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BioAssay: AID 623919

Late stage assay provider Counterscreen for AddAB inhibitors: Cell-based colony formation assay to identify inhibitors of the recombination-promoting activity of RecBCD in V66 E. coli (100uM)

Name: Late stage assay provider Counterscreen for AddAB inhibitors: Cell-based colony formation assay to identify inhibitors of the recombination-promoting activity of RecBCD in V66 E. coli (100uM). ..more
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 Tested Compounds
 Tested Compounds
All(49)
 
 
Active(7)
 
 
Inactive(43)
 
 
 Tested Substances
 Tested Substances
All(50)
 
 
Active(7)
 
 
Inactive(43)
 
 
AID: 623919
Data Source: The Scripps Research Institute Molecular Screening Center (HFR-RECOMBINATION_INH_ABS_PETRI MCSRUN 100uM)
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
Deposit Date: 2012-03-29
Hold-until Date: 2013-03-27
Modify Date: 2013-03-27

Data Table ( Complete ):           View Active Data    View All Data
Targets
BioActive Compounds: 7
Related Experiments
Show more
AIDNameTypeComment
435030Absorbance-based primary bacterial cell-based high throughput screening assay to identify inhibitors of AddAB recombination protein complexScreeningdepositor-specified cross reference: Primary screen (AddAB inhibitors in singlicate)
449728Counterscreen for inhibitors of AddAB: absorbance-based bacterial cell-based high throughput screening assay to identify inhibitors of bacterial viabilityScreeningdepositor-specified cross reference: Counterscreen (bacterial viability inhibitors in singlicate)
449731Summary of the probe development effort to identify inhibitors of AddAB recombination protein complexSummarydepositor-specified cross reference: Summary (AddAB inhibitors)
488942Absorbance-based bacterial cell-based high throughput confirmation assay for inhibitors of AddAB recombination protein complexScreeningdepositor-specified cross reference: Confirmation (AddAB inhibitors in triplicate)
488955Counterscreen for AddAB inhibitors: absorbance-based high throughput cell-based assay to identify inhibitors of RecBCDScreeningdepositor-specified cross reference: Counterscreen (RecBCD inhibitors in triplicate)
488956Counterscreen for AddAB inhibitors: absorbance-based bacterial cell-based high throughput confirmation assay for inhibitors of bacterial viabilityScreeningdepositor-specified cross reference: Counterscreen (bacterial viability inhibitors in triplicate)
492957Counterscreen for AddAB inhibitors: absorbance-based bacterial cell-based high throughput dose response assay to identify inhibitors of RecBCDConfirmatorydepositor-specified cross reference: Dose response counterscreen (RecBCD inhibitors in triplicate)
492958Counterscreen for AddAB inhibitors: absorbance-based bacterial cell-based high throughput dose response assay for inhibitors of bacterial viabilityConfirmatorydepositor-specified cross reference: Dose response counterscreen (bacterial viablity in triplicate)
492959Absorbance-based bacterial cell-based high throughput dose response assay for inhibitors of AddAB recombination protein complexConfirmatorydepositor-specified cross reference: Dose response (AddAB inhibitors in triplicate)
602421Late stage assay provider assay for AddAB inhibitors: Radioactivity-based biochemical dose response assay to identify inhibitors of the nuclease activity of purified AddABConfirmatorydepositor-specified cross reference: Late stage dose response (AddAB nuclease in triplicate)
602422Late stage assay provider assay for AddAB inhibitors: Radioactivity-based biochemical assay to identify inhibitors of the nuclease activity of purified AddAB (100 micromolar dose)Otherdepositor-specified cross reference: Late stage assay (AddAB nuclease in triplicate)
651942Late stage results for the probe development effort to identify inhibitors of AddAB recombination protein complex: Absorbance-based bacterial cell-based dose response assay for inhibitors of AddAB recombination protein complex (ROUND 2)Confirmatorydepositor-specified cross reference
651943Late stage counterscreen results for the probe development effort to identify inhibitors of AddAB recombination protein complexes: absorbance-based bacterial cell-based dose response assay for inhibitors of bacterial viability (ROUND 2)Confirmatorydepositor-specified cross reference
651944Late stage counterscreen results for the probe development effort to identify inhibitors of AddAB recombination protein complex: absorbance-based bacterial cell-based dose response assay to identify inhibitors of RecBCD (ROUND 2)Confirmatorydepositor-specified cross reference
504677Late stage results for the probe development effort to identify inhibitors of AddAB recombination protein complex: Absorbance-based bacterial cell-based dose response assay for inhibitors of AddAB recombination protein complexConfirmatorysame project related to Summary assay
504678Late stage counterscreen results for the probe development effort to identify inhibitors of AddAB recombination protein complexes: absorbance-based bacterial cell-based dose response assay for inhibitors of bacterial viabilityConfirmatorysame project related to Summary assay
504679Late stage counterscreen results for the probe development effort to identify inhibitors of AddAB recombination protein complex: absorbance-based bacterial cell-based dose response assay to identify inhibitors of RecBCDConfirmatorysame project related to Summary assay
623916Late stage assay provider Counterscreen for AddAB inhibitors: Cell-based colony-formation assay to identify inhibitors of the viability of V66 E. coli in Hfr recombination assaysOthersame project related to Summary assay
623917Late stage assay provider Counterscreen for AddAB inhibitors: Cell-based colony-formation dose response assay to identify inhibitors of the viability of V66 E. coli in Hfr recombination assaysOthersame project related to Summary assay
623918Late stage assay provider Counterscreen for AddAB inhibitors: Cell-based colony formation assay to identify inhibitors of the recombination-promoting activity of RecBCD in V66 E. coli (dose response)Confirmatorysame project related to Summary assay
623920Late stage assay provider counterscreen for AddAB inhibitors: Radioactivity-based biochemical dose response assay to identify inhibitors of the nuclease activity of purified RecBCD enzymeConfirmatorysame project related to Summary assay
623921Late stage assay provider counterscreen for AddAB inhibitors: Radioactivity-based biochemical assay to identify inhibitors of the nuclease activity of purified RecBCD enzyme (at 100 micromolar)Othersame project related to Summary assay
623922Late stage assay provider counterscreen for AddAB inhibitors: Radioactivity-based biochemical assay to identify inhibitors of the nuclease activity of purified RecBCD enzyme (at 50 micromolar)Othersame project related to Summary assay
623937Late stage assay provider counterscreen for AddAB inhibitors: Radioactivity-based biochemical assay to identify inhibitors of the helicase/Chi cutting activity of purified RecBCDOthersame project related to Summary assay
623942Late stage assay provider counterscreen for AddAB inhibitors: Radioactivity-based biochemical dose response assay to identify inhibitors of the helicase/Chi cutting activity of purified RecBCDConfirmatorysame project related to Summary assay
623954Late stage assay provider Counterscreen for AddAB inhibitors: Cell-based colony-formation assay to identify inhibitors of recombination of E. coli transformed with p(addAB) and p(recA) (100 micromolar compound dose)Othersame project related to Summary assay
623956Late stage assay provider Counterscreen for AddAB inhibitors: Cell-based colony-formation assay to identify inhibitors of recombination of E. coli transformed with p(addAB) and p(recA) (20 micromolar compound dose)Othersame project related to Summary assay
Description:
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Gerald R. Smith, Fred Hutchinson Cancer Research Center
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number: GM031693
Grant Proposal PI: Gerald R. Smith
External Assay ID: HFR-RECOMBINATION_INH_ABS_PETRI MCSRUN 100uM

