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<script type="text/javascript" src="/corehtml/pmc/jatsreader/ptpmc_3.22/js/jr.boots.min.js"> </script><title>Identification of a small molecule inhibitor of Pseudomonas aeruginosa PvdQ acylase, an enzyme involved in siderophore pyoverdine synthesis - Probe Reports from the NIH Molecular Libraries Program - NCBI Bookshelf</title>
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<meta name="citation_inbook_title" content="Probe Reports from the NIH Molecular Libraries Program [Internet]">
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<meta name="citation_title" content="Identification of a small molecule inhibitor of Pseudomonas aeruginosa PvdQ acylase, an enzyme involved in siderophore pyoverdine synthesis">
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<meta name="citation_publisher" content="National Center for Biotechnology Information (US)">
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<meta name="citation_date" content="2013/02/28">
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<meta name="citation_author" content="Jimmy R. Theriault">
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<meta name="citation_author" content="Jacqueline Wurst">
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<meta name="citation_author" content="Ivan Jewett">
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<meta name="citation_author" content="Lynn Verplank">
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<meta name="citation_author" content="Jose R. Perez">
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<meta name="citation_author" content="Andrew M. Gulick">
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<meta name="citation_author" content="Eric J. Drake">
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<meta name="citation_author" content="Michelle Palmer">
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<meta name="citation_author" content="Sam Moskowitz">
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<meta name="citation_author" content="Nandini Dasgupta">
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<meta name="citation_author" content="Mark K. Brannon">
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<meta name="citation_author" content="Sivaraman Dandapani">
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<meta name="citation_author" content="Ben Munoz">
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<meta name="citation_author" content="Stuart Schreiber">
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<meta name="citation_pmid" content="23658940">
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<meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK133446/">
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<meta name="DC.Title" content="Identification of a small molecule inhibitor of Pseudomonas aeruginosa PvdQ acylase, an enzyme involved in siderophore pyoverdine synthesis">
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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="Jimmy R. Theriault">
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<meta name="DC.Contributor" content="Jacqueline Wurst">
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<meta name="DC.Contributor" content="Ivan Jewett">
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<meta name="DC.Contributor" content="Lynn Verplank">
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<meta name="DC.Contributor" content="Jose R. Perez">
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<meta name="DC.Contributor" content="Andrew M. Gulick">
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<meta name="DC.Contributor" content="Eric J. Drake">
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<meta name="DC.Contributor" content="Michelle Palmer">
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<meta name="DC.Contributor" content="Sam Moskowitz">
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<meta name="DC.Contributor" content="Nandini Dasgupta">
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<meta name="DC.Contributor" content="Mark K. Brannon">
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<meta name="DC.Contributor" content="Sivaraman Dandapani">
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<meta name="DC.Contributor" content="Ben Munoz">
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<meta name="DC.Contributor" content="Stuart Schreiber">
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<meta name="DC.Date" content="2013/02/28">
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<meta name="DC.Identifier" content="https://www.ncbi.nlm.nih.gov/books/NBK133446/">
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<meta name="description" content="The bacteria Pseudomonas aeruginosa produces a peptide siderophore known as pyoverdine, which has a great affinity for iron and is used to acquire iron from the external environment. Several enzymes such as the PvdQ acylase are required for the biosynthesis of pyoverdine. Deletion of the PvdQ gene disrupts pyoverdine production and hinders P. aeruginosa proliferation. Bacteria defective in pyoverdine synthesis are not infectious implying that disrupting this siderophore production through PvdQ inhibition could be exploited as a putative target for the development of novel antibiotic compounds. This report describes the development of a small molecule inhibitor (ML318, CID 56604881) of PvdQ acylase. ML318 inhibits PvdQ in vitro with an IC50 of 6 nM, has no apparent toxicity in mammalian HeLa cells up to a concentration of 100 μM, and inhibits growth and pyoverdine production in P. aeruginosa exposed to iron-limiting conditions with an IC50 < 50 μM. ML318 can also significantly reduce the intracellular uptake of iron inside the bacteria. This probe is a useful tool for ongoing characterization of pyoverdine’s role in P. aeruginosa biology and could serve as a starting point for the development of a novel antibiotic.">
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<meta name="og:title" content="Identification of a small molecule inhibitor of Pseudomonas aeruginosa PvdQ acylase, an enzyme involved in siderophore pyoverdine synthesis">
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<meta name="og:description" content="The bacteria Pseudomonas aeruginosa produces a peptide siderophore known as pyoverdine, which has a great affinity for iron and is used to acquire iron from the external environment. Several enzymes such as the PvdQ acylase are required for the biosynthesis of pyoverdine. Deletion of the PvdQ gene disrupts pyoverdine production and hinders P. aeruginosa proliferation. Bacteria defective in pyoverdine synthesis are not infectious implying that disrupting this siderophore production through PvdQ inhibition could be exploited as a putative target for the development of novel antibiotic compounds. This report describes the development of a small molecule inhibitor (ML318, CID 56604881) of PvdQ acylase. ML318 inhibits PvdQ in vitro with an IC50 of 6 nM, has no apparent toxicity in mammalian HeLa cells up to a concentration of 100 μM, and inhibits growth and pyoverdine production in P. aeruginosa exposed to iron-limiting conditions with an IC50 < 50 μM. ML318 can also significantly reduce the intracellular uptake of iron inside the bacteria. This probe is a useful tool for ongoing characterization of pyoverdine’s role in P. aeruginosa biology and could serve as a starting point for the development of a novel antibiotic.">
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1,0-2,2l170,170m-91-117 110,110-26,26-110-110"></path></svg></a><a id="jr-fip-done" class="wsprkl btn" title="Dismiss find">✘</a></nav><nav id="jr-fip-info-p"><a id="jr-fip-prev" class="wsprkl btn" title="Jump to previuos match">◀</a><button id="jr-fip-matches">no matches yet</button><a id="jr-fip-next" class="wsprkl btn" title="Jump to next match">▶</a></nav></nav></div><div id="jr-epub-interstitial" class="hidden"></div><div id="jr-content"><article data-type="main"><div class="main-content lit-style" itemscope="itemscope" itemtype="http://schema.org/CreativeWork"><div class="meta-content fm-sec"><div class="fm-sec"><h1 id="_NBK133446_"><span class="title" itemprop="name">Identification of a small molecule inhibitor of Pseudomonas aeruginosa PvdQ acylase, an enzyme involved in siderophore pyoverdine synthesis</span></h1><p class="contribs">Theriault JR, Wurst J, Jewett I, et al.</p><p class="fm-aai"><a href="#_NBK133446_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>The bacteria <i>Pseudomonas aeruginosa</i> produces a peptide siderophore known as pyoverdine, which has a great affinity for iron and is used to acquire iron from the external environment. Several enzymes such as the PvdQ acylase are required for the biosynthesis of pyoverdine. Deletion of the PvdQ gene disrupts pyoverdine production and hinders <i>P. aeruginosa</i> proliferation. Bacteria defective in pyoverdine synthesis are not infectious implying that disrupting this siderophore production through PvdQ inhibition could be exploited as a putative target for the development of novel antibiotic compounds. This report describes the development of a small molecule inhibitor (<a href="/pcsubstance/?term=ML318[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML318</a>, CID 56604881) of PvdQ acylase. <a href="/pcsubstance/?term=ML318[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML318</a> inhibits PvdQ <i>in vitro</i> with an IC<sub>50</sub> of 6 nM, has no apparent toxicity in mammalian HeLa cells up to a concentration of 100 μM, and inhibits growth and pyoverdine production in <i>P. aeruginosa</i> exposed to iron-limiting conditions with an IC<sub>50</sub> < 50 μM. <a href="/pcsubstance/?term=ML318[synonym]" ref="pagearea=abstract&targetsite=entrez&targetcat=term&targettype=pubchem">ML318</a> can also significantly reduce the intracellular uptake of iron inside the bacteria. This probe is a useful tool for ongoing characterization of pyoverdine’s role in <i>P. aeruginosa</i> biology and could serve as a starting point for the development of a novel antibiotic.</p></div><div class="h2"></div><p><b>Assigned Assay Grant #:</b> 1 R03 MH092076-01</p><p><b>Screening Center Name & PI:</b> Broad Institute Probe Development Center, Dr. Stuart Schreiber</p><p><b>Chemistry Center Name & PI:</b> Broad Institute Probe Development Center, Dr. Stuart Schreiber</p><p><b>Assay Submitter & Institution:</b> Dr. Andrew Gulick, Hauptman-Woodward Medical Research Institute, State University of New York, Buffalo, NY</p><p><b>PubChem Summary Bioassay Identifier (AID):</b>
|
|
<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/488968" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">488968</a></p><div id="ml318.s1"><h2 id="_ml318_s1_">Probe Structure & Characteristics</h2><div id="ml318.fu1" class="figure bk_fig"><div class="graphic"><img src="/books/NBK133446/bin/ml318fu1.jpg" alt="ML318." /></div><h3><span class="title">ML318</span></h3></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml318tu1"><a href="/books/NBK133446/table/ml318.tu1/?report=objectonly" target="object" title="Table" class="img_link icnblk_img figpopup" rid-figpopup="figml318tu1" rid-ob="figobml318tu1"><img class="small-thumb" src="/books/NBK133446/table/ml318.tu1/?report=thumb" src-large="/books/NBK133446/table/ml318.tu1/?report=previmg" alt="Image " /></a><div class="icnblk_cntnt"><h4 id="ml318.tu1"><a href="/books/NBK133446/table/ml318.tu1/?report=objectonly" target="object" rid-ob="figobml318tu1">Table</a></h4><p class="float-caption no_bottom_margin">wild type (PAO) AbsAC<sub>-35</sub> = 41.2/23.3 [SID 134356610, AID 624011, AID 624016, AID 624018 and AID 624020]</p></div></div></div><div id="ml318.s2"><h2 id="_ml318_s2_">1. Recommendations for Scientific Use of the Probe</h2><p><i>Pseudomonas aeruginosa</i> is an opportunistic Gram-negative human pathogen causing nosocomial and chronic lung infections. Pyoverdine is the primary iron siderophore produced by <i>P. aerugin</i>osa and its production has been associated with infection in multiple disease models. The aim of this project was to discover a small molecule inhibitor of the PvdQ acylase, an enzyme involved in pyoverdine production that could ultimately be used in the treatment of <i>P. aeruginosa</i> infection.</p><p>This screening project identified a probe (<a href="/pcsubstance/?term=ML318[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML318</a>) achieving potent inhibition of the <i>P. aeruginosa</i> PvdQ activity <i>in vitro</i> in the nM range. It significantly decreased pyoverdine production and iron uptake in the cell. These effects make the probe a useful tool for the assay for the scientific community interested in the role of the PvdQ/pyoverdine in iron metabolism, biofilm formation, swarming, and quorum sensing. This small molecule could facilitate the identification and understanding of other members of the broader family of N-terminal nucleophile (NTN) hydrolases beyond PvdQ. Ultimately, this chemical probe may serve as lead compound for the development of a novel class of antibiotics, which exploits a non-essential pathway for bacteria survival relevant to <i>P. aeruginosa</i> virulence.</p></div><div id="ml318.s3"><h2 id="_ml318_s3_">2. Materials and Methods</h2><div id="ml318.s4"><h3>2.1. Assays</h3><div id="ml318.s5"><h4>Key Assay Materials</h4><p>High-Base 1536-well black plates (00019180BK), 384-well black clear bottom assay plates (32441), low flange white flat bottom polystyrene tissue culture-treated 384-well microplates (8867BC) were obtained from Aurora Biotechnologies and Corning respectively. The positive control Isopropyl Dodecylfluorophosphonate (IDFP) used in the <i>in vitro</i> primary assay was purchased from Cayman Chemical. The PvdQ acylase protein preparation was provided by Dr. Andrew Gulick (Hauptman-Woodward Medical Research Institute, Buffalo, NY). 4-methylumbelliferyl laurate (4-MU laurate) (0183M) was from Research Organics. CellTiter-Glo (G7573) was obtained from Promega. Metal chelator ethylenediamine-N,N′-bis(2-hydroxyphenylacetic acid) (EDDHA) utilized in <i>P. aeruginosa</i> growth assay was purchased from Complete Green Company. Chrome azurol S (SC-252601) was bought from Santa Cruz Biotech.</p></div><div id="ml318.s6"><h4>Growth media</h4><p>For <i>P. aeruginosa</i> cell growth and replication, cells were seeded in Succinate M9 (<i>P. aeruginosa</i> EDDHA-dependent growth delay assay) or Casamino Acids media (Chrome Azurol assay). For normal mammalian cell propagation (cell toxicity assay), HeLa cells were cultured in DMEM media with phenol red (Invitrogen, 16140089) containing 10% heat inactivated fetal bovine serum (HyClone, (Thermo Scientific), SH30071.03), 1% penicillin/streptomycin/glutamine (Invitrogen, 10378-016) and 1 mM Sodium pyruvate solution (Gibco, 11360). For cell toxicity studies, the DMEM with phenol red was replaced DMEM medium without phenol red with all the other components (e.g. FBS). Cells were trypsinized using Trypsin-EDTA 0.25% (25-053-Cl) from Cellgro, Mediatech.</p><p><b>Microorganism and mammalian cell line:</b> The following bacteria and mammalian cell line were used for all of the assays in this project:</p><ul><li class="half_rhythm"><div><i>Pseudomonas aeruginosa</i> (Schroeter) Migula (ATCC, 15692), PAO.</div></li><li class="half_rhythm"><div><i>Pseudomonas aeruginosa</i> PAO PvdQ deletion mutant (Provided by Dr. Andrew Gulick, Hauptman-Woodward Medical Research Institute, Buffalo, NY).</div></li><li class="half_rhythm"><div><i>Pseudomonas aeruginosa</i> PAK and PAK mexAB pump mutant (Provided by Dr. Sam Moskowitz, Massachusetts General Hospital, Harvard Medical School, Cambridge, MA). (Detailed description of the generation of the PAK mexAB pump mutant strain in appendix B, section 4.2 (pp.50–51)).</div></li><li class="half_rhythm"><div>Human adenocarcinoma epithelial HeLa cell line (ATCC, CCL-2).</div></li></ul></div><div id="ml318.s7"><h4>Fluorescent biochemical assay-PvdQ-mediated cleavage of 4-Methylumbelliferyl-laurate</h4><div id="ml318.s8"><h5>Reagent and enzyme preparation</h5><p>144 mg (16 mM) of 4-methylumbelliferyl laurate (4-MU laurate) (Research Organics, 0183M) is dissolved in 25 mL isopropanol and is mixed with 2.5 mL of Triton X-100 (Sigma, T8787) (0.1 volume), vortexed for 10 sec and gently mixed with 375 mL TNT 50 mM Tris-HCl pH 8.0, 50 mM NaCl, and 0.2 mM Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) buffer (15 volumes). The <i>P. aeruginosa</i> PvdQ was provided by Dr. Andrew Gulick, Hauptman-Woodward Medical Research Institute, Buffalo, NY at a concentration of 10.06 mg/mL (126.6 μM stock concentration). The PvdQ preparation comes as 40 μL droplets. 80 μL of the PvdQ preparation is added in 50.2 mL TNT buffer (final concentration of 0.2 μM).</p></div><div id="ml318.s9"><h5>Reaction</h5><p>The PvdQ (0.75 μL) and 4-MU laurate (6.75 μL) solutions are dispensed using BioRaptr<sup>TM</sup> FRD microfluidic workstation (Beckman Coulter) at a final concentration 0.02 μM (PvdQ) and 0.8 mM (4-MU laurate) at room temperature in 1536-well high base black bar-coded plate square well assay plate (Aurora Biotechnologies, 00019180BK) where 7.5 or 15 nL (8 concentrations, 3-fold dilution with a final concentration of 10 μM) of the MLPCN compound library was previously dispensed in the assay plate. The positive control Isopropyl Dodecylfluorophosphonate (IDFP) (Cayman chemicals, 0215) is added in 24 selective wells (1.7 mM stock solution in TNT buffer to a 200 μM final concentration). The plates are then incubated at room temperature for 60 min.</p><p>The assay plates are read at time 0 and 60 min using the ViewLux (Perkin Elmer) with 303–367 nm excitation filter and 440–460 nm emission filter. The fluorescence generated by the enzymatic reaction was calculated as the difference between the reads (60 min vs. 0 min).</p><div id="ml318.s10"><h5>Cell toxicity assay</h5><div id="ml318.s11"><h5>Day 1 (Cell plating)</h5><dl class="temp-labeled-list"><dl class="bkr_refwrap"><dt>1.</dt><dd><p class="no_top_margin">HeLa cells are harvested and resuspended in DMEM without phenol red (Lonza, 12-917F) with 10% Heat inactivated fetal bovine serum (HyClone (Thermo Scientific, SH30071.03) and 1% penicillin/streptomycin/glutamine (Invitrogen, 10378-016). HeLa cells (from an initial cell suspension of 62,500 or 125,000 cells/mL) are dispensed using a MultiDrop Combi/Standard tube dispensing cassette (Thermo Scientific) in white bottom 384 well assay plates (Corning, 8867BC) at a final density of 2,500 or 5,000 cells per well in final volume of 40 μL. The cells are kept in suspension using a magnetic bar and a stirrer during the dispensing.</p></dd></dl><dl class="bkr_refwrap"><dt>2.</dt><dd><p class="no_top_margin">The assay plate (cell plate) are placed in Liconic Instruments cassettes (22 plates/cassette) and incubated for 24 hours at 37°C in the Liconic CO<sub>2</sub> incubator (Liconic Instruments) calibrated at 5% CO<sub>2</sub>, 21% O<sub>2</sub>, and 95% humidity.</p></dd></dl></dl></div><div id="ml318.s12"><h5>Day 2 (Pin copmounds into assay plates)</h5><dl class="temp-labeled-list"><dl class="bkr_refwrap"><dt>3.</dt><dd><p class="no_top_margin">The dose response compound plate (8 concentrations, 3 fold dilution, starting concentration 10 mM) are pinned 2 to 5 times using 384 well pin tool (100 nL) on pin table (Cybi Well) and transferred to the assay plate. Pins are washed with methanol and DMSO between each transfer.</p></dd></dl><dl class="bkr_refwrap"><dt>4.</dt><dd><p class="no_top_margin">The assay plates treated with compounds are moving back to Liconic CO<sub>2</sub> incubator to be incubated for an additional 24 or 48 hours.</p></dd></dl></dl></div><div id="ml318.s13"><h5>Day 3 or 4 (Reading luminescence from assay plates with EnVision)</h5><dl class="temp-labeled-list"><dl class="bkr_refwrap"><dt>5.</dt><dd><p class="no_top_margin">Each assay plate is pulled out of the incubator and cooled down at room temperature for 30 min. Then, 30 μL/well (384 well) of CellTiter-Glo luciferase reagent 0.5× (diluted in H<sub>2</sub>O) (Promega, G7573) is dispensed using the MultiDrop Combi dispensing cassette from Thermo Scientific. The assay plate returned to room temperature for 30 min to allow a complete cellular lysis.</p></dd></dl><dl class="bkr_refwrap"><dt>6.</dt><dd><p class="no_top_margin">Luminescence is measured using the EnVision multi-mode reader (Perkin Elmer) at 0.2 seconds/well in the 384-well format.</p></dd></dl></dl></div></div><div id="ml318.s14"><h5><i>P. aeruginosa</i> PAO EDDHA-dependent growth delay assay</h5><div id="ml318.s15"><h5>Day 1 (Thaw bacteria)</h5><dl class="temp-labeled-list"><dl class="bkr_refwrap"><dt>1.</dt><dd><p class="no_top_margin">Thaw frozen vial of <i>P. aeruginosa</i> PAO and grow overnight bacterial culture in Succinate M9 media pH 7.0 (34.5 mM KH<sub>2</sub>PO<sub>4</sub>, 22 mM K<sub>2</sub>HPO<sub>4</sub>, 7.5 mM (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub>, 34 mM Succinic acid and MgSO<sub>4</sub>) at 37°C with shaking.</p></dd></dl></dl></div><div id="ml318.s16"><h5>Day 2 (Pin compounds and dispense bacteria)</h5><dl class="temp-labeled-list"><dl class="bkr_refwrap"><dt>2.</dt><dd><p class="no_top_margin">Dispense 30 μL of Succinate M9 media with 2 μM (final concentration) of ethylenediamine-di (o-hydroxyphenulacetic acid) EDDHA (Complete Green Company) in 384-well black clear bottom assay plates (Aurora Biotechnologies, 32441) using MultiDrop Combi/Standard tube dispensing cassette (Thermo Scientific). The dose response compound plates (8 concentrations, 3 fold dilution, starting concentration 10 mM) are pinned twice using 384 well pin tool (100 nL) on pin table (Walkup Cybi Well) and transferred to assay plate. Pins are washed with methanol and DMSO between each pinning. Then, 10 μL of <i>P. aeruginosa</i> (diluted to a calculated OD<sub>600</sub> of 0.000002) with 2 μM EDDHA is dispensed with the MultiDrop Combi/Standard tube dispensing cassette (Thermo Scientific) in the same assay plates.</p></dd></dl><dl class="bkr_refwrap"><dt>3.</dt><dd><p class="no_top_margin">Incubate bacteria in humidified environment at 37°C for 24–30 h.</p></dd></dl></dl></div><div id="ml318.s17"><h5>Day 3 (Read on EnVision)</h5><dl class="temp-labeled-list"><dl class="bkr_refwrap"><dt>4.</dt><dd><p class="no_top_margin">Read absorbance (600 nm, growth and 405 nm, pyoverdine production) on the EnVision multi-mode reader (Perkin Elmer).</p></dd></dl></dl></div></div><div id="ml318.s18"><h5><i>P. aeruginosa</i> PAK (WT and pump mutant mexAB-OprM) growth delay assay in presence of iron chelator EDDHA</h5><p>Procedure:</p><div id="ml318.s19"><h5>Day 1 (Overnight culture)</h5><dl class="temp-labeled-list"><dl class="bkr_refwrap"><dt>1.</dt><dd><p class="no_top_margin">Starter cultures of <i>P.aeruginosa</i> PAK wild-type or pump mutant mexAB-OprM (kindly provided by Dr. Sam Moskowitz, Massachusetts General Hospital, Harvard Medical School, Boston, MA) were grown overnight in Succinate M9 media, at 37°C. (Detailed description of the generation of the PAK mexAB pump mutant strain in appendix B, section 4.2 (pp.50–51).</p></dd></dl></dl></div><div id="ml318.s20"><h5>Day 2 (Pin the compounds and add bacteria in assay plates)</h5><dl class="temp-labeled-list"><dl class="bkr_refwrap"><dt>2.</dt><dd><p class="no_top_margin">The next day, Succinate M9 media (34.5 mM Potassium phosphate dibasic, 22 mM Potassium phosphate monobasic, 7.5 mM Ammonium sulfate, 34 mM Succinic acid and 2 mM Magnesuin sulfate) was prepared with 2 μM ethylenediamine-<i>N,N</i>′-bis(2-hydroxyphenylacetic) acid (EDDHA). 20 μL was added to each well of 384-well, black, clear bottom plates (Aurora, 32441).</p></dd></dl><dl class="bkr_refwrap"><dt>3.</dt><dd><p class="no_top_margin">The dose response compound plates (9 concentrations, 3 fold dilution, starting concentration 10 mM) were pinned once or twice using 384 well pin tool (300 nL) on pin table (Walkup Cybi Well) and transferred into assay plate.</p></dd></dl><dl class="bkr_refwrap"><dt>4.</dt><dd><p class="no_top_margin">The absorbance at 600 nm (OD600) was measured for the overnight cultures. These were then diluted to an OD<sub>600</sub> of 0.05 in Succinate M9 media with 2 μM EDDHA.</p></dd></dl><dl class="bkr_refwrap"><dt>5.</dt><dd><p class="no_top_margin">10 μL of the diluted <i>P.aeruginosa</i> wild-type or pump mutant mexAB-OprM culture was added to each well of the plates containing the compounds, bringing the final compound concentrations to a range of 100 μM to 15 nM.</p></dd></dl><dl class="bkr_refwrap"><dt>6.</dt><dd><p class="no_top_margin">The plates were incubated for 28 hours at 30°C, in a humidified environment.</p></dd></dl></dl></div><div id="ml318.s21"><h5>Day 3 (Read absorbance)</h5><dl class="temp-labeled-list"><dl class="bkr_refwrap"><dt>7.</dt><dd><p class="no_top_margin">Following the 28h incubation, absorbance values were read with a plate reader at 600 nm (cell growth) and 405 nm (pyoverdine production).</p></dd></dl></dl></div></div><div id="ml318.s22"><h5>Chrome Azurol assay</h5><p>Protocol was derived from: Owen and Ackerley [<a class="bibr" href="#ml318.r27" rid="ml318.r27">27</a>].</p><div id="ml318.s23"><h5>Day 1 (Overnight culture)</h5><p><i>P. aeruginosa</i> PAO and PvdQ mutant (Dr. Andrew Gulick) cells are grown overnight in 5 mL CAA Media (5 g/L Casamino Acid growth powder, 1.54 g/L, K<sub>2</sub>HPO<sub>4</sub> and 0.25 g/L MgSO<sub>4</sub>) to an OD of ~0.6–0.8.</p></div><div id="ml318.s24"><h5>Day 2 (Assay media)</h5><p>The <i>P. aeruginosa</i> cells are collected by centrifugation and washed twice with CAA media. At the same time, the test compounds are dissolved in media in dose (starting concentration 125 μM, 2-fold dilutions, 10 dose points). 185 μL of cells diluted to an optical density (OD) of a 0.1 at 595 nm in presence of the test compound are then dispensed into 96 well plates in triplicate. The bacteria are grown at 30°C with slow shaking for 6 hours to an OD<sub>600</sub> between 0.5–0.6. The plate is then spun down (3,000 rpm, 25°C) for 15 min to pellet the cells. 150 μL of the media supernatant is collected and transferred into a flat bottom 96-well plate. 30 μL of the CAZS solution (1mM Chrome Azurol S, 0.1 mM FeCl<sub>3</sub> and 2 mM hexadecyltrimethyl-ammonium bromide) is added and incubated for 30 min.</p><p>Absorbance is read at 665 nm using a spectrophotometer. Cell number is normalized by measuring absorbance at 595 nm. Activity is determined as a percentage of the positive control (PvdQ mutant, set to 100%).</p></div></div></div></div></div><div id="ml318.s25"><h3>2.2. Probe Chemical Characterization</h3><p>The probe (<a href="/pcsubstance/?term=ML318[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML318</a>) was prepared as described in <a href="#ml318.s26">Section 2.3</a>, and was 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 spectrometry were consistent with the structure of the probe, and UPLC analysis showed purity of >90%.</p><p>The solubility of the probe (<a href="/pcsubstance/?term=ML318[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML318</a>) was determined to be >100 μM in Phosphate buffered saline (PBS; pH 7.4, 23°C) solution with 1% DMSO. The stability of the probe in PBS (1% DMSO) was measured over 48 hours and >82% was remaining after that time (<a class="figpopup" href="/books/NBK133446/figure/ml318.f1/?report=objectonly" target="object" rid-figpopup="figml318f1" rid-ob="figobml318f1">Figure 1</a>). Additionally, the stability of the probe in PBS in the presence of 50 μM glutathione nucleophile was measured over 48 hours and >53% was remaining after that time. The stability of the probe was confirmed by measuring stability in human plasma, with 70% remaining after a 5-hour incubation period. Plasma protein binding (PPB) studies showed that it was 66% bound in human plasma.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml318f1" co-legend-rid="figlgndml318f1"><a href="/books/NBK133446/figure/ml318.f1/?report=objectonly" target="object" title="Figure 1" class="img_link icnblk_img figpopup" rid-figpopup="figml318f1" rid-ob="figobml318f1"><img class="small-thumb" src="/books/NBK133446/bin/ml318f1.gif" src-large="/books/NBK133446/bin/ml318f1.jpg" alt="Figure 1. Stability of Probe (ML318) in PBS Buffer (pH 7.4, 23°C)." /></a><div class="icnblk_cntnt" id="figlgndml318f1"><h4 id="ml318.f1"><a href="/books/NBK133446/figure/ml318.f1/?report=objectonly" target="object" rid-ob="figobml318f1">Figure 1</a></h4><p class="float-caption no_bottom_margin">Stability of Probe (ML318) in PBS Buffer (pH 7.4, 23°C). </p></div></div><p><a class="figpopup" href="/books/NBK133446/table/ml318.t1/?report=objectonly" target="object" rid-figpopup="figml318t1" rid-ob="figobml318t1">Table 1</a> summarizes known properties of the probe.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figml318t1"><a href="/books/NBK133446/table/ml318.t1/?report=objectonly" target="object" title="Table 1" class="img_link icnblk_img figpopup" rid-figpopup="figml318t1" rid-ob="figobml318t1"><img class="small-thumb" src="/books/NBK133446/table/ml318.t1/?report=thumb" src-large="/books/NBK133446/table/ml318.t1/?report=previmg" alt="Table 1. Summary of Probe (ML318) Properties Computed from Structure." /></a><div class="icnblk_cntnt"><h4 id="ml318.t1"><a href="/books/NBK133446/table/ml318.t1/?report=objectonly" target="object" rid-ob="figobml318t1">Table 1</a></h4><p class="float-caption no_bottom_margin">Summary of Probe (ML318) Properties Computed from Structure. </p></div></div></div><div id="ml318.s26"><h3>2.3. Probe Preparation</h3><p>The racemic version of the probe (<a href="/pcsubstance/?term=ML318[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML318</a>) and most of the analogs prepared for SAR analysis were conveniently synthesized via arylations of benzyl nitirles with bromo or choro pyridines, and alkylations with bromo alkanes. The probe (<a href="/pcsubstance/?term=ML318[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML318</a>) was assembled via arylation of 4-fluoro benzyl nitrile using potassium <i>tert</i>-butoxide and 2-bromo-5-trifluoromethyl pyridine shown in <a class="figpopup" href="/books/NBK133446/figure/ml318.f5/?report=objectonly" target="object" rid-figpopup="figml318f5" rid-ob="figobml318f5">Scheme 1</a>. Tetrazoles were synthesized from benzyl nitriles using a [3+2] cycloaddition. Amino nitriles were synthesized from 4-chloro benzaldehyde using the Strecker reaction.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml318f5" co-legend-rid="figlgndml318f5"><a href="/books/NBK133446/figure/ml318.f5/?report=objectonly" target="object" title="Scheme 1" class="img_link icnblk_img figpopup" rid-figpopup="figml318f5" rid-ob="figobml318f5"><img class="small-thumb" src="/books/NBK133446/bin/ml318f5.gif" src-large="/books/NBK133446/bin/ml318f5.jpg" alt="Scheme 1. Synthesis of Probe (ML318)." /></a><div class="icnblk_cntnt" id="figlgndml318f5"><h4 id="ml318.f5"><a href="/books/NBK133446/figure/ml318.f5/?report=objectonly" target="object" rid-ob="figobml318f5">Scheme 1</a></h4><p class="float-caption no_bottom_margin">Synthesis of Probe (ML318). </p></div></div></div></div><div id="ml318.s27"><h2 id="_ml318_s27_">3. Results</h2><div id="ml318.s28"><h3>3.1. Dose Response Curves for Probe</h3><p>The dose response curves for the molecular probe (<a href="/pcsubstance/?term=ML318[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML318</a>) described in this report is presented in <a class="figpopup" href="/books/NBK133446/figure/ml318.f2/?report=objectonly" target="object" rid-figpopup="figml318f2" rid-ob="figobml318f2">Figure 3</a>. In the primary assay, the inhibition of a fluorescent signal mediated by enzymatic cleavage initiated by PvdQ acylase on the fatty acid laurate and a fluorescent molecule known as 4-methylumbelliferone by the probe was evaluated. The molecular probe (<a href="/pcsubstance/?term=ML318[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML318</a>) was able to decrease the fluorescent signal to a level similar to the positive control IDFP with an IC<sub>50</sub> of 6 nM (<a class="figpopup" href="/books/NBK133446/figure/ml318.f2/?report=objectonly" target="object" rid-figpopup="figml318f2" rid-ob="figobml318f2">Figure 3a</a>) (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/623951" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 623951</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/623985" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 623985</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/623991" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 623991</a> and <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/623993" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 623993</a>). The probe appeared not to aggregate or denature PvdQ in a non-specific fashion since a pre-incubation (30 min) of the enzyme with the probe did not affect the level of inhibition (data not shown). However, the positive control IDFP did inhibit to a greater extent PvdQ enzymatic activity when pre-incubated with the enzyme. The probe was not toxic to HeLa cells up to a concentration of 100 μM (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/623950" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 623950</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/623990" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 623990</a> and <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/623987" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 623987</a>) (<a class="figpopup" href="/books/NBK133446/figure/ml318.f2/?report=objectonly" target="object" rid-figpopup="figml318f2" rid-ob="figobml318f2">Figure 3b</a>). In the secondary assay, the ability of the molecular probe to delay growth in wild type <i>P. aeruginosa</i> PAO (absorbance at 600 nm) and inhibit pyoverdine production (absorbance at 405 nm) was tested in presence of the iron chelator EDDHA at 37°C. The role of EDDHA in the media was to reduce the level of available iron, thus, forcing the bacteria to rely on the pyoverdine pathway for iron uptake and growth. The molecular probe (<a href="/pcsubstance/?term=ML318[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML318</a>) decreased the absorbance at both 600 nm and 405 nm with an AbsAC<sub>-35</sub> of 41 μM and 23 μM, respectively (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/624018" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 624018</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/624011" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 624011</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/624020" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 624020</a> and <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/624016" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 624016</a>) (<a class="figpopup" href="/books/NBK133446/figure/ml318.f2/?report=objectonly" target="object" rid-figpopup="figml318f2" rid-ob="figobml318f2">Figure 3c</a>). The same experiment was performed in the wild-type <i>P. aeruginosa</i> PAK and the pump deletion mutant (mexAB-OprM) strains at 30°C. The probe (<a href="/pcsubstance/?term=ML318[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML318</a>) demonstrated similar potency in the wild-type <i>P. aeruginosa</i> PAK strain at 30°C with an AbsAC<sub>-35</sub> of 66.9 μM at 600 nm and AbsAC<sub>-35</sub> of 12.6 μM at 405 nm (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/624073" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 624073</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/624075" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 624075</a>). However, the probe (<a href="/pcsubstance/?term=ML318[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML318</a>) showed a significant increase in potency when tested in the pump mutant strain with an AbsAC<sub>-35</sub> of 16.2 μM and 1.4 μM at 600 nm and 405 nm, respectively (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/624072" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 624072</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/624074" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 624074</a>) (<a class="figpopup" href="/books/NBK133446/figure/ml318.f2/?report=objectonly" target="object" rid-figpopup="figml318f2" rid-ob="figobml318f2">Figure 3d and e</a>). Finally, an assay using chrome azurol S, an iron (III)-complexed dye, was performed to assess pyoverdine production/secretion from <i>P. aeruginosa</i> grown in the presence of the probe <a href="/pcsubstance/?term=ML318[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML318</a>. Normally, pyoverdine produced by the <i>P. aeruginosa</i> removes the iron from the dye causing a distinct color change (blue to orange) and a subsequent reduction in absorbance at 665 nm. <a href="/pcsubstance/?term=ML318[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML318</a> was able to block pyoverdine production as compared to the PvdQ mutant with an IC<sub>50</sub> around 6 μM (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/624033" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 624033</a>) (<a class="figpopup" href="/books/NBK133446/figure/ml318.f2/?report=objectonly" target="object" rid-figpopup="figml318f2" rid-ob="figobml318f2">Figure 3F</a>). The dose response curves in the primary assay for the probe and selected analogs submitted to the SMR collection are captured in <a class="figpopup" href="/books/NBK133446/figure/ml318.f3/?report=objectonly" target="object" rid-figpopup="figml318f3" rid-ob="figobml318f3">Figure 4</a>.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml318f2" co-legend-rid="figlgndml318f2"><a href="/books/NBK133446/figure/ml318.f2/?report=objectonly" target="object" title="Figure 3" class="img_link icnblk_img figpopup" rid-figpopup="figml318f2" rid-ob="figobml318f2"><img class="small-thumb" src="/books/NBK133446/bin/ml318f2a.gif" src-large="/books/NBK133446/bin/ml318f2a.jpg" alt="Figure 3. Dose-dependent activity of the probe (ML318)." /></a><div class="icnblk_cntnt" id="figlgndml318f2"><h4 id="ml318.f2"><a href="/books/NBK133446/figure/ml318.f2/?report=objectonly" target="object" rid-ob="figobml318f2">Figure 3</a></h4><p class="float-caption no_bottom_margin">Dose-dependent activity of the probe (ML318). (<i>A</i>) Fluorescent biochemical assay- PvdQ-mediated cleavage of 4-Methylumbelliferyl-laurate (Primary assay and Retest at dose) (AID 623951, AID 623985, AID 623991 and AID 623993). (<i>B</i>) HeLa cell toxicity assay <a href="/books/NBK133446/figure/ml318.f2/?report=objectonly" target="object" rid-ob="figobml318f2">(more...)</a></p></div></div><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml318f3" co-legend-rid="figlgndml318f3"><a href="/books/NBK133446/figure/ml318.f3/?report=objectonly" target="object" title="Figure 4" class="img_link icnblk_img figpopup" rid-figpopup="figml318f3" rid-ob="figobml318f3"><img class="small-thumb" src="/books/NBK133446/bin/ml318f3.gif" src-large="/books/NBK133446/bin/ml318f3.jpg" alt="Figure 4. Dose response curves of probe and analogs submitted to SMR collection." /></a><div class="icnblk_cntnt" id="figlgndml318f3"><h4 id="ml318.f3"><a href="/books/NBK133446/figure/ml318.f3/?report=objectonly" target="object" rid-ob="figobml318f3">Figure 4</a></h4><p class="float-caption no_bottom_margin">Dose response curves of probe and analogs submitted to SMR collection. Fluorescent biochemical assay- PvdQ-mediated cleavage of 4-Methylumbelliferyl-laurate (Retest at dose). (AID: 623951, 623985, 623991 and 623993). </p></div></div></div><div id="ml318.s29"><h3>3.2. Cellular Activity</h3><p>The HeLa cellular toxicity assay performed with the probe revealed that the compound was not toxic in mammalian cells up to a concentration of 100 uM.</p></div><div id="ml318.s30"><h3>3.3. Profiling Assays</h3><p>No profiling assays were performed.</p></div></div><div id="ml318.s31"><h2 id="_ml318_s31_">4. Discussion</h2><div id="ml318.s32"><h3>4.1. Comparison to Existing Art and How the New Probe is an Improvement</h3><p>A series of searches were performed to find previously studied small molecule inhibitors of PvdQ. The current literature revealed only two small molecule inhibitors of <i>P. aeruginosa</i> a/b heterodimeric NTN-hydrolase PvdQ, which originated from the assay provider’s lab (<a class="figpopup" href="/books/NBK133446/figure/ml318.f4/?report=objectonly" target="object" rid-figpopup="figml318f4" rid-ob="figobml318f4">Figure 5</a>) [<a class="bibr" href="#ml318.r24" rid="ml318.r24">24</a>]. These compounds were discovered from a biochemical screen with the commercially available LOPAC library containing 1280 compounds. The small molecules shown in <a class="figpopup" href="/books/NBK133446/figure/ml318.f4/?report=objectonly" target="object" rid-figpopup="figml318f4" rid-ob="figobml318f4">Figure 5</a> were not very potent in an <i>in vitro</i> assay of PvdQ yielding IC<sub>50</sub> values of 130 μM and 65 μM, respectively. Therefore, our development of a potent, nanomolar inhibitor of PvdQ (<a href="/pcsubstance/?term=ML318[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML318</a>) to attenuate bacterial virulence via inhibition of siderophore biosynthesis (pyoverdine) pathways significantly improves upon the prior art.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figml318f4" co-legend-rid="figlgndml318f4"><a href="/books/NBK133446/figure/ml318.f4/?report=objectonly" target="object" title="Figure 5" class="img_link icnblk_img figpopup" rid-figpopup="figml318f4" rid-ob="figobml318f4"><img class="small-thumb" src="/books/NBK133446/bin/ml318f4.gif" src-large="/books/NBK133446/bin/ml318f4.jpg" alt="Figure 5. Comparison of Probe to Prior Art Compounds." /></a><div class="icnblk_cntnt" id="figlgndml318f4"><h4 id="ml318.f4"><a href="/books/NBK133446/figure/ml318.f4/?report=objectonly" target="object" rid-ob="figobml318f4">Figure 5</a></h4><p class="float-caption no_bottom_margin">Comparison of Probe to Prior Art Compounds. </p></div></div></div></div><div id="ml318.s33"><h2 id="_ml318_s33_">5. References</h2><dl class="temp-labeled-list"><dl class="bkr_refwrap"><dt>1.</dt><dd><div class="bk_ref" id="ml318.r1">Ochsner UA, Wilderman PJ, Vasil AI, Vasil ML. GeneChip expression analysis of the iron starvation response in Pseudomonas aeruginosa: identification of novel pyoverdine biosynthesis genes. <span><span class="ref-journal">Mol Microbiol. </span>2002;<span class="ref-vol">45</span>:1277–1287.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/12207696" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 12207696</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>2.</dt><dd><div class="bk_ref" id="ml318.r2">Meyer JM, Neely A, Stintzi A, Georges C, Holder IA. Pyoverdine is essential for virulence of Pseudomonas aeruginosa. <span><span class="ref-journal">Infect Immun. </span>1996;<span class="ref-vol">64</span>:518–523.</span> [<a href="/pmc/articles/PMC173795/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC173795</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/8550201" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 8550201</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>3.</dt><dd><div class="bk_ref" id="ml318.r3">Lamont IL, Beare PA, Ochsner U, Vasil AI, Vasil ML. Siderophore-mediated signaling regulates virulence factor production in Pseudomonas aeruginosa. <span><span class="ref-journal">Proc Natl Acad Sci U S A. </span>2002;<span class="ref-vol">99</span>:7072–7077.</span> [<a href="/pmc/articles/PMC124530/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC124530</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/11997446" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 11997446</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>4.</dt><dd><div class="bk_ref" id="ml318.r4">Nadal Jimenez P, Koch G, Papaioannou E, Wahjudi M, Krzeslak J, Coenye T, Cool RH, Quax WJ. Role of PvdQ in Pseudomonas aeruginosa virulence under iron limiting conditions. <span><span class="ref-journal">Microbiology. </span>2010;<span class="ref-vol">156</span>:49–59.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/19778968" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19778968</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>5.</dt><dd><div class="bk_ref" id="ml318.r5">Hentzer M, Eberl L, Givskov M. Transcriptome analysis of Pseudomonas aeruginosa biofilm development: anaerobic respiration and iron limitation. <span><span class="ref-journal">Biofilms. </span>2005;<span class="ref-vol">2</span>:37–61.</span></div></dd></dl><dl class="bkr_refwrap"><dt>6.</dt><dd><div class="bk_ref" id="ml318.r6">Sauer K, Cullen MC, Rickard AH, Zeef LA, Davies DG, Gilbert P. Characterization of nutrient-induced dispersion in Pseudomonas aeruginosa PAO1 biofilm. <span><span class="ref-journal">J Bacteriol. </span>2004;<span class="ref-vol">186</span>:7312–7326.</span> [<a href="/pmc/articles/PMC523207/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC523207</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/15489443" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 15489443</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>7.</dt><dd><div class="bk_ref" id="ml318.r7">Wu W, Badrane H, Arora S, Baker HV, Jin S. MucA-mediated coordination of type III secretion and alginate synthesis in Pseudomonas aeruginosa. <span><span class="ref-journal">J Bacteriol. </span>2004;<span class="ref-vol">186</span>:7575–7585.</span> [<a href="/pmc/articles/PMC524895/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC524895</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/15516570" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 15516570</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>8.</dt><dd><div class="bk_ref" id="ml318.r8">Banin E, Vasil ML, Greenberg EP. Iron and Pseudomonas aeruginosa biofilm formation. <span><span class="ref-journal">Proc Natl Acad Sci U S A. </span>2005;<span class="ref-vol">102</span>:11076–11081.</span> [<a href="/pmc/articles/PMC1182440/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC1182440</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/16043697" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 16043697</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>9.</dt><dd><div class="bk_ref" id="ml318.r9">Challis GL, Naismith JH. Structural aspects of non-ribosomal peptide biosynthesis. <span><span class="ref-journal">Curr Opin Struct Biol. </span>2004;<span class="ref-vol">14</span>:748–756.</span> [<a href="/pmc/articles/PMC3326538/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3326538</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/15582399" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 15582399</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>10.</dt><dd><div class="bk_ref" id="ml318.r10">Artymiuk PJ. A sting in the (N-terminal) tail. <span><span class="ref-journal">Nat Struct Biol. </span>1995;<span class="ref-vol">2</span>:1035–1037.</span> 1995. [<a href="https://pubmed.ncbi.nlm.nih.gov/8846211" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 8846211</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>11.</dt><dd><div class="bk_ref" id="ml318.r11">Brannigan JA, Dodson G, Duggleby HJ, Moody PC, Smith JL, Tomchick DR, Murzin AG. A protein catalytic framework with an N-terminal nucleophile is capable of self-activation. <span><span class="ref-journal">Nature. </span>1995;<span class="ref-vol">378</span>:416–419.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/7477383" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 7477383</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>12.</dt><dd><div class="bk_ref" id="ml318.r12">Iqbal A, Clifton IJ, Bagonis M, Kershaw NJ, Domene C, Claridge TD, Wharton CW, Schofield CJ. Anatomy of a simple acyl intermediate in enzyme catalysis: combined biophysical and modeling studies on ornithine acetyl transferase. <span><span class="ref-journal">J Am Chem Soc. </span>2009;<span class="ref-vol">131</span>:749–757.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/19105697" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19105697</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>13.</dt><dd><div class="bk_ref" id="ml318.r13">Lehoux DE, Sanschagrin F, Levesque RC. Genomics of the 35-kb pvd locus and analysis of novel pvdIJK genes implicated in pyoverdine biosynthesis in Pseudomonas aeruginosa. <span><span class="ref-journal">FEMS Microbiol Lett. </span>2000;<span class="ref-vol">190</span>:141–146.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/10981704" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 10981704</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>14.</dt><dd><div class="bk_ref" id="ml318.r14">Taguchi F, Suzuki T, Inagaki Y, Toyoda K, Shiraishi T, Ichinose Y. Siderophore pyoverdine of Pseudomonas syringae pv. tabaci 6605 is intrinsic virulence factor in host tobacco infection. <span><span class="ref-journal">J Bacteriol. </span>2010;<span class="ref-vol">192</span>(1):117–26.</span> [<a href="/pmc/articles/PMC2798240/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2798240</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/19854904" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19854904</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>15.</dt><dd><div class="bk_ref" id="ml318.r15">Papaioannou E, Wahjudi M, Nadal-Jimenez P, Koch G, Setroikromo R, Quax WJ. Quorum-quenching acylase reduces the virulence of Pseudomonas aeruginosa in a Caenorhabditis elegans infection model. <span><span class="ref-journal">Antimicrob Agents Chemother. </span>2009;<span class="ref-vol">53</span>:4891–4897.</span> [<a href="/pmc/articles/PMC2772301/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2772301</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/19721066" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19721066</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>16.</dt><dd><div class="bk_ref" id="ml318.r16">Somu RV, Boshoff H, Qiao C, Bennett EM, Barry CE 3rd, Aldrich CC. Rationally designed nucleoside antibiotics that inhibit siderophore biosynthesis of Mycobacterium tuberculosis. <span><span class="ref-journal">J Med Chem. </span>2006;<span class="ref-vol">49</span>:31–34.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/16392788" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 16392788</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>17.</dt><dd><div class="bk_ref" id="ml318.r17">Vannada J, Bennett EM, Wilson DJ, Boshoff HI, Barry CE 3rd, Aldrich CC. Design, synthesis, and biological evaluation of b-ketosulfonamide adenylation inhibitors as potential antitubercular agents. <span><span class="ref-journal">Org. Lett. </span>2006;<span class="ref-vol">8</span>:4707–4710.</span> [<a href="/pmc/articles/PMC2596716/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2596716</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/17020283" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17020283</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>18.</dt><dd><div class="bk_ref" id="ml318.r18">Qiao C, Gupte A, Boshoff HI, Wilson DJ, Bennett EM, Somu RV, Barry CE 3rd, Aldrich CC. 5′-O-[(N-Acyl)sulfamoyl]adenosines as Antitubercular Agents that Inhibit MbtA: An Adenylation Enzyme Required for Siderophore Biosynthesis of the Mycobactins. <span><span class="ref-journal">J Med Chem. </span>2007;<span class="ref-vol">50</span>:6080–6094.</span> [<a href="/pmc/articles/PMC2539069/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2539069</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/17967002" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17967002</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>19.</dt><dd><div class="bk_ref" id="ml318.r19">Miethke M, Bisseret P, Beckering CL, Vignard D, Eustache J, Marahiel MA. Inhibition of aryl acid adenylation domains involved in bacterial siderophore synthesis. <span><span class="ref-journal">Febs J. </span>2006;<span class="ref-vol">273</span>:409–419.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/16403027" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 16403027</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>20.</dt><dd><div class="bk_ref" id="ml318.r20">Cisar JS, Ferreras JA, Soni RK, Quadri LE, Tan DS. Exploiting ligand conformation in selective inhibition of non-ribosomal peptide synthetase amino acid adenylation with designed macrocyclic small molecules. <span><span class="ref-journal">J Am Chem Soc. </span>2007;<span class="ref-vol">129</span>:7752–7753.</span> [<a href="/pmc/articles/PMC2565600/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2565600</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/17542590" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17542590</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>21.</dt><dd><div class="bk_ref" id="ml318.r21">Clatworthy AE, Pierson E, Hung DT. Targeting virulence: a new paradigm for antimicrobial therapy. <span><span class="ref-journal">Nat Chem Biol. </span>2007;<span class="ref-vol">3</span>:541–548.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/17710100" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17710100</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>22.</dt><dd><div class="bk_ref" id="ml318.r22">Yeterian E, Martin LW, Guillon L, Journet L, Lamont IL, Schalk IJ. Synthesis of the siderophore pyoverdine in Pseudomonas aeruginosa involves a periplasmic maturation. <span><span class="ref-journal">Amino Acids. </span>2009 2010 May;<span class="ref-vol">38</span>(5):1447–59.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/19787431" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19787431</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>23.</dt><dd><div class="bk_ref" id="ml318.r23">Lomovskaya O, Warren MS, Lee A, Galazzo J, Fronko R, Lee M, Blais J, Cho D, Chamberland S, Renau T, Leger R, Hecker S, Watkins W, Hoshino K, Ishida H, Lee VJ. Identification and characterization of inhibitors of multidrug resistance efflux pumps in Pseudomonas aeruginosa: novel agents for combination therapy. <span><span class="ref-journal">Antimicrob Agents Chemother. </span>2001;<span class="ref-vol">45</span>(1):105–16.</span> [<a href="/pmc/articles/PMC90247/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC90247</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/11120952" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 11120952</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>24.</dt><dd><div class="bk_ref" id="ml318.r24">Drake EJ, Gulick AM. Structural characterization and high-throughput screening of inhibitors of PvdQ, an NTN hydrolase involved in pyoverdine synthesis. <span><span class="ref-journal">ACS Chem Biol. </span>2011;<span class="ref-vol">6</span>(11):1277–86.</span> [<a href="/pmc/articles/PMC3220798/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3220798</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/21892836" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 21892836</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>25.</dt><dd><div class="bk_ref" id="ml318.r25">Imperi F, Tiburzi F, Visca P. Molecular basis of pyoverdine siderophore recycling in Pseudomonas aeruginosa. <span><span class="ref-journal">Proc Natl Acad Sci U S A. </span>2009;<span class="ref-vol">1;106</span>(48):20440–5.</span> [<a href="/pmc/articles/PMC2787144/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2787144</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/19906986" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19906986</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>26.</dt><dd><div class="bk_ref" id="ml318.r26">Zuhl AM, Mohr JT, Bachovchin DA, Niessen S, Hsu KL, Berlin JM, Dochnahl M, López-Alberca MP, Fu GC, Cravatt BF. Competitive Activity-Based Protein Profiling Identifies Aza-β-Lactams as a Versatile Chemotype for Serine Hydrolase Inhibition. <span><span class="ref-journal">J Am Chem Soc. </span>2012;<span class="ref-vol">134</span>(11):5068–71.