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BioAssay: AID 651943

Late 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)

Name: Late 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). ..more
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 Related BioAssays
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AID: 651943
Data Source: The Scripps Research Institute Molecular Screening Center (ECOLIV66VBLTY_INH_ABS_1536_3XIC50 MDCSRUN SAR Round 2)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2012-12-16
Hold-until Date: 2013-06-27
Modify Date: 2013-06-27

Data Table ( Complete ):           View All Data
Tested Compounds:
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 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 (Cytotoxicity 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 screen (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 viability inhibitors 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)
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: Late stage dose response (AddAB recombination protein complex inhibitors in triplicate)
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: Late stage dose response counterscreen (bacterial viability inhibitors in triplicate)
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: Late stage dose response counterscreen (RecBCD 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 (purified AddAB nuclease activity inhibitors)
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 (purified AddAB nuclease activity inhibitors)
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: Late stage counterscreen (viability of V66 E. coli in Hfr recombination assays inhibitors)
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: Late stage dose response counterscreen (viability of V66 E. coli in Hfr recombination assays inhibit
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: Late stage dose response counterscreen (recombination-promoting activity of RecBCD in V66 E. coli in
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: Late stage counterscreen (recombination-promoting activity of RecBCD in V66 E. coli inhibitors at 10
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: Late stage dose response counterscreen (nuclease activity of purified RecBCD enzyme inhibitors)
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: Late stage counterscreen (nuclease activity of purified RecBCD enzyme inhibitors at 100 uM)
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: Late stage counterscreen (nuclease activity of purified RecBCD enzyme inhibitors at 50 uM)
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: Late stage counterscreen (helicase/Chi cutting activity of purified RecBCD inhibitors)
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: Late stage dose response counterscreen (helicase/Chi cutting activity of purified RecBCD inhibitors)
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: Late stage counterscreen (recombination of E. coli transformed with p(addAB) and p(recA) inhibitors)
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: Late stage counterscreen (recombination of E. coli transformed with p(addAB) and p(recA) inhibitors)
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)Confirmatorysame project related to Summary assay
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)Confirmatorysame 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: ECOLIV66VBLTY_INH_ABS_1536_3XIC50 MDCSRUN SAR Round 2

Name: Late 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).

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. Smith, G. (2012) How RecBCD enzyme and Chi promote DNA break repair and recombination - A molecular biologist's view. Microbiol Mol Biol Rev, in press.
5. 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.

Keywords:

late stage, late stage AID, chemistry, purchased, synthesis, synthesized, powders, helicase, nuclease, exonuclease, AddAB, ADDAB, AddAB complex, RecBCD enzyme, beta subunit, gamma chain, alpha chain, Escherichia coli, E. coli, bacteria, phage, DNA, dsDNA, DNA damage, DNA repair, DNA binding, ATP-binding, homologous recombination, recombination, Chi, inhibition, inhibitor, optical density, OD, absorbance, exonuclease V, helicase, nuclease, RecBCD, RecBCD complex, recB, recC, recD, beta subunit, gamma chain, alpha chain, HTS, high throughput screen, counterscreen, dose response, triplicate, viability, turbidity, cytotoxicity, secondary, 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 bacterial viability dose response curves for powder samples of compounds identified as possible AddAB probe candidates. In this assay, V66 bacteria are not infected with phage but, are incubated in the presence of test compounds alone, followed by measurement of well optical density as an indicator of bacterial growth. As designed, compounds that inhibit bacterial growth will reduce 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, V66 bacterial culture was grown at 37 C until it reached an OD600 of 0.05 or 2.5e07 cfu/mL. V66 was diluted in assay buffer to achieve 1.25e07 cfu/mL. To start the assay, 3 uL of Assay Buffer (0.1% Glycerol + 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.

Next, 2 uL of V66 (RecBCD+) bacterial culture was dispensed into the appropriate wells. Plates were incubated for 18 hours at 37 C and 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 V66 + Ciprofloxacin
Low_Control is defined as wells containing V66 + DMSO
Test_Compound is defined as wells containing V66 in the presence of test compound

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 inactive compounds is 0-0. There are no active compounds.

List of Reagents:

V66 E.coli bacteria (supplied by Assay Provider)
Ciprofloxacin (Sigma, part 17850)
Cation-Adjusted Mueller Hinton II Broth (BD, part 297963)
1536-well plates (Aurora, part 19326)
Comment
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.
Categorized Comment
Assay: Dictionary: Version: 0.1

Assay: CurveFit [1]: Equation: =( ( [Maximal Response] * [Concentration]^[Hill Slope] ) / ( [Inflection Point Concentration]^[Hill Slope] + [Concentration]^[Hill Slope] ) ) + [Baseline Response]