Name: Late stage assay provider Counterscreen for AddAB inhibitors: Cell-based colony formation assay to identify inhibitors of the recombination-promoting activity of RecBCD in V66 E. coli (100uM).

Description:

Helicobacter pylori infects approximately half of the world's population and is responsible for inducing chronic gastric inflammation that can progress to gastric cancer (1). At the cellular level, Helicobacter pylori infection of the human stomach is associated with inflammation that elicits DNA damage in both bacterial and host cells (2). This DNA damage must be repaired in order for the bacteria to persist. The H. pylori AddAB helicase-exonuclease is required for DNA repair and efficient stomach colonization (3), and inhibitors of this enzyme may be useful antibacterial drugs for treating these infections. The AddAB class of enzymes is closely related to the RecBCD class of helicase-nucleases; both classes are widely distributed in bacteria but appear to be absent in eukaryotes (4). The protein complex functions in DNA repair by directing free DNA ends into the homologous recombination pathway (5). As a result, the identification of inhibitors of AddAB may be useful tools for elucidating the role of AddAB and RecBCD in bacterial recombination and as potential novel antibiotics with few off-target effects.

References:

1. Fox JG, Wang TC. Inflammation, atrophy, and gastric cancer. J Clin Invest. 2007 Jan;117(1):60-9.
2. Ernst P. Aliment Pharmacol Ther. 1999 Mar;13 Suppl 1:13-8. Review article: the role of inflammation in the pathogenesis of gastric cancer.
3. Dillingham MS, Kowalczykowski SC. RecBCD enzyme and the repair of double-stranded DNA breaks.
Microbiol Mol Biol Rev. 2008 Dec;72(4):642-71.
4. Amundsen SK, Fero J, Hansen LM, Cromie GA, Solnick JV, Smith GR, Salama NR, Helicobacter pylori AddAB helicase-nuclease and RecA promote recombination-related DNA repair and survival during stomach colonization. Mol Microbiol, 2008. 69(4): p. 994-1007.
5. Chedin F. and Kowalczykowski S.C. A novel family of regulated helicases/nucleases from Gram-positive bacteria: insights into the initiation of DNA recombination, Mol. Microbiol. 43 (2002), pp. 823-834.

Keywords:

assay provider, V66, phage, T4, T4 phage, gene 2, mutant, growth, RecBCD, chi cutting, late stage, late stage AID, chemistry, purchased, synthesis, synthesized, powders, helicase, nuclease, exonuclease, ATP-dependent nuclease, AddAB, ADDAB, AddAB complex, RecBCD enzyme, beta subunit, gamma chain, alpha chain, Escherichia coli, E. coli, bacteria, Helicobacter pylori, phage, T4, DNA, dsDNA, DNA damage, DNA repair, DNA binding, ATP-binding, homologous recombination, recombination, Chi, inhibition, inhibitor, plate, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
Protocol
Assay Overview:

The purpose of this assay is to determine whether powder samples of compounds identified as AddAB inhibitor probe candidates can inhibit recombination in Hfr conjugational crosses. In this assay a test strain is mated with an Hfr strain in the presence of compound, and the frequency of selected recombinants (His+ StrR) in the mixture is determined by differential plating. The donor and recipient are also plated on non-selective media to measure viability. As designed, compounds that inhibit RecBCD will reduce the frequency of recombinants. This assay is designed to determine if inhibitors of RecBCD can enter living cells and inhibit the recombination-promoting function of RecBCD. E. coli are exposed to test compounds for 1.5 hours. Probes should be active in this assay. Compounds are tested at a nominal concentration of 100 uM in duplicate experiments.

Protocol Summary:

Recipient cells are grown for 1 hr in LB broth with or without compound. Donor and recipient cells are plated separately on non-selective media to measure viability. Donor (Hfr) and recipient (F-) cells are mixed in a ratio of 1:10, incubated for 30 min, vortexed to separate mating cells, and plated differentially to determine the frequency of recombinants (His+ StrR).

The relative viability of each culture was calculated as follows:

Relative_Viability = the number of colony forming units per ml of culture treated with compound normalized to the number of colony forming units per ml of culture treated with DMSO only.

The fold reduction in recombinant frequency for each compound was calculated as follows:

Fold_Reduction = [Recombinant frequency of DMSO control] / [Recombinant frequency in presence of compound adjusted for relative viability of the culture]

PubChem Activity Outcome and Score:

Compounds that reduced V66 E. coli recombinant frequency by less than or equal to 4-fold compared to the DMSO control were considered inactive. Compounds that reduced V66 E. coli recombinant frequency by greater than 4-fold compared to DMSO control were considered active.

Compounds were ranked by fold reduction, with the largest fold reduction receiving a score of 100.

The PubChem Activity Score range for active compounds is 100-0, and for inactive compounds 0-0.

List of Reagents:

E . coli strains V66 and V1306
Comment
This assay was run in the assay provider's lab. This assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample.
Categorized Comment
BAO: version: 1.4b1090

BAO: bioassay specification: assay stage: secondary: counter screening

BAO: bioassay specification: assay biosafety level: bsl1

BAO: assay format: cell-based format

BAO: bioassay specification: assay measurement type: endpoint assay

BAO: bioassay specification: assay readout content: assay readout method: regular screening

BAO: bioassay specification: assay readout content: content readout type: single readout

BAO: meta target: molecular target: protein target: enzyme: generic hydrolase

BAO: meta target: biological process target: cell proliferation

BAO: meta target detail: binding reporter specification: interaction: protein-small molecule

BAO: assay design: viability reporter: cell number

BAO: detection technology: spectrophotometry: absorbance

BAO: bioassay specification: bioassay type: functional: viability

BAO: bioassay specification: assay measurement throughput quality: single concentration multiple replicates

Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Fold Reduction at 100 uM (100μM**)Fold reduction of Hfr RecombinationFloatratio

** Test Concentration.
Additional Information
Grant Number: GM031693

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
Classification
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