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BioAssay: AID 623918

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 (dose response)

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 (dose response). ..more
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 Tested Compounds
 Tested Compounds
All(7)
 
 
Active(7)
 
 
 Tested Substances
 Tested Substances
All(7)
 
 
Active(7)
 
 
AID: 623918
Data Source: The Scripps Research Institute Molecular Screening Center (HFR-RECOMBINATION_INH_ABS_PETRI MDCSRUN)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
Deposit Date: 2012-03-28
Hold-until Date: 2013-03-28
Modify Date: 2013-03-29

Data Table ( Complete ):           Active    All
Targets
BioActive Compounds: 7
Depositor Specified Assays
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AIDNameTypeComment
435030Absorbance-based primary bacterial cell-based high throughput screening assay to identify inhibitors of AddAB recombination protein complexscreeningPrimary screen (AddAB inhibitors in singlicate)
449731Summary of the probe development effort to identify inhibitors of AddAB recombination protein complexsummarySummary (AddAB inhibitors)
449728Counterscreen for inhibitors of AddAB: absorbance-based bacterial cell-based high throughput screening assay to identify inhibitors of bacterial viabilityscreeningCounterscreen (bacterial viability inhibitors in singlicate)
488942Absorbance-based bacterial cell-based high throughput confirmation assay for inhibitors of AddAB recombination protein complexscreeningConfirmation (AddAB inhibitors in triplicate)
488955Counterscreen for AddAB inhibitors: absorbance-based high throughput cell-based assay to identify inhibitors of RecBCDscreeningCounterscreen (RecBCD inhibitors in triplicate)
488956Counterscreen for AddAB inhibitors: absorbance-based bacterial cell-based high throughput confirmation assay for inhibitors of bacterial viabilityscreeningCounterscreen (bacterial viability inhibitors in triplicate)
492957Counterscreen for AddAB inhibitors: absorbance-based bacterial cell-based high throughput dose response assay to identify inhibitors of RecBCDconfirmatoryDose response counterscreen (RecBCD inhibitors in triplicate)
492958Counterscreen for AddAB inhibitors: absorbance-based bacterial cell-based high throughput dose response assay for inhibitors of bacterial viabilityconfirmatoryDose response counterscreen (bacterial viablity in triplicate)
492959Absorbance-based bacterial cell-based high throughput dose response assay for inhibitors of AddAB recombination protein complexconfirmatoryDose response (AddAB inhibitors 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)otherLate stage assay (AddAB nuclease 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 AddABconfirmatoryLate stage dose response (AddAB nuclease in triplicate)
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)confirmatory
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)confirmatory
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)confirmatory
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 MDCSRUN

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 (dose response).

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 provdier, 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, dose response, triplicate, 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. E. coli are exposed to test compounds for 1.5 hours. 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. Probes should be active in this assay. Compounds were tested using a 5-point dilution series of doses ranging from 0.03 to 100 uM.

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 = 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 with an IC50 value greater than or equal to 1 uM were considered inactive. Compounds with an IC50 value less than 1 uM were considered active.

Activity score was then ranked by the potency of the compounds with fitted curves, with the most potent compounds assigned the highest activity scores.

The PubChem Activity Score range for active compounds is 100-1. There are no inactive compounds.

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: concentration response multiple replicates

Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1QualifierActivity Qualifier identifies if the resultant data IC50 came from a fitted curve or was determined manually to be less than or greater than its listed IC50 concentration.String
2IC50*The concentration at which 50 percent of the activity in the inhibitor assay is observed; (IC50) shown in micromolar.FloatμM

* Activity Concentration.
Additional Information
Grant Number: GM031693

Data Table (Concise)
Classification
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