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BioAssay: AID 488968

Broad Institute Inhibitors of Pyoverdine Production Project

The human pathogen Pseudomonas aeruginosa uses a biosynthetic cluster to produce a peptide siderophore called pyoverdine. Pyoverdine is produced by non-ribosomal peptide synthetases, and utilizes 10 other proteins that are co-regulated and catalyze chemical modifications to the amino acid substrates or peptide intermediates. Immature pyoverdine is first produced with an amide-linked fatty acid at more ..
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AID: 488968
Data Source: Broad Institute (2091-01_Inhibitors_Project)
BioAssay Type: Summary, Candidate Probes/Leads with Supporting Evidence
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-10-28
Modify Date: 2012-04-11
Target
Depositor Specified Assays
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AIDNameTypeComment
488965Fluorescent Biochemical Primary HTS to Identify Inhibitors of P. aeruginosa PvdQ acylase Measured in Biochemical System Using Plate Reader and Imaging Combination - 2091-01_Inhibitor_SinglePoint_HTS_Activityscreening337881 hts compounds at singlepoint in Primary Assay measuring activity
493231Fluorescent Biochemical Primary HTS to Identify Inhibitors of P. aeruginosa PvdQ acylase Measured in Biochemical System Using Plate Reader and Imaging Combination - 2091-01_Inhibitor_Dose_CherryPick_Activityconfirmatory396 cherrypick compounds at dose in Primary Assay PvdQ 4-MU measuring activity
504942Mammalian cell toxicity screen in HeLa cells 48h Measured in Cell-Based System Using Plate Reader - 2091-03_Inhibitor_Dose_CherryPick_Activityconfirmatory105 cherrypick compounds at dose in Cell toxicity assay (counterscreen) measuring activity
504944Mammalian cell toxicity screen in HeLa cells 48h Measured in Cell-Based System Using Plate Reader - 2091-03_Inhibitor_Dose_CherryPick_Activity_Set2confirmatory105 cherrypick compounds at dose in Cell toxicity assay (counterscreen) measuring activity set 2
602389Whole cell secondary assay to determine compounds inducing growth delay in presence of metal chelator in Pseudomonas Aeruginosa Measured in Whole Organism System Using Plate Reader - 2091-05_Inhibitor_Dose_CherryPick_Activityconfirmatory105 cherrypick compounds at dose in In vivo P. aeruginosa survival toxicity with metal chelator measuring activity
623951Fluorescent Biochemical Primary HTS to Identify Inhibitors of P. aeruginosa PvdQ acylase Measured in Biochemical System Using Plate Reader - 2091-02_Inhibitor_Dose_DryPowder_Activityconfirmatory41 drypowder compounds at dose in Cell toxicity assay (counterscreen) measuring activity
623950Counterscreen measuring mammalian cell toxicity screen in HeLa cells of pyoverdine inhibitors Measured in Cell-Based System Using Plate Reader - 2091-03_Inhibitor_Dose_DryPowder_Activityconfirmatory
624058Whole cell secondary assay to determine compounds inducing growth delay in presence of metal chelator in Pseudomonas Aeruginosa (absorbance 600 nm) Measured in Whole Organism System Using Plate Reader - 2091-05_Inhibitor_Dose_CherryPick_Activity_Set2confirmatory105 cherrypick compounds at dose in In vivo P. aeruginosa survival toxicity with metal chelator measuring activity set 2
623985Fluorescent Biochemical Primary HTS to Identify Inhibitors of P. aeruginosa PvdQ acylase Measured in Biochemical System Using Plate Reader - 2091-02_Inhibitor_Dose_DryPowder_Activity_Set2confirmatory42 drypowder compounds at dose in Primary Assay PvdQ 4-MU measuring activity set 2
624018Whole cell secondary assay to determine compounds inducing growth delay in presence of metal chelator in Pseudomonas Aeruginosa (absorbance 405nm) Measured in Whole Organism System Using Plate Reader - 2091-05_Inhibitor_Dose_DryPowder_Activityconfirmatory41 drypowder compounds at dose in In vivo P. aeruginosa survival toxicity with metal chelator measuring activity
623991Fluorescent Biochemical Primary HTS to Identify Inhibitors of P. aeruginosa PvdQ acylase Measured in Biochemical System Using Plate Reader - 2091-02_Inhibitor_Dose_DryPowder_Activity_Set3confirmatory25 drypowder compounds at dose in Primary Assay PvdQ 4-MU measuring activity set 3
623993Fluorescent Biochemical Primary HTS to Identify Inhibitors of P. aeruginosa PvdQ acylase Measured in Biochemical System Using Plate Reader - 2091-02_Inhibitor_Dose_DryPowder_Activity_Set4confirmatory26 drypowder compounds at dose in Primary Assay PvdQ 4-MU measuring activity set 4
623987Counterscreen measuring mammalian cell toxicity screen in HeLa cells of pyoverdine inhibitors Measured in Cell-Based System Using Plate Reader - 2091-03_Inhibitor_Dose_DryPowder_Activity_Set3confirmatory26 drypowder compounds at dose in Cell toxicity assay (counterscreen) measuring activity set 3
623990Counterscreen measuring mammalian cell toxicity screen in HeLa cells of pyoverdine inhibitors Measured in Cell-Based System Using Plate Reader - 2091-03_Inhibitor_Dose_DryPowder_Activity_Set2confirmatory26 drypowder compounds at dose in Cell toxicity assay (counterscreen) measuring activity set 2
