Bookmark and Share
BioAssay: AID 504679

Late 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

Name: Late 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. ..more
_
   
 Tested Compounds
 Tested Compounds
All(12)
 
 
Inactive(12)
 
 
 Tested Substances
 Tested Substances
All(12)
 
 
Inactive(12)
 
 
AID: 504679
Data Source: The Scripps Research Institute Molecular Screening Center (RECBCD_INH_ABS_1536_3XIC50 MDCSRUN + phage)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2011-04-18
Hold-until Date: 2012-04-14
Modify Date: 2012-04-15

Data Table ( Complete ):           All
Targets
Tested Compounds:
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)
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_INH_ABS_1536_3XIC50 MDCSRUN + phage

Name: Late 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.

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:

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 RecBCD inhibition dose response curves for powder samples of compounds identified as possible AddAB probe candidates. Compounds that also inhibit E. coli RecBCD may have potential to elicit a broad-spectrum antibiotic like effect. This assay involves infecting E. coli with a T4 bacteriophage that carries three nonsense mutations in gene 2, whose wild-type protein product protects viral DNA from RecBCD-mediated degradation after infection. The mutant T4 phage is able to infect and block the growth of V67 E. coli (recB21, a recBCD null mutation), which lack RecBCD nuclease activity. The mutant phage also infects V66 E. coli (recBCD+), but V66 proliferate because of the RecBCD helicase and nuclease activity against the unprotected mutant phage. In this assay, the V66 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 RecBCD will allow the virus to replicate and block 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, V66 and V67 bacterial cultures were grown at 37 C until it reached an OD600 of 0.05 or 2.5e07 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. Then, 1 uL of V66 (recBCD+) or V67 (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 V66 + Ciprofloxacin + phage
Low_Control is defined as wells containing V66 + DMSO + phage.
Test_Compound is defined as wells containing V66 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 inactive compounds is 100-0. There are no active compounds.

List of Reagents:

V66 (recBCD+) and V67 (recB21) E. coli bacteria (supplied by Assay Provider)
T4 2 149 mutant bacteriophage (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 provided.
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]

Result Definitions
Show more
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
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
6Inflection Point ConcentrationThe concentration value for the inflection point of the curve.FloatμM
7Hill 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
8Hill S0Y-min of the curve.Float
9Hill SinfY-max of the curve.Float
10Hill dSThe range of Y.Float
11Chi 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
12RsquareThis 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
13Number 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
14Inhibition at 0.006 uM [1] (0.006μM**)Value of % Inhibition at 0.006 uM inhibitor concentration: replicate [1]Float%
15Inhibition at 0.006 uM [2] (0.006μM**)Value of % Inhibition at 0.006 uM inhibitor concentration: replicate [2]Float%
16Inhibition at 0.006 uM [3] (0.006μM**)Value of % Inhibition at 0.006 uM inhibitor concentration: replicate [3]Float%
17Inhibition at 0.018 uM [1] (0.018μM**)Value of % Inhibition at 0.018 uM inhibitor concentration: replicate [1]Float%
18Inhibition at 0.018 uM [2] (0.018μM**)Value of % Inhibition at 0.018 uM inhibitor concentration: replicate [2]Float%
19Inhibition at 0.018 uM [3] (0.018μM**)Value of % Inhibition at 0.018 uM inhibitor concentration: replicate [3]Float%
20Inhibition at 0.054 uM [1] (0.054μM**)Value of % Inhibition at 0.054 uM inhibitor concentration: replicate [1]Float%
21Inhibition at 0.054 uM [2] (0.054μM**)Value of % Inhibition at 0.054 uM inhibitor concentration: replicate [2]Float%
22Inhibition at 0.054 uM [3] (0.054μM**)Value of % Inhibition at 0.054 uM inhibitor concentration: replicate [3]Float%
23Inhibition at 0.163 uM [1] (0.163μM**)Value of % Inhibition at 0.163 uM inhibitor concentration: replicate [1]Float%
24Inhibition at 0.163 uM [2] (0.163μM**)Value of % Inhibition at 0.163 uM inhibitor concentration: replicate [2]Float%
25Inhibition at 0.163 uM [3] (0.163μM**)Value of % Inhibition at 0.163 uM inhibitor concentration: replicate [3]Float%
26Inhibition at 0.488 uM [1] (0.488μM**)Value of % Inhibition at 0.488 uM inhibitor concentration: replicate [1]Float%
27Inhibition at 0.488 uM [2] (0.488μM**)Value of % Inhibition at 0.488 uM inhibitor concentration: replicate [2]Float%
28Inhibition at 0.488 uM [3] (0.488μM**)Value of % Inhibition at 0.488 uM inhibitor concentration: replicate [3]Float%
29Inhibition at 1.5 uM [1] (1.5μM**)Value of % Inhibition at 1.5 uM inhibitor concentration: replicate [1]Float%
30Inhibition at 1.5 uM [2] (1.5μM**)Value of % Inhibition at 1.5 uM inhibitor concentration: replicate [2]Float%
31Inhibition at 1.5 uM [3] (1.5μM**)Value of % Inhibition at 1.5 uM inhibitor concentration: replicate [3]Float%
32Inhibition at 4.4 uM [1] (4.4μM**)Value of % Inhibition at 4.4 uM inhibitor concentration: replicate [1]Float%
33Inhibition at 4.4 uM [2] (4.4μM**)Value of % Inhibition at 4.4 uM inhibitor concentration: replicate [2]Float%
34Inhibition at 4.4 uM [3] (4.4μM**)Value of % Inhibition at 4.4 uM inhibitor concentration: replicate [3]Float%
35Inhibition at 13.2 uM [1] (13.2μM**)Value of % Inhibition at 13.2 uM inhibitor concentration: replicate [1]Float%
36Inhibition at 13.2 uM [2] (13.2μM**)Value of % Inhibition at 13.2 uM inhibitor concentration: replicate [2]Float%
37Inhibition at 13.2 uM [3] (13.2μM**)Value of % Inhibition at 13.2 uM inhibitor concentration: replicate [3]Float%
38Inhibition at 39.5 uM [1] (39.5μM**)Value of % Inhibition at 39.5 uM inhibitor concentration: replicate [1]Float%
39Inhibition at 39.5 uM [2] (39.5μM**)Value of % Inhibition at 39.5 uM inhibitor concentration: replicate [2]Float%
40Inhibition at 39.5 uM [3] (39.5μM**)Value of % Inhibition at 39.5 uM inhibitor concentration: replicate [3]Float%
41Inhibition at 118.6 uM [1] (118.6μM**)Value of % Inhibition at 118.6 uM inhibitor concentration: replicate [1]Float%
42Inhibition at 118.6 uM [2] (118.6μM**)Value of % Inhibition at 118.6 uM inhibitor concentration: replicate [2]Float%
43Inhibition 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)
PageFrom: