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BioAssay: AID 651983

Counterscreen for inhibitors of RecBCD: Absorbance-based cell-based high throughput assay to identify inhibitors of bacterial viability

Name: Counterscreen for inhibitors of RecBCD: Absorbance-based cell-based high throughput assay