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BioAssay: AID 492959

Absorbance-based bacterial cell-based high throughput dose response assay for inhibitors of AddAB recombination protein complex

Name: Absorbance-based bacterial cell-based high throughput dose response assay for inhibitors of AddAB recombination protein complex. ..more
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Active(7)
 
 
Inactive(218)
 
 
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 Tested Substances
All(225)
 
 
Active(7)
 
 
Inactive(218)
 
 
 Related BioAssays
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AID: 492959
Data Source: The Scripps Research Institute Molecular Screening Center (ADDAB_INH_ABS_1536_3XIC50 DRUN + phage)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-12-02

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 7
Related Experiments
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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 recombination protein complex 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 recombination protein complex inhibitors)
488942Absorbance-based bacterial cell-based high throughput confirmation assay for inhibitors of AddAB recombination protein complexScreeningdepositor-specified cross reference: Confirmation screen (AddAB recombination protein complex 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)
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 complexConfirmatorydepositor-specified cross reference
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 viabilityConfirmatorydepositor-specified cross reference
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 RecBCDConfirmatorydepositor-specified cross reference
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
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
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 assaysOtherdepositor-specified cross reference
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 assaysOtherdepositor-specified cross reference
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)Confirmatorydepositor-specified cross reference
623919Late 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)Otherdepositor-specified cross reference
623920Late stage assay provider counterscreen for AddAB inhibitors: Radioactivity-based biochemical dose response assay to identify inhibitors of the nuclease activity of purified RecBCD enzymeConfirmatorydepositor-specified cross reference
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)Otherdepositor-specified cross reference
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)Otherdepositor-specified cross reference
623937Late stage assay provider counterscreen for AddAB inhibitors: Radioactivity-based biochemical assay to identify inhibitors of the helicase/Chi cutting activity of purified RecBCDOtherdepositor-specified cross reference
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 RecBCDConfirmatorydepositor-specified cross reference
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)Otherdepositor-specified cross reference
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)Otherdepositor-specified cross reference
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
492957Counterscreen for AddAB inhibitors: absorbance-based bacterial cell-based high throughput dose response assay to identify inhibitors of RecBCDConfirmatorysame project related to Summary assay
492958Counterscreen for AddAB inhibitors: absorbance-based bacterial cell-based high throughput dose response assay for inhibitors of bacterial viabilityConfirmatorysame 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: ADDAB_INH_ABS_1536_3XIC50 DRUN + phage

Name: Absorbance-based bacterial cell-based high throughput dose response assay for inhibitors of AddAB recombination protein complex.

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:

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, optical density, OD, absorbance, HTS, high throughput screen, dose response, triplicate, 1536, 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 the efficacy of compounds in AddAB dose response assays for those that confirmed activity in a set of previous experiments entitled, "Absorbance-based bacterial cell-based high throughput confirmation assay for inhibitors of AddAB recombination protein complex" (AID 488942), and were active in a set of experiments entitled, "Counterscreen for AddAB inhibitors: absorbance-based high throughput cell-based assay to identify inhibitors of RecBCD" (AID 488955) and inactive in a set of experiments entitled, "Counterscreen for AddAB inhibitors: absorbance-based bacterial cell-based high throughput confirmation assay for inhibitors of bacterial viability" (AID 488956).

This bacterial cell-based assay employs E. coli that express the Helicobacter pylori AddAB+ genes. The bacteria are infected with a mutant T4 bacteriophage that carries three nonsense mutations in gene 2, whose protein product normally protects viral DNA from AddAB-mediated degradation after infection. The mutant phage infects and blocks the growth of V3069 E. coli, which lack AddAB nuclease activity (AddAB-). The mutant phage also infect V3065 E. coli, which contain a plasmid expressing the H. pylori addAB+ genes , but V3065 proliferate because of the AddAB directed nuclease and helicase activity against the unprotected mutant phage. In this assay, the V3065 E. coli cells are infected with mutant T4 phage in the presence of test compounds, followed by measurement of well optical density as an indicator of bacterial growth. As designed, compounds that inhibit AddAB will allow the virus to replicate and inhibit bacterial growth, leading to reduced well absorbance. Compounds are tested in triplicate in a 10-point 1:3 dilution series starting at a nominal test concentration of 118.6 uM.

Protocol Summary:

Prior to the start of the assay, V3065 and V3069 bacterial cultures were grown at 37 C until it reached an OD600 of 0.05 or 2.5e07 cfu/mL. To start the assay, 3uL of Assay Buffer (0.1% Glycerol + 100ug/mL Ampicillin + Cation Mueller Hinton Broth) was dispensed into all wells. Next, 60 nL of test compound in DMSO, Ciprofloxacin (0.95 ug/ml final concentration) or DMSO alone (1.2% final concentration) were added to the appropriate wells. Then, 1 uL of V3065 (addAB+) or V3069 (phage control) bacterial cultures were dispensed into the appropriate wells and plates were incubated for 60 minutes at 37 C.

Next, 1 uL of mutant T4 2 149 bacteriophage was dispensed to the appropriate wells at a multiplicity of infection (MOI) of 0.02. Plates were centrifuged and after 18 hours of incubation at 37 C, absorbance (OD600) was read on a Envision microplate reader (PerkinElmer, Turku, Finland) using 10 flashes per well.

The percent inhibition for each compound was calculated as follows:

% Inhibition = 100 * ( ( Test_Compound - Median_Low_Control ) / ( Median_High_Control - Median_Low_Control ) )

Where:

High_Control is defined as wells containing V3065 + Ciprofloxacin + phage.
Low_Control is defined as wells containing V3065 + DMSO + phage.
Test_Compound is defined as wells containing V3065 in the presence of test compound + phage

For each test compound, percent inhibition was plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Assay Explorer software (Symyx Technologies 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. In cases where the highest concentration tested (i.e. 118.6 uM) did not result in greater than 50% inhibition, the IC50 was determined manually as greater than 118.6 uM.

PubChem Activity Outcome and Score:

Compounds with an IC50 greater than 10 uM were considered inactive. Compounds with an IC50 equal to or less than 10 uM were considered active.

Any compound with a percent activity value < 50% at all test concentrations was assigned an activity score of zero. Any compound with a percent activity value >= 50% at any test concentration was assigned an activity score greater than zero. Activity score was then ranked by the potency, with the most potent compounds assigned the highest activity scores.

The PubChem Activity Score range for active compounds is 100-93, and for inactive compounds 93-0.

List of Reagents:

