,
|
|
1× pen strep, 1× pyruvate</p></div></dd></dl></dl></div></div></div></article><article data-type="table-wrap" id="figobml104t3"><div id="ml104.t3" class="table"><h3><span class="label">Table 4</span><span class="title">Purified Luciferase Enzyme Assay Protocol</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK47357/table/ml104.t3/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml104.t3_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml104.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Sequence</th><th id="hd_h_ml104.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Parameter</th><th id="hd_h_ml104.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Value</th><th id="hd_h_ml104.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Description</th></tr></thead><tbody><tr><td headers="hd_h_ml104.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">
|
|
<b>1</b>
|
|
</td><td headers="hd_h_ml104.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
|
|
<b>Reagent</b>
|
|
</td><td headers="hd_h_ml104.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
|
|
<b>2.5 uL</b>
|
|
</td><td headers="hd_h_ml104.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
|
|
<b>Enzyme In buffer</b>
|
|
<sup>A</sup>
|
|
</td></tr><tr><td headers="hd_h_ml104.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">2</td><td headers="hd_h_ml104.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Compound</td><td headers="hd_h_ml104.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">23 nL</td><td headers="hd_h_ml104.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">dilutions of Libraries</td></tr><tr><td headers="hd_h_ml104.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">
|
|
<b>3</b>
|
|
</td><td headers="hd_h_ml104.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
|
|
<b>Time</b>
|
|
</td><td headers="hd_h_ml104.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
|
|
<b>5 minutes</b>
|
|
</td><td headers="hd_h_ml104.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
|
|
<b>Room temp</b>
|
|
</td></tr><tr><td headers="hd_h_ml104.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">4</td><td headers="hd_h_ml104.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Reagent</td><td headers="hd_h_ml104.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">2.5 ul</td><td headers="hd_h_ml104.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Luciferase detection
|
|
reagent</td></tr><tr><td headers="hd_h_ml104.t3_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">
|
|
<b>5</b>
|
|
</td><td headers="hd_h_ml104.t3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
|
|
<b>Detector</b>
|
|
</td><td headers="hd_h_ml104.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
|
|
<b>2 sec Integrate</b>
|
|
</td><td headers="hd_h_ml104.t3_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">
|
|
<b>Luminescent read on Viewlux</b>
|
|
<sup>B</sup>
|
|
</td></tr></tbody></table></div></div></article><article data-type="table-wrap" id="figobml104t4"><div id="ml104.t4" class="table"><h3><span class="label">Table 5</span><span class="title">Reagents for purified luciferase counterscreen</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK47357/table/ml104.t4/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml104.t4_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml104.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;">Reagents</th><th id="hd_h_ml104.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;">Resources</th><th id="hd_h_ml104.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;">Catalog Number</th></tr></thead><tbody><tr><td headers="hd_h_ml104.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">OneGlo Luicferase assay
|
|
system</td><td headers="hd_h_ml104.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Promega</td><td headers="hd_h_ml104.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">E6130</td></tr><tr><td headers="hd_h_ml104.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Tris Acetate</td><td headers="hd_h_ml104.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Sigma</td><td headers="hd_h_ml104.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">T1258</td></tr><tr><td headers="hd_h_ml104.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Mg Acetate</td><td headers="hd_h_ml104.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Sigma</td><td headers="hd_h_ml104.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">M2545</td></tr><tr><td headers="hd_h_ml104.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">BSA</td><td headers="hd_h_ml104.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Sigma</td><td headers="hd_h_ml104.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">B4287</td></tr><tr><td headers="hd_h_ml104.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Tween-20</td><td headers="hd_h_ml104.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Sigma</td><td headers="hd_h_ml104.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">P7949</td></tr><tr><td headers="hd_h_ml104.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Firefly Luciferase</td><td headers="hd_h_ml104.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Sigma</td><td headers="hd_h_ml104.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">L9009</td></tr><tr><td headers="hd_h_ml104.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">AMP</td><td headers="hd_h_ml104.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Sigma</td><td headers="hd_h_ml104.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">A2252</td></tr></tbody></table></div></div></article><article data-type="fig" id="figobml104fu2"><div id="ml104.fu2" class="figure"><div class="graphic"><img data-src="/books/NBK47357/bin/ml104fu2.jpg" alt="Image ml104fu2" /></div></div></article><article data-type="fig" id="figobml104f3"><div id="ml104.f3" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK47357/bin/ml104f3.jpg" alt="Scheme 1. Synthesis of 5-methoxy-3,6-diphenyl-1,2,4-triazine." /></div><h3><span class="label">Scheme 1</span><span class="title">Synthesis of 5-methoxy-3,6-diphenyl-1,2,4-triazine</span></h3></div></article><article data-type="table-wrap" id="figobml104tu2"><div id="ml104.tu2" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK47357/table/ml104.tu2/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml104.tu2_lrgtbl__"><table><tbody><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;">263.29392
|
|
[g/mol]</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">XLogP3-AA</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">2.8</td></tr><tr><td rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">tPSA:</td><td rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">65.85</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;">0</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></tbody></table></div></div></article><article data-type="table-wrap" id="figobml104t5"><div id="ml104.t5" class="table"><h3><span class="label">Table 6</span><span class="title">Activity of MLS-000763654 and related compounds in reporter assays of
|
|
SMN2 and SMN1 expression</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK47357/table/ml104.t5/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml104.t5_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml104.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Sample ID</th><th id="hd_h_ml104.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">PUBCHEM_SID</th><th id="hd_h_ml104.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SMN2 AC50 (µM)</th><th id="hd_h_ml104.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SMN2 Max Response Observed
|
|
(%)</th><th id="hd_h_ml104.t5_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SMN1 AC50 (µM)</th><th id="hd_h_ml104.t5_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">SMN1 Max Response Observed
|
|
(%)</th><th id="hd_h_ml104.t5_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Luciferase IC50 (µM)</th><th id="hd_h_ml104.t5_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Luciferase Max Response Observed
|
|
(%)</th></tr></thead><tbody><tr><td headers="hd_h_ml104.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;"><div class="graphic"><img src="/books/NBK47357/bin/ml104fu3.jpg" alt="Image ml104fu3.jpg" /></div><br />MLS000763654-01</td><td headers="hd_h_ml104.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/24819285" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">24819285</a></td><td headers="hd_h_ml104.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">2.5</td><td headers="hd_h_ml104.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">98</td><td headers="hd_h_ml104.t5_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Inactive</td><td headers="hd_h_ml104.t5_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">15</td><td headers="hd_h_ml104.t5_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">12.6</td><td headers="hd_h_ml104.t5_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">−50</td></tr><tr><td headers="hd_h_ml104.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;"><div class="graphic"><img src="/books/NBK47357/bin/ml104fu4.jpg" alt="Image ml104fu4.jpg" /></div><br />MLS000755163-01</td><td headers="hd_h_ml104.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/24819589" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">24819589</a></td><td headers="hd_h_ml104.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Inactive</td><td headers="hd_h_ml104.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">2</td><td headers="hd_h_ml104.t5_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Inactive</td><td headers="hd_h_ml104.t5_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">−2</td><td headers="hd_h_ml104.t5_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Inactive</td><td headers="hd_h_ml104.t5_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">13</td></tr><tr><td headers="hd_h_ml104.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;"><div class="graphic"><img src="/books/NBK47357/bin/ml104fu5.jpg" alt="Image ml104fu5.jpg" /></div><br />MLS000546870-01</td><td headers="hd_h_ml104.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/17409493" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">17409493</a></td><td headers="hd_h_ml104.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Inactive</td><td headers="hd_h_ml104.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">−1</td><td headers="hd_h_ml104.t5_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Inactive</td><td headers="hd_h_ml104.t5_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">0</td><td headers="hd_h_ml104.t5_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">11.2</td><td headers="hd_h_ml104.t5_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">−73</td></tr><tr><td headers="hd_h_ml104.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;"><div class="graphic"><img src="/books/NBK47357/bin/ml104fu6.jpg" alt="Image ml104fu6.jpg" /></div><br />MLS000546479-01</td><td headers="hd_h_ml104.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/17403029" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">17403029</a></td><td headers="hd_h_ml104.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Inactive</td><td headers="hd_h_ml104.