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<script type="text/javascript" src="/corehtml/pmc/jatsreader/ptpmc_3.22/js/jr.boots.min.js"> </script><title>ML346: A Novel Modulator of Proteostasis for Protein Conformational Diseases - 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="ML346: A Novel Modulator of Proteostasis for Protein Conformational Diseases">
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<meta name="citation_publisher" content="National Center for Biotechnology Information (US)">
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<meta name="citation_date" content="2013/04/05">
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<meta name="citation_author" content="Barbara Calamini">
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<meta name="citation_author" content="Maria Catarina Silva">
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<meta name="citation_author" content="Franck Madoux">
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<meta name="citation_author" content="Darren M. Hutt">
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<meta name="citation_author" content="Shilpi Khanna">
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<meta name="citation_author" content="Monica A. Chalfant">
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<meta name="citation_author" content="Christophe Allais">
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<meta name="citation_author" content="Souad Ouizem">
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<meta name="citation_author" content="Sanjay A. Saldanha">
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<meta name="citation_author" content="Jill Ferguson">
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<meta name="citation_author" content="Becky A. Mercer">
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<meta name="citation_author" content="Cameron Michael">
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<meta name="citation_author" content="Bradley D. Tait">
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<meta name="citation_author" content="Dan Garza">
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<meta name="citation_author" content="William E. Balch">
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<meta name="citation_author" content="William R. Roush">
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<meta name="citation_author" content="Richard I. Morimoto">
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<meta name="citation_author" content="Peter Hodder">
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<meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK148494/">
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<meta name="DC.Title" content="ML346: A Novel Modulator of Proteostasis for Protein Conformational Diseases">
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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="Barbara Calamini">
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<meta name="DC.Contributor" content="Maria Catarina Silva">
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<meta name="DC.Contributor" content="Franck Madoux">
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<meta name="DC.Contributor" content="Darren M. Hutt">
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<meta name="DC.Contributor" content="Shilpi Khanna">
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<meta name="DC.Contributor" content="Monica A. Chalfant">
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<meta name="DC.Contributor" content="Christophe Allais">
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<meta name="DC.Contributor" content="Souad Ouizem">
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<meta name="DC.Contributor" content="Sanjay A. Saldanha">
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<meta name="DC.Contributor" content="Jill Ferguson">
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<meta name="DC.Contributor" content="Becky A. Mercer">
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<meta name="DC.Contributor" content="Cameron Michael">
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<meta name="DC.Contributor" content="Bradley D. Tait">
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<meta name="DC.Contributor" content="Dan Garza">
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<meta name="DC.Contributor" content="William E. Balch">
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<meta name="DC.Contributor" content="William R. Roush">
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<meta name="DC.Contributor" content="Richard I. Morimoto">
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<meta name="DC.Contributor" content="Peter Hodder">
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<meta name="DC.Date" content="2013/04/05">
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<meta name="description" content="Protein homeostasis, also called proteostasis, is critical for cellular health and its dysregulation is implicated in aging, cancer, metabolic disease, and neurodegenerative disorders. Proteostasis involves compartmentalized cellular responses (e.g. Heat Shock Response in the cytoplasm, Unfolded Protein Response in the mitochondria and endoplasmic reticulum) that limit protein misfolding and aggregation. Diseases of protein conformation are characterized by inefficient induction of these responses. As a result, identification of molecules that activate cellular stress responses and increase proteostasis may be useful for maintaining cell health. Here, we report on high throughput screening efforts that resulted in identification of a novel activator of heat shock protein 70 (Hsp70): ML346. Probe ML346 belongs to the barbituric acid scaffold. ML346 induces HSF-1-dependent chaperone expression and restores protein folding in conformational disease models. These effects are mediated by novel mechanisms involving FOXO, HSF-1, and Nfr-2.">
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<meta name="og:description" content="Protein homeostasis, also called proteostasis, is critical for cellular health and its dysregulation is implicated in aging, cancer, metabolic disease, and neurodegenerative disorders. Proteostasis involves compartmentalized cellular responses (e.g. Heat Shock Response in the cytoplasm, Unfolded Protein Response in the mitochondria and endoplasmic reticulum) that limit protein misfolding and aggregation. Diseases of protein conformation are characterized by inefficient induction of these responses. As a result, identification of molecules that activate cellular stress responses and increase proteostasis may be useful for maintaining cell health. Here, we report on high throughput screening efforts that resulted in identification of a novel activator of heat shock protein 70 (Hsp70): ML346. Probe ML346 belongs to the barbituric acid scaffold. ML346 induces HSF-1-dependent chaperone expression and restores protein folding in conformational disease models. These effects are mediated by novel mechanisms involving FOXO, HSF-1, and Nfr-2.">
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match">◀</a><button id="jr-fip-matches">no matches yet</button><a id="jr-fip-next" class="wsprkl btn" title="Jump to next match">▶</a></nav></nav></div><div id="jr-epub-interstitial" class="hidden"></div><div id="jr-content"><article data-type="main"><div class="main-content lit-style" itemscope="itemscope" itemtype="http://schema.org/CreativeWork"><div class="meta-content fm-sec"><div class="fm-sec"><h1 id="_NBK148494_"><span class="title" itemprop="name">ML346: A Novel Modulator of Proteostasis for Protein Conformational Diseases</span></h1><p class="contribs">Calamini B, Silva MC, Madoux F, et al.</p><p class="fm-aai"><a href="#_NBK148494_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>Protein homeostasis, also called proteostasis, is critical for cellular health and its dysregulation is implicated in aging, cancer, metabolic disease, and neurodegenerative disorders. Proteostasis involves compartmentalized cellular responses (e.g. Heat Shock Response in the cytoplasm, Unfolded Protein Response in the mitochondria and endoplasmic reticulum) that limit protein misfolding and aggregation. Diseases of protein conformation are characterized by inefficient induction of these responses. As a result, identification of molecules that activate cellular stress responses and increase proteostasis may be useful for maintaining cell health. Here, we report on high throughput screening efforts that resulted in identification of a novel activator of heat shock protein 70 (Hsp70): <a href="/pcsubstance/?term=ML346[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML346</a>. Probe <a href="/pcsubstance/?term=ML346[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML346</a> belongs to the barbituric acid scaffold. <a href="/pcsubstance/?term=ML346[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML346</a> induces HSF-1-dependent chaperone expression and restores protein folding in conformational disease models. These effects are mediated by novel mechanisms involving FOXO, HSF-1, and Nfr-2.</p></div><div class="h2"></div><p><b>Assigned Assay Grant #:</b> 5 R21 NS056337-02</p><p><b>Screening Center Name & PI:</b> The Scripps Research Institute Molecular Screening Center (SRIMSC), H. Rosen</p><p><b>Chemistry Center Name & PI:</b> SRIMSC, H. Rosen</p><p><b>Assay Submitter & Institution:</b> R. Morimoto, Northwestern University</p><p><b>PubChem Summary Bioassay Identifier (AID):</b>
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<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/588815" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">588815</a></p><div id="ml346.rp"><h2 id="_ml346_rp_">Resulting Publications</h2><dl class="temp-labeled-list"><dl class="bkr_refwrap"><dt>1.</dt><dd><div class="bk_ref" id="ml246.rp1">Calamini B, et al. Small-molecule
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proteostasis regulators for protein conformational diseases. <span><span class="ref-journal">Nat Chem
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Biol. </span>2012;<span class="ref-vol">8</span>(2):185–96.</span> [<a href="/pmc/articles/PMC3262058/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3262058</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/22198733" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 22198733</span></a>]</div></dd></dl></dl></div><div id="ml346.s1"><h2 id="_ml346_s1_">Probe Structure & Characteristics</h2><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml346tu1"><a href="/books/NBK148494/table/ml346.tu1/?report=objectonly" target="object" title="Table" class="img_link icnblk_img figpopup" rid-figpopup="figml346tu1" rid-ob="figobml346tu1"><img class="small-thumb" src="/books/NBK148494/table/ml346.tu1/?report=thumb" src-large="/books/NBK148494/table/ml346.tu1/?report=previmg" alt="Image " /></a><div class="icnblk_cntnt"><h4 id="ml346.tu1"><a href="/books/NBK148494/table/ml346.tu1/?report=objectonly" target="object" rid-ob="figobml346tu1">Table</a></h4><p class="float-caption no_bottom_margin">Hsp70 QPCR: 2.4-fold induction (Active) [SID 152186720] HSP70 downstream target QPCR [SID 152186720]</p></div></div></div><div id="ml346.s2"><h2 id="_ml346_s2_">1. Recommendations for Scientific Use of the Probe</h2><p>As an activator of Hsp70 expression and HSF-1 activity, <a href="/pcsubstance/?term=ML346[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML346</a> is immediately useful in understanding how the heat shock responses in general, and these proteins in particular regulate the processing, folding, and recycling of misfolded proteins. <a href="/pcsubstance/?term=ML346[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML346</a> has good chemical stability, significantly high water solubility, is not reactive with excess glutathione, and is cell permeable. The probe <a href="/pcsubstance/?term=ML346[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML346</a> and its analogs, and any improved derivatives that emerge, will help elucidate roles for activation of Hsp70 and HSF-1 in the prevention and progression of cancers, cellular aging, and metabolic and neurodegenerative disorders.