624020Whole cell secondary assay to determine compounds inducing growth delay in presence of metal chelator in Pseudomonas Aeruginosa (absorbance 405nm) Measured in Whole Organism System Using Plate Reader - 2091-05_Inhibitor_Dose_DryPowder_Activity_Set3confirmatory42 drypowder compounds at dose in In vivo P. aeruginosa survival toxicity with metal chelator measuring activity set 3
624016Whole cell secondary assay to determine compounds inducing growth delay in presence of metal chelator in Pseudomonas Aeruginosa (absorbance 405nm) Measured in Whole Organism System Using Plate Reader - 2091-05_Inhibitor_Dose_DryPowder_Activity_Set4confirmatory40 drypowder compounds at dose in In vivo P. aeruginosa survival toxicity with metal chelator measuring activity set 4
624011Whole cell secondary assay to determine compounds inducing growth delay in presence of metal chelator in Pseudomonas Aeruginosa (absorbance 405nm) Measured in Whole Organism System Using Plate Reader - 2091-05_Inhibitor_Dose_DryPowder_Activity_Set2confirmatory42 drypowder compounds at dose in In vivo P. aeruginosa survival toxicity with metal chelator measuring activity set 2
624033Whole cell secondary assay to identify compounds reducing the production of iron chelator pyoverdine using Chromeazurol S dye in Pseudomonas Aeruginosa Measured in Whole Organism System Using Imaging - 2091-11_Inhibitor_Dose_DryPowder_Activityconfirmatory11 drypowder compounds at dose in 11 Chrome azurol assay measuring activity
624070Whole cell secondary assay to determine compounds inducing growth delay in presence of metal chelator in Pseudomonas Aeruginosa (absorbance 405 nm) Measured in Whole Organism System Using Plate Reader - 2091-05_Inhibitor_Dose_DryPowder_Activity_Set5confirmatory
624102Whole cell secondary assay to determine compounds inducing growth delay in presence of metal chelator in Pseudomonas Aeruginosa (absorbance 405 nm) Measured in Whole Organism System Using Plate Reader - 2091-05_Inhibitor_Dose_DryPowder_Activity_Set8confirmatory
624107Whole cell secondary assay to determine compounds inducing growth delay in presence of metal chelator in Pseudomonas Aeruginosa PAK strain (absorbance 405nm) Measured in Whole Organism System Using Plate Reader - 2091-08_Inhibitor_Dose_DryPowder_Activityconfirmatory
624064Whole cell secondary assay to determine compounds inducing growth delay in presence of metal chelator in Pseudomonas Aeruginosa (absorbance 600 nm) Measured in Whole Organism System Using Plate Reader - 2091-05_Inhibitor_Dose_DryPowder_Activity_Set7confirmatory
624105Whole cell secondary assay to determine compounds inducing growth delay in presence of metal chelator in Pseudomonas Aeruginosa PAK pump mutant mexAB-OprM strain (absorbance 600nm) Measured in Whole Organism System Using Plate Reader - 2091-10_Inhibitor_Dose_DryPowder_Activityconfirmatory
624074Whole cell secondary assay to determine compounds inducing growth delay in presence of metal chelator in Pseudomonas Aeruginosa PAK pump mutant mexAB-OprM strain (absorbance 600nm) Measured in Whole Organism System Using Plate Reader - 2091-10_Inhibitor_Dose_DryPowder_Activity_Set4confirmatory
624072Whole cell secondary assay to determine compounds inducing growth delay in presence of metal chelator in Pseudomonas Aeruginosa PAK pump mutant mexAB-OprM strain (absorbance 600nm) Measured in Whole Organism System Using Plate Reader - 2091-10_Inhibitor_Dose_DryPowder_Activity_Set3confirmatory
624075Whole cell secondary assay to determine compounds inducing growth delay in presence of metal chelator in Pseudomonas Aeruginosa PAK strain (absorbance 600nm) Measured in Whole Organism System Using Plate Reader - 2091-08_Inhibitor_Dose_DryPowder_Activity_Set4confirmatory
624104Whole cell secondary assay to determine compounds inducing growth delay in presence of metal chelator in Pseudomonas Aeruginosa PAK pump mutant mexAB-OprM strain (absorbance 600nm) Measured in Whole Organism System Using Plate Reader - 2091-10_Inhibitor_Dose_DryPowder_Activity_Set2confirmatory
624106Whole cell secondary assay to determine compounds inducing growth delay in presence of metal chelator in Pseudomonas Aeruginosa PAK strain (absorbance 405nm) Measured in Whole Organism System Using Plate Reader - 2091-08_Inhibitor_Dose_DryPowder_Activity_Set2confirmatory
624109Whole cell secondary assay to determine compounds inducing growth delay in presence of metal chelator in Pseudomonas Aeruginosa (absorbance 405 nm) Measured in Whole Organism System Using Plate Reader - 2091-05_Inhibitor_Dose_DryPowder_Activity_Set6confirmatory
624073Whole cell secondary assay to determine compounds inducing growth delay in presence of metal chelator in Pseudomonas Aeruginosa PAK strain (absorbance 600nm) Measured in Whole Organism System Using Plate Reader - 2091-08_Inhibitor_Dose_DryPowder_Activity_Set3confirmatory
Description:
Keywords: pyoverdine