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">−1</td><td headers="hd_h_ml104.t5_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Inactive</td><td headers="hd_h_ml104.t5_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">7</td><td headers="hd_h_ml104.t5_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">11.2</td><td headers="hd_h_ml104.t5_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">−80</td></tr><tr><td headers="hd_h_ml104.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;"><div class="graphic"><img src="/books/NBK47357/bin/ml104fu7.jpg" alt="Image ml104fu7.jpg" /></div><br />MLS000763610-01</td><td headers="hd_h_ml104.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/24319999" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">24319999</a></td><td headers="hd_h_ml104.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Inactive</td><td headers="hd_h_ml104.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">1</td><td headers="hd_h_ml104.t5_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Inactive</td><td headers="hd_h_ml104.t5_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">−20</td><td headers="hd_h_ml104.t5_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"></td><td headers="hd_h_ml104.t5_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"></td></tr><tr><td headers="hd_h_ml104.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;"><div class="graphic"><img src="/books/NBK47357/bin/ml104fu8.jpg" alt="Image ml104fu8.jpg" /></div><br />MLS000546874-01</td><td headers="hd_h_ml104.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/17411152" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">17411152</a></td><td headers="hd_h_ml104.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Inactive</td><td headers="hd_h_ml104.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">−2</td><td headers="hd_h_ml104.t5_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Inactive</td><td headers="hd_h_ml104.t5_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">−3</td><td headers="hd_h_ml104.t5_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">8.9</td><td headers="hd_h_ml104.t5_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">−76</td></tr><tr><td headers="hd_h_ml104.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;"><div class="graphic"><img src="/books/NBK47357/bin/ml104fu9.jpg" alt="Image ml104fu9.jpg" /></div><br />MLS000543740-01</td><td headers="hd_h_ml104.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/17401426" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">17401426</a></td><td headers="hd_h_ml104.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Inactive</td><td headers="hd_h_ml104.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">5</td><td headers="hd_h_ml104.t5_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Inactive</td><td headers="hd_h_ml104.t5_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">17</td><td headers="hd_h_ml104.t5_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">10.0</td><td headers="hd_h_ml104.t5_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">−44</td></tr><tr><td headers="hd_h_ml104.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;"><div class="graphic"><img src="/books/NBK47357/bin/ml104fu10.jpg" alt="Image ml104fu10.jpg" /></div><br />MLS000702665-01</td><td headers="hd_h_ml104.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/22401957" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">22401957</a></td><td headers="hd_h_ml104.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Inactive</td><td headers="hd_h_ml104.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">−2</td><td headers="hd_h_ml104.t5_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Inactive</td><td headers="hd_h_ml104.t5_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">1</td><td headers="hd_h_ml104.t5_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Inactive</td><td headers="hd_h_ml104.t5_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">−2</td></tr><tr><td headers="hd_h_ml104.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;"><div class="graphic"><img src="/books/NBK47357/bin/ml104fu11.jpg" alt="Image ml104fu11.jpg" /></div><br />MLS000698881-01</td><td headers="hd_h_ml104.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/22404512" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">22404512</a></td><td headers="hd_h_ml104.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Inactive</td><td headers="hd_h_ml104.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">5</td><td headers="hd_h_ml104.t5_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Inactive</td><td headers="hd_h_ml104.t5_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">10</td><td headers="hd_h_ml104.t5_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Inactive</td><td headers="hd_h_ml104.t5_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">−20</td></tr><tr><td headers="hd_h_ml104.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;"><div class="graphic"><img src="/books/NBK47357/bin/ml104fu12.jpg" alt="Image ml104fu12.jpg" /></div><br />MLS000327664-01</td><td headers="hd_h_ml104.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/17411413" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">17411413</a></td><td headers="hd_h_ml104.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Inactive</td><td headers="hd_h_ml104.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">−1</td><td headers="hd_h_ml104.t5_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Inactive</td><td headers="hd_h_ml104.t5_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">−12</td><td headers="hd_h_ml104.t5_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Inactive</td><td headers="hd_h_ml104.t5_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">1</td></tr><tr><td headers="hd_h_ml104.