</span> [<a href="/pmc/articles/PMC3326416/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3326416</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/22400490" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 22400490</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>27.</dt><dd><div class="bk_ref" id="ml318.r27">Owen JG, Ackerley DF. Characterization of Pseudomonas achromobactin in Pseudomonas syringae pv. phaseolicola 1448a. <span><span class="ref-journal">BMC Microbiology. </span>2011;<span class="ref-vol">11</span>:218.</span> [<a href="/pmc/articles/PMC3207962/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3207962</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/21967163" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 21967163</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>28.</dt><dd><div class="bk_ref" id="ml318.r28">Ho SN, Horton RM. Method for gene splicing by overlap extension using the polymerase chain reaction. 5023171. <span><span class="ref-journal">United States patent. </span>1991</span></div></dd></dl><dl class="bkr_refwrap"><dt>29.</dt><dd><div class="bk_ref" id="ml318.r29">Wolfgang MC, Lee VT, Gilmore ME, Lory S. Coordinate regulation of bacterial virulence genes by a novel adenylate cyclase-dependent signaling pathway. <span><span class="ref-journal">Dev Cell. </span>2003;<span class="ref-vol">4</span>:253–263.</span> 2003. [<a href="https://pubmed.ncbi.nlm.nih.gov/12586068" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 12586068</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>30.</dt><dd><div class="bk_ref" id="ml318.r30">Hoang TT, Karkhoff-Schweizer RR, Kutchma AJ, Schweizer HP. A broad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants. <span><span class="ref-journal">Gene. </span>1998;<span class="ref-vol">212</span>:77–86.</span> 1998. [<a href="https://pubmed.ncbi.nlm.nih.gov/9661666" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 9661666</span></a>]</div></dd></dl></dl></div><div id="bk_toc_contnr"></div></div></div><div class="fm-sec"><h2 id="_NBK133446_pubdet_">Publication Details</h2><h3>Author Information and Affiliations</h3><p class="contrib-group"><h4>Authors</h4><span itemprop="author">Jimmy R. Theriault</span>,<sup>1</sup> <span itemprop="author">Jacqueline Wurst</span>,<sup>1</sup> <span itemprop="author">Ivan Jewett</span>,<sup>1</sup> <span itemprop="author">Lynn Verplank</span>,<sup>1</sup> <span itemprop="author">Jose R. Perez</span>,<sup>1</sup> <span itemprop="author">Andrew M. Gulick</span>,<sup>2</sup> <span itemprop="author">Eric J. Drake</span>,<sup>2</sup> <span itemprop="author">Michelle Palmer</span>,<sup>1</sup> <span itemprop="author">Sam Moskowitz</span>,<sup>3</sup> <span itemprop="author">Nandini Dasgupta</span>,<sup>3</sup> <span itemprop="author">Mark K. Brannon</span>,<sup>3</sup> <span itemprop="author">Sivaraman Dandapani</span>,<sup>1</sup> <span itemprop="author">Ben Munoz</span>,<sup>1</sup> and <span itemprop="author">Stuart Schreiber</span><sup>1</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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Hauptman-Woodward Medical Research Institute, State University of New York, Buffalo, NY</div><div class="affiliation"><sup>3</sup>
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Department of Pediatrics, Massachusetts General Hospital, Boston, MA</div><div class="affiliation"><sup>4</sup>
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Department of Pediatrics, Harvard Medical School, Boston, MA</div><h3>Publication History</h3><p class="small">Received: <span itemprop="datePublished">April 16, 2012</span>; Last Update: <span itemprop="dateModified">February 28, 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>Theriault JR, Wurst J, Jewett I, et al. Identification of a small molecule inhibitor of Pseudomonas aeruginosa PvdQ acylase, an enzyme involved in siderophore pyoverdine synthesis. 2012 Apr 16 [Updated 2013 Feb 28]. 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/ml320/?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/ml317/?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="figobml318fu1"><div id="ml318.fu1" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK133446/bin/ml318fu1.jpg" alt="ML318." /></div><h3><span class="title">ML318</span></h3></div></article><article data-type="table-wrap" id="figobml318tu1"><div id="ml318.tu1" class="table"><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK133446/table/ml318.tu1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml318.tu1_lrgtbl__"><table class="no_bottom_margin"><thead><tr><th id="hd_h_ml318.tu1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CID/ML</th><th id="hd_h_ml318.tu1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Target</th><th id="hd_h_ml318.tu1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">IC<sub>50</sub> (μM) [SID, AID]</th><th id="hd_h_ml318.tu1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Anti-target</th><th id="hd_h_ml318.tu1_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">IC<sub>50</sub> (μM) [SID, AID]</th><th id="hd_h_ml318.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Fold Selective</th><th id="hd_h_ml318.tu1_1_1_1_7" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">Secondary Assays (600 nm/405 nm) AbsAC<sub>-35</sub> (μM)<sup>a</sup> and <sup>d</sup> [SID, AID]</th></tr></thead><tbody><tr><td headers="hd_h_ml318.tu1_1_1_1_1" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">CID 56604881/<a href="/pcsubstance/?term=ML318[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML318</a></td><td headers="hd_h_ml318.tu1_1_1_1_2" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">PvdQ pyoverdine synthesis</td><td headers="hd_h_ml318.tu1_1_1_1_3" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">IC<sub>50</sub> = 0.006<sup>b</sup> [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/134356610" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 134356610</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/623951" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 623951</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/623985" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 623985</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/623991" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 623991</a> and <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/623993" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 623993</a>]</td><td headers="hd_h_ml318.tu1_1_1_1_4" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">24-hour HeLa cell cytotoxicity</td><td headers="hd_h_ml318.tu1_1_1_1_5" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">IC<sub>50</sub> = >100<sup>c</sup> [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/134356610" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 134356610</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/623950" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 623950</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/623990" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 623990</a> and <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/623987" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 623987</a>]</td><td headers="hd_h_ml318.tu1_1_1_1_6" rowspan="1" colspan="1" style="text-align:center;vertical-align:middle;">>3200×</td><td headers="hd_h_ml318.tu1_1_1_1_7" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">
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<ol><li class="half_rhythm"><div>wild type (PAO)</div><div>AbsAC<sub>-35</sub> = 41.2/23.3 [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/134356610" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 134356610</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/624011" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 624011</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/624016" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 624016</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/624018" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 624018</a> and <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/624020" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 624020</a>]</div></li><li class="half_rhythm"><div>wild type (PAK)</div><div>AbsAC<sub>-35</sub> = 66.9/12.6 [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/134356610" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 134356610</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/624073" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID624073</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/624075" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID624075</a>]</div></li><li class="half_rhythm"><div>pump mutant (PAK)</div><div>bsAC<sub>-35</sub> = 16.2/1.4 [<a href="https://pubchem.ncbi.nlm.nih.gov/substance/134356610" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">SID 134356610</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/624072" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 624072</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/624074" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 624074</a>]</div></li></ol></td></tr></tbody></table></div><div class="tblwrap-foot"><div><dl class="temp-labeled-list small"><dl class="bkr_refwrap"><dt>a</dt><dd><div id="ml318.tfn1"><p class="no_margin">AbsAc<sub>-35</sub> represents a normalized activity where compounds are compared at a common threshold (-35) (see section 3.1.1).