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BioAssay: AID 623942

Late stage assay provider counterscreen for AddAB inhibitors: Radioactivity-based biochemical dose response assay to identify inhibitors of the helicase/Chi cutting activity of purified RecBCD

Name: Late stage assay provider counterscreen for AddAB inhibitors: Radioactivity-based biochemical dose response assay to identify inhibitors of the helicase/Chi cutting activity of purified RecBCD ..more
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 Tested Compounds
 Tested Compounds
All(15)
 
 
Active(8)
 
 
Inactive(7)
 
 
 Tested Substances
 Tested Substances
All(15)
 
 
Active(8)
 
 
Inactive(7)
 
 
AID: 623942
Data Source: The Scripps Research Institute Molecular Screening Center (RECBCD-CHI-CUTTING_INH_RADIOACTIVITY_FILM_IC50 MDCSRUN)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
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 ):           Active    All
Targets
BioActive Compounds: 8
Depositor Specified Assays
Show more
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)
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
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
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: RECBCD-CHI-CUTTING_INH_RADIOACTIVITY_FILM_IC50 MDCSRUN

Name: Late stage assay provider counterscreen for AddAB inhibitors: Radioactivity-based biochemical dose response assay to identify inhibitors of the helicase/Chi cutting activity of purified RecBCD

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:

RecBCD, chi cutting, counterscreen, dose response, 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, T4, DNA, dsDNA, DNA damage, DNA repair, DNA binding, ATP-binding, homologous recombination, recombination, Chi, inhibition, inhibitor, triplicate, 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 the ability of E. coli RecBCD enzyme to cut DNA at Chi sequences during DNA unwinding. In this assay linear, 5'end-labeled duplex DNA with Chi is incubated with RecBCD in the absence and presence of compound, and the products are analyzed by gel electrophoresis and autoradiography. A labeled fragment ending at or near Chi, but absent in reactions with DNA lacking Chi, reflects Chi-specific cutting. Controls include native and boiled DNA without RecBCD. As designed an inhibitor of RecBCD will decrease the amount of the Chi-specific fragment. If the compound inhibits DNA unwinding, less full length single-stranded DNA, with mobility equal to that of boiled DNA, will be seen. This assay is designed to determine if compounds inhibit either the unwinding of DNA or specific cutting at Chi hotspots of recombination. Compounds were tested using a dilution series starting at a maximum nominal concentration of 500 uM.

Protocol Summary:

Helicase assays measured the formation of ss DNA from 5'[32P] pBR322 Chi+F (or Chi_o control) DNA (0.1 nM molecules) linearized by digestion with HindIII enzyme.RecBCD unwinding assays were in 25 mM Tris-acetate (pH 7.5), 2.0 mM Mg(OAc)2, 5 mM ATP. RecBCD assays used 0.15 nM enzyme and without SSB.

Compounds were added to the reaction mixture containing all the reagents except ATP; final DMSO concentration was 2.5% (v/v) for each assay. (All other percentages in this paragraph are w/v.) Reactions were started by addition of ATP and were at 37 C for 1 min . Reactions were stopped by addition of 1/3 vol of stop buffer (2.5% SDS, 100 mM EDTA, 0.125% bromophenol blue, 0.125% xylene cyanol FF, and 10% Ficoll), and the products were subjected to electrophoresis in a 1.25% agarose gel at 5 V/cm for 2.5 h in TAE buffer (40 mM Tris base, 20 mM acetic acid, 1 mM EDTA). Gels were dried under vacuum, and the products were detected by autoradiography or analyzed with a Typhoon Trio PhosphorImager (GE Lifesciences) and ImageQuant TL software (Amersham). With RecBCD, this assay also detects cutting of DNA at the Chi site Chi+F, which produces a 1.46 kb fragment.

The fold reduction in RecBCD activity for each compound was determined according to the degree of reduction of Chi cutting as observed on the X-Ray film of the dried gels.

Fold_Reduction = ( Test_Compound_Chi_Band_Intensity )/( DMSO_Control_Chi_Band_Intensity)

Where:

DMSO_Control_Chi_Band_Intensity is defined as the intensity of the Chi band formed with the presence of 5% DMSO control.
Test_Compound_Chi_Band_Intensity is defined as the intensity of the Chi band formed in the presence of test compound dissolved in 5% final concentration of DMSO in the assay mixture.

For each test compound, inhibition of RecBCD Chi cutting was measured as the reduction of Chi band intensity in the presence of test compound compared to DMSO control and was plotted against compound concentration or manually estimated. Values were calculated using Prism 5 (GraphPad Software Inc). The reported IC50 values were generated from fitted curves by solving for the X-intercept value at the 50% inhibition level of the Y-intercept value or manual estimation of the reaction product band intensities on X-ray images of the dried gels. In cases where the highest concentration tested (i.e. 500 uM) did not result in greater than 50% inhibition, the IC50 was determined manually as greater than 500 uM.

PubChem Activity Outcome and Score:

Compounds with an IC50 greater than 50 uM were considered inactive. Compounds with an IC50 equal to or less than 50 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 100-71, and for inactive compounds 24-0.

List of Reagents:

E. coli RecBCD enzyme; 5' [32P] linear DNA with Chi (5' GCTGGTGG 3')
Comment
This assay was run by 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 provided.
Categorized Comment
BAO: version: 1.4b1090

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

BAO: bioassay specification: assay biosafety level: bsl1

BAO: assay format: biochemical format: protein format: single protein 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: regulation of molecular function

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

BAO: assay design: binding reporter: radioligand binding

BAO: detection technology: radiometry: scintillation counting: filter assay

BAO: bioassay specification: bioassay type: functional: enzyme activity

BAO: bioassay specification: assay footprint: vial

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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