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;"><div class="graphic"><img src="/books/NBK47357/bin/ml104fu13.jpg" alt="Image ml104fu13.jpg" /></div><br />MLS000324922-01</td><td headers="hd_h_ml104.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/17386414" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">17386414</a></td><td headers="hd_h_ml104.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Inactive</td><td headers="hd_h_ml104.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">2</td><td headers="hd_h_ml104.t5_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Inactive</td><td headers="hd_h_ml104.t5_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">17</td><td headers="hd_h_ml104.t5_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">11.2</td><td headers="hd_h_ml104.t5_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">−69</td></tr><tr><td headers="hd_h_ml104.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;"><div class="graphic"><img src="/books/NBK47357/bin/ml104fu14.jpg" alt="Image ml104fu14.jpg" /></div><br />MLS000550587-01</td><td headers="hd_h_ml104.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/14745133" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">14745133</a></td><td headers="hd_h_ml104.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Inactive</td><td headers="hd_h_ml104.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">0</td><td headers="hd_h_ml104.t5_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Inactive</td><td headers="hd_h_ml104.t5_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">2</td><td headers="hd_h_ml104.t5_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Inactive</td><td headers="hd_h_ml104.t5_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">3</td></tr><tr><td headers="hd_h_ml104.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;"><div class="graphic"><img src="/books/NBK47357/bin/ml104fu15.jpg" alt="Image ml104fu15.jpg" /></div><br />MLS000545041-01</td><td headers="hd_h_ml104.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/17406633" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">17406633</a></td><td headers="hd_h_ml104.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Inactive</td><td headers="hd_h_ml104.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">0</td><td headers="hd_h_ml104.t5_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Inactive</td><td headers="hd_h_ml104.t5_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">11</td><td headers="hd_h_ml104.t5_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">15.8</td><td headers="hd_h_ml104.t5_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">−42</td></tr><tr><td headers="hd_h_ml104.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;"><div class="graphic"><img src="/books/NBK47357/bin/ml104fu16.jpg" alt="Image ml104fu16.jpg" /></div><br />MLS000705838-01</td><td headers="hd_h_ml104.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/22413062" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">22413062</a></td><td headers="hd_h_ml104.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Inactive</td><td headers="hd_h_ml104.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">−3</td><td headers="hd_h_ml104.t5_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Inactive</td><td headers="hd_h_ml104.t5_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">−19</td><td headers="hd_h_ml104.t5_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Inactive</td><td headers="hd_h_ml104.t5_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">−2</td></tr><tr><td headers="hd_h_ml104.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;"><div class="graphic"><img src="/books/NBK47357/bin/ml104fu17.jpg" alt="Image ml104fu17.jpg" /></div><br />MLS000698884-01</td><td headers="hd_h_ml104.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/22404510" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">22404510</a></td><td headers="hd_h_ml104.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Inactive</td><td headers="hd_h_ml104.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">3</td><td headers="hd_h_ml104.t5_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">4.5</td><td headers="hd_h_ml104.t5_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">−35</td><td headers="hd_h_ml104.t5_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">11.2</td><td headers="hd_h_ml104.t5_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">−81</td></tr><tr><td headers="hd_h_ml104.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;"><div class="graphic"><img src="/books/NBK47357/bin/ml104fu18.jpg" alt="Image ml104fu18.jpg" /></div><br />MLS000544733-01</td><td headers="hd_h_ml104.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/17406729" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">17406729</a></td><td headers="hd_h_ml104.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Inactive</td><td headers="hd_h_ml104.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">2</td><td headers="hd_h_ml104.t5_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Inactive</td><td headers="hd_h_ml104.t5_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">−5</td><td headers="hd_h_ml104.t5_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">11.2</td><td headers="hd_h_ml104.t5_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">−32</td></tr><tr><td headers="hd_h_ml104.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;"><div class="graphic"><img src="/books/NBK47357/bin/ml104fu19.jpg" alt="Image ml104fu19.jpg" /></div><br />MLS000544701-01</td><td headers="hd_h_ml104.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/17406794" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">17406794</a></td><td headers="hd_h_ml104.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Inactive</td><td headers="hd_h_ml104.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">5</td><td headers="hd_h_ml104.t5_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Inactive</td><td headers="hd_h_ml104.t5_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">15</td><td headers="hd_h_ml104.t5_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">10.0</td><td headers="hd_h_ml104.t5_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">−91</td></tr><tr><td headers="hd_h_ml104.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;"><div class="graphic"><img src="/books/NBK47357/bin/ml104fu20.jpg" alt="Image ml104fu20.jpg" /></div><br />MLS000327681-01</td><td headers="hd_h_ml104.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/17411491" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">17411491</a></td><td headers="hd_h_ml104.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Inactive</td><td headers="hd_h_ml104.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">−5</td><td headers="hd_h_ml104.t5_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Inactive</td><td headers="hd_h_ml104.t5_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">5</td><td headers="hd_h_ml104.t5_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Inactive</td><td headers="hd_h_ml104.t5_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">−6</td></tr><tr><td headers="hd_h_ml104.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;"><div class="graphic"><img src="/books/NBK47357/bin/ml104fu21.jpg" alt="Image ml104fu21.jpg" /></div><br />MLS000764193-01</td><td headers="hd_h_ml104.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/24819516" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">24819516</a></td><td headers="hd_h_ml104.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Inactive</td><td headers="hd_h_ml104.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">1</td><td headers="hd_h_ml104.t5_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Inactive</td><td headers="hd_h_ml104.t5_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">−5</td><td headers="hd_h_ml104.t5_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"></td><td headers="hd_h_ml104.t5_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"></td></tr><tr><td headers="hd_h_ml104.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;"><div class="graphic"><img src="/books/NBK47357/bin/ml104fu22.jpg" alt="Image ml104fu22.jpg" /></div><br />MLS000594527-01</td><td headers="hd_h_ml104.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/14744810" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">14744810</a></td><td headers="hd_h_ml104.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Inactive</td><td headers="hd_h_ml104.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">2</td><td headers="hd_h_ml104.t5_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Inactive</td><td headers="hd_h_ml104.t5_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">−8</td><td headers="hd_h_ml104.t5_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Inactive</td><td headers="hd_h_ml104.t5_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">−1</td></tr><tr><td headers="hd_h_ml104.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;"><div class="graphic"><img src="/books/NBK47357/bin/ml104fu23.jpg" alt="Image ml104fu23.jpg" /></div><br />MLS000554266-01</td><td headers="hd_h_ml104.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/14742197" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">14742197</a></td><td headers="hd_h_ml104.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Inactive</td><td headers="hd_h_ml104.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">1</td><td headers="hd_h_ml104.t5_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Inactive</td><td headers="hd_h_ml104.t5_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">3</td><td headers="hd_h_ml104.t5_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Inactive</td><td headers="hd_h_ml104.t5_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">4</td></tr><tr><td headers="hd_h_ml104.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;"><div class="graphic"><img src="/books/NBK47357/bin/ml104fu24.jpg" alt="Image ml104fu24.jpg" /></div><br />MLS000589349-01</td><td headers="hd_h_ml104.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/17512835" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">17512835</a></td><td headers="hd_h_ml104.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Inactive</td><td headers="hd_h_ml104.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">1</td><td headers="hd_h_ml104.t5_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Inactive</td><td headers="hd_h_ml104.t5_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">−6</td><td headers="hd_h_ml104.t5_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Inactive</td><td headers="hd_h_ml104.t5_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">−17</td></tr><tr><td headers="hd_h_ml104.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;"><div class="graphic"><img src="/books/NBK47357/bin/ml104fu25.jpg" alt="Image ml104fu25.jpg" /></div><br />MLS000544732-01</td><td headers="hd_h_ml104.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"><a href="https://pubchem.ncbi.nlm.nih.gov/substance/17406985" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">17406985</a></td><td headers="hd_h_ml104.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Inactive</td><td headers="hd_h_ml104.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">−5</td><td headers="hd_h_ml104.t5_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">Inactive</td><td headers="hd_h_ml104.t5_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">−7</td><td headers="hd_h_ml104.t5_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">12.6</td><td headers="hd_h_ml104.t5_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">−33</td></tr></tbody></table></div></div></article><article data-type="fig" id="figobml104f4"><div id="ml104.f4" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK47357/bin/ml104f4.jpg" alt="Figure 3. Activity of the probe molecule in SMN2, SMN1 reporter cell lines and in an in vitro assay against purified luciferase enzyme." /></div><h3><span class="label">Figure 3</span><span class="title">Activity of the probe molecule in SMN2, SMN1 reporter cell lines and
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in an <i>in vitro</i> assay against purified luciferase
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enzyme</span></h3></div></article><article data-type="fig" id="figobml104f5"><div id="ml104.f5" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK47357/bin/ml104f5.jpg" alt="Figure 4. SMN protein abundance in patient fibroblasts after incubation with probe molecule." /></div><h3><span class="label">Figure 4</span><span class="title">SMN protein abundance in patient fibroblasts after incubation with
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probe molecule</span></h3><div class="caption"><p>Sodium butyrate was used as a positive control; it has been observed to
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increase SMN protein roughly 10-fold. The probe molecule effected an
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approximately 2-fold increase in protein abundance at 1 micromolar
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compound concentration.</p></div></div></article><article data-type="table-wrap" id="figobml104t6"><div id="ml104.t6" class="table"><h3><span class="label">Table 7</span><span class="title">Summary of data in PubChem</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK47357/table/ml104.t6/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml104.t6_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml104.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;">PubChem AID</th><th id="hd_h_ml104.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;">Type</th><th id="hd_h_ml104.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;">Target</th><th id="hd_h_ml104.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;">Conc. Range</th><th id="hd_h_ml104.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;">Samples Tested</th></tr></thead><tbody><tr><td headers="hd_h_ml104.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1458" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1458</a></td><td headers="hd_h_ml104.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Primary DR</td><td headers="hd_h_ml104.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">SMN2 Luciferase cell line</td><td headers="hd_h_ml104.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0.5nM–46μM</td><td headers="hd_h_ml104.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">208516</td></tr><tr><td headers="hd_h_ml104.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1740" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1740</a></td><td headers="hd_h_ml104.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Confirmation</td><td headers="hd_h_ml104.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">SMN2 Luciferase cell line</td><td headers="hd_h_ml104.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0.5nM–46μM</td><td headers="hd_h_ml104.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">84</td></tr><tr><td headers="hd_h_ml104.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1739" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1739</a></td><td headers="hd_h_ml104.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Counterscreen</td><td headers="hd_h_ml104.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">SMN1 Luciferase cell line</td><td headers="hd_h_ml104.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0.5nM–46μM</td><td headers="hd_h_ml104.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">84</td></tr><tr><td headers="hd_h_ml104.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1733" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1733</a></td><td headers="hd_h_ml104.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Counterscreen</td><td headers="hd_h_ml104.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Luciferase enzyme</td><td headers="hd_h_ml104.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0.5nM–46μM</td><td headers="hd_h_ml104.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">77</td></tr><tr><td headers="hd_h_ml104.t6_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1474" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1474</a></td><td headers="hd_h_ml104.t6_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Summary</td><td headers="hd_h_ml104.t6_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">N/A</td><td headers="hd_h_ml104.t6_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">N/A</td><td headers="hd_h_ml104.t6_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">N/A</td></tr></tbody></table></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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