</p></div></dd></dl><dl class="bkr_refwrap"><dt>b</dt><dd><div id="ml318.tfn2"><p class="no_margin">Average of n=4,</p></div></dd></dl><dl class="bkr_refwrap"><dt>c</dt><dd><div id="ml318.tfn3"><p class="no_margin">Average of n=3,</p></div></dd></dl><dl class="bkr_refwrap"><dt>d</dt><dd><div id="ml318.tfn4"><p class="no_margin">Average of n=2.</p></div></dd></dl></dl></div></div></div></article><article data-type="fig" id="figobml318f1"><div id="ml318.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%20Probe%20(ML318)%20in%20PBS%20Buffer%20(pH%207.4%2C%2023%B0C).&p=BOOKS&id=133446_ml318f1.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/NBK133446/bin/ml318f1.jpg" alt="Figure 1. Stability of Probe (ML318) 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 Probe (<a href="/pcsubstance/?term=ML318[synonym]" ref="pagearea=body&targetsite=entrez&targetcat=term&targettype=pubchem">ML318</a>) in PBS Buffer (pH 7.4, 23°C)</span></h3></div></article><article data-type="table-wrap" id="figobml318t1"><div id="ml318.t1" class="table"><h3><span class="label">Table 1</span><span class="title">Summary of Probe (ML318) Properties Computed from Structure</span></h3><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK133446/table/ml318.t1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__ml318.t1_lrgtbl__"><table class="no_top_margin"><tbody><tr><th id="hd_b_ml318.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:middle;">IUPAC Chemical Name</th><td headers="hd_b_ml318.t1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">2-(4-fluorophenyl)-2-[6-(trifluoromethyl)pyridin-2-yl] acetonitrile</td></tr><tr><th id="hd_b_ml318.t1_1_1_2_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">PubChem CID</th><td headers="hd_b_ml318.t1_1_1_2_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">56604881</td></tr><tr><th id="hd_b_ml318.t1_1_1_3_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Molecular Weight</th><td headers="hd_b_ml318.t1_1_1_3_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">280.220333 [g/mol]</td></tr><tr><th id="hd_b_ml318.t1_1_1_4_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Molecular Formula</th><td headers="hd_b_ml318.t1_1_1_4_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">C<sub>14</sub>H<sub>8</sub>F<sub>4</sub>N<sub>2</sub></td></tr><tr><th id="hd_b_ml318.t1_1_1_5_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">H-Bond Donor</th><td headers="hd_b_ml318.t1_1_1_5_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">0</td></tr><tr><th id="hd_b_ml318.t1_1_1_6_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">H-Bond Acceptor</th><td headers="hd_b_ml318.t1_1_1_6_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">6</td></tr><tr><th id="hd_b_ml318.t1_1_1_7_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Rotatable Bond Count</th><td headers="hd_b_ml318.t1_1_1_7_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">2</td></tr><tr><th id="hd_b_ml318.t1_1_1_8_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Exact Mass</th><td headers="hd_b_ml318.t1_1_1_8_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">280.062361 [g/mol]</td></tr><tr><th id="hd_b_ml318.t1_1_1_9_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Topological Polar Surface Area</th><td headers="hd_b_ml318.t1_1_1_9_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">36.7</td></tr></tbody></table></div></div></article><article data-type="fig" id="figobml318f5"><div id="ml318.f5" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK133446/bin/ml318f5.jpg" alt="Scheme 1. Synthesis of Probe (ML318)." /></div><h3><span class="label">Scheme 1</span><span class="title">Synthesis of Probe (ML318)</span></h3></div></article><article data-type="fig" id="figobml318f2"><div id="ml318.f2" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%203.%20Dose-dependent%20activity%20of%20the%20probe%20(ML318).&p=BOOKS&id=133446_ml318f2a.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/NBK133446/bin/ml318f2a.jpg" alt="Figure 3. Dose-dependent activity of the probe (ML318)." class="tileshop" title="Click on image to zoom" /></a></div><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%203.%20Dose-dependent%20activity%20of%20the%20probe%20(ML318).&p=BOOKS&id=133446_ml318f2b.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/NBK133446/bin/ml318f2b.jpg" alt="Figure 3. Dose-dependent activity of the probe (ML318)." class="tileshop" title="Click on image to zoom" /></a></div><div class="graphic"><img data-src="/books/NBK133446/bin/ml318f2c.jpg" alt="Figure 3. Dose-dependent activity of the probe (ML318)." /></div><h3><span class="label">Figure 3</span><span class="title">Dose-dependent activity of the probe (ML318)</span></h3><div class="caption"><p>(<b>A</b>) Fluorescent biochemical assay- PvdQ-mediated cleavage of 4-Methylumbelliferyl-laurate (Primary assay and Retest at dose) (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/623951" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 623951</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/623985" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 623985</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/623991" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 623991</a> and <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/623993" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 623993</a>). (<b>B</b>) HeLa cell toxicity assay (Counter screen) (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/623950" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 623950</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/623990" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 623990</a> and <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/623987" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 623987</a>). (<b>C</b>) <i>P. aeruginosa</i> (P.aeru) PAO EDDHA-dependent growth delay assay (Secondary assay), at absorbance 405 nm (pyoverdine production, Pyo) (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/624020" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 624020</a> and <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/624018" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 624018</a>) and at absorbance 600 nm (growth) (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/624011" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 624011</a> and <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/624016" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 624016</a>). (<b>D</b>) <i>P. aeruginosa</i> (P.aeru) PAK Wild-Type (PAK) and (<b>E</b>) pump mutant mexAB-OprM (Pump Mut.) growth delay assay in presence of iron chelator EDDHA (Secondary assay), at absorbance 405 nm (pyoverdine production, Pyo) (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/624072" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 624072</a> and <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/624073" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 624073</a>) and absorbance 600 nm (growth) (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/624074" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 624074</a> and <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/624075" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 624075</a>). (<b>F</b>) Chrome azurol assay. (<a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/624033" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">AID 624033</a>).</p></div></div></article><article data-type="fig" id="figobml318f3"><div id="ml318.f3" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%204.%20Dose%20response%20curves%20of%20probe%20and%20analogs%20submitted%20to%20SMR%20collection.&p=BOOKS&id=133446_ml318f3.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/NBK133446/bin/ml318f3.jpg" alt="Figure 4. Dose response curves of probe and analogs submitted to SMR collection." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 4</span><span class="title">Dose response curves of probe and analogs submitted to SMR collection</span></h3><div class="caption"><p>Fluorescent biochemical assay- PvdQ-mediated cleavage of 4-Methylumbelliferyl-laurate (Retest at dose). (AID: <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/623951" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">623951</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/623985" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">623985</a>, <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/623991" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">623991</a> and <a href="https://pubchem.ncbi.nlm.nih.gov/bioassay/623993" ref="pagearea=body&targetsite=entrez&targetcat=link&targettype=pubchem">623993</a>).</p></div></div></article><article data-type="fig" id="figobml318f4"><div id="ml318.f4" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK133446/bin/ml318f4.jpg" alt="Figure 5. Comparison of Probe to Prior Art Compounds." /></div><h3><span class="label">Figure 5</span><span class="title">Comparison of Probe to Prior Art Compounds</span></h3></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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