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<script type="text/javascript" src="/corehtml/pmc/jatsreader/ptpmc_3.22/js/jr.boots.min.js"> </script><title>Discovery of Two, Structurally Distinct Agonists of Vibrio cholerae Quorum Sensing Acting via the CqsS Membrane Receptor - 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="Discovery of Two, Structurally Distinct Agonists of Vibrio cholerae Quorum Sensing Acting via the CqsS Membrane Receptor">
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<meta name="citation_publisher" content="National Center for Biotechnology Information (US)">
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<meta name="citation_date" content="2013/09/03">
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<meta name="citation_author" content="Patrick Faloon">
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<meta name="citation_author" content="Willmen Youngsaye">
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<meta name="citation_author" content="Melissa Bennion">
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<meta name="citation_author" content="Wai-Leung Ng">
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<meta name="citation_author" content="Amanda Hurley">
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<meta name="citation_author" content="Timothy A. Lewis">
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<meta name="citation_author" content="Rahul V. Edwankar">
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<meta name="citation_author" content="Evan Yao">
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<meta name="citation_author" content="Jun Pu">
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<meta name="citation_author" content="Partha P. Nag">
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<meta name="citation_author" content="Jacqueline M. Wurst">
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<meta name="citation_author" content="Melissa Bennion">
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<meta name="citation_author" content="Hanh Le">
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<meta name="citation_author" content="Carrie M. Mosher">
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<meta name="citation_author" content="Stephen Johnston">
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<meta name="citation_author" content="Sivaraman Dandapani">
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<meta name="citation_author" content="Benito Munoz">
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<meta name="citation_author" content="Michelle Palmer">
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<meta name="citation_author" content="Bonnie L. Bassler">
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<meta name="citation_author" content="Stuart L. Schreiber">
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<meta name="citation_pmid" content="24260780">
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<meta name="DC.Title" content="Discovery of Two, Structurally Distinct Agonists of Vibrio cholerae Quorum Sensing Acting via the CqsS Membrane Receptor">
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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="Patrick Faloon">
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<meta name="DC.Contributor" content="Willmen Youngsaye">
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<meta name="DC.Contributor" content="Melissa Bennion">
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<meta name="DC.Contributor" content="Wai-Leung Ng">
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<meta name="DC.Contributor" content="Amanda Hurley">
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<meta name="DC.Contributor" content="Timothy A. Lewis">
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<meta name="DC.Contributor" content="Rahul V. Edwankar">
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<meta name="DC.Contributor" content="Evan Yao">
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<meta name="DC.Contributor" content="Jun Pu">
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<meta name="DC.Contributor" content="Partha P. Nag">
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<meta name="DC.Contributor" content="Jacqueline M. Wurst">
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<meta name="DC.Contributor" content="Melissa Bennion">
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<meta name="DC.Contributor" content="Hanh Le">
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<meta name="DC.Contributor" content="Carrie M. Mosher">
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<meta name="DC.Contributor" content="Stephen Johnston">
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<meta name="DC.Contributor" content="Sivaraman Dandapani">
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<meta name="DC.Contributor" content="Benito Munoz">
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<meta name="DC.Contributor" content="Michelle Palmer">
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<meta name="DC.Contributor" content="Bonnie L. Bassler">
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<meta name="DC.Contributor" content="Stuart L. Schreiber">
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<meta name="DC.Date" content="2013/09/03">
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<meta name="description" content="Quorum sensing (QS) is a process of bacterial cell-to-cell communication that relies upon recognition of extracellular signaling molecules called autoinducers. QS allows bacteria to synchronize their behavior in response to changes in the population density and species composition of the proximal bacterial community. Known behaviors regulated by QS include bioluminescence, sporulation, virulence factor production, and biofilm formation. We carried out a high throughput screen (HTS) to identify small molecules that modulate QS in a modified V. cholerae strain carrying a luciferase operon; activation of the quorum pathway is accompanied by light production. 352,083 compounds from the NIH-MLPCN compound library were evaluated. Potential QS modulators were characterized via additional bacterium-based epistatic assays to elucidate their mode of action. We report the discovery and development of two, structurally distinct, small-molecule probes (ML343 and ML344) shown to be agonists of Vibrio cholerae CqsS, a transmembrane receptor. ML343 and ML344 should greatly expand the general understanding of how QS membrane receptors productively interact with ligands and how they relay information from the external environment. In addition these compounds could lead to the development of antibacterial drugs designed to interfere with QS which could have enormous ramifications for improving human health.">
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<meta name="og:description" content="Quorum sensing (QS) is a process of bacterial cell-to-cell communication that relies upon recognition of extracellular signaling molecules called autoinducers. QS allows bacteria to synchronize their behavior in response to changes in the population density and species composition of the proximal bacterial community. Known behaviors regulated by QS include bioluminescence, sporulation, virulence factor production, and biofilm formation. We carried out a high throughput screen (HTS) to identify small molecules that modulate QS in a modified V. cholerae strain carrying a luciferase operon; activation of the quorum pathway is accompanied by light production. 352,083 compounds from the NIH-MLPCN compound library were evaluated. Potential QS modulators were characterized via additional bacterium-based epistatic assays to elucidate their mode of action. We report the discovery and development of two, structurally distinct, small-molecule probes (ML343 and ML344) shown to be agonists of Vibrio cholerae CqsS, a transmembrane receptor. ML343 and ML344 should greatly expand the general understanding of how QS membrane receptors productively interact with ligands and how they relay information from the external environment. In addition these compounds could lead to the development of antibacterial drugs designed to interfere with QS which could have enormous ramifications for improving human health.">
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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="_NBK169447_"><span class="title" itemprop="name">Discovery of Two, Structurally Distinct Agonists of <i>Vibrio cholerae</i> Quorum Sensing Acting <i>via</i> the CqsS Membrane Receptor</span></h1><p class="contribs">Faloon P, Youngsaye W, Bennion M, et al.</p><p class="fm-aai"><a href="#_NBK169447_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>Quorum sensing (QS) is a process of bacterial cell-to-cell communication that relies upon recognition of extracellular signaling molecules called autoinducers. QS allows bacteria to synchronize their behavior in response to changes in the population density and species composition of the proximal bacterial community. Known behaviors regulated by QS include bioluminescence, sporulation, virulence factor production, and biofilm formation. We carried out a high throughput screen (HTS) to identify small molecules that modulate QS in a modified <i>V. cholerae</i> strain carrying a luciferase operon; activation of the quorum pathway is accompanied by light production. 352,083 compounds from the NIH-MLPCN compound library were evaluated. Potential QS modulators were characterized <i>via</i> additional bacterium-based epistatic assays to elucidate their mode of action. We report the discovery and development of two, structurally distinct, small-molecule probes (<a href="/pcsubstance/?term=ML343[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML343</a> and <a href="/pcsubstance/?term=ML344[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML344</a>) shown to be agonists of <i>Vibrio cholerae</i> CqsS, a transmembrane receptor. <a href="/pcsubstance/?term=ML343[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML343</a> and <a href="/pcsubstance/?term=ML344[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML344</a> should greatly expand the general understanding of how QS membrane receptors productively interact with ligands and how they relay information from the external environment. In addition these compounds could lead to the development of antibacterial drugs designed to interfere with QS which could have enormous ramifications for improving human health.</p></div><div class="h2"></div><p><b>Assigned Assay Grant No:</b> R03 MH094166-01</p><p><b>Screening Center Name & PI:</b> Broad institute Probe Development Center, Stuart L. Schreiber, PhD</p><p><b>Chemistry Center Name & PI:</b> Broad institute Probe Development Center, Stuart L. Schreiber, PhD</p><p><b>Assay Submitter & Institution:</b> Bonnie L. Bassler, PhD., Princeton University, Princeton, NJ</p><p><b>PubChem Summary Bioassay Identifier (AID):</b>