</p><div id="ml346.s3"><h3>Introduction</h3><p>The human heat shock protein 70 (Hsp70) family is evolutionarily conserved among all organisms from <i>archaebacteria</i> to humans, suggesting an essential role in cell survival [<a class="bibr" href="#ml346.r1" rid="ml346.r1">1</a>, <a class="bibr" href="#ml346.r2" rid="ml346.r2">2</a>]. Under circumstances of transient cell stress, the heat shock response and activities of molecular chaperones can restore protein homeostasis. In human disease, however, misfolded proteins can accumulate when polyglutamine-expansion proteins are chronically expressed over the life of the cell. Elevated expression of molecular chaperones suppresses protein misfolding/aggregation and toxicity phenotypes in various model systems of disease. Mutations in the respective proteins huntingtin, tau, alpha-synuclein, and superoxide dismutase (SOD1), associated with these diseases, result in the appearance of misfolded species that adopt alternate conformations. These observations led to the proposal that a common feature of diverse diseases of protein conformation is the appearance of alternate folded states that self-associate and form toxic species and protein aggregates.</p><p>A role for Hsp70 family proteins in controlling these events has been widely studied. Studies with mammalian tissue culture cells, transgenic mice, Drosophila, and <i>C. elegans</i> have established that the heat shock response can be activated in cells expressing aggregation-prone proteins, suggesting a role for molecular chaperones as an adaptive survival response [<a class="bibr" href="#ml346.r3" rid="ml346.r3">3</a>, <a class="bibr" href="#ml346.r4" rid="ml346.r4">4</a>]. Moreover, a direct relationship with polyglutamine diseases is suggested by the co-localization of several heat shock proteins, including Hdj-1, Hdj-2, Hsp70 and ubiquitin with polyglutamine aggregates in the tissues of affected individuals, transgenic mice and tissue culture cells [<a class="bibr" href="#ml346.r5" rid="ml346.r5">5</a>]. Finally, overexpression of Hsp70 can suppress the toxicity associated with the accumulation of misfolded proteins [<a class="bibr" href="#ml346.r6" rid="ml346.r6">6</a>–<a class="bibr" href="#ml346.r8" rid="ml346.r8">8</a>]. High throughput screening initiatives aimed at the identification of compounds that enhance the heat shock response, in particular Hsp70, will provide insights into this conserved cellular process and may lead to novel therapeutics for these devastating disorders.</p><p>We developed a high throughput screen that measures activation of the heat shock response in HeLa cells stably transfected with a heat-shock–inducible reporter containing the proximal human Hsp70.1 promoter sequence upstream of a luciferase (luc) reporter gene [<a class="bibr" href="#ml346.r9" rid="ml346.r9">9</a>]. The SRIMSC screened this assay against over 196,000 compounds from the Molecular Libraries Probe Production Center Network (MLPCN) library and another 600,000 compounds from the Scripps institutional drug discovery library.</p><p>Our strategy was designed to identify cell-permeable small molecule activators. Our HTS effort was complemented by a medicinal chemistry and molecular biology effort intended to optimize properties of the active compounds that were identified, to define their mode of action, to show their relevance, and finally to deliver a potent and selective probe. The resulting probe compound, <a href="/pcsubstance/?term=ML346[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML346</a>, belongs to the barbituric acid scaffold. <a href="/pcsubstance/?term=ML346[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML346</a> has been extensively tested by the assay provider in several mechanism-of-action and protein folding assays. Of note, <a href="/pcsubstance/?term=ML346[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML346</a> activates transcription of the Hsp70 promoter and suppresses aggregation of poly-glutamines in a <i>C. elegans</i> model, suggesting the probe has efficacy in modifying protein aggregation and associated toxicity.</p><p>The simplicity of the structure of <a href="/pcsubstance/?term=ML346[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML346</a>, its relative ease of preparation, its favorable physical characteristics, and its impressive biological properties all contribute to its use as a molecular probe useful for understanding the role(s) of heat shock response in cellular growth and aging.</p><p><b><i>Much of the data in this probe report has been published in our Nature Chemical Biology manuscript [<a class="bibr" href="#ml346.r9" rid="ml346.r9">9</a>] and we refer readers to that publication for details and insights beyond those presented in this probe report.</i></b></p></div></div><div id="ml346.s4"><h2 id="_ml346_s4_">2. Materials and Methods</h2><p><b>Chemistry:</b> All chemical reagents and solvents were acquired from commercial vendors. Reactions were monitored by LC/MS (Thermo/Finnegan LCQ Duo system with MS/MS capability). An Agilent 1200 analytical HPLC was used for quantitative purity assessment. Teledyne-Isco “combiflash” automated silica gel MPLC instruments were used for chromatographic purifications. A 400 Brüker MHz NMR instrument was used for NMR analysis.</p><p><b>Biology:</b> All protocols are reported in the relevant PubChem AIDs and in [<a class="bibr" href="#ml346.r9" rid="ml346.r9">9</a>].</p><p><b>Evaluation of compound properties:</b> Solubility, stability, and glutathione reactivity analyses were conducted in accordance with NIH guidelines.</p><div id="ml346.s5"><h3>2.1. Assays</h3><p><a class="figpopup" href="/books/NBK148494/table/ml346.t1/?report=objectonly" target="object" rid-figpopup="figml346t1" rid-ob="figobml346t1">Table 1</a> shows Hsp70-related PubChem AIDs. Descriptions of the assays follow the table.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml346t1"><a href="/books/NBK148494/table/ml346.t1/?report=objectonly" target="object" title="Table 1" class="img_link icnblk_img figpopup" rid-figpopup="figml346t1" rid-ob="figobml346t1"><img class="small-thumb" src="/books/NBK148494/table/ml346.t1/?report=thumb" src-large="/books/NBK148494/table/ml346.t1/?report=previmg" alt="Table 1. Hsp70 Activators PubChem Summary." /></a><div class="icnblk_cntnt"><h4 id="ml346.t1"><a href="/books/NBK148494/table/ml346.t1/?report=objectonly" target="object" rid-ob="figobml346t1">Table 1</a></h4><p class="float-caption no_bottom_margin">Hsp70 Activators PubChem Summary. </p></div></div><div id="ml346.s6"><h4>Assay Descriptions</h4><p>Click on the hyperlinked text to see the assay details in PubChem.</p><div id="ml346.s7"><h5>Hsp70 Activation Assays (AID 1203, AID 1252)</h5><p>The purpose of this assay is to determine the ability of compounds from the MLPCN library to act as activators of Hsp70 expression. Induction of the heat shock response by test compound is measured in a HeLa cell line stably expressing a luciferase reporter under control of the human Hsp70 promoter. As designed, a compound that acts as an activator of Hsp70 expression will activate the Hsp70 promoter, which will increase luciferase transcription, and thus increase well luminescence as detected with the appropriate substrate. Compounds were tested in singlicate at a final nominal concen<i>t</i>ration of 10 micromolar (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1203" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1203</a>), and in triplicate using a 10-point 1:3 dilution series starting at a nominal test concentration of 100 μM (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1252" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1252</a>).</p><p>The hsp70.1pr-luc HeLa cell line was grown in tissue culture flasks in Dulbecco’s Modified Eagle’s Media supplemented with 10% v/v fetal bovine serum, 1% pen-strep-neomycin antibiotic mixture and 1% Geneticin at 37 degrees C in an atmosphere of 5% CO2 and 95% relative humidity (RH).</p><p>Prior to the start of the assay, cells were resuspended in growth media as above at a concentration of 750,000 cells/mL. Next, 5 μL of well-mixed cell suspension were dispensed into each well of 1536-well plates (3,750 cells per well). After incubation for 4 hours at 37 degrees C, 5% CO2 and 95% (RH), the assay was started by dispensing 50 nL of test compound in DMSO to sample wells, DMSO alone (1% final concentration) to negative control wells, or MG132 (final nominal EC100 concentration of 30 μM, set as 100% activation) to positive control wells. The plates were then incubated for 16 hours at 37 degrees C (5% CO2, 95% RH). The assay was stopped by dispensing 5 μL of SteadyLite HTS luciferase substrate to each well, followed by incubation at room temperature for 15 minutes. Luminescence was measured on the ViewLux plate reader. The percent activity was defined using the following mathematical formula:</p><div class="pmc_disp_formula whole_rhythm clearfix" id="ml346.eq1"><div class="inline_block pmc_inline_block pmc_va_middle pmc_hide_overflow twelve_col">% Activity = 100*((Test_Compound − Median_Low_Control) / (Median_High_Control −<br />Median_Low_Control))</div><div class="inline_block pmc_inline_block pmc_va_middle pmc_hide_overflow last bk_equ_label "><div><span class="nowrap"></span></div></div></div><p>Where:</p><ul><li class="half_rhythm"><div>Test_Compound is defined as wells containing test compound</div></li><li class="half_rhythm"><div>High_Control is defined as wells containing MG132</div></li><li class="half_rhythm"><div>and Low_Control is define as wells containing DMSO</div></li></ul><p>A mathematical algorithm was used to determine nominally activating compounds in the primary screen. Two values were calculated: (1) the average percent activation of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % activation than the cutoff parameter was declared active.</p><p>List of reagents:</p><ul><li class="half_rhythm"><div>Dulbecco’s Modified Eagle’s Media (Invitrogen, part 11965-092)</div></li><li class="half_rhythm"><div>Fetal Bovine Serum (Hyclone, part SH 30088.03)</div></li><li class="half_rhythm"><div>Geneticin (Invitrogen, part 10131-027)</div></li><li class="half_rhythm"><div>Penicillin-Streptomycin-Neomycin antibiotic mix (Invitrogen, part 15640-055)</div></li><li class="half_rhythm"><div>SteadyLite HTS luciferase substrate (PerkinElmer, part 6016989)</div></li><li class="half_rhythm"><div>1536-well plates (Greiner, part 789173)</div></li><li class="half_rhythm"><div>T175 HYPERflasks (Corning, part 10010)</div></li><li class="half_rhythm"><div>Reference agonist MG132 (American Peptide, part 81-5-15)</div></li></ul></div><div id="ml346.s8"><h5>HeLa Cytotoxicity Assays (AID 1259)</h5><p>The purpose of this assay is to determine the cytotoxicity of test compounds from the MLPCN library identified as active in a previous set of experiments entitled, “Primary cell-based high-throughput screening assay to identify transcriptional activators of heat shock protein 70 (Hsp70)” (PubChem <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1203" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1203</a>) and confirmed activity via dose-response assays entitled, “Dose response cell-based high-throughput screening assay to identify transcriptional activators of heat shock protein 70 (Hsp70)” (PubChem <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1252" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 1252</a>). The assay utilizes the CellTiter-Glo luminescent reagent to measure intracellular ATP found in viable cells. Luciferase present in the reagent catalyzes the oxidation of beetle luciferin to oxyluciferin and light in the presence of ATP. Thus, well luminescence is directly proportional to ATP levels and cell viability. As designed, compounds that induce cell death will reduce ATP levels, and therefore reduce well luminescence. Compounds were assayed in a 10-point 1:3 dilution series starting at a nominal concentration of 99 μM. Compounds active in the assays above and inactive in this cytotoxicity counterscreen are considered nontoxic inducers of Hsp70 transcription. <a class="figpopup" href="/books/NBK148494/figure/ml346.f1/?report=objectonly" target="object" rid-figpopup="figml346f1" rid-ob="figobml346f1">Figure 1</a> indicates that the probe was not toxic.