Assay: CurveFit [1]: Mask: Excluded Points

Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
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
4Maximal ResponseThe maximal or asymptotic response above the baseline as concentration increases without bound.Float
5Baseline ResponseAdjustable baseline of the curve fit, minimal response value.Float
6Response RangeThe range of y.Float
7Inflection Point ConcentrationThe concentration value for the inflection point of the curve.FloatμM
8Hill 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
9Chi 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
10RsquareThis 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
11Number 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
12Excluded PointsFlags to indicate which of the dose-response points were excluded from analysis. (1) means the point was excluded and (0) means the point was not excluded.String
13Inhibition at 0.006 uM [1] (0.006μM**)Value of % Inhibition at 0.006 uM inhibitor concentration: replicate [1]Float%
14Inhibition at 0.006 uM [2] (0.006μM**)Value of % Inhibition at 0.006 uM inhibitor concentration: replicate [2]Float%
15Inhibition at 0.006 uM [3] (0.006μM**)Value of % Inhibition at 0.006 uM inhibitor concentration: replicate [3]Float%
16Inhibition at 0.018 uM [1] (0.018μM**)Value of % Inhibition at 0.018 uM inhibitor concentration: replicate [1]Float%
17Inhibition at 0.018 uM [2] (0.018μM**)Value of % Inhibition at 0.018 uM inhibitor concentration: replicate [2]Float%
18Inhibition at 0.018 uM [3] (0.018μM**)Value of % Inhibition at 0.018 uM inhibitor concentration: replicate [3]Float%
19Inhibition at 0.054 uM [1] (0.054μM**)Value of % Inhibition at 0.054 uM inhibitor concentration: replicate [1]Float%
20Inhibition at 0.054 uM [2] (0.054μM**)Value of % Inhibition at 0.054 uM inhibitor concentration: replicate [2]Float%
21Inhibition at 0.054 uM [3] (0.054μM**)Value of % Inhibition at 0.054 uM inhibitor concentration: replicate [3]Float%
22Inhibition at 0.163 uM [1] (0.163μM**)Value of % Inhibition at 0.163 uM inhibitor concentration: replicate [1]Float%
23Inhibition at 0.163 uM [2] (0.163μM**)Value of % Inhibition at 0.163 uM inhibitor concentration: replicate [2]Float%
24Inhibition at 0.163 uM [3] (0.163μM**)Value of % Inhibition at 0.163 uM inhibitor concentration: replicate [3]Float%
25Inhibition at 0.488 uM [1] (0.488μM**)Value of % Inhibition at 0.488 uM inhibitor concentration: replicate [1]Float%
26Inhibition at 0.488 uM [2] (0.488μM**)Value of % Inhibition at 0.488 uM inhibitor concentration: replicate [2]Float%
27Inhibition at 0.488 uM [3] (0.488μM**)Value of % Inhibition at 0.488 uM inhibitor concentration: replicate [3]Float%
28Inhibition at 1.5 uM [1] (1.5μM**)Value of % Inhibition at 1.5 uM inhibitor concentration: replicate [1]Float%
29Inhibition at 1.5 uM [2] (1.5μM**)Value of % Inhibition at 1.5 uM inhibitor concentration: replicate [2]Float%
30Inhibition at 1.5 uM [3] (1.5μM**)Value of % Inhibition at 1.5 uM inhibitor concentration: replicate [3]Float%
31Inhibition at 4.4 uM [1] (4.4μM**)Value of % Inhibition at 4.4 uM inhibitor concentration: replicate [1]Float%
32Inhibition at 4.4 uM [2] (4.4μM**)Value of % Inhibition at 4.4 uM inhibitor concentration: replicate [2]Float%
33Inhibition at 4.4 uM [3] (4.4μM**)Value of % Inhibition at 4.4 uM inhibitor concentration: replicate [3]Float%
34Inhibition at 13.2 uM [1] (13.2μM**)Value of % Inhibition at 13.2 uM inhibitor concentration: replicate [1]Float%
35Inhibition at 13.2 uM [2] (13.2μM**)Value of % Inhibition at 13.2 uM inhibitor concentration: replicate [2]Float%
36Inhibition at 13.2 uM [3] (13.2μM**)Value of % Inhibition at 13.2 uM inhibitor concentration: replicate [3]Float%
37Inhibition at 39.5 uM [1] (39.5μM**)Value of % Inhibition at 39.5 uM inhibitor concentration: replicate [1]Float%
38Inhibition at 39.5 uM [2] (39.5μM**)Value of % Inhibition at 39.5 uM inhibitor concentration: replicate [2]Float%
39Inhibition at 39.5 uM [3] (39.5μM**)Value of % Inhibition at 39.5 uM inhibitor concentration: replicate [3]Float%
40Inhibition at 118.6 uM [1] (118.6μM**)Value of % Inhibition at 118.6 uM inhibitor concentration: replicate [1]Float%
41Inhibition at 118.6 uM [2] (118.6μM**)Value of % Inhibition at 118.6 uM inhibitor concentration: replicate [2]Float%
42Inhibition at 118.6 uM [3] (118.6μM**)Value of % Inhibition at 118.6 uM inhibitor concentration: replicate [3]Float%

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

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