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<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/588521" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">588521</a></p><div id="ml344.s1"><h2 id="_ml344_s1_">Probe Structure & Characteristics</h2><div id="ml344.fu1" class="figure bk_fig"><div class="graphic"><img src="/books/NBK169447/bin/ml344fu1.jpg" alt="ML344." /></div><h3><span class="title">ML344</span></h3></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml344tu1"><a href="/books/NBK169447/table/ml344.tu1/?report=objectonly" target="object" title="Table" class="img_link icnblk_img figpopup" rid-figpopup="figml344tu1" rid-ob="figobml344tu1"><img class="small-thumb" src="/books/NBK169447/table/ml344.tu1/?report=thumb" src-large="/books/NBK169447/table/ml344.tu1/?report=previmg" alt="Image " /></a><div class="icnblk_cntnt"><h4 id="ml344.tu1"><a href="/books/NBK169447/table/ml344.tu1/?report=objectonly" target="object" rid-ob="figobml344tu1">Table</a></h4></div></div><div id="ml344.fu2" class="figure bk_fig"><div class="graphic"><img src="/books/NBK169447/bin/ml344fu2.jpg" alt="ML343." /></div><h3><span class="title">ML343</span></h3></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml344tu2"><a href="/books/NBK169447/table/ml344.tu2/?report=objectonly" target="object" title="Table" class="img_link icnblk_img figpopup" rid-figpopup="figml344tu2" rid-ob="figobml344tu2"><img class="small-thumb" src="/books/NBK169447/table/ml344.tu2/?report=thumb" src-large="/books/NBK169447/table/ml344.tu2/?report=previmg" alt="Image " /></a><div class="icnblk_cntnt"><h4 id="ml344.tu2"><a href="/books/NBK169447/table/ml344.tu2/?report=objectonly" target="object" rid-ob="figobml344tu2">Table</a></h4></div></div></div><div id="ml344.s2"><h2 id="_ml344_s2_">1. Recommendations for the Scientific Use of these Probes</h2><p>Discovery of quorum sensing (QS) receptor agonists will permit more precise control of <i>Vibrio cholerae</i> QS through the perturbation of distinct steps within the signaling cascade. This, in turn, would enable elucidation of the underlying principles governing ligand-receptor interactions utilized by transmembrane histidine kinases, such as the <i>V. cholerae</i> QS membrane receptor CqsS. Upon ligand binding, CqsS undergoes a number of conformational changes that are critical to its numerous functions. The newly identified probes may shed light on the species-specific nature of QS ligands and possibly provide a clearer understanding of the inherent kinase and phosphatase activity of CqsS [<a class="bibr" href="#ml344.r1" rid="ml344.r1">1</a>].</p><p>Crystallography efforts to visualize the CqsS transmembrane domain are underway, and these high-quality probes will be evaluated for possible co-crystallization. Concurrently, directed mutagenesis will be applied to understand how these probes interact with CqsS. Since there is some capacity for interspecies communication amongst the different <i>Vibrio</i> species [<a class="bibr" href="#ml344.r2" rid="ml344.r2">2</a>], <a href="/pcsubstance/?term=ML343[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML343</a> and <a href="/pcsubstance/?term=ML344[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML344</a> can be tested for QS agonism with other <i>Vibrio</i> species. Identification of CqsS-agonist binding motifs will establish a framework for the development of antibacterial drugs designed to interfere with quorum sensing. These probes can also test the utility of quorum pathway activation as a means of treating cholera <i>via</i> reduction of pathogen’s virulence <i>in vivo</i>. There are several animal models available with which such studies may be conducted [<a class="bibr" href="#ml344.r3" rid="ml344.r3">3</a>–<a class="bibr" href="#ml344.r5" rid="ml344.r5">5</a>]. If such an approach is efficacious, these potential therapeutics, operating in a completely unexploited target space, would have enormous ramifications upon improving human health.</p></div><div id="ml344.s3"><h2 id="_ml344_s3_">2. Materials and Methods</h2><p>See subsections for a description of the materials and methods used for each assay.</p><div id="ml344.s4"><h3>Materials and Reagents</h3><ul><li class="half_rhythm"><div>CellTiter-Glo<sup>®</sup> Luminescent Cell Viability Assay was purchased from Promega (Catalog No. G7573; Madison, WI)</div></li></ul></div><div id="ml344.s5"><h3>Bacterial strains & Cell Lines</h3><p>The following cell lines were used in this study:</p><ul><li class="half_rhythm"><div><i>Vibrio cholerae</i> BH1578; a genetically modified strain lacking LuxS and CqsA autoinducer synthases that was provided by the Bassler lab. This strain was used in the primary assay.</div></li><li class="half_rhythm"><div><i>Vibrio cholerae</i> BH1651; a genetically modified strain with constitutively active LuxO that was provided by the Bassler lab.</div></li><li class="half_rhythm"><div><i>Vibrio cholerae</i> DH231; a genetically modified strain lacking the CqsS receptor that was provided by the Bassler lab.</div></li><li class="half_rhythm"><div><i>Vibrio cholerae</i> WN1103; a genetically modified strain lacking the LuxQ receptor that was provided by the Bassler lab.</div></li><li class="half_rhythm"><div>HeLa obtained from ATCC (Catalog Number CCL-2; Manassas, VA) is a human epithelial adenocarcinoma cell line used for mammalian cytotoxicity profiling</div></li></ul><p>Note: All of the <i>V. cholerae</i> strains also carry the heterologous <i>V. harveyi luxCDABE</i> luciferase operon</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml344t1"><a href="/books/NBK169447/table/ml344.t1/?report=objectonly" target="object" title="Table 1" class="img_link icnblk_img figpopup" rid-figpopup="figml344t1" rid-ob="figobml344t1"><img class="small-thumb" src="/books/NBK169447/table/ml344.t1/?report=thumb" src-large="/books/NBK169447/table/ml344.t1/?report=previmg" alt="Table 1. Strains of V. cholera used in assays." /></a><div class="icnblk_cntnt"><h4 id="ml344.t1"><a href="/books/NBK169447/table/ml344.t1/?report=objectonly" target="object" rid-ob="figobml344t1">Table 1</a></h4><p class="float-caption no_bottom_margin">Strains of <i>V. cholera</i> used in assays. </p></div></div></div><div id="ml344.s6"><h3>2.1. Assays</h3><div id="ml344.s7"><h4>2.1.1. Primary assay – <i>V. cholerae</i> BH1578 bioluminescence inducer assay (AID 588346, AID 602243, AID 624270, AID 651809, AID 651816)</h4><p>A modified strain of <i>Vibrio cholerae</i> used in this assay uses light production to indicate quorum sensing induction. <i>Vibrio cholerae</i> is not naturally bioluminescent but the closely related species <i>Vibrio harveyi</i> produces light when the population is at a high density (i.e. a quorum is sensed). The heterologous <i>Vibrio harveyi</i> luciferase operon (<i>luxCDABE</i>) was cloned into the <i>Vibrio cholerae</i> C6706 O1 El Tor bacterial strain on the pBB1 cosmid to create a bioluminescence assay strain. This operon is activated by the endogenous <i>V. cholerae</i> quorum sensing pathway [<a class="bibr" href="#ml344.r2" rid="ml344.r2">2</a>]. The BH1578 strain is a cqsA, luxS double mutant that lacks both autoinducer synthases (CAI-1 and AI-2). BH1578 does not generate light in the absence of exogenous autoinducers but bioluminescence can be stimulated up to 10,000-fold by adding 1 μM (saturating) CAI-1. On day 0, a colony of bacteria was picked into 50 mL Luria Broth with 10 μg/mL tetracycline and cultured overnight at 30 °C. On day 1, bacterial density was determined by spectrometry, and the OD600 was adjusted to 0.3. 20 μL of Luria Broth with 10 μg/mL tetracycline was added per well into white, opaque 384 well plates. Compounds and controls were added by pin transfer method. The HTS used 150 nL of compound for screening at 20 μM, and the retest assays used 100 nL of compound. After 6 hours, the luminescence signal was determined with the Perkin-Elmer EnVision plate reader. In addition to luminescence, the confluency of each well was measured at an absorbance of 600 nM. Primary HTS data were analyzed in Genedata Screener Assay Analyzer. All values were normalized against DMSO treated samples and the positive control (1 μM CAI-1, CID 24892809). For the HTS, the average of two replicates was used to rank order activity and to choose compounds for retests. For dose studies, percent (%) activity was determined for each concentration, and the concentration response curves (CRCs) were generated with Genedata Screener’s Condoseo.</p></div><div id="ml344.s8"><h4>2.1.2. Secondary assay – HeLa cytotoxicity assay (AID 624140, AID 651774, AID 651864)</h4><p>HeLa cells were treated with compounds for 24 hours, and then cell viability was measured using the CellTiter-Glo Assay (Promega), a luciferase-based reagent that measures cellular ATP levels. The compounds were tested at different concentrations to determine IC<sub>50</sub> values. Compounds that were inactive (IC<sub>50</sub> ≥ 30 μM) in this assay were considered for probe development. Data were normalized against DMSO in Genedata Screener’s Assay Analyzer. Curves were generated with Genedata Screener’s Condoseo and showed percent (%) activity for the individual doses.</p></div><div id="ml344.s9"><h4>2.1.3. Secondary assay – <i>V. cholerae</i> BH1651 LuxO inhibitor assay (AID 624254, AID 624269, AID 651847)</h4><p>A modified strain of <i>Vibrio cholerae</i> used in this assay uses light production to indicate quorum sensing induction. <i>Vibrio cholerae</i> is not naturally bioluminescent but the closely related species <i>Vibrio harveyi</i> produces light when the population is at a high density (i.e. a quorum is sensed). The heterologous <i>Vibrio harveyi</i> luciferase operon (<i>luxCDABE</i>) was cloned into the <i>Vibrio cholerae</i> C6706 O1 El Tor bacterial strain on the pBB1 cosmid to create a bioluminescence assay strain. This operon is activated by the endogenous <i>V. cholerae</i> quorum sensing pathway [<a class="bibr" href="#ml344.r2" rid="ml344.r2">2</a>]. The BH1651 strain is a luxO<sup>D47E</sup> mutant where LuxO<sup>D47E</sup> mimics the behavior of phosphorylated LuxO, rendering LuxO<sup>D47E</sup> constitutively active within the QS pathway. BH1651 does not generate light but any compound that inhibits LuxO or works downstream of LuxO will induce light production. On day 0, a colony of bacteria was picked into 50 mL Luria Broth with 10 μg/mL tetracycline and cultured overnight at 30 °C. On day 1, bacterial density was determined by spectrometry, and the OD<sub>600</sub> was adjusted to 0.3. 