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml346f1" co-legend-rid="figlgndml346f1"><a href="/books/NBK148494/figure/ml346.f1/?report=objectonly" target="object" title="Figure 1" class="img_link icnblk_img figpopup" rid-figpopup="figml346f1" rid-ob="figobml346f1"><img class="small-thumb" src="/books/NBK148494/bin/ml346f1.gif" src-large="/books/NBK148494/bin/ml346f1.jpg" alt="Figure 1. Probe ML346 is not toxic to HeLa cells." /></a><div class="icnblk_cntnt" id="figlgndml346f1"><h4 id="ml346.f1"><a href="/books/NBK148494/figure/ml346.f1/?report=objectonly" target="object" rid-ob="figobml346f1">Figure 1</a></h4><p class="float-caption no_bottom_margin">Probe ML346 is not toxic to HeLa cells. HeLa-luc cells were treated with increasing concentrations of probe ML346 for 24 h. Cell viability is shown. </p></div></div></div><div id="ml346.s9"><h5>QPCR Hsp70 Assays (AID 651945)</h5><p>The purpose of this assay is to determine whether powder samples of a compound identified as transcriptional activators of heat shock protein 70 (Hsp70) modulates the gene expression of Hsp70 involved in the heat shock response and protein homeostasis. In this assay, HeLa cells are incubated with test compound and harvested. RNA is purified and subjected to quantitative reverse transcription (qRT-PCR) of Hsp70 and GAPDH (control). Gene expression is normalized to GAPDH and compared to levels in cells treated with DMSO only. As designed, test compounds that induce an increase in Hsp70 gene expression will result in an increase in amplified RNA product.</p><p>HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with phenol red buffered with HEPES and supplemented with 10% v/v fetal bovine serum (FBS), 1% L-glutamine, and 100 U/ml penicillin/streptomycin. The cells were treated with test compound, the positive controls celastrol (3 μM), CdCl2 (50 μM) and MG132 (10 μM), or left in vehicle (DMSO). The cells were harvested 4 hours after compound addition for analysis of chaperone expression by quantitative reverse transcription (qRT)-PCR. RNA was purified using the RNeasy Mini kit according to the manufacturer’s instructions. After the reverse transcription reaction, PCR was performed using PCR primers specific for human Hsp70 and GAPDH. PCR products were amplified with Taq polymerase by using standard cycling conditions. Gene expression was normalized to GAPDH and compared to levels in cells treated with DMSO (mRNA levels set as 1.0) using ImageJ. Compounds that induced a minimum of 1.5-fold change in gene expression compared to the DMSO levels (set at 1.0) were active in this assay.</p><p>List of Reagents: HeLa cells (provided by Assay Provider); DMEM (Invitrogen); RNeasy Mini kit (Qiagen, part 74106); Taq polymerase (Promega, part M3001); PCR primers were ordered as appropriate. The primer pair sequences for the human Hsp70 and GAPDH gene targets are indicated below:</p><ul><li class="half_rhythm"><div>Hsp70 forward: 5′-AGAGCCGAGCCGACAGAG-3′</div></li><li class="half_rhythm"><div>Hsp70 reverse: 5′-CACCTTGCCGTGTTGGAA-3′</div></li><li class="half_rhythm"><div>GAPDH forward: 5′-GTCGGAGTCAACGGATT-3′</div></li><li class="half_rhythm"><div>GAPDH reverse: 5′-AAGCTTCCCGTTCTCAG-3′</div></li></ul><p>The results of this assay reveal that probe <a href="/pcsubstance/?term=ML346[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML346</a> increases the expression of Hsp70 mRNA by 2.4-fold over DMSO control levels (<a class="figpopup" href="/books/NBK148494/figure/ml346.f2/?report=objectonly" target="object" rid-figpopup="figml346f2" rid-ob="figobml346f2">Figure 2</a>), further validating the HTS results. The expression levels are normalized to the GAPDH housekeeping gene.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml346f2" co-legend-rid="figlgndml346f2"><a href="/books/NBK148494/figure/ml346.f2/?report=objectonly" target="object" title="Figure 2" class="img_link icnblk_img figpopup" rid-figpopup="figml346f2" rid-ob="figobml346f2"><img class="small-thumb" src="/books/NBK148494/bin/ml346f2.gif" src-large="/books/NBK148494/bin/ml346f2.jpg" alt="Figure 2. Probe ML346 (“F1”) induces Hsp70 mRNA expression." /></a><div class="icnblk_cntnt" id="figlgndml346f2"><h4 id="ml346.f2"><a href="/books/NBK148494/figure/ml346.f2/?report=objectonly" target="object" rid-ob="figobml346f2">Figure 2</a></h4><p class="float-caption no_bottom_margin">Probe ML346 (“F1”) induces Hsp70 mRNA expression. “D” indicates DMSO control. The other lanes are for other positive controls. See [9] for details. </p></div></div></div><div id="ml346.s10"><h5>Chaperone Protein Western blots (AID 651948)</h5><p>The purpose of this assay is to determine whether powder sample of a test compound identified as transcriptional activators of heat shock protein 70 (Hsp70) induces a change in the protein levels of three chaperones–Hsp70, Hsp40, and Hsp27. This will provide further evidence that the probe can induce the HSR. In this assay, HeLa cells are incubated with test compound, following by harvesting and western blot analysis of the reaction products using standard western blotting techniques. As designed, test compounds that induce a change in protein expression will result in a change in protein signal (compared to DMSO control) detected on the Western blot. Compound was tested at 10 μM.</p><p>HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with phenol red buffered with HEPES and supplemented with 10% v/v fetal bovine serum (FBS), 1% L-glutamine, and 100 U/ml penicillin/streptomycin. The cells were treated with test compound (10 μM), the positive controls celastrol (3 μM) and MG132 (10 μM), or left in vehicle (DMSO). The cells were harvested 8 hours after compound addition for analysis of chaperone expression by western blot analysis. Cells were lysed in a buffer containing 20 mM HEPES (N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid; pH 7.9), 25% v/v glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol and 2 mg/ml of complete protease inhibitor cocktail for 30 minutes on ice. 15 ug of whole cell extracts were run on 7.5% SDS-PAGE gels and transferred to nitrocellulose. Primary antibody incubations were for 12 hours at 4 degrees C in 10% BSA. The following primary antibodies were used: a mouse monoclonal Hsp70 antibody, a mouse monoclonal Hsp40 (alphaHdj-1 clone 25), and a mouse monoclonal Hsp27. All primary antibodies were used at a dilution of 1:10,000, except for the Hsp27 antibody, which was diluted 1:500. The anti-beta-tubulin antibody was diluted 1:5,000 and used to verify equal protein loading. The secondary antibody was an Alexa Fluor 680 goat anti mouse IgG diluted 1:5,000. Western analysis was performed with the Odyssey system. Protein expression levels were normalized to tubulin and compared to levels in cells treated with DMSO (mRNA levels set as 1.0) using ImageJ on an Odyssey LI-COR imaging system. Compounds that induced a minimum of 1.5-fold change in protein expression compared to the DMSO levels (set at 1.0) were active in this assay. As shown in <a class="figpopup" href="/books/NBK148494/figure/ml346.f3/?report=objectonly" target="object" rid-figpopup="figml346f3" rid-ob="figobml346f3">Figure 3</a>, these assays reveal that probe <a href="/pcsubstance/?term=ML346[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML346</a> induces the levels of all three chaperone proteins. There was no effect on tubulin, showing that the probe did not act non-specifically.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml346f3" co-legend-rid="figlgndml346f3"><a href="/books/NBK148494/figure/ml346.f3/?report=objectonly" target="object" title="Figure 3" class="img_link icnblk_img figpopup" rid-figpopup="figml346f3" rid-ob="figobml346f3"><img class="small-thumb" src="/books/NBK148494/bin/ml346f3.gif" src-large="/books/NBK148494/bin/ml346f3.jpg" alt="Figure 3. Probe ML346 (“F1”) induces chaperone protein expression." /></a><div class="icnblk_cntnt" id="figlgndml346f3"><h4 id="ml346.f3"><a href="/books/NBK148494/figure/ml346.f3/?report=objectonly" target="object" rid-ob="figobml346f3">Figure 3</a></h4><p class="float-caption no_bottom_margin">Probe ML346 (“F1”) induces chaperone protein expression. “D” indicates DMSO control. The other lanes are for additional compounds. See [9] for details. </p></div></div></div><div id="ml346.s11"><h5>QPCR Hsp70 Downstream Target Assays</h5><p>The purpose of this assay is to determine whether powder samples of compounds identified as transcriptional activators of heat shock protein 70 (Hsp70) modulate the endogenous gene expression of Hsp70 target genes and other stress-responsive proteostasis network pathways (such as the UPR and the anti-oxidant stress response). This assay confirms the effects of HSR activation by test compounds. We measured the expression of the UPR-inducible gene GRP78/BiP, the antioxidant responsive genes heme oxygenase 1 (HO1) and the regulatory subunit of glutamate-cysteine ligase (GCLM), and the proapoptotic growth arrest- and DNA damage-inducible gene 153 (GADD153, also known as CHOP). This assay employed WT and hsf-1 null mouse embryonic fibroblasts (MEFs). The probe (compound F1) induced multiple responses and strongly induced Hsp70 (<a class="figpopup" href="/books/NBK148494/figure/ml346.f4/?report=objectonly" target="object" rid-figpopup="figml346f4" rid-ob="figobml346f4">Figure 4</a>), the oxidative stress response genes (HO1 and GCLM), and a 2.5-fold upregulation of BiP. This assay also suggests that induction of HO1 by probe <a href="/pcsubstance/?term=ML246[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML246</a> was due to the generation of oxidative stress, as shown by a concerted upregulation of the antioxidant GCLM gene. Further, we observed potent induction of the antioxidant responsive gene HO1 in absence of HSF-1, suggesting that the probe’s action is hsf1-independent, elucidating the probe’s mechanism of action.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml346f4" co-legend-rid="figlgndml346f4"><a href="/books/NBK148494/figure/ml346.f4/?report=objectonly" target="object" title="Figure 4" class="img_link icnblk_img figpopup" rid-figpopup="figml346f4" rid-ob="figobml346f4"><img class="small-thumb" src="/books/NBK148494/bin/ml346f4.gif" src-large="/books/NBK148494/bin/ml346f4.jpg" alt="Figure 4. a, Wild type (hsf-1+/+) and b, HSF-1 null (hsf-1−/−) (MEFs) were treated for 4 h with probe ML346 (compound F1) at the indicated concentrations." /></a><div class="icnblk_cntnt" id="figlgndml346f4"><h4 id="ml346.f4"><a href="/books/NBK148494/figure/ml346.f4/?report=objectonly" target="object" rid-ob="figobml346f4">Figure 4</a></h4><p class="float-caption no_bottom_margin"><i>a,</i> Wild type (hsf-1+/+) and <i>b,</i> HSF-1 null (<i>hsf-1−/−</i>) (MEFs) were treated for 4 h with probe ML346 (compound F1) at the indicated concentrations. Relative levels of multiple cytoprotective genes were measured by real-time PCR (qPCR). See <a href="/books/NBK148494/figure/ml346.f4/?report=objectonly" target="object" rid-ob="figobml346f4">(more...)</a></p></div></div></div><div id="ml346.s12"><h5>CFTR Model (QFRET Assay)</h5><p>Human cystic fibrosis bronchial epithelial cells (CFBE41o-) stably expressing ΔF508-CFTR as well as H148Q/I152L-YFP (CFBE41o- -YFP) were added to a 96-well black walled plate and grown to confluency in growth media (α-MEM containing 100 U/ml penicillin, 100 U/ml streptomycin, 10% v/v FBS, 2mM L-glutamine, 2 μg/ml puromycin and 0.75 μg/ml G418). Cells were treated with the indicated concentration of compounds in complete growth media and incubated at 37 degrees C, 5% CO2 for 24 hours. Cells were subsequently washed three times with 200 μL of PBS pH 7.4 (137 mM NaCl, 2.7 mM KCl, 0.7 mM CaCl2, 1.1 mM MgCl2, 1.5 mM KH2PO4, 8.1 mM Na2HPO4) and equilibrated in 40 μL of PBS pH 7.4 and maintained at 37 degrees C throughout. Cells were stimulated with a final concentration of 10 μM forskolin (fsk) and 50 μM genistein (gen) for 15 minutes prior to addition of PBS + NaI (replacement of NaCl with 137 mM NaI). Fluorescence was monitored every second for a total of 30 seconds (3 seconds prior to addition of NaI and 27 seconds after addition of NaI). Data were normalized to the initial fluorescence to account for variations in the overall starting fluorescence. To ensure that the observed H148Q/I152L-YFP fluorescence quenching was the result of ΔF508-CFTR activation and not the action of additional halide channels, the CFTR specific inhibitor (CFInh-172) was used. This assay shows that probe <a href="/pcsubstance/?term=ML246[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML246</a> is able to restore proteostasis reduce the aggregation and misfolding of ΔF508-CFTR (<a class="figpopup" href="/books/NBK148494/figure/ml346.f5/?report=objectonly" target="object" rid-figpopup="figml346f5" rid-ob="figobml346f5">Figure 5</a>). Probe <a href="/pcsubstance/?term=ML346[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML346</a> also restored CFTR-mediated iodide conductance. Probe <a href="/pcsubstance/?term=ML346[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML346</a> is the first small molecule capable of enhancing the correct folding of proteins expressed in two different cellular compartments.