V3065 & V3069 E.coli bacteria (supplied by Assay Provider)
T4 2 149 mutant bacteriophage (supplied by Assay Provider)
Ciprofloxacin (Sigma, part 17850)
Ampicillin (Fisher, part BP1760-5)
Cation-Adjusted Mueller Hinton II Broth (BD, part 297963)
1536-well plates (Aurora, part 19326)
Comment
Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that modulate well fluorescence. 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 by the MLSMR. The MLSMR was not able to provide all compounds selected for testing in this assay.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Cell-based
From ChEMBL:
Assay Format: Cell-based
Assay Type: Functional
Result Definitions
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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
3LogIC50Log10 of the qualified IC50 (IC50) from the inhibitor assay in M concentrationFloat
4Hill SlopeThe variable HillSlope describes the steepness of the curve. This variable is called the Hill slope, the slope factor, or the Hill coefficient. If it is positive, the curve increases as X increases. If it is negative, the curve decreases as X increases. A standard sigmoid dose-response curve (previous equation) has a Hill Slope of 1.0. When HillSlope is less than 1.0, the curve is more shallow. When HillSlope is greater than 1.0, the curve is steeper. The Hill slope has no units.Float
5Hill S0Y-min of the curve.Float
6Hill SinfY-max of the curve.Float
7Hill dSThe range of Y.Float
8Chi SquareA measure for the 'goodness' of a fit. The chi-square test (Snedecor and Cochran, 1989) is used to test if a sample of data came from a population with a specific distribution.Float
9RsquareThis statistic measures how successful the fit explains the variation of the data; R-square is the square of the correlation between the response values and the predicted response values.Float
10Number of DataPointsOverall number of data points of normalized percent activation that was used for calculations (includes all concentration points); in some cases a data point can be excluded as outlier.Integer
11Inhibition at 0.006 uM [1] (0.006μM**)Value of % inhibition at 0.006 micromolar inhibitor concentration; replicate one.Float%
12Inhibition at 0.006 uM [2] (0.006μM**)Value of % inhibition at 0.006 micromolar inhibitor concentration; replicate two.Float%
13Inhibition at 0.006 uM [3] (0.006μM**)Value of % inhibition at 0.006 micromolar inhibitor concentration; replicate three.Float%
14Inhibition at 0.018 uM [1] (0.018μM**)Value of % inhibition at 0.018 micromolar inhibitor concentration; replicate one.Float%
15Inhibition at 0.018 uM [2] (0.018μM**)Value of % inhibition at 0.018 micromolar inhibitor concentration; replicate two.Float%
16Inhibition at 0.018 uM [3] (0.018μM**)Value of % inhibition at 0.018 micromolar inhibitor concentration; replicate three.Float%
17Inhibition at 0.054 uM [1] (0.054μM**)Value of % inhibition at 0.054 micromolar inhibitor concentration; replicate one.Float%
18Inhibition at 0.054 uM [2] (0.054μM**)Value of % inhibition at 0.054 micromolar inhibitor concentration; replicate two.Float%
19Inhibition at 0.054 uM [3] (0.054μM**)Value of % inhibition at 0.054 micromolar inhibitor concentration; replicate three.Float%
20Inhibition at 0.163 uM [1] (0.163μM**)Value of % inhibition at 0.163 micromolar inhibitor concentration; replicate one.Float%
21Inhibition at 0.163 uM [2] (0.163μM**)Value of % inhibition at 0.163 micromolar inhibitor concentration; replicate two.Float%
22Inhibition at 0.163 uM [3] (0.163μM**)Value of % inhibition at 0.163 micromolar inhibitor concentration; replicate three.Float%
23Inhibition at 0.488 uM [1] (0.488μM**)Value of % inhibition at 0.488 micromolar inhibitor concentration; replicate one.Float%
24Inhibition at 0.488 uM [2] (0.488μM**)Value of % inhibition at 0.488 micromolar inhibitor concentration; replicate two.Float%
25Inhibition at 0.488 uM [3] (0.488μM**)Value of % inhibition at 0.488 micromolar inhibitor concentration; replicate three.Float%
26Inhibition at 1.5 uM [1] (1.5μM**)Value of % inhibition at 1.5 micromolar inhibitor concentration; replicate one.Float%
27Inhibition at 1.5 uM [2] (1.5μM**)Value of % inhibition at 1.5 micromolar inhibitor concentration; replicate two.Float%
28Inhibition at 1.5 uM [3] (1.5μM**)Value of % inhibition at 1.5 micromolar inhibitor concentration; replicate three.Float%
29Inhibition at 4.4 uM [1] (4.4μM**)Value of % inhibition at 4.4 micromolar inhibitor concentration; replicate one.Float%
30Inhibition at 4.4 uM [2] (4.4μM**)Value of % inhibition at 4.4 micromolar inhibitor concentration; replicate two.Float%
31Inhibition at 4.4 uM [3] (4.4μM**)Value of % inhibition at 4.4 micromolar inhibitor concentration; replicate three.Float%
32Inhibition at 13.2 uM [1] (13.2μM**)Value of % inhibition at 13.2 micromolar inhibitor concentration; replicate one.Float%
33Inhibition at 13.2 uM [2] (13.2μM**)Value of % inhibition at 13.2 micromolar inhibitor concentration; replicate two.Float%
34Inhibition at 13.2 uM [3] (13.2μM**)Value of % inhibition at 13.2 micromolar inhibitor concentration; replicate three.Float%
35Inhibition at 39.5 uM [1] (39.5μM**)Value of % inhibition at 39.5 micromolar inhibitor concentration; replicate one.Float%
36Inhibition at 39.5 uM [2] (39.5μM**)Value of % inhibition at 39.5 micromolar inhibitor concentration; replicate two.Float%
37Inhibition at 39.5 uM [3] (39.5μM**)Value of % inhibition at 39.5 micromolar inhibitor concentration; replicate three.Float%
38Inhibition at 118.6 uM [1] (118.6μM**)Value of % inhibition at 118.6 micromolar inhibitor concentration; replicate one.Float%
39Inhibition at 118.6 uM [2] (118.6μM**)Value of % inhibition at 118.6 micromolar inhibitor concentration; replicate two.Float%
40Inhibition at 118.6 uM [3] (118.6μM**)Value of % inhibition at 118.6 micromolar inhibitor concentration; replicate three.Float%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: GM031693

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