20 μL of Luria Broth with 10 μg/mL tetracycline was added per well into white, opaque 384 well plates. Compounds and controls were added by pin transfer method. After 6 hours, the luminescence signal was determined with the Perkin-Elmer EnVision plate reader. In addition to luminescence, the confluency of each well was measured at an absorbance of 600 nM. Data were analyzed in Genedata Screener Assay Analyzer. All values were normalized against DMSO treated samples. Percent (%) activity was determined for each concentration, and the concentration response curves (CRCs) were generated with Genedata Screener’s Condoseo.</p></div><div id="ml344.s10"><h4>2.1.4. Secondary assay – <i>V. cholerae</i> DH231 sensor mechanism assay (AID 624281, AID 651808)</h4><p>A modified strain of <i>Vibrio cholerae</i> used in this assay uses light production to indicate quorum sensing induction. <i>Vibrio cholerae</i> is not naturally bioluminescent but the closely related species <i>Vibrio harveyi</i> produces light when the population is at a high density (i.e. a quorum is sensed). The heterologous <i>Vibrio harveyi</i> luciferase operon (<i>luxCDABE</i>) was cloned into the <i>Vibrio cholerae</i> C6706 O1 El Tor bacterial strain on the pBB1 cosmid to create a bioluminescence assay strain. This operon is activated by the endogenous <i>V. cholerae</i> quorum sensing pathway [<a class="bibr" href="#ml344.r2" rid="ml344.r2">2</a>]. The DH231 strain is a luxS and cqsS double deletion mutant. DH231 does not generate light but any compound that agonizes the receptor LuxQ will induce light production, and CqsS agonists will have no activity in this assay. On day 0, a colony of bacteria was picked into 50 mL Luria Broth with 10 μg/mL tetracycline and cultured overnight at 30 °C. On day 1, bacterial density was determined by spectrometry, and the OD<sub>600</sub> was adjusted to 0.3. 20 μL of Luria Broth with 10 μg/mL tetracycline was added per well into white, opaque 384 well plates. Compounds and controls were added by pin transfer method. After 6 hours, the luminescence signal was determined with the Perkin-Elmer EnVision plate reader. In addition to luminescence, the confluency of each well was measured at an absorbance of 600 nM. Data were analyzed in Genedata Screener Assay Analyzer. All values were normalized against DMSO treated samples. Percent (%) activity was determined for each concentration, and the concentration response curves (CRCs) were generated with Genedata Screener’s Condoseo.</p></div><div id="ml344.s11"><h4>2.1.5. Secondary assay – <i>V. cholerae</i> WN1103 sensor mechanism assay (AID 624275, AID 651841)</h4><p>A modified strain of <i>Vibrio cholerae</i> used in this assay uses light production to indicate quorum sensing induction. <i>Vibrio cholerae</i> is not naturally bioluminescent but the closely related species <i>Vibrio harveyi</i> produces light when the population is at a high density (i.e. a quorum is sensed). The heterologous <i>Vibrio harveyi</i> luciferase operon (<i>luxCDABE</i>) was cloned into the <i>Vibrio cholerae</i> C6706 O1 El Tor bacterial strain on the pBB1 cosmid to create a bioluminescence assay strain. This operon is activated by the endogenous <i>V. cholerae</i> quorum sensing pathway [<a class="bibr" href="#ml344.r2" rid="ml344.r2">2</a>]. The WN1103 strain is a luxQ and cqsA double deletion mutant. DH231 does not generate light but any compound that agonizes the CqsS receptor will induce light production and LuxQ agonists will have no activity in this assay. On day 0, a colony of bacteria was picked into 50 mL Luria Broth with 10 μg/mL tetracycline and cultured overnight at 30 °C. On day 1, bacterial density was determined by spectrometry, and the OD<sub>600</sub> was adjusted to 0.3. 20 μL of Luria Broth with 10 μg/mL tetracycline was added per well into white, opaque 384 well plates. Compounds and controls were added by pin transfer method. After 6 hours, the luminescence signal was determined with the Perkin-Elmer EnVision plate reader. In addition to luminescence, the confluency of each well was measured at an absorbance of 600 nM. Data were analyzed in Genedata Screener Assay Analyzer. All values were normalized against DMSO and 1 μM CAI-1 treated samples. Percent (%) activity was determined for each concentration, and the concentration response curves (CRCs) were generated with Genedata Screener’s Condoseo.</p></div><div id="ml344.s12"><h4>2.1.6. Secondary assay – CqsS <i>in vitro</i> auto-phosphorylation assay (AID 651905)</h4><p>Kinase assays were performed with inverted <i>E. coli</i> membranes expressing recombinant <i>Vibrio cholerae</i> wildtype CqsS. Reactions were carried out in phosphorylation buffer (50 mM Tris pH 8.0, 100 mM KCl, 5 mM MgCl<sub>2</sub>, and 10% (v/v) glycerol) and were initiated with the addition of 100 μM ATP and 2 uCi [γ-<sup>32</sup>ATP] (stock 3000 Ci mmol<sup>−1</sup>, Perkin Elmer). DMSO, CAI-1 or CqS agonists were added for a final concentration of 100 μM ten minutes prior to ATP addition. Reactions were incubated at room temperature and terminated after two minutes with SDS-PAGE loading buffer. Reaction products were separated using SDS-PAGE and the gels were dried at 80 °C on filter paper under a vacuum. The gels were exposed to a phosphoscreen for two hours and subsequently analyzed using Typhoon 9400 scanner and ImageQuant software [<a class="bibr" href="#ml344.r10" rid="ml344.r10">10</a>].</p></div></div><div id="ml344.s13"><h3>2.2. Probe Chemical Characterization</h3><p>After preparation as described in <a href="#ml344.s14">Section 2.3</a>, the probes <a href="/pcsubstance/?term=ML344[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML344</a> and <a href="/pcsubstance/?term=ML343[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML343</a> were analyzed by UPLC, <sup>1</sup>H and <sup>13</sup>C NMR spectroscopy, and high-resolution mass spectrometry. The data obtained from NMR and mass spectroscopy are consistent with the structure of the probe, and UPLC indicates an isolated purity of greater than 99% for both probes. The 1,5-substitution pattern of <a href="/pcsubstance/?term=ML344[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML344</a> was unambiguously established by 2D-NMR spectroscopy. The solubility of probe <a href="/pcsubstance/?term=ML344[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML344</a> was experimentally determined to be >100 μM in phosphate buffered saline (PBS) with 1% (v/v) DMSO. Under identical conditions, the solubility of probe <a href="/pcsubstance/?term=ML343[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML343</a> was measured to be 1.4 μM. The solubility of <a href="/pcsubstance/?term=ML343[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML343</a> was also measured using the Luria Broth (LB) media utilized for the primary assay. <a class="figpopup" href="/books/NBK169447/table/ml344.t2/?report=objectonly" target="object" rid-figpopup="figml344t2" rid-ob="figobml344t2">Table 2</a> summarizes the solubility of <a href="/pcsubstance/?term=ML343[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML343</a> and several analogs in both PBS and LB media. In general, LB media is a better solvent than PBS for the biphenyl compounds.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml344t2"><a href="/books/NBK169447/table/ml344.t2/?report=objectonly" target="object" title="Table 2" class="img_link icnblk_img figpopup" rid-figpopup="figml344t2" rid-ob="figobml344t2"><img class="small-thumb" src="/books/NBK169447/table/ml344.t2/?report=thumb" src-large="/books/NBK169447/table/ml344.t2/?report=previmg" alt="Table 2. Solubility of ML343 and Analogs in PBS and LB Assay Media." /></a><div class="icnblk_cntnt"><h4 id="ml344.t2"><a href="/books/NBK169447/table/ml344.t2/?report=objectonly" target="object" rid-ob="figobml344t2">Table 2</a></h4><p class="float-caption no_bottom_margin">Solubility of ML343 and Analogs in PBS and LB Assay Media. </p></div></div><p>Both probes are stable in PBS solution (>99% remaining after a 48-hour incubation at 23 °C). The data from the PBS stability assay is provided in <a class="figpopup" href="/books/NBK169447/figure/ml344.f1/?report=objectonly" target="object" rid-figpopup="figml344f1" rid-ob="figobml344f1">Figure 1</a>. The probes are also stable to human and murine plasma exposure, with greater than 84% remaining after incubation at 37 °C for 5 hours. No significant reaction with glutathione (GSH) was observed for either compound. <a class="figpopup" href="/books/NBK169447/table/ml344.t3/?report=objectonly" target="object" rid-figpopup="figml344t3" rid-ob="figobml344t3">Table 3</a> summarizes the various stability assays performed. The physical properties of probes <a href="/pcsubstance/?term=ML344[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML344</a> and <a href="/pcsubstance/?term=ML343[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML343</a> are summarized in <a class="figpopup" href="/books/NBK169447/table/ml344.t4/?report=objectonly" target="object" rid-figpopup="figml344t4" rid-ob="figobml344t4">Table 4</a>.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml344f1" co-legend-rid="figlgndml344f1"><a href="/books/NBK169447/figure/ml344.f1/?report=objectonly" target="object" title="Figure 1" class="img_link icnblk_img figpopup" rid-figpopup="figml344f1" rid-ob="figobml344f1"><img class="small-thumb" src="/books/NBK169447/bin/ml344f1.gif" src-large="/books/NBK169447/bin/ml344f1.jpg" alt="Figure 1. Stability of Probes ML344 and ML343 in PBS Buffer (pH 7.4, 23°C)." /></a><div class="icnblk_cntnt" id="figlgndml344f1"><h4 id="ml344.f1"><a href="/books/NBK169447/figure/ml344.f1/?report=objectonly" target="object" rid-ob="figobml344f1">Figure 1</a></h4><p class="float-caption no_bottom_margin">Stability of Probes ML344 and ML343 in PBS Buffer (pH 7.4, 23°C). </p></div></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml344t3"><a href="/books/NBK169447/table/ml344.t3/?report=objectonly" target="object" title="Table 3" class="img_link icnblk_img figpopup" rid-figpopup="figml344t3" rid-ob="figobml344t3"><img class="small-thumb" src="/books/NBK169447/table/ml344.t3/?report=thumb" src-large="/books/NBK169447/table/ml344.t3/?report=previmg" alt="Table 3. Plasma Stability and Plasma Binding of Probes ML344 and ML343." /></a><div class="icnblk_cntnt"><h4 id="ml344.t3"><a href="/books/NBK169447/table/ml344.t3/?report=objectonly" target="object" rid-ob="figobml344t3">Table 3</a></h4><p class="float-caption no_bottom_margin">Plasma Stability and Plasma Binding of Probes ML344 and ML343. </p></div></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml344t4"><a href="/books/NBK169447/table/ml344.t4/?report=objectonly" target="object" title="Table 4" class="img_link icnblk_img figpopup" rid-figpopup="figml344t4" rid-ob="figobml344t4"><img class="small-thumb" src="/books/NBK169447/table/ml344.t4/?report=thumb" src-large="/books/NBK169447/table/ml344.t4/?report=previmg" alt="Table 4. Summary of Probe Properties Computed for Probes ML344 and ML343." /></a><div class="icnblk_cntnt"><h4 id="ml344.t4"><a href="/books/NBK169447/table/ml344.t4/?report=objectonly" target="object" rid-ob="figobml344t4">Table 4</a></h4><p class="float-caption no_bottom_margin">Summary of Probe Properties Computed for Probes ML344 and ML343. </p></div></div></div><div id="ml344.s14"><h3>2.3. Probe Preparation</h3><p>Probe 1 (<a href="/pcsubstance/?term=ML344[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML344</a>) and most associated analogs were prepared according to the route outlined below in <a class="figpopup" href="/books/NBK169447/figure/ml344.f5/?report=objectonly" target="object" rid-figpopup="figml344f5" rid-ob="figobml344f5">Scheme 1</a>. 4-Isopropylbenzaldehyde (<b>2</b>) was stirred with 5-aminotetrazole (<b>3</b>) in the presence of sodium cyanoborohydride and acetic acid to give the coupled amine <b>4</b>. The tetrazole adduct was then reacted with ethyl iodide and potassium carbonate to give a mixture of regioisomers. Column chromatography removed the undesired 2,5-regioisomer to afford the probe <a href="/pcsubstance/?term=ML344[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML344</a>.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml344f5" co-legend-rid="figlgndml344f5"><a href="/books/NBK169447/figure/ml344.f5/?report=objectonly" target="object" title="Scheme 1" class="img_link icnblk_img figpopup" rid-figpopup="figml344f5" rid-ob="figobml344f5"><img class="small-thumb" src="/books/NBK169447/bin/ml344f5.gif" src-large="/books/NBK169447/bin/ml344f5.jpg" alt="Scheme 1. Synthesis of Probe ML344." /></a><div class="icnblk_cntnt" id="figlgndml344f5"><h4 id="ml344.f5"><a href="/books/NBK169447/figure/ml344.f5/?report=objectonly" target="object" rid-ob="figobml344f5">Scheme 1</a></h4><p class="float-caption no_bottom_margin">Synthesis of Probe ML344. </p></div></div><p>Probe 2 (<a href="/pcsubstance/?term=ML343[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML343</a>) and its analogs were prepared by amide coupling of the appropriate phenyl acetic acids with the corresponding amine (<a class="figpopup" href="/books/NBK169447/figure/ml344.f6/?report=objectonly" target="object" rid-figpopup="figml344f6" rid-ob="figobml344f6">Scheme 2</a>). 4-Biphenyl acetic acid <b>6</b> was coupled to cyclobutylamine <b>7</b> with the assistance of 4-(<i>N</i>,<i>N</i>-dimethylamino)pyridine and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. <a href="/pcsubstance/?term=ML343[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML343</a> was isolated by selective precipitation from the reaction mixture.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml344f6" co-legend-rid="figlgndml344f6"><a href="/books/NBK169447/figure/ml344.f6/?report=objectonly" target="object" title="Scheme 2" class="img_link icnblk_img figpopup" rid-figpopup="figml344f6" rid-ob="figobml344f6"><img class="small-thumb" src="/books/NBK169447/bin/ml344f6.gif" src-large="/books/NBK169447/bin/ml344f6.jpg" alt="Scheme 2. Synthesis of Probe ML343." /></a><div class="icnblk_cntnt" id="figlgndml344f6"><h4 id="ml344.f6"><a href="/books/NBK169447/figure/ml344.f6/?report=objectonly" target="object" rid-ob="figobml344f6">Scheme 2</a></h4><p class="float-caption no_bottom_margin">Synthesis of Probe ML343. </p></div></div></div></div><div id="ml344.s15"><h2 id="_ml344_s15_">3. Results</h2><div id="ml344.s16"><h3>3.1. Dose Response Curves for Probe</h3><div id="ml344.f2" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%202.%20ML344%20and%20ML343%20were%20tested%20across%20a%20range%20of%20concentrations%20up%20to%2035%20%003BCM%20in%20the%20primary%20assay%20and%20a%20HeLa%20cytotoxicity%20assay.&p=BOOKS&id=169447_ml344f2.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 src="/books/NBK169447/bin/ml344f2.jpg" alt="Figure 2. ML344 and ML343 were tested across a range of concentrations up to 35 μM in the primary assay and a HeLa cytotoxicity assay." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 2</span><span class="title">ML344 and ML343 were tested across a range of concentrations up to 35 μM in the primary assay and a HeLa cytotoxicity assay</span></h3><div class="caption"><p>Concentration response curves were generated with Genedata Screener Condeseo and show normalized percent activity for the individual doses. <a href="/pcsubstance/?term=ML344[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML344</a> BH1578 assay (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651816" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651816</a>), IC<sub>50</sub> = 0.17 μM (<b><i>A</i></b>); <a href="/pcsubstance/?term=ML343[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML343</a> BH1578 assay (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651816" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651816</a>), IC<sub>50</sub> = 0.56 μM (<b><i>B</i></b>); <a href="/pcsubstance/?term=ML344[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML344</a> HeLa CellTiter-Glo (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651864" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651864</a>), IC<sub>50</sub> ≥ 35 μM (<i>C</i>); <a href="/pcsubstance/?term=ML343[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML343</a> HeLa CellTiter-Glo (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651864" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651864</a>), IC<sub>50</sub> ≥ 35 μM (<b><i>D</i></b>). O = replicate 1, Δ = replicate 2.</p></div></div></div><div id="ml344.s17"><h3>3.2. Cellular Activity</h3><p>The primary assay was performed with whole bacterial cells. A secondary screen evaluating toxicity utilized mammalian HeLa cells, and additional phenotypic assays evaluated the activation of QS pathways in live <i>Vibrio cholerae</i> mutants. An overview of these assays is provided above in <a href="#ml344.s6">Section 2.1</a>. The probes <a href="/pcsubstance/?term=ML344[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML344</a> and <a href="/pcsubstance/?term=ML343[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML343</a> satisfy the cellular activity criteria specified for this project.</p></div><div id="ml344.s18"><h3>3.3. Profiling Assays</h3><p>Both probes were screened for binding to a broad panel of receptors and ion channels using the Eurofins Panlabs’ LeadProfilingScreen (Catalog No. 68). The results of this screen are summarized below in <a class="figpopup" href="/books/NBK169447/figure/ml344.f3/?report=objectonly" target="object" rid-figpopup="figml344f3" rid-ob="figobml344f3">Figures 3</a> and <a class="figpopup" href="/books/NBK169447/figure/ml344.f4/?report=objectonly" target="object" rid-figpopup="figml344f4" rid-ob="figobml344f4">4</a>. <a href="/pcsubstance/?term=ML344[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML344</a> shows less than 50% inhibition of all 67 targets evaluated (<a class="figpopup" href="/books/NBK169447/figure/ml344.f3/?report=objectonly" target="object" rid-figpopup="figml344f3" rid-ob="figobml344f3">Figure 3</a>). Its strongest activity is against the melatonin MT1 receptor, which exhibits 44% inhibition when treated with 10 μM <a href="/pcsubstance/?term=ML344[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML344</a>. Similarly, <a href="/pcsubstance/?term=ML343[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML343</a> does not show significant activity in this screening panel (<a class="figpopup" href="/books/NBK169447/figure/ml344.f4/?report=objectonly" target="object" rid-figpopup="figml344f4" rid-ob="figobml344f4">Figure 4</a>). The serotonin 5-HT2B receptor is inhibited by 45% using 10 μM <a href="/pcsubstance/?term=ML343[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML343</a>, and this represents the most potent interaction between <a href="/pcsubstance/?term=ML343[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML343</a> and the 67 targets screened.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml344f3" co-legend-rid="figlgndml344f3"><a href="/books/NBK169447/figure/ml344.f3/?report=objectonly" target="object" title="Figure 3" class="img_link icnblk_img figpopup" rid-figpopup="figml344f3" rid-ob="figobml344f3"><img class="small-thumb" src="/books/NBK169447/bin/ml344f3.gif" src-large="/books/NBK169447/bin/ml344f3.jpg" alt="Figure 3. Probe ML344 was evaluated for inhibition of 67 mammalian membrane receptors, ion channels, and membrane transporters." /></a><div class="icnblk_cntnt" id="figlgndml344f3"><h4 id="ml344.f3"><a href="/books/NBK169447/figure/ml344.f3/?report=objectonly" target="object" rid-ob="figobml344f3">Figure 3</a></h4><p class="float-caption no_bottom_margin">Probe ML344 was evaluated for inhibition of 67 mammalian membrane receptors, ion channels, and membrane transporters. A test concentration of 10 μM was used, and each experiment was performed in duplicate (n=1). </p></div></div><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml344f4" co-legend-rid="figlgndml344f4"><a href="/books/NBK169447/figure/ml344.f4/?report=objectonly" target="object" title="Figure 4" class="img_link icnblk_img figpopup" rid-figpopup="figml344f4" rid-ob="figobml344f4"><img class="small-thumb" src="/books/NBK169447/bin/ml344f4.gif" src-large="/books/NBK169447/bin/ml344f4.jpg" alt="Figure 4. Probe ML343 was evaluated for inhibition of 67 mammalian membrane receptors, ion channels, and membrane transporters." /></a><div class="icnblk_cntnt" id="figlgndml344f4"><h4 id="ml344.f4"><a href="/books/NBK169447/figure/ml344.f4/?report=objectonly" target="object" rid-ob="figobml344f4">Figure 4</a></h4><p class="float-caption no_bottom_margin">Probe ML343 was evaluated for inhibition of 67 mammalian membrane receptors, ion channels, and membrane transporters. A test concentration of 10 μM was used, and each experiment was performed in duplicate (n=1). </p></div></div></div></div><div id="ml344.s19"><h2 id="_ml344_s19_">4. Discussion</h2><div id="ml344.s20"><h3>4.1. Comparison to Existing Art and How the New Probe is an Improvement</h3><p>Currently, the only known agonists of CqsS are the natural ligand CAI-1 and structurally related analogs [<a class="bibr" href="#ml344.r2" rid="ml344.r2">2</a>,<a class="bibr" href="#ml344.r13" rid="ml344.r13">13</a>]. While <a href="/pcsubstance/?term=ML344[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML344</a> and <a href="/pcsubstance/?term=ML343[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML343</a> are less potent than CAI-1 in the primary assay (relative to EC<sub>50</sub> values), they elicit a stronger response from BH1578 cells as indicated by the stronger observed luminescence (<a class="figpopup" href="/books/NBK169447/table/ml344.t5/?report=objectonly" target="object" rid-figpopup="figml344t5" rid-ob="figobml344t5">Table 5</a>). The SAR associated with CAI-1 is steep [<a class="bibr" href="#ml344.r2" rid="ml344.r2">2</a>,<a class="bibr" href="#ml344.r13" rid="ml344.r13">13</a>], and not much structural information is known regarding its CqsS binding pocket. Additionally, the underlying structure of CAI-1 promotes micelle formation that complicates its usage <i>in vitro</i> and leads to increased variability in the cell-based assays. In contrast, the distinct architectures of <a href="/pcsubstance/?term=ML344[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML344</a> and <a href="/pcsubstance/?term=ML343[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML343</a> do not suggest that either compound will form micelles <i>in vitro.</i> It is anticipated that these probes will provide valuable information regarding available binding pockets of CqsS and guide the development of the next generation of quorum sensing focused therapeutics.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml344t5"><a href="/books/NBK169447/table/ml344.t5/?report=objectonly" target="object" title="Table 5" class="img_link icnblk_img figpopup" rid-figpopup="figml344t5" rid-ob="figobml344t5"><img class="small-thumb" src="/books/NBK169447/table/ml344.t5/?report=thumb" src-large="/books/NBK169447/table/ml344.t5/?report=previmg" alt="Table 5. Comparison of Probes ML344 and ML343 to Natural CqsS Ligand CAI-1." /></a><div class="icnblk_cntnt"><h4 id="ml344.t5"><a href="/books/NBK169447/table/ml344.t5/?report=objectonly" target="object" rid-ob="figobml344t5">Table 5</a></h4><p class="float-caption no_bottom_margin">Comparison of Probes ML344 and ML343 to Natural CqsS Ligand CAI-1. </p></div></div></div></div><div id="ml344.s21"><h2 id="_ml344_s21_">5. References</h2><dl class="temp-labeled-list"><dl class="bkr_refwrap"><dt>1.</dt><dd><div class="bk_ref" id="ml344.r1">Ng WL, Wei Y, Perez LJ, Cong J, Long T, Koch M, Semmelhack MF, Wingreen NS, Bassler BL. Probing bacterial transmembrane histidine kinase receptor-ligand interactions with natural and synthetic molecules. <span><span class="ref-journal">Proc Natl Acad Sci U S A. </span>2010 Mar 23;<span class="ref-vol">107</span>(12):5575–80.</span> Epub 2010 Mar 8. [<a href="/pmc/articles/PMC2851778/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2851778</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/20212168" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 20212168</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>2.</dt><dd><div class="bk_ref" id="ml344.r2">Ng WL, Perez LJ, Wei Y, Kraml C, Semmelhack MF, Bassler BL. Signal production and detection specificity in Vibrio CqsA/CqsS quorum sensing systems. <span><span class="ref-journal">Mol Microbiol. </span>2011 Mar;<span class="ref-vol">79</span>(6):1407–17.</span> Epub 2011 Jan 26. [<a href="/pmc/articles/PMC3285556/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3285556</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/21219472" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 21219472</span></a>] [<a href="http://dx.crossref.org/10.1111/j.1365-958.2011.07548.x" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd></dl><dl class="bkr_refwrap"><dt>3.</dt><dd><div class="bk_ref" id="ml344.r3">Dutta NK, Habbu MK. Experimental cholera in infant rabbits: a method for chemotherapeutic investigation. <span><span class="ref-journal">Br J Pharmacol Chemother. </span>1955 Jun;<span class="ref-vol">10</span>(2):153–9.</span> [<a href="/pmc/articles/PMC1509487/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC1509487</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/14389652" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 14389652</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>4.</dt><dd><div class="bk_ref" id="ml344.r4">Aziz KM, Mohsin AK, Hare WK, Phillips RA. Using the rat as a cholera “model” <span><span class="ref-journal">Nature. </span>1968 Nov 23;<span class="ref-vol">220</span>(5169):814–5.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/5725345" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 5725345</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>5.</dt><dd><div class="bk_ref" id="ml344.r5">Klose KE. The suckling mouse model of cholera. <span><span class="ref-journal">Trends Microbiol. </span>2000 Apr;<span class="ref-vol">8</span>(4):189–91.</span> Review. [<a href="https://pubmed.ncbi.nlm.nih.gov/10754579" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 10754579</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>6.</dt><dd><div class="bk_ref" id="ml344.r6">Casino P, Rubio V, Marina A. The mechanism of signal transduction by two-component systems. <span><span class="ref-journal">Curr Opin Struct Biol. </span>2010 Dec;<span class="ref-vol">20</span>(6):763–71.</span> Epub 2010 Oct 13. Review. [<a href="https://pubmed.ncbi.nlm.nih.gov/20951027" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 20951027</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>7.</dt><dd><div class="bk_ref" id="ml344.r7">Rutherford ST, Bassler BL. Bacterial quorum sensing: Its role in virulence and possibilities for its control. <span><span class="ref-journal">Cold Spring Harb. Perspect. Med. </span>2012;<span class="ref-vol">2</span>:a012427.</span> [<a href="/pmc/articles/PMC3543102/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3543102</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/23125205" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 23125205</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>8.</dt><dd><div class="bk_ref" id="ml344.r8">Miller MB, Skorupski K, Lenz DH, Taylor RK, Bassler BL. Parallel quorum sensing systems converge to regulate virulence in Vibrio cholerae. <span><span class="ref-journal">Cell. </span>2002 Aug 9;<span class="ref-vol">110</span>(3):303–14.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/12176318" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 12176318</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>9.</dt><dd><div class="bk_ref" id="ml344.r9">Higgins DA, Pomianek ME, Kraml CM, Taylor RK, Semmelhack MF, Bassler BL. The major Vibrio cholerae autoinducer and its role in virulence factor production. <span><span class="ref-journal">Nature. </span>2007 Dec 6;<span class="ref-vol">450</span>(7171):883–6.</span> Epub 2007 Nov 14. [<a href="https://pubmed.ncbi.nlm.nih.gov/18004304" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18004304</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>10.</dt><dd><div class="bk_ref" id="ml344.r10">Wei Y, Ng WL, Cong J, Bassler BL. Ligand and antagonist driven regulation of the Vibrio cholerae quorum sensing receptor CqsS. <span><span class="ref-journal">Mol Microbiol. </span>2012 Mar;<span class="ref-vol">83</span>(6):1095–108.</span> Epub 2012 Feb 14. [<a href="/pmc/articles/PMC3310172/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3310172</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/22295878" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 22295878</span></a>] [<a href="http://dx.crossref.org/10.1111/j.1365-2958.2012.07992.x" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd></dl><dl class="bkr_refwrap"><dt>11.</dt><dd><div class="bk_ref" id="ml344.r11">Lenz DH, Mok KC, Linney BN, Kulkarni RV, Wingreen NS, Bassler BL. The small chaperone Hfq and multiple small RNAs control quorum sensing in Vibrio harveyi and Vibrio cholerae. <span><span class="ref-journal">Cell. </span>2004;<span class="ref-vol">118</span>:69–82.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/15242645" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 15242645</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>12.</dt><dd><div class="bk_ref" id="ml344.r12">Zhu J, Mekalanos JJ. Quorum sensing-dependent biofilms enhance colonization in Vibrio cholerae. <span><span class="ref-journal">Dev Cell. </span>2003 Oct;<span class="ref-vol">5</span>(4):647–56.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/14536065" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 14536065</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>13.</dt><dd><div class="bk_ref" id="ml344.r13">Perez LJ, Ng WL, Marano P, Brook K, Bassler BL, Semmelhack MF. Role of the CAI-1 Fatty Acid tail in the Vibrio cholerae quorum sensing response. <span><span class="ref-journal">J Med Chem. </span>2012 Nov 8;</span> [Epub ahead of print] [<a href="/pmc/articles/PMC3798069/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3798069</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/23092313" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 23092313</span></a>]</div></dd></dl></dl></div><div id="bk_toc_contnr"></div></div></div><div class="fm-sec"><h2 id="_NBK169447_pubdet_">Publication Details</h2><h3>Author Information and Affiliations</h3><p class="contrib-group"><h4>Authors</h4><span itemprop="author">Patrick Faloon</span>,<sup><img src="/corehtml/pmc/pmcgifs/corrauth.gif" alt="corresponding author" /></sup><sup>1</sup> <span itemprop="author">Willmen Youngsaye</span>,<sup>1</sup> <span itemprop="author">Melissa Bennion</span>,<sup>1</sup> <span itemprop="author">Wai-Leung Ng</span>,<sup>2,3</sup> <span itemprop="author">Amanda Hurley</span>,<sup>2</sup> <span itemprop="author">Timothy A. Lewis</span>,<sup>1</sup> <span itemprop="author">Rahul V. Edwankar</span>,<sup>1</sup> <span itemprop="author">Evan Yao</span>,<sup>1</sup> <span itemprop="author">Jun Pu</span>,<sup>1</sup> <span itemprop="author">Partha P. Nag</span>,<sup>1</sup> <span itemprop="author">Jacqueline M. Wurst</span>,<sup>1</sup> <span itemprop="author">Melissa Bennion</span>,<sup>1</sup> <span itemprop="author">Hanh Le</span>,<sup>1</sup> <span itemprop="author">Carrie M. Mosher</span>,<sup>1</sup> <span itemprop="author">Stephen Johnston</span>,<sup>1</sup> <span itemprop="author">Sivaraman Dandapani</span>,<sup>1</sup> <span itemprop="author">Benito Munoz</span>,<sup>1</sup> <span itemprop="author">Michelle Palmer</span>,<sup>1</sup> <span itemprop="author">Bonnie L. Bassler</span>,<sup>2</sup> and <span itemprop="author">Stuart L. Schreiber</span><sup>1,4,5</sup>.</p><h4>Affiliations</h4><div class="affiliation"><sup>1</sup>