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml346f5" co-legend-rid="figlgndml346f5"><a href="/books/NBK148494/figure/ml346.f5/?report=objectonly" target="object" title="Figure 5" class="img_link icnblk_img figpopup" rid-figpopup="figml346f5" rid-ob="figobml346f5"><img class="small-thumb" src="/books/NBK148494/bin/ml346f5.gif" src-large="/books/NBK148494/bin/ml346f5.jpg" alt="Figure 5. Probe ML346 restores proteostasis." /></a><div class="icnblk_cntnt" id="figlgndml346f5"><h4 id="ml346.f5"><a href="/books/NBK148494/figure/ml346.f5/?report=objectonly" target="object" rid-ob="figobml346f5">Figure 5</a></h4><p class="float-caption no_bottom_margin">Probe ML346 restores proteostasis. CFBE41o- YFP cells were treated with 0.1% DMSO (black), the positive controls 5 μM SAHA (purple), 10 μM Corrector 4a (Corr4a) (grey) and the PRs A3 (dark blue), C1 (royal blue) and Probe ML346 (F1; cyan) <a href="/books/NBK148494/figure/ml346.f5/?report=objectonly" target="object" rid-ob="figobml346f5">(more...)</a></p></div></div></div></div></div><div id="ml346.s13"><h3>2.2. Probe Chemical Characterization</h3><p>The chemical structure of the probe was verified by analysis of its 400 MHz <sup>1</sup>H NMR spectra (<a class="figpopup" href="/books/NBK148494/figure/ml346.f6/?report=objectonly" target="object" rid-figpopup="figml346f6" rid-ob="figobml346f6">Figure 6</a>) obtained on a Brüker 400 MHz instrument. The chemical structure was also corroborated by high resolution mass spectroscopy (calc for M+: 273.0875, found: 273.0891). Purity was measured at >98% (LC/MS analysis, confirmed by analytical HLPC analysis. The mass spectrum is shown in <a class="figpopup" href="/books/NBK148494/figure/ml346.f7/?report=objectonly" target="object" rid-figpopup="figml346f7" rid-ob="figobml346f7">Figure 7</a>. HPLC purity data is shown in <a class="figpopup" href="/books/NBK148494/figure/ml346.f8/?report=objectonly" target="object" rid-figpopup="figml346f8" rid-ob="figobml346f8">Figure 8</a>. HPLC data was obtained using an Agilent 1200 analytical HPLC with an Agilent Eclipse XDB-C18 column, 4.6×150mm. The HPLC solvents used were acetonitrile and water with 0.1% formic acid added to each mobile phase as the pH modifier.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml346f6" co-legend-rid="figlgndml346f6"><a href="/books/NBK148494/figure/ml346.f6/?report=objectonly" target="object" title="Figure 6" class="img_link icnblk_img figpopup" rid-figpopup="figml346f6" rid-ob="figobml346f6"><img class="small-thumb" src="/books/NBK148494/bin/ml346f6.gif" src-large="/books/NBK148494/bin/ml346f6.jpg" alt="Figure 6. 1H NMR spectrum." /></a><div class="icnblk_cntnt" id="figlgndml346f6"><h4 id="ml346.f6"><a href="/books/NBK148494/figure/ml346.f6/?report=objectonly" target="object" rid-ob="figobml346f6">Figure 6</a></h4><p class="float-caption no_bottom_margin"><sup>1</sup>H NMR spectrum. </p></div></div><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml346f7" co-legend-rid="figlgndml346f7"><a href="/books/NBK148494/figure/ml346.f7/?report=objectonly" target="object" title="Figure 7" class="img_link icnblk_img figpopup" rid-figpopup="figml346f7" rid-ob="figobml346f7"><img class="small-thumb" src="/books/NBK148494/bin/ml346f7.gif" src-large="/books/NBK148494/bin/ml346f7.jpg" alt="Figure 7. Mass spectrum, calc for M+1: 273.08, found 273.10." /></a><div class="icnblk_cntnt" id="figlgndml346f7"><h4 id="ml346.f7"><a href="/books/NBK148494/figure/ml346.f7/?report=objectonly" target="object" rid-ob="figobml346f7">Figure 7</a></h4><p class="float-caption no_bottom_margin">Mass spectrum, calc for M+1: 273.08, found 273.10. </p></div></div><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml346f8" co-legend-rid="figlgndml346f8"><a href="/books/NBK148494/figure/ml346.f8/?report=objectonly" target="object" title="Figure 8" class="img_link icnblk_img figpopup" rid-figpopup="figml346f8" rid-ob="figobml346f8"><img class="small-thumb" src="/books/NBK148494/bin/ml346f8.gif" src-large="/books/NBK148494/bin/ml346f8.jpg" alt="Figure 8. HPLC spectrum; purity >95%." /></a><div class="icnblk_cntnt" id="figlgndml346f8"><h4 id="ml346.f8"><a href="/books/NBK148494/figure/ml346.f8/?report=objectonly" target="object" rid-ob="figobml346f8">Figure 8</a></h4><p class="float-caption no_bottom_margin">HPLC spectrum; purity >95%. </p></div></div><p>The solubility of the probe <a href="/pcsubstance/?term=ML346[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML346</a> in PBS at pH 7.4 was determined to be 0.64 μM, while in DMES with 10% FBS the solubility was 21.45 μM (see <a class="figpopup" href="/books/NBK148494/table/ml346.t2/?report=objectonly" target="object" rid-figpopup="figml346t2" rid-ob="figobml346t2">Table 2</a>). Its solubility is fully adequate to provide the high potency seen in cell-based assays and is also adequate for broad use as a biological probe to be used in a variety of aqueous-based media.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml346t2"><a href="/books/NBK148494/table/ml346.t2/?report=objectonly" target="object" title="Table 2" class="img_link icnblk_img figpopup" rid-figpopup="figml346t2" rid-ob="figobml346t2"><img class="small-thumb" src="/books/NBK148494/table/ml346.t2/?report=thumb" src-large="/books/NBK148494/table/ml346.t2/?report=previmg" alt="Table 2. Solubility at 100 μM; 1% DMSO." /></a><div class="icnblk_cntnt"><h4 id="ml346.t2"><a href="/books/NBK148494/table/ml346.t2/?report=objectonly" target="object" rid-ob="figobml346t2">Table 2</a></h4><p class="float-caption no_bottom_margin">Solubility at 100 μM; 1% DMSO. </p></div></div><p>The probe <a href="/pcsubstance/?term=ML346[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML346</a> has an apparent half-life of 16 hours in 30% PBS-70% DMSO at room temperature when tested at 10 μM protected from light (<a class="figpopup" href="/books/NBK148494/figure/ml346.f9/?report=objectonly" target="object" rid-figpopup="figml346f9" rid-ob="figobml346f9">Figure 9</a>). The apparent half-life was 6 h when this study was performed in 10% PBS-30% DMSO (absence of light) or 2 h under light exposure. Based on these data, exposure of solutions of the probe to light should be minimized (this can be accomplished by wrapping the glassware in foil or by using dark-glass vessels). Concerning stability of <a href="/pcsubstance/?term=ML346[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML346</a>, the probe was completely stable in a 7:1 mixture of <i>d</i><sup><i>6</i></sup>-DMSO:D<sub>2</sub>O) over 7 days as determined by NMR (<a class="figpopup" href="/books/NBK148494/figure/ml346.f6/?report=objectonly" target="object" rid-figpopup="figml346f6" rid-ob="figobml346f6">Figure 6</a>). Therefore, the apparent instability of the probe compound in PBS (when studied in the dark) is believed to be due to poor solubility under these condition. The potential light-sensitivity of <a href="/pcsubstance/?term=ML346[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML346</a> when in PBS buffer was not an issue in the cell-based and <i>in vivo</i> studies reported in the 2012 Nature Chem. Biol. paper that preceded the submission of this probe report [<a class="bibr" href="#ml346.r9" rid="ml346.r9">9</a>].</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml346f9" co-legend-rid="figlgndml346f9"><a href="/books/NBK148494/figure/ml346.f9/?report=objectonly" target="object" title="Figure 9" class="img_link icnblk_img figpopup" rid-figpopup="figml346f9" rid-ob="figobml346f9"><img class="small-thumb" src="/books/NBK148494/bin/ml346f9.gif" src-large="/books/NBK148494/bin/ml346f9.jpg" alt="Figure 9. Stability of Probe ML346." /></a><div class="icnblk_cntnt" id="figlgndml346f9"><h4 id="ml346.f9"><a href="/books/NBK148494/figure/ml346.f9/?report=objectonly" target="object" rid-ob="figobml346f9">Figure 9</a></h4><p class="float-caption no_bottom_margin">Stability of Probe ML346. </p></div></div><p>The probe is stable (>95%) over a 4 hour period in the presence excess glutathione (50 μM) as determined by HPLC analysis. After 6 hours, 79%–89% of the probe remained unchanged. Because glutathione adducts were not detected by LCMS analysis of these experiments, it is believed that the ‘decrease’ in the amount of probe <a href="/pcsubstance/?term=ML346[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML346</a> after 6 hours is due to solubility issues. These data show that the presence of the highly conjugated diene unit does not render the probe highly susceptible to Michael addition.</p><p>The probe is stable in DMSO solution at room temperature (no erosion of peak intensity over 48 hours) and is also stable as a free base dry powder. It is also stable under assay conditions, as indicated by potency in various secondary assays that is independent of incubation time.</p></div><div id="ml346.s14"><h3>2.3. Probe Preparation</h3><p>Probe <a href="/pcsubstance/?term=ML346[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML346</a> was synthesized in a straightforward fashion in a single step using commercially available reagents, as shown in <a class="figpopup" href="/books/NBK148494/figure/ml346.f10/?report=objectonly" target="object" rid-figpopup="figml346f10" rid-ob="figobml346f10">Figure 10</a>. The yield was 87%. Analogs for SAR evaluation were prepared by similar methods.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml346f10" co-legend-rid="figlgndml346f10"><a href="/books/NBK148494/figure/ml346.f10/?report=objectonly" target="object" title="Figure 10" class="img_link icnblk_img figpopup" rid-figpopup="figml346f10" rid-ob="figobml346f10"><img class="small-thumb" src="/books/NBK148494/bin/ml346f10.gif" src-large="/books/NBK148494/bin/ml346f10.jpg" alt="Figure 10. One Step Synthesis of ML346 (CID 767276)." /></a><div class="icnblk_cntnt" id="figlgndml346f10"><h4 id="ml346.f10"><a href="/books/NBK148494/figure/ml346.f10/?report=objectonly" target="object" rid-ob="figobml346f10">Figure 10</a></h4><p class="float-caption no_bottom_margin">One Step Synthesis of ML346 (CID 767276). </p></div></div><p>A solution of barbituric acid (647 mg, 5.05 mmol, 1.01 equiv; purchased from Fluka, #11709) in water (10 mL) was heated at 100 °C until homogeneous. The resulting solution was cooled to 90 °C, and then 4-methoxycinnamaldehyde (811 mg, 5.00 mmol, 1 equiv; purchased from TCI America, #M1012) was added portion-wise. An orange precipitate appeared almost instantly. The mixture was stirred for 12 h at 90 °C, then was allowed to cool to room temperature. The orange precipitate was filtered off, washed three times with water (10 mL) and one time with cold ethanol (10 mL). The solid was dried under vacuum affording the probe (CID 767276) as an orange powder (1.18 g, 87 % yield, >97% purity by NMR and HPLC).</p><p><b><sup>1</sup>H NMR</b> (400 MHz, <i>d</i><sup><i>6</i></sup>-DMSO): δ 10.70 (s, 1H), 10.65 (s, 1H), 7.82 (dd, <i>J</i> = 12.0, 15.4 Hz, 1H), 7.50 (d, <i>J</i> = 12.1 Hz, 1H), 7.19 (d, <i>J</i> = 15.2 Hz, 1H), 7.16 (d, <i>J</i> = 8.8 Hz, 2H), 6.57 (d, <i>J</i> = 8.8 Hz, 2H), 2.83 (s, 3H); <sup><b>13</b></sup><b>C NMR</b> (100 MHz, CDCl<sub>3</sub>): δ 163.3, 163.1, 162.1, 154.6, 153.4, 150.4, 130.8 (2C), 128.1, 122.1, 114.9 (2C), 114.0, 55.5; <b>HRMS</b> (ESI) calculated for C<sub>14</sub>H<sub>13</sub>N<sub>2</sub>O<sub>4</sub> [M+H]<sup>+</sup>: 273.0875, found: 273.0891; <b>IR</b> (cm<sup>−1</sup>): 3173, 3059, 2827, 1724, 1657, 1588, 1537, 1505, 1428, 1373, 1317, 1306, 1258, 1232, 1213, 1159, 1047, 1020, 993, 946, 937, 880, 842, 820, 793, 756, 702.</p></div></div><div id="ml346.s15"><h2 id="_ml346_s15_">3. Results</h2><div id="ml346.s16"><h3>3.1. Dose Response Curves for Probe</h3><p>This HeLa cell-based assay measures the activation of the heat shock response (HSR) in HeLa cells stably transfected with a heat-shock–inducible reporter containing the proximal human Hsp70.1 promoter sequence upstream of a luciferase (luc) reporter gene. The assay was used in the uHTS campaign and in follow-up runs to aid probe development. Using our standard calculation methods, the probe compound yielded an EC<sub>50</sub> of 4.6 μM in this assay (<a href="https://pubchem.ncbi.nlm.nih.gov/substance/152186720" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 152186720</a>).</p><p>However, instead of graded, sigmoidal dose-response curves, the dose-response profiles (see <a class="figpopup" href="/books/NBK148494/figure/ml346.f12/?report=objectonly" target="object" rid-figpopup="figml346f12" rid-ob="figobml346f12">Figure 11</a>) of all compounds assayed in the HSE-Luc assay were characterized by an increasing luminescence response with increasing test compound dose, followed by a sharp decrease in response at higher dose concentrations. This type of dose-response profile has been previously reported for this cell line [<a class="bibr" href="#ml346.r10" rid="ml346.r10">10</a>]. Therefore the maximum average percent activity measured, and the corresponding concentration at which the maximal activity was observed was used to rank compound potency. As this assay was run in an activation mode, it is not unusual to observe more than 100% activation: it indicates that the compound was more active than the positive control (the proteasome inhibitor MG132 at its EC<sub>100</sub>, 30 micromolar).</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml346f12" co-legend-rid="figlgndml346f12"><a href="/books/NBK148494/figure/ml346.f12/?report=objectonly" target="object" title="Figure 11" class="img_link icnblk_img figpopup" rid-figpopup="figml346f12" rid-ob="figobml346f12"><img class="small-thumb" src="/books/NBK148494/bin/ml346f12.gif" src-large="/books/NBK148494/bin/ml346f12.jpg" alt="Figure 11. Concentration response curve for probe ML346." /></a><div class="icnblk_cntnt" id="figlgndml346f12"><h4 id="ml346.f12"><a href="/books/NBK148494/figure/ml346.f12/?report=objectonly" target="object" rid-ob="figobml346f12">Figure 11</a></h4><p class="float-caption no_bottom_margin">Concentration response curve for probe ML346. </p></div></div><p>We also profiled the probe compound in a variety of secondary assays, including Cytotoxicity assays, and <i>in vitro</i> and <i>in vivo</i> efficacy assays. These results are discussed in <a href="#ml346.s18">section 3.3</a> under profiling assays.</p></div><div id="ml346.s17"><h3>3.2. Cellular Activity</h3><p>The HTS assays and follow-up primary and secondary assays are cell-based, so the probe has confirmed cellular permeability.</p></div><div id="ml346.s18"><h3>3.3. Profiling Assays</h3><p>We recently published a communication describing the screening effort and the uHTS results [<a class="bibr" href="#ml346.r9" rid="ml346.r9">9</a>]. In that communication a great deal of profiling of the screening hit and related leads was disclosed. In particular relevance to this probe report, the assay provider performed an <i>in vivo</i> assay to determine the effect of the probe on protein aggregation. This assay is described in the following sections.</p><div id="ml346.s19"><h4><i>C. elegans</i> Assays for Aggregation and Motility Defects</h4><p>The purpose of this assay is to determine whether powder samples of the compounds identified as transcriptional activators of heat shock protein 70 (Hsp70) can modulate the characteristics of polyglutamine diseases such as Huntington’s. These assays employed a model organism: <i>C. elegans</i> worms that exhibit age-dependent aggregation of polyQ35-YFP in body wall muscles. Worms with polyQ35 aggregation will exhibit decreased motility. As designed, compounds that reduce the aggregation of polyQ35 without reducing levels of the protein are considered active. This assay also monitored aggregation-associated toxicity.</p><p>Worms were maintained according to standard methods, at 20°C on nematode growth media (NGM) with OP50 E. coli (Brenner 1974). The following strains were obtained from the <i>C. elegans</i> Genetic Center (CGC): wild-type (wt) Bristol strain N2, HSF-1 mutant hsf-1(sy441) (PS3551), temperature sensitive strains unc- 52(e669su250) and unc-45(e286) (HE250 and CB286, respectively). The polyglutamine strain expressing 35 CAG-repeats fused with YFP (Q35::YFP) was described elsewhere (AM140 in CGC)[<a class="bibr" href="#ml346.r11" rid="ml346.r11">11</a>].</p><p>Treatment with compounds was performed in a 96-well plate format (final volume 60 μL),
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comprising 20 to 25 L2 (larval 2 stage) age-synchronized animals, compound at the appropriate
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concentration, and OP50 bacteria to a final OD595nm of 0.8 in the microtiter plate. Animals and bacteria were resuspended in S-medium supplemented with streptomycin, penicillin, and nystatin (Sigma, St. Louis, MO). To obtain the age synchronized population of L2 larvae, gravid adults were bleached with a NaOCl solution [250 mM NaOH and 1:4 (v/v) dilution of commercial bleach] and the eggs were allowed to hatch in M9 buffer overnight at 20°C. The first larval stage (L1) animals were transferred to OP50 plates to develop into L2 stage. Animals were washed with M9 buffer, resuspended in S medium, and transferred into 96-well plates. Compounds were dissolved and diluted in 100% DMSO, and animals were incubated at a maximum concentration of 1.5% DMSO to avoid solvent-specific developmental defects and toxicity. The range of final concentrations tested was 0, 1, 5, 10 and 15 μM. OP50-only and DMSO-only controls were used. In addition, 17-(allylamino)-17-demethoxygeldanamycin (17-AAG, Biomol, Plymouth Meeting, PA) (0.5, 1, 5 and 50 μM) was used as positive control for HSR induction. Plates were incubated at 20°C for 4 days. Animals were scored for changes in aggregation (number of fluorescent foci) using the stereomicroscope Leica MZ16FA equipped for epi-fluorescence. Suppression of aggregation was scored positive when ≥50% of worms had a reduction in fluorescent foci, without loss of body-wall fluorescence, compared to DMSO. As shown in the results of <a class="figpopup" href="/books/NBK148494/figure/ml346.f11/?report=objectonly" target="object" rid-figpopup="figml346f11" rid-ob="figobml346f11">Figure 12</a>, probe <a href="/pcsubstance/?term=ML346[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML346</a> is able to suppress the aggregation of polyQ35, as demonstrated by statistically significant reductions in the protein foci. These reductions in protein aggregation correlated with restorations of animal motility (<a class="figpopup" href="/books/NBK148494/figure/ml346.f11/?report=objectonly" target="object" rid-figpopup="figml346f11" rid-ob="figobml346f11">Figure 12c</a>). This result shows the <i>in vivo</i> efficacy of probe <a href="/pcsubstance/?term=ML346[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML346</a> for reducing protein aggregation and associated toxicity.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml346f11" co-legend-rid="figlgndml346f11"><a href="/books/NBK148494/figure/ml346.f11/?report=objectonly" target="object" title="Figure 12" class="img_link icnblk_img figpopup" rid-figpopup="figml346f11" rid-ob="figobml346f11"><img class="small-thumb" src="/books/NBK148494/bin/ml346f11.gif" src-large="/books/NBK148494/bin/ml346f11.jpg" alt="Figure 12. Probe ML346 (compound F1) reduces aggregation/toxicity in C. elegans models of diseases associated with polyQ expansions." /></a><div class="icnblk_cntnt" id="figlgndml346f11"><h4 id="ml346.f11"><a href="/books/NBK148494/figure/ml346.f11/?report=objectonly" target="object" rid-ob="figobml346f11">Figure 12</a></h4><p class="float-caption no_bottom_margin">Probe ML346 (compound F1) reduces aggregation/toxicity in <i>C. elegans</i> models of diseases associated with polyQ expansions. (<i>a</i>) <i>C. elegans</i> expressing YFP-tagged Q35 protein were treated with either DMSO (panel I) or PRs (panels III–V) at different <a href="/books/NBK148494/figure/ml346.f11/?report=objectonly" target="object" rid-ob="figobml346f11">(more...)</a></p></div></div></div><div id="ml346.s20"><h4>PubChem Promiscuity Analyses</h4><p>Analysis of the PubChem database of assays and small molecules shows that the screening hit (CID 1045135) is active in only a small percentage of all assays in which it has been screened (~1.2%), indicating a lack of non-specific protein binding or polypharmacology, characteristics that likely extend to the probe <a href="/pcsubstance/?term=ML346[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML346</a>, a positional isomer of the screening hit. There are no serious issues with regard to the drug-like attributes of the probe [<a class="bibr" href="#ml346.r12" rid="ml346.r12">12</a>–<a class="bibr" href="#ml346.r14" rid="ml346.r14">14</a>]. There are also no concerns with respect to toxicity structure alerts [<a class="bibr" href="#ml346.r15" rid="ml346.r15">15</a>].</p></div></div></div><div id="ml346.s21"><h2 id="_ml346_s21_">4. Discussion</h2><div id="ml346.s22"><h3>4.1. Comparison to Existing Art and How the New Probe is an Improvement</h3><p>Because of the anti-apoptotic and anti-inflammatory properties of Hsp70, research efforts have attempted to increase Hsp70 expression and activity through the use of small molecules. Several activators of the HSR are available, including natural product derivatives. Radicicol (CID 6323491) and geldanamycin (CID 5288382) that inhibit formation of huntingtin protein aggregates [<a class="bibr" href="#ml346.r16" rid="ml346.r16">16</a>]. Celastrol is neuroprotective in animal models of neurodegeneration [<a class="bibr" href="#ml346.r17" rid="ml346.r17">17</a>]. Compounds such as bimoclomol (CID 9576891) [<a class="bibr" href="#ml346.r18" rid="ml346.r18">18</a>] and arimoclomol (CID 208924), promising candidates for the treatment of ischemia and ALS, respectively, demonstrate the ability as coinducers of the HSR and Hsp70 [<a class="bibr" href="#ml346.r19" rid="ml346.r19">19</a>,<a class="bibr" href="#ml346.r20" rid="ml346.r20">20</a>]. Although registered, neither of these compounds has been tested in PubChem bioassays.</p><p>Protein synthesis inhibitors, amino acid analogues [<a class="bibr" href="#ml346.r21" rid="ml346.r21">21</a>,<a class="bibr" href="#ml346.r22" rid="ml346.r22">22</a>], proteasome inhibitors such as MG132 (CID 462382; reversible) and lactacystin (CID 6610292; irreversible), and certain NSAIDs increase the HSR, in part by inducing hyperphosphorylation and/or DNA binding activity of HSF-1 [<a class="bibr" href="#ml346.r23" rid="ml346.r23">23</a>,<a class="bibr" href="#ml346.r24" rid="ml346.r24">24</a>]. However, many of these compounds either do not increase Hsp70 expression or fail to be selective [<a class="bibr" href="#ml346.r24" rid="ml346.r24">24</a>]. However, MG132, used as a High Control for Hsp70 activation is not selective for the HSR.</p><p>In comparison to the above prior art and control compounds, probe <a href="/pcsubstance/?term=ML346[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML346</a> is the first small molecule shown to restore the correct folding of proteins in both cellular and animal models, without significant cytotoxicity or lack of specificity. The probe is cell permeable and induces specific increases in genes and protein effectors of the HSR, including chaperones such as Hsp70, Hsp40, and Hsp27.</p><div id="ml346.s23"><h4>Publications</h4><p>A communication describing the screening effort and the HTS results has been published in 2012 in