Primary Collaborators:
Andrew Gulick,Hauptman-Woodward Medical Research Inst.,Buffalo, NY,gulick@hwi.buffalo.edu

Project Goal:

The human pathogen Pseudomonas aeruginosa uses a biosynthetic cluster to produce a peptide siderophore called pyoverdine. Pyoverdine is produced by non-ribosomal peptide synthetases, and utilizes 10 other proteins that are co-regulated and catalyze chemical modifications to the amino acid substrates or peptide intermediates. Immature pyoverdine is first produced with an amide-linked fatty acid at the N-terminus that is removed during pyoverdine maturation and prior to export. Multiple lines of evidence suggest that the protein that is responsible for removal of the fatty acid is the PvdQ acylase. Mutant strains of P. aeruginosa with a deletion in the pvdQ gene do not make pyoverdine and cannot grow on iron limiting media. Chemical probes that inhibit the PvdQ protein therefore would be useful tools in further characterizing the pyoverdine synthetic pathway and may provide lead compounds for the development of novel anti-Pseudomonal drugs that prevent mature pyoverdine production.

Aims and goals specified in corresponding grant
- To find compounds that can be used to study the pyoverdine synthetic pathway
- To find compounds that may be leads for the development of novel anti-pseudomonal drugs

Probe attributes:

Desired Probe
IC50 < 10 uM in vitro
Active in EDDHA-dependent growth measurement of P. aeruginosa at 3 - 5 X the in vitro IC50
Not toxic to mammalian cells at 3X effective IC50
- Functional Groups to be avoided 1. Electrophiles such as (a) epoxides, (b) alpha-activated carbonyls, (c) aldehydes, (d) ketones, (e) tosylates and mesylates, (f) benzyl halides, (g) Michael acceptors 2. Reactive heterocycles such as furan and thiophene. 3. Anilines, oximes and hydrazones
- Chemical Solubility Criteria in Assay buffer
Other Characteristics (BBB, Profiling, Plasma level, etc.) LogD < 3.5, PPB (free fraction) > 2%, Solubility > 10 uM, PAMPA > 10-6 cm/s

Biological Relevance:

Bacteria typically require total iron concentrations in the uM range to support growth. The low solubility of Fe(III) and the ability to cause damage to proteins and nucleic acids through Fenton chemistry has led to the evolution of elaborate mechanisms that eukaryotic organisms employ to maintain serum concentrations of free iron as low as 10-24 M. This poses a serious challenge to pathogenic bacteria that attempt to establish an infection and presents an opportunity to develop antibiotics that prevent iron acquisition. Many pathogens produce siderophores with molecular weights below 1500 Da that bind to iron with remarkably high affinities. The iron-siderophore complex is then taken into the cell through the activity of specific siderophore receptors. Studies with mutant strains of bacteria with defects in siderophore biosynthesis have demonstrated that these pathways are valid targets for the development of novel antibiotic compounds.
Additional Information
Grant Number: 1 R03 MH092076-01

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