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The Broad Institute Probe Development Center, Cambridge, MA</div><div class="affiliation"><sup>2</sup>
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Princeton University, Princeton, NJ</div><div class="affiliation"><sup>3</sup>
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Tufts University, Sackler School of Graduate Biomedical Sciences, Boston, MA</div><div class="affiliation"><sup>4</sup>
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Chemical Biology Program, Broad Institute</div><div class="affiliation"><sup>5</sup>
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Department of Chemistry & Chemical Biology, Harvard University</div><div class="affiliation"><sup><img src="/corehtml/pmc/pmcgifs/corrauth.gif" alt="corresponding author" /></sup>Corresponding author email:
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<a href="mailto:dev@null" data-email="gro.etutitsnidaorb@noolafp" class="oemail">gro.etutitsnidaorb@noolafp</a></div><h3>Publication History</h3><p class="small">Received: <span itemprop="datePublished">December 14, 2012</span>; Last Update: <span itemprop="dateModified">September 3, 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>Faloon P, Youngsaye W, Bennion M, et al. Discovery of Two, Structurally Distinct Agonists of Vibrio cholerae Quorum Sensing Acting via the CqsS Membrane Receptor. 2012 Dec 14 [Updated 2013 Sep 3]. 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/ml345/?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/ml342/?report=reader"><svg xmlns="http://www.w3.org/2000/svg" viewBox="0 0 100 100" preserveAspectRatio="none"><path d="M25,30c80,60 80,0 0,60 c30,-60 30,0 0,-60"></path><text x="20" y="28" textLength="60" style="font-size:25px">Next</text></svg></a></div></article><article data-type="fig" id="figobml344fu1"><div id="ml344.fu1" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK169447/bin/ml344fu1.jpg" alt="ML344." /></div><h3><span class="title">ML344</span></h3></div></article><article data-type="table-wrap" id="figobml344tu1"><div id="ml344.tu1" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK169447/table/ml344.tu1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml344.tu1_lrgtbl__"><table class="no_bottom_margin"><thead><tr><th id="hd_h_ml344.tu1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">PubChem<br />CID</th><th id="hd_h_ml344.tu1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Targets</th><th id="hd_h_ml344.tu1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">EC<sub>50</sub> (μM)<br />[SID, AID]</th><th id="hd_h_ml344.tu1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Anti-Target</th><th id="hd_h_ml344.tu1_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">IC<sub>50</sub> (μM)<br />[SID, AID]</th><th id="hd_h_ml344.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Fold Selective<sup>*</sup></th></tr></thead><tbody><tr><td headers="hd_h_ml344.tu1_1_1_1_1" rowspan="4" colspan="1" style="text-align:center;vertical-align:middle;">CID 4727493</td><td headers="hd_h_ml344.tu1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><i>V. cholerae</i> BH1578</td><td headers="hd_h_ml344.tu1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.2<br />[<a href="https://pubchem.ncbi.nlm.nih.gov/substance/152199102" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 152199102</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651816" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651816</a>]</td><td headers="hd_h_ml344.tu1_1_1_1_4" rowspan="4" colspan="1" style="text-align:center;vertical-align:middle;">HeLa<br />cytotoxicity</td><td headers="hd_h_ml344.tu1_1_1_1_5" rowspan="4" colspan="1" style="text-align:center;vertical-align:middle;">>30<br />[<a href="https://pubchem.ncbi.nlm.nih.gov/substance/152199102" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 152199102</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651864" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651864</a>]</td><td headers="hd_h_ml344.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">150x</td></tr><tr><td headers="hd_h_ml344.tu1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><i>V. cholerae</i> BH1651</td><td headers="hd_h_ml344.tu1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>30<br />[<a href="https://pubchem.ncbi.nlm.nih.gov/substance/152199102" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 152199102</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651847" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651847</a>]</td><td headers="hd_h_ml344.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">N/A</td></tr><tr><td headers="hd_h_ml344.tu1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><i>V. cholerae</i> DH231</td><td headers="hd_h_ml344.tu1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>30<br />[<a href="https://pubchem.ncbi.nlm.nih.gov/substance/152199102" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 152199102</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651808" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651808</a>]</td><td headers="hd_h_ml344.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">N/A</td></tr><tr><td headers="hd_h_ml344.tu1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><i>V. cholerae</i> WN1103</td><td headers="hd_h_ml344.tu1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.06<br />[<a href="https://pubchem.ncbi.nlm.nih.gov/substance/152199102" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 152199102</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651841" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651841</a>]</td><td headers="hd_h_ml344.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">500x</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="ml344.tfn1"><p class="no_margin">Selectivity = Anti-target IC<sub>50</sub>/Target EC<sub>50</sub></p></div></dd></dl></dl></div></div></div></article><article data-type="fig" id="figobml344fu2"><div id="ml344.fu2" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK169447/bin/ml344fu2.jpg" alt="ML343." /></div><h3><span class="title">ML343</span></h3></div></article><article data-type="table-wrap" id="figobml344tu2"><div id="ml344.tu2" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK169447/table/ml344.tu2/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml344.tu2_lrgtbl__"><table class="no_bottom_margin"><thead><tr><th id="hd_h_ml344.tu2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">PubChem<br />CID</th><th id="hd_h_ml344.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Targets</th><th id="hd_h_ml344.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">EC<sub>50</sub> (μM)<br />[SID, AID]</th><th id="hd_h_ml344.tu2_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Anti-Target</th><th id="hd_h_ml344.tu2_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">IC<sub>50</sub> (μM)<br />[SID, AID]</th><th id="hd_h_ml344.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Fold Selective<sup>*</sup></th></tr></thead><tbody><tr><td headers="hd_h_ml344.tu2_1_1_1_1" rowspan="4" colspan="1" style="text-align:center;vertical-align:middle;">CID 57525036</td><td headers="hd_h_ml344.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><i>V. cholerae</i> BH1578</td><td headers="hd_h_ml344.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.5<br />[<a href="https://pubchem.ncbi.nlm.nih.gov/substance/137282727" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 137282727</a>,<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651816" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651816</a>]</td><td headers="hd_h_ml344.tu2_1_1_1_4" rowspan="4" colspan="1" style="text-align:center;vertical-align:middle;">HeLa<br />cytotoxicity</td><td headers="hd_h_ml344.tu2_1_1_1_5" rowspan="4" colspan="1" style="text-align:center;vertical-align:middle;">>30<br />[<a href="https://pubchem.ncbi.nlm.nih.gov/substance/137282727" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 137282727</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651864" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651864</a>]</td><td headers="hd_h_ml344.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">60x</td></tr><tr><td headers="hd_h_ml344.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><i>V. cholerae</i> BH1651</td><td headers="hd_h_ml344.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>30<br />[<a href="https://pubchem.ncbi.nlm.nih.gov/substance/137282727" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 137282727</a>,<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651847" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651847</a></td><td headers="hd_h_ml344.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">N/A</td></tr><tr><td headers="hd_h_ml344.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><i>V. cholerae</i> DH231</td><td headers="hd_h_ml344.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>30<br />[<a href="https://pubchem.ncbi.nlm.nih.gov/substance/137282727" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 137282727</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651808" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651808</a>]</td><td headers="hd_h_ml344.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">N/A</td></tr><tr><td headers="hd_h_ml344.tu2_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><i>V. cholerae</i> WN1103</td><td headers="hd_h_ml344.tu2_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.3<br />[<a href="https://pubchem.ncbi.nlm.nih.gov/substance/137282727" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 137282727</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651841" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651841</a>]</td><td headers="hd_h_ml344.tu2_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">100x</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="ml344.tfn2"><p class="no_margin">Selectivity = Anti-target IC<sub>50</sub>/Target EC<sub>50</sub></p></div></dd></dl></dl></div></div></div></article><article data-type="table-wrap" id="figobml344t1"><div id="ml344.t1" class="table"><h3><span class="label">Table 1</span><span class="title">Strains of <i>V. cholera</i> used in assays</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK169447/table/ml344.t1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml344.t1_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml344.