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the journal <i>Nature Chemical Biology</i> [<a class="bibr" href="#ml346.r9" rid="ml346.r9">9</a>].</p></div></div></div><div id="ml346.s24"><h2 id="_ml346_s24_">5. References</h2><dl class="temp-labeled-list"><dl class="bkr_refwrap"><dt>1.</dt><dd><div class="bk_ref" id="ml346.r1">Gupta RS, Singh B. Phylogenetic analysis of 70 kD heat shock protein sequences suggests a chimeric origin for the eukaryotic cell nucleus. <span><span class="ref-journal">Curr Biol. </span>1994;<span class="ref-vol">4</span>(12):1104–14.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/7704574" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 7704574</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>2.</dt><dd><div class="bk_ref" id="ml346.r2">Lindquist S, Craig EA. The heat-shock proteins. <span><span class="ref-journal">Annu Rev Genet. </span>1988;<span class="ref-vol">22</span>:631–77.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/2853609" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 2853609</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>3.</dt><dd><div class="bk_ref" id="ml346.r3">Satyal SH, et al. Polyglutamine aggregates alter protein folding homeostasis in Caenorhabditis elegans. <span><span class="ref-journal">Proc Natl Acad Sci U S A. </span>2000;<span class="ref-vol">97</span>(11):5750–5.</span> [<a href="/pmc/articles/PMC18505/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC18505</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/10811890" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 10811890</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>4.</dt><dd><div class="bk_ref" id="ml346.r4">Wyttenbach A, et al. Effects of heat shock, heat shock protein 40 (HDJ-2), and proteasome inhibition on protein aggregation in cellular models of Huntington’s disease. <span><span class="ref-journal">Proc Natl Acad Sci U S A. </span>2000;<span class="ref-vol">97</span>(6):2898–903.</span> [<a href="/pmc/articles/PMC16027/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC16027</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/10717003" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 10717003</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>5.</dt><dd><div class="bk_ref" id="ml346.r5">Cummings CJ, et al. Chaperone suppression of aggregation and altered subcellular proteasome localization imply protein misfolding in SCA1. <span><span class="ref-journal">Nat Genet. </span>1998;<span class="ref-vol">19</span>(2):148–54.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/9620770" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 9620770</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>6.</dt><dd><div class="bk_ref" id="ml346.r6">Krobitsch S, Lindquist S. Aggregation of huntingtin in yeast varies with the length of the polyglutamine expansion and the expression of chaperone proteins. <span><span class="ref-journal">Proc Natl Acad Sci U S A. </span>2000;<span class="ref-vol">97</span>(4):1589–94.</span> [<a href="/pmc/articles/PMC26479/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC26479</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/10677504" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 10677504</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>7.</dt><dd><div class="bk_ref" id="ml346.r7">Kazemi-Esfarjani P, Benzer S. Genetic suppression of polyglutamine toxicity in Drosophila. <span><span class="ref-journal">Science. </span>2000;<span class="ref-vol">287</span>(5459):1837–40.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/10710314" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 10710314</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>8.</dt><dd><div class="bk_ref" id="ml346.r8">Warrick JM, et al. Suppression of polyglutamine-mediated neurodegeneration in Drosophila by the molecular chaperone HSP70. <span><span class="ref-journal">Nat Genet. </span>1999;<span class="ref-vol">23</span>(4):425–8.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/10581028" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 10581028</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>9.</dt><dd><div class="bk_ref" id="ml346.r9">Calamini B, et al. Small-molecule proteostasis regulators for protein conformational diseases. <span><span class="ref-journal">Nat Chem Biol. </span>2012;<span class="ref-vol">8</span>(2):185–96.</span> [<a href="/pmc/articles/PMC3262058/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3262058</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/22198733" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 22198733</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>10.</dt><dd><div class="bk_ref" id="ml346.r10">Westerheide SD, et al. Celastrols as inducers of the heat shock response and cytoprotection. <span><span class="ref-journal">J Biol Chem. </span>2004 Dec 31;<span class="ref-vol">279</span>(53):56053–60.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/15509580" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 15509580</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>11.</dt><dd><div class="bk_ref" id="ml346.r11">Morley JF, et al. The threshold for polyglutamine-expansion protein aggregation and cellular toxicity is dynamic and influenced by aging in Caenorhabditis elegans. <span><span class="ref-journal">Proc Natl Acad Sci U S A. </span>2002;<span class="ref-vol">99</span>(16):10417–22.</span> [<a href="/pmc/articles/PMC124929/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC124929</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/12122205" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 12122205</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>12.</dt><dd><div class="bk_ref" id="ml346.r12">Rishton GM. Nonleadlikeness and leadlikeness in biochemical screening. <span><span class="ref-journal">Drug Discov Today. </span>2003;<span class="ref-vol">8</span>(2):86–96.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/12565011" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 12565011</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>13.</dt><dd><div class="bk_ref" id="ml346.r13">Walters WP. Going further than Lipinski’s rule in drug design. <span><span class="ref-journal">Expert Opin Drug Discov. </span>2012;<span class="ref-vol">7</span>(2):99–107.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/22468912" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 22468912</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>14.</dt><dd><div class="bk_ref" id="ml346.r14">Lipinski CA, et al. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. <span><span class="ref-journal">Adv Drug Deliv Rev. </span>2001;<span class="ref-vol">46</span>(1-3):3–26.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/11259830" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 11259830</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>15.</dt><dd><div class="bk_ref" id="ml346.r15">Benigni R, Bossa C. Mechanisms of chemical carcinogenicity and mutagenicity: a review with implications for predictive toxicology. <span><span class="ref-journal">Chem Rev. </span>2011;<span class="ref-vol">111</span>(4):2507–36.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/21265518" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 21265518</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>16.</dt><dd><div class="bk_ref" id="ml346.r16">Hay DG, et al. Progressive decrease in chaperone protein levels in a mouse model of Huntington’s disease and induction of stress proteins as a therapeutic approach. <span><span class="ref-journal">Hum Mol Genet. </span>2004;<span class="ref-vol">13</span>(13):1389–405.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/15115766" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 15115766</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>17.</dt><dd><div class="bk_ref" id="ml346.r17">Cleren C, et al. Celastrol protects against MPTP- and 3-nitropropionic acid-induced neurotoxicity. <span><span class="ref-journal">J Neurochem. </span>2005;<span class="ref-vol">94</span>(4):995–1004.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/16092942" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 16092942</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>18.</dt><dd><div class="bk_ref" id="ml346.r18">Jednakovits A, et al. In vivo and in vitro acute cardiovascular effects of bimoclomol. <span><span class="ref-journal">Gen Pharmacol. </span>2000;<span class="ref-vol">34</span>(5):363–9.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/11368893" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 11368893</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>19.</dt><dd><div class="bk_ref" id="ml346.r19">Nanasi PP, Jednakovits A. Multilateral in vivo and in vitro protective effects of the novel heat shock protein coinducer, bimoclomol: results of preclinical studies. <span><span class="ref-journal">Cardiovasc Drug Rev. </span>2001;<span class="ref-vol">19</span>(2):133–51.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/11484067" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 11484067</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>20.</dt><dd><div class="bk_ref" id="ml346.r20">Brown IR. Heat shock proteins and protection of the nervous system. <span><span class="ref-journal">Ann N Y Acad Sci. </span>2007;<span class="ref-vol">1113</span>:147–58.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/17656567" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17656567</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>21.</dt><dd><div class="bk_ref" id="ml346.r21">Lee YJ, Dewey WC. Effect of cycloheximide or puromycin on induction of thermotolerance by heat in Chinese hamster ovary cells: dose fractionation at 45.5 degrees C1. <span><span class="ref-journal">Cancer Res. </span>1987;<span class="ref-vol">47</span>(22):5960–6.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/3664499" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 3664499</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>22.</dt><dd><div class="bk_ref" id="ml346.r22">Hightower LE. Cultured animal cells exposed to amino acid analogues or puromycin rapidly synthesize several polypeptides. <span><span class="ref-journal">J Cell Physiol. </span>1980;<span class="ref-vol">102</span>(3):407–27.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/6901532" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 6901532</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>23.</dt><dd><div class="bk_ref" id="ml346.r23">Kim D, Li GC. Proteasome inhibitors lactacystin and MG132 inhibit the dephosphorylation of HSF1 after heat shock and suppress thermal induction of heat shock gene expression. <span><span class="ref-journal">Biochem Biophys Res Commun. </span>1999;<span class="ref-vol">264</span>(2):352–8.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/10529368" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 10529368</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>24.</dt><dd><div class="bk_ref" id="ml346.r24">Lee BS, et al. Pharmacological modulation of heat shock factor 1 by antiinflammatory drugs results in protection against stress-induced cellular damage. <span><span class="ref-journal">Proc Natl Acad Sci U S A. </span>1995;<span class="ref-vol">92</span>(16):7207–11.</span> [<a href="/pmc/articles/PMC41308/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC41308</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/7638169" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 7638169</span></a>]</div></dd></dl></dl></div><div id="bk_toc_contnr"></div></div></div><div class="fm-sec"><h2 id="_NBK148494_pubdet_">Publication Details</h2><h3>Author Information and Affiliations</h3><p class="contrib-group"><h4>Authors</h4><span itemprop="author">Barbara Calamini</span>,<sup>1</sup> <span itemprop="author">Maria Catarina Silva</span>,<sup>1,2</sup> <span itemprop="author">Franck Madoux</span>,<sup>3</sup> <span itemprop="author">Darren M. Hutt</span>,<sup>4</sup> <span itemprop="author">Shilpi Khanna</span>,<sup>5</sup> <span itemprop="author">Monica A. Chalfant</span>,<sup>4</sup> <span itemprop="author">Christophe Allais</span>,<sup>6</sup> <span itemprop="author">Souad Ouizem</span>,<sup>6</sup> <span itemprop="author">Sanjay A. Saldanha</span>,<sup>3</sup> <span itemprop="author">Jill Ferguson</span>,<sup>3</sup> <span itemprop="author">Becky A. Mercer</span>,<sup>3</sup> <span itemprop="author">Cameron Michael</span>,<sup>7</sup> <span itemprop="author">Bradley D. Tait</span>,<sup>5</sup> <span itemprop="author">Dan Garza</span>,<sup>5</sup> <span itemprop="author">William E. Balch</span>,<sup>4</sup> <span itemprop="author">William R. Roush</span>,<sup>6</sup> <span itemprop="author">Richard I. Morimoto</span>,<sup>1</sup> and <span itemprop="author">Peter Hodder</span><sup>3</sup><sup>,*</sup>.</p><h4>Affiliations</h4><div class="affiliation"><sup>1</sup>
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Department of Molecular Biosciences, Rice Institute for Biomedical Research, Northwestern University, Evanston, IL, USA</div><div class="affiliation"><sup>2</sup>
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Faculty of Sciences, Centre for Biodiversity, Functional and Integrative Genomics; (BioFIG), University of Lisboa, Lisboa, Portugal</div><div class="affiliation"><sup>3</sup>
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Scripps Research Institute Molecular Screening Center, Lead Identification Division, The Scripps Research Institute, Scripps Florida, Jupiter, Florida, USA</div><div class="affiliation"><sup>4</sup>
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Department of Cell Biology and Chemical Physiology, Institute for Childhood and Neglected Diseases, The Scripps Research Institute, La Jolla, CA, USA</div><div class="affiliation"><sup>5</sup>
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Proteostasis Therapeutics Inc., Cambridge, MA, USA</div><div class="affiliation"><sup>6</sup>
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Department of Chemistry, The Scripps Research Institute, Scripps Florida</div><div class="affiliation"><sup>7</sup>
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Discovery Biology, Drug Metabolism and Pharmacokinetics, The Scripps Research Institute Florida</div><div class="affiliation">
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<sup>*</sup> Corresponding author:
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<span class="before-email-separator"></span><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="ude.sppircs@preddoh" class="oemail">ude.sppircs@preddoh</a></div><h3>Publication History</h3><p class="small">Received: <span itemprop="datePublished">December 17, 2012</span>; Last Update: <span itemprop="dateModified">April 5, 2013</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>Calamini B, Silva MC, Madoux F, et al. ML346: A Novel Modulator of Proteostasis for Protein Conformational Diseases. 2012 Dec 17 [Updated 2013 Apr 5]. 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/ml347/?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/ml345/?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="table-wrap" id="figobml346tu1"><div id="ml346.tu1" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK148494/table/ml346.tu1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml346.tu1_lrgtbl__"><table><thead><tr><th id="hd_h_ml346.tu1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CID/ML#/SR#</th><th id="hd_h_ml346.tu1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Structure</th><th id="hd_h_ml346.tu1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Target Name</th><th id="hd_h_ml346.tu1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">EC<sub>50</sub> (nM) [SID]</th><th id="hd_h_ml346.tu1_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Anti-target Name</th><th id="hd_h_ml346.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">EC<sub>50</sub> (μM) [SID]</th><th id="hd_h_ml346.tu1_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Fold Selective</th><th id="hd_h_ml346.tu1_1_1_1_8" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Secondary Assay(s): [SID]</th></tr></thead><tbody><tr><td headers="hd_h_ml346.tu1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CID 767276<br /><br /><a href="/pcsubstance/?term=ML346[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML346</a><br /><br />SR-01000219514</td><td headers="hd_h_ml346.tu1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK148494/bin/ml346fu1.jpg" alt="Image ml346fu1.jpg" /></div></td><td headers="hd_h_ml346.tu1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Hsp70</td><td headers="hd_h_ml346.tu1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">4600<br />[<a href="https://pubchem.ncbi.nlm.nih.gov/substance/152186720" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 152186720</a>]</td><td headers="hd_h_ml346.tu1_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">HeLa Cell Toxicity</td><td headers="hd_h_ml346.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>25 μM<br />[<a href="https://pubchem.ncbi.nlm.nih.gov/substance/152186720" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 152186720</a>]</td><td headers="hd_h_ml346.tu1_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>5-fold</td><td headers="hd_h_ml346.tu1_1_1_1_8" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">
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<ul><li class="half_rhythm"><div>Hsp70 QPCR: 2.4-fold induction (Active) [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/152186720" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 152186720</a>]</div></li><li class="half_rhythm"><div>HSP70 downstream target QPCR [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/152186720" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 152186720</a>]</div></li><li class="half_rhythm"><div>CFTR Model [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/152186720" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 152186720</a>]</div></li><li class="half_rhythm"><div>Hsp70 Western Blot</div></li><li class="half_rhythm"><div>1.8-fold induction of chaperone protein (Active) [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/152186720" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 152186720</a>]</div></li><li class="half_rhythm"><div><i>C. elegans</i> Aggregation Model: Active [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/152186720" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 152186720</a>]</div></li></ul></td></tr></tbody></table></div></div></article><article data-type="table-wrap" id="figobml346t1"><div id="ml346.t1" class="table"><h3><span class="label">Table 1</span><span class="title">Hsp70 Activators PubChem Summary<sup>*</sup></span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK148494/table/ml346.t1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml346.t1_lrgtbl__"><table class="no_margin"><thead><tr><th id="hd_h_ml346.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Stage</th><th id="hd_h_ml346.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Assay Name</th><th id="hd_h_ml346.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Tested</th><th id="hd_h_ml346.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Active</th><th id="hd_h_ml346.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">PubChem AID</th></tr></thead><tbody><tr><td headers="hd_h_ml346.t1_1_1_1_1" rowspan="3" colspan="1" style="text-align:left;vertical-align:middle;"><b>HTS</b> (run by Scripps: MLSMR Liquids)</td><td headers="hd_h_ml346.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Primary Hsp70 Assay (1X%ACT; cell-based)</td><td headers="hd_h_ml346.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">196,255</td><td headers="hd_h_ml346.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">82</td><td headers="hd_h_ml346.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1203" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1203</a></td></tr><tr><td headers="hd_h_ml346.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Hsp70 Titration Assay (3XEC50; cell-based)</td><td headers="hd_h_ml346.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">72</td><td headers="hd_h_ml346.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">12</td><td headers="hd_h_ml346.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1252" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1252</a></td></tr><tr><td headers="hd_h_ml346.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">HeLa Cytotoxicity Counterscreen (3XIC50, cell-based)</td><td headers="hd_h_ml346.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">72</td><td headers="hd_h_ml346.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">21</td><td headers="hd_h_ml346.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/1259" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">1259</a></td></tr><tr><td headers="hd_h_ml346.t1_1_1_1_1" rowspan="6" colspan="1" style="text-align:left;vertical-align:middle;"><b>SAR</b> (run by Scripps or the Assay Provider: powders)</td><td headers="hd_h_ml346.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Hsp70 Titration Assay (3XEC50; cell-based)</td><td headers="hd_h_ml346.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">16</td><td headers="hd_h_ml346.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">11</td><td headers="hd_h_ml346.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651950" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">651950</a></td></tr><tr><td headers="hd_h_ml346.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Hsp70 Gene Expression QPCR Assay (cell-based)</td><td headers="hd_h_ml346.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">1</td><td headers="hd_h_ml346.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">1</td><td headers="hd_h_ml346.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651945" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">651945</a></td></tr><tr><td headers="hd_h_ml346.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Hsp70 Western Blot Assay (cell-based)</td><td headers="hd_h_ml346.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">1</td><td headers="hd_h_ml346.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">1</td><td headers="hd_h_ml346.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651948" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">651948</a></td></tr><tr><td headers="hd_h_ml346.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Hsp70 Downstream Target Gene Expression QPCR Assay (cell-based)</td><td headers="hd_h_ml346.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">1</td><td headers="hd_h_ml346.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">1</td><td headers="hd_h_ml346.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651964" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">651964</a></td></tr><tr><td headers="hd_h_ml346.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">HBEC QFRET ΔF508-CFTR Activation (cell-based)</td><td headers="hd_h_ml346.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">1</td><td headers="hd_h_ml346.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">1</td><td headers="hd_h_ml346.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651992" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">651992</a></td></tr><tr><td headers="hd_h_ml346.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Aggregation suppression Assay (<i>C. elegans</i> larvae)</td><td headers="hd_h_ml346.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">1</td><td headers="hd_h_ml346.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">1</td><td headers="hd_h_ml346.t1_1_1_1_5" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;"><a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651963" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">651963</a></td></tr></tbody></table></div><div class="tblwrap-foot"><div><dl class="temp-labeled-list small"><dl class="bkr_refwrap"><dt>*</dt><dd><div id="ml346.tfn1"><p class="no_margin">See also <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/588815" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">Summary AID 588815</a>.</p></div></dd></dl></dl></div></div></div></article><article data-type="fig" id="figobml346f1"><div id="ml346.f1" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK148494/bin/ml346f1.jpg" alt="Figure 1. Probe ML346 is not toxic to HeLa cells." /></div><h3><span class="label">Figure 1</span><span class="title">Probe ML346 is not toxic to HeLa cells</span></h3><div class="caption"><p>HeLa-luc cells were treated with increasing concentrations of probe <a href="/pcsubstance/?term=ML346[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML346</a> for 24 h. Cell viability is shown.