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Strain</th><th id="hd_h_ml344.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Genotype</th><th id="hd_h_ml344.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Reporter</th><th id="hd_h_ml344.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Suggested Mechanism for Inducers</th></tr></thead><tbody><tr><td headers="hd_h_ml344.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BH1578</td><td headers="hd_h_ml344.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Δ<i>luxS</i> Δ<i>cqsA</i></td><td headers="hd_h_ml344.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><i>V. harveyi</i> luciferase operon</td><td headers="hd_h_ml344.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Multiple Mechanisms possible</td></tr><tr><td headers="hd_h_ml344.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BH1651</td><td headers="hd_h_ml344.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><i>luxO</i><sup><i>D47E</i></sup></td><td headers="hd_h_ml344.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><i>V. harveyi</i> luciferase operon</td><td headers="hd_h_ml344.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">LuxO inhibitor or downstream</td></tr><tr><td headers="hd_h_ml344.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">DH231</td><td headers="hd_h_ml344.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Δ<i>luxS</i> Δ<i>cqsS</i></td><td headers="hd_h_ml344.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><i>V. harveyi</i> luciferase operon</td><td headers="hd_h_ml344.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">LuxPQ agonist or downstream</td></tr><tr><td headers="hd_h_ml344.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">WN1103</td><td headers="hd_h_ml344.t1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Δ<i>luxQ</i> Δ<i>cqsA</i></td><td headers="hd_h_ml344.t1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><i>V. harveyi</i> luciferase operon</td><td headers="hd_h_ml344.t1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CqsS agonist or LuxO inhibition</td></tr></tbody></table></div></div></article><article data-type="table-wrap" id="figobml344t2"><div id="ml344.t2" class="table"><h3><span class="label">Table 2</span><span class="title">Solubility of ML343 and Analogs in PBS and LB Assay Media</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK169447/table/ml344.t2/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml344.t2_lrgtbl__"><table class="no_top_margin"><thead><tr><th id="hd_h_ml344.t2_1_1_1_1" rowspan="2" colspan="1" headers="hd_h_ml344.t2_1_1_1_1" style="text-align:center;vertical-align:middle;">PubChem CID/ML</th><th id="hd_h_ml344.t2_1_1_1_2" rowspan="2" colspan="1" headers="hd_h_ml344.t2_1_1_1_2" style="text-align:center;vertical-align:middle;">Structure</th><th id="hd_h_ml344.t2_1_1_1_3" colspan="2" rowspan="1" style="text-align:center;vertical-align:middle;">Solubility</th></tr><tr><th headers="hd_h_ml344.t2_1_1_1_3" id="hd_h_ml344.t2_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><i>PBS</i><br /><i>w/1% DMSO</i></th><th headers="hd_h_ml344.t2_1_1_1_3" id="hd_h_ml344.t2_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;"><i>LB Media</i><br /><i>w/1% DMSO</i></th></tr></thead><tbody><tr><td headers="hd_h_ml344.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CID 57525036/<a href="/pcsubstance/?term=ML343[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML343</a></td><td headers="hd_h_ml344.t2_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/NBK169447/bin/ml344fu3.jpg" alt="Image ml344fu3.jpg" /></div></td><td headers="hd_h_ml344.t2_1_1_1_3 hd_h_ml344.t2_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1.4</td><td headers="hd_h_ml344.t2_1_1_1_3 hd_h_ml344.t2_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">6.7</td></tr><tr><td headers="hd_h_ml344.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CID 2530160</td><td headers="hd_h_ml344.t2_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/NBK169447/bin/ml344fu4.jpg" alt="Image ml344fu4.jpg" /></div></td><td headers="hd_h_ml344.t2_1_1_1_3 hd_h_ml344.t2_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.4</td><td headers="hd_h_ml344.t2_1_1_1_3 hd_h_ml344.t2_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">17.7</td></tr><tr><td headers="hd_h_ml344.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CID 4811370</td><td headers="hd_h_ml344.t2_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/NBK169447/bin/ml344fu5.jpg" alt="Image ml344fu5.jpg" /></div></td><td headers="hd_h_ml344.t2_1_1_1_3 hd_h_ml344.t2_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.4</td><td headers="hd_h_ml344.t2_1_1_1_3 hd_h_ml344.t2_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2.0</td></tr><tr><td headers="hd_h_ml344.t2_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CID 57525050</td><td headers="hd_h_ml344.t2_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/NBK169447/bin/ml344fu6.jpg" alt="Image ml344fu6.jpg" /></div></td><td headers="hd_h_ml344.t2_1_1_1_3 hd_h_ml344.t2_1_1_2_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.5</td><td headers="hd_h_ml344.t2_1_1_1_3 hd_h_ml344.t2_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">38.4</td></tr></tbody></table></div></div></article><article data-type="fig" id="figobml344f1"><div id="ml344.f1" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%201.%20Stability%20of%20Probes%20ML344%20and%20ML343%20in%20PBS%20Buffer%20(pH%207.4%2C%2023%B0C).&p=BOOKS&id=169447_ml344f1.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/NBK169447/bin/ml344f1.jpg" alt="Figure 1. Stability of Probes ML344 and ML343 in PBS Buffer (pH 7.4, 23°C)." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 1</span><span class="title">Stability of Probes ML344 and ML343 in PBS Buffer (pH 7.4, 23°C)</span></h3></div></article><article data-type="table-wrap" id="figobml344t3"><div id="ml344.t3" class="table"><h3><span class="label">Table 3</span><span class="title">Plasma Stability and Plasma Binding of Probes ML344 and ML343</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK169447/table/ml344.t3/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml344.t3_lrgtbl__"><table class="no_top_margin"><tbody><tr><th id="hd_b_ml344.t3_1_1_1_1" colspan="2" rowspan="1" style="text-align:center;vertical-align:middle;"></th><th id="hd_b_ml344.t3_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/NBK169447/bin/ml344fu7.jpg" alt="Image ml344fu7.jpg" /></div></th><th id="hd_b_ml344.t3_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">
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<div class="graphic"><img src="/books/NBK169447/bin/ml344fu3.jpg" alt="Image ml344fu3.jpg" /></div></th></tr><tr><th headers="hd_b_ml344.t3_1_1_1_1" id="hd_b_ml344.t3_1_1_2_1" colspan="2" rowspan="1" style="text-align:center;vertical-align:middle;">PubChem CID<br />ML#</th><th headers="hd_b_ml344.t3_1_1_1_2" id="hd_b_ml344.t3_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">4727493<br /><a href="/pcsubstance/?term=ML344[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML344</a></th><th headers="hd_b_ml344.t3_1_1_1_3" id="hd_b_ml344.t3_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">57525036<br /><a href="/pcsubstance/?term=ML343[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML343</a></th></tr><tr><th id="hd_b_ml344.t3_1_1_3_1" colspan="2" rowspan="1" style="text-align:left;vertical-align:middle;">GSH stability<br />(% remaining after 6 hours)</th><td headers="hd_b_ml344.t3_1_1_3_1 hd_b_ml344.t3_1_1_1_2 hd_b_ml344.t3_1_1_2_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">91%</td><td headers="hd_b_ml344.t3_1_1_3_1 hd_b_ml344.t3_1_1_1_3 hd_b_ml344.t3_1_1_2_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">80%</td></tr><tr><th id="hd_b_ml344.t3_1_1_4_1" rowspan="2" colspan="1" style="text-align:left;vertical-align:middle;">Plasma stability<br />(% remaining after 5 hours)</th><td headers="hd_b_ml344.t3_1_1_4_1 hd_b_ml344.t3_1_1_3_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Human</td><td headers="hd_b_ml344.t3_1_1_4_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>99%</td><td headers="hd_b_ml344.t3_1_1_4_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">88%</td></tr><tr><td headers="hd_b_ml344.t3_1_1_4_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Murine</td><td headers="hd_b_ml344.t3_1_1_4_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">87%</td><td headers="hd_b_ml344.t3_1_1_4_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">84%</td></tr><tr><th id="hd_b_ml344.t3_1_1_6_1" rowspan="2" colspan="1" style="text-align:left;vertical-align:middle;">Plasma protein binding<br />(% bound)</th><td headers="hd_b_ml344.t3_1_1_6_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Human</td><td headers="hd_b_ml344.t3_1_1_6_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">92%</td><td headers="hd_b_ml344.t3_1_1_6_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">98%</td></tr><tr><td headers="hd_b_ml344.t3_1_1_6_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">Murine</td><td headers="hd_b_ml344.t3_1_1_6_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">91%</td><td headers="hd_b_ml344.t3_1_1_6_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">98%</td></tr></tbody></table></div></div></article><article data-type="table-wrap" id="figobml344t4"><div id="ml344.t4" class="table"><h3><span class="label">Table 4</span><span class="title">Summary of Probe Properties Computed for Probes ML344 and ML343</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK169447/table/ml344.t4/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml344.t4_lrgtbl__"><table class="no_margin"><thead><tr><th id="hd_h_ml344.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:bottom;">Probe</th><th id="hd_h_ml344.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"><a href="/pcsubstance/?term=ML344[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML344</a></th><th id="hd_h_ml344.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:bottom;"><a href="/pcsubstance/?term=ML343[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML343</a></th></tr></thead><tbody><tr><td headers="hd_h_ml344.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;"><b>IUPAC Chemical Name</b></td><td headers="hd_h_ml344.