</p></div></div></article><article data-type="fig" id="figobml346f2"><div id="ml346.f2" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK148494/bin/ml346f2.jpg" alt="Figure 2. Probe ML346 (“F1”) induces Hsp70 mRNA expression." /></div><h3><span class="label">Figure 2</span><span class="title">Probe ML346 (“F1”) induces Hsp70 mRNA expression</span></h3><div class="caption"><p>“D” indicates DMSO control. The other lanes are for other positive controls. See [<a class="bibr" href="#ml346.r9" rid="ml346.r9">9</a>] for details.</p></div></div></article><article data-type="fig" id="figobml346f3"><div id="ml346.f3" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK148494/bin/ml346f3.jpg" alt="Figure 3. Probe ML346 (“F1”) induces chaperone protein expression." /></div><h3><span class="label">Figure 3</span><span class="title">Probe ML346 (“F1”) induces chaperone protein expression</span></h3><div class="caption"><p>“D” indicates DMSO control. The other lanes are for additional compounds. See [<a class="bibr" href="#ml346.r9" rid="ml346.r9">9</a>] for details.</p></div></div></article><article data-type="fig" id="figobml346f4"><div id="ml346.f4" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK148494/bin/ml346f4.jpg" alt="Figure 4. a, Wild type (hsf-1+/+) and b, HSF-1 null (hsf-1−/−) (MEFs) were treated for 4 h with probe ML346 (compound F1) at the indicated concentrations." /></div><h3><span class="label">Figure 4</span></h3><div class="caption"><p><b>a,</b> Wild type (hsf-1+/+) and <b>b,</b> HSF-1 null (<i>hsf-1−/−</i>) (MEFs) were treated for 4 h with probe <a href="/pcsubstance/?term=ML346[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML346</a> (compound F1) at the indicated concentrations. Relative levels of multiple cytoprotective genes were measured by real-time PCR (qPCR). See [<a class="bibr" href="#ml346.r9" rid="ml346.r9">9</a>] for details.</p></div></div></article><article data-type="fig" id="figobml346f5"><div id="ml346.f5" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK148494/bin/ml346f5.jpg" alt="Figure 5. Probe ML346 restores proteostasis." /></div><h3><span class="label">Figure 5</span><span class="title">Probe ML346 restores proteostasis</span></h3><div class="caption"><p>CFBE41o- YFP cells were treated with 0.1% DMSO (black), the positive controls 5 μM SAHA (purple), 10 μM Corrector 4a (Corr4a) (grey) and the PRs A3 (dark blue), C1 (royal blue) and Probe <a href="/pcsubstance/?term=ML346[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML346</a> (F1; cyan) at 10 μM for 24 h. Fluorescence quenching is indicative of restored ΔF508-CFTR trafficking (mean ± s.e.m.; n = 3). Color-coded asterisks indicate statistically significant differences from DMSO control at the 30 s time point. See [<a class="bibr" href="#ml346.r9" rid="ml346.r9">9</a>] for details.</p></div></div></article><article data-type="fig" id="figobml346f6"><div id="ml346.f6" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%206.%201H%20NMR%20spectrum.&p=BOOKS&id=148494_ml346f6.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img data-src="/books/NBK148494/bin/ml346f6.jpg" alt="Figure 6. 1H NMR spectrum." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 6</span><span class="title"><sup>1</sup>H NMR spectrum</span></h3></div></article><article data-type="fig" id="figobml346f7"><div id="ml346.f7" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%207.%20Mass%20spectrum%2C%20calc%20for%20M%2B1%3A%20273.08%2C%20found%20273.10.&p=BOOKS&id=148494_ml346f7.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img data-src="/books/NBK148494/bin/ml346f7.jpg" alt="Figure 7. Mass spectrum, calc for M+1: 273.08, found 273.10." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 7</span><span class="title">Mass spectrum, calc for M+1: 273.08, found 273.10</span></h3></div></article><article data-type="fig" id="figobml346f8"><div id="ml346.f8" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK148494/bin/ml346f8.jpg" alt="Figure 8. HPLC spectrum; purity >95%." /></div><h3><span class="label">Figure 8</span><span class="title">HPLC spectrum; purity >95%</span></h3></div></article><article data-type="table-wrap" id="figobml346t2"><div id="ml346.t2" class="table"><h3><span class="label">Table 2</span><span class="title">Solubility at 100 μM; 1% DMSO</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK148494/table/ml346.t2/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml346.t2_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml346.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;"></th><th id="hd_h_ml346.t2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;"></th><th id="hd_h_ml346.t2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;"></th><th id="hd_h_ml346.t2_1_1_1_4" colspan="3" rowspan="1" style="text-align:center;vertical-align:bottom;"><i>Analyzed on API6500 instrument</i></th></tr><tr><th headers="hd_h_ml346.t2_1_1_1_1" id="hd_h_ml346.t2_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;">SR1-219514 8hr no light</th><th headers="hd_h_ml346.t2_1_1_1_2" id="hd_h_ml346.t2_1_1_2_2" rowspan="1" colspan="1" style="text-align:right;vertical-align:bottom;">20μM Std - PA</th><th headers="hd_h_ml346.t2_1_1_1_3" id="hd_h_ml346.t2_1_1_2_3" rowspan="1" colspan="1" style="text-align:right;vertical-align:bottom;">pH 7.4 - PA</th><th headers="hd_h_ml346.t2_1_1_1_4" id="hd_h_ml346.t2_1_1_2_4" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">Solubility (μM)</th><th headers="hd_h_ml346.t2_1_1_1_4" id="hd_h_ml346.t2_1_1_2_5" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">DMES+10% FBS</th><th headers="hd_h_ml346.t2_1_1_1_4" id="hd_h_ml346.t2_1_1_2_6" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">DMES+10% FBS Solubility (μM)</th></tr></thead><tbody><tr><td headers="hd_h_ml346.t2_1_1_1_1 hd_h_ml346.t2_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">#1</td><td headers="hd_h_ml346.t2_1_1_1_2 hd_h_ml346.t2_1_1_2_2" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">5.26E+06</td><td headers="hd_h_ml346.t2_1_1_1_3 hd_h_ml346.t2_1_1_2_3" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">1.73E+05</td><td headers="hd_h_ml346.t2_1_1_1_4 hd_h_ml346.t2_1_1_2_4" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;"></td><td headers="hd_h_ml346.t2_1_1_1_4 hd_h_ml346.t2_1_1_2_5" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">5.62E+06</td><td headers="hd_h_ml346.t2_1_1_1_4 hd_h_ml346.t2_1_1_2_6" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;"></td></tr><tr><td headers="hd_h_ml346.t2_1_1_1_1 hd_h_ml346.t2_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">#2</td><td headers="hd_h_ml346.t2_1_1_1_2 hd_h_ml346.t2_1_1_2_2" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">5.18E+06</td><td headers="hd_h_ml346.t2_1_1_1_3 hd_h_ml346.t2_1_1_2_3" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">1.66E+05</td><td headers="hd_h_ml346.t2_1_1_1_4 hd_h_ml346.t2_1_1_2_4" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;"></td><td headers="hd_h_ml346.t2_1_1_1_4 hd_h_ml346.t2_1_1_2_5" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">5.84E+06</td><td headers="hd_h_ml346.t2_1_1_1_4 hd_h_ml346.t2_1_1_2_6" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;"></td></tr><tr><td headers="hd_h_ml346.t2_1_1_1_1 hd_h_ml346.t2_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;">#3</td><td headers="hd_h_ml346.t2_1_1_1_2 hd_h_ml346.t2_1_1_2_2" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">5.29E+06</td><td headers="hd_h_ml346.t2_1_1_1_3 hd_h_ml346.t2_1_1_2_3" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">1.69E+05</td><td headers="hd_h_ml346.t2_1_1_1_4 hd_h_ml346.t2_1_1_2_4" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;"></td><td headers="hd_h_ml346.t2_1_1_1_4 hd_h_ml346.t2_1_1_2_5" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;">5.41E+06</td><td headers="hd_h_ml346.t2_1_1_1_4 hd_h_ml346.t2_1_1_2_6" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;"></td></tr><tr><td headers="hd_h_ml346.t2_1_1_1_1 hd_h_ml346.t2_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>Avg.</b></td><td headers="hd_h_ml346.t2_1_1_1_2 hd_h_ml346.t2_1_1_2_2" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;"><b>5.2E+06</b></td><td headers="hd_h_ml346.t2_1_1_1_3 hd_h_ml346.t2_1_1_2_3" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;"><b>1.7E+05</b></td><td headers="hd_h_ml346.t2_1_1_1_4 hd_h_ml346.t2_1_1_2_4" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;"><b>0.6459</b></td><td headers="hd_h_ml346.t2_1_1_1_4 hd_h_ml346.t2_1_1_2_5" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;"><b>5.6E+06</b></td><td headers="hd_h_ml346.t2_1_1_1_4 hd_h_ml346.t2_1_1_2_6" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;"><b>21.45</b></td></tr><tr><td headers="hd_h_ml346.t2_1_1_1_1 hd_h_ml346.t2_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:top;"><b>%RSD</b></td><td headers="hd_h_ml346.t2_1_1_1_2 hd_h_ml346.t2_1_1_2_2" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;"><b>1.1</b></td><td headers="hd_h_ml346.t2_1_1_1_3 hd_h_ml346.t2_1_1_2_3" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;"><b>2.1</b></td><td headers="hd_h_ml346.t2_1_1_1_4 hd_h_ml346.t2_1_1_2_4" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;"></td><td headers="hd_h_ml346.t2_1_1_1_4 hd_h_ml346.t2_1_1_2_5" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;"><b>3.8</b></td><td headers="hd_h_ml346.t2_1_1_1_4 hd_h_ml346.t2_1_1_2_6" rowspan="1" colspan="1" style="text-align:right;vertical-align:top;"></td></tr></tbody></table></div></div></article><article data-type="fig" id="figobml346f9"><div id="ml346.f9" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%209.%20Stability%20of%20Probe%20ML346.&p=BOOKS&id=148494_ml346f9.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img data-src="/books/NBK148494/bin/ml346f9.jpg" alt="Figure 9. Stability of Probe ML346." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 9</span><span class="title">Stability of Probe ML346</span></h3></div></article><article data-type="fig" id="figobml346f10"><div id="ml346.f10" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK148494/bin/ml346f10.jpg" alt="Figure 10. One Step Synthesis of ML346 (CID 767276)." /></div><h3><span class="label">Figure 10</span><span class="title">One Step Synthesis of ML346 (CID 767276)</span></h3></div></article><article data-type="fig" id="figobml346f12"><div id="ml346.f12" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK148494/bin/ml346f12.jpg" alt="Figure 11. Concentration response curve for probe ML346." /></div><h3><span class="label">Figure 11</span><span class="title">Concentration response curve for probe ML346</span></h3></div></article><article data-type="fig" id="figobml346f11"><div id="ml346.f11" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK148494/bin/ml346f11.jpg" alt="Figure 12. Probe ML346 (compound F1) reduces aggregation/toxicity in C. elegans models of diseases associated with polyQ expansions." /></div><h3><span class="label">Figure 12</span><span class="title">Probe ML346 (compound F1) reduces aggregation/toxicity in <i>C. elegans</i> models of diseases associated with polyQ expansions</span></h3><div class="caption"><p>(<b>a</b>) <i>C. elegans</i> expressing YFP-tagged Q35 protein were treated with either DMSO (panel I) or PRs (panels III–V) at different concentrations (1, 5, 10 and 15 μM) for 4 days. 17-AAG was used as positive control (50 μM, panel II). Fluorescence microscopy shows proteostasis regulators that reduced Q35 aggregation (10 μM) in 6-day old animals. Panels VI–X show higher magnification images. Scale bar: 0.1 mm. (<b>b</b>) Proteostasis regulators suppress Q35 aggregation as shown by the quantification of fluorescent foci in 6-day old animals, relative to DMSO. (<b>c</b>) Rescue from polyQ-associated toxicity was determined by comparing the motility of Q35 animals treated with either DMSO alone or the candidate PRs compounds (10 μM) to that of WT animals in DMSO. Standard error is shown. (<i>t-test</i> ***<i>p</i>-value<0.001).</p></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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