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1-ethyl-<i>N</i>-(4-isopropylbenzyl)-1<i>H</i>-tetrazol-5-amine</td><td headers="hd_h_ml344.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2-([1,1′-biphenyl]-4-yl)-<i>N</i>-cyclobutylacetamide</td></tr><tr><td headers="hd_h_ml344.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;"><b>PubChem CID</b></td><td headers="hd_h_ml344.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">4727493</td><td headers="hd_h_ml344.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">57525036</td></tr><tr><td headers="hd_h_ml344.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;"><b>Molecular Formula</b></td><td headers="hd_h_ml344.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">C<sub>13</sub>H<sub>19</sub>N<sub>5</sub></td><td headers="hd_h_ml344.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">C<sub>18</sub>H<sub>19</sub>NO</td></tr><tr><td headers="hd_h_ml344.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;"><b>Molecular Weight (g/mol)</b></td><td headers="hd_h_ml344.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">245.32</td><td headers="hd_h_ml344.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">265.35</td></tr><tr><td headers="hd_h_ml344.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;"><b>Exact Mass (amu)</b></td><td headers="hd_h_ml344.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">245.1640</td><td headers="hd_h_ml344.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">265.1467</td></tr><tr><td headers="hd_h_ml344.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;"><b>XlogP3-AA</b></td><td headers="hd_h_ml344.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2.8</td><td headers="hd_h_ml344.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3.5<sup>*</sup></td></tr><tr><td headers="hd_h_ml344.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;"><b>Topological Surface Area</b></td><td headers="hd_h_ml344.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">55.6</td><td headers="hd_h_ml344.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">29.1</td></tr><tr><td headers="hd_h_ml344.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;"><b>H-Bond Donors</b></td><td headers="hd_h_ml344.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1</td><td headers="hd_h_ml344.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1</td></tr><tr><td headers="hd_h_ml344.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;"><b>H-Bond Acceptors</b></td><td headers="hd_h_ml344.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">4</td><td headers="hd_h_ml344.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1</td></tr><tr><td headers="hd_h_ml344.t4_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;"><b>Rotatable Bond Count</b></td><td headers="hd_h_ml344.t4_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">5</td><td headers="hd_h_ml344.t4_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">4</td></tr></tbody></table></div><div class="tblwrap-foot"><div><dl class="temp-labeled-list small"><dl class="bkr_refwrap"><dt>*</dt><dd><div id="ml344.tfn3"><p class="no_margin">This value represents ClogP as calculated with ChemBioDraw Ultra, version 12.0.</p></div></dd></dl></dl></div></div></div></article><article data-type="fig" id="figobml344f5"><div id="ml344.f5" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK169447/bin/ml344f5.jpg" alt="Scheme 1. Synthesis of Probe ML344." /></div><h3><span class="label">Scheme 1</span><span class="title">Synthesis of Probe ML344</span></h3></div></article><article data-type="fig" id="figobml344f6"><div id="ml344.f6" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK169447/bin/ml344f6.jpg" alt="Scheme 2. Synthesis of Probe ML343." /></div><h3><span class="label">Scheme 2</span><span class="title">Synthesis of Probe ML343</span></h3></div></article><article data-type="fig" id="figobml344f2"><div id="ml344.f2" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%202.%20ML344%20and%20ML343%20were%20tested%20across%20a%20range%20of%20concentrations%20up%20to%2035%20%003BCM%20in%20the%20primary%20assay%20and%20a%20HeLa%20cytotoxicity%20assay.&p=BOOKS&id=169447_ml344f2.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/NBK169447/bin/ml344f2.jpg" alt="Figure 2. ML344 and ML343 were tested across a range of concentrations up to 35 μM in the primary assay and a HeLa cytotoxicity assay." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 2</span><span class="title">ML344 and ML343 were tested across a range of concentrations up to 35 μM in the primary assay and a HeLa cytotoxicity assay</span></h3><div class="caption"><p>Concentration response curves were generated with Genedata Screener Condeseo and show normalized percent activity for the individual doses. <a href="/pcsubstance/?term=ML344[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML344</a> BH1578 assay (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651816" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651816</a>), IC<sub>50</sub> = 0.17 μM (<b><i>A</i></b>); <a href="/pcsubstance/?term=ML343[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML343</a> BH1578 assay (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651816" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651816</a>), IC<sub>50</sub> = 0.56 μM (<b><i>B</i></b>); <a href="/pcsubstance/?term=ML344[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML344</a> HeLa CellTiter-Glo (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651864" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651864</a>), IC<sub>50</sub> ≥ 35 μM (<i>C</i>); <a href="/pcsubstance/?term=ML343[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML343</a> HeLa CellTiter-Glo (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/651864" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 651864</a>), IC<sub>50</sub> ≥ 35 μM (<b><i>D</i></b>). O = replicate 1, Δ = replicate 2.</p></div></div></article><article data-type="fig" id="figobml344f3"><div id="ml344.f3" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%203.%20Probe%20ML344%20was%20evaluated%20for%20inhibition%20of%2067%20mammalian%20membrane%20receptors%2C%20ion%20channels%2C%20and%20membrane%20transporters.&p=BOOKS&id=169447_ml344f3.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/NBK169447/bin/ml344f3.jpg" alt="Figure 3. Probe ML344 was evaluated for inhibition of 67 mammalian membrane receptors, ion channels, and membrane transporters." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 3</span><span class="title">Probe ML344 was evaluated for inhibition of 67 mammalian membrane receptors, ion channels, and membrane transporters</span></h3><div class="caption"><p>A test concentration of 10 μM was used, and each experiment was performed in duplicate (n=1).</p></div></div></article><article data-type="fig" id="figobml344f4"><div id="ml344.f4" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%204.%20Probe%20ML343%20was%20evaluated%20for%20inhibition%20of%2067%20mammalian%20membrane%20receptors%2C%20ion%20channels%2C%20and%20membrane%20transporters.&p=BOOKS&id=169447_ml344f4.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/NBK169447/bin/ml344f4.jpg" alt="Figure 4. Probe ML343 was evaluated for inhibition of 67 mammalian membrane receptors, ion channels, and membrane transporters." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 4</span><span class="title">Probe ML343 was evaluated for inhibition of 67 mammalian membrane receptors, ion channels, and membrane transporters</span></h3><div class="caption"><p>A test concentration of 10 μM was used, and each experiment was performed in duplicate (n=1).</p></div></div></article><article data-type="table-wrap" id="figobml344t5"><div id="ml344.t5" class="table"><h3><span class="label">Table 5</span><span class="title">Comparison of Probes ML344 and ML343 to Natural CqsS Ligand CAI-1</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK169447/table/ml344.t5/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml344.t5_lrgtbl__"><table class="no_margin"><thead><tr><th id="hd_h_ml344.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">No.</th><th id="hd_h_ml344.t5_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Compound</th><th id="hd_h_ml344.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">BH1578 EC<sub>50</sub> (μM)</th><th id="hd_h_ml344.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Max. Response<sup>†</sup></th></tr></thead><tbody><tr><td headers="hd_h_ml344.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">1</td><td headers="hd_h_ml344.t5_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/NBK169447/bin/ml344fu8.jpg" alt="Image ml344fu8.jpg" /></div><br />(<i>S</i>)<b>-CAI-1</b></td><td headers="hd_h_ml344.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.04<sup>‡</sup></td><td headers="hd_h_ml344.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">100%</td></tr><tr><td headers="hd_h_ml344.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">2</td><td headers="hd_h_ml344.t5_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/NBK169447/bin/ml344fu9.jpg" alt="Image ml344fu9.jpg" /></div><br /><b><a href="/pcsubstance/?term=ML344[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML344</a></b></td><td headers="hd_h_ml344.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.2</td><td headers="hd_h_ml344.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">146%</td></tr><tr><td headers="hd_h_ml344.t5_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">3</td><td headers="hd_h_ml344.t5_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/NBK169447/bin/ml344fu3.jpg" alt="Image ml344fu3.jpg" /></div><br /><b><a href="/pcsubstance/?term=ML343[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML343</a></b></td><td headers="hd_h_ml344.t5_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">0.5</td><td headers="hd_h_ml344.t5_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">128%</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="ml344.tfn4"><p class="no_margin">Percent maximal bioluminescence relative to CAI-1, which is set at 100%.</p></div></dd></dl><dl class="bkr_refwrap"><dt>‡</dt><dd><div id="ml344.tfn5"><p class="no_margin">Reference <a class="bibr" href="#ml344.r7" rid="ml344.r7">7</a>.</p></div></dd></dl></dl></div></div></div></article></div><div id="jr-scripts"><script src="/corehtml/pmc/jatsreader/ptpmc_3.22/js/libs.min.js"> </script><script src="/corehtml/pmc/jatsreader/ptpmc_3.22/js/jr.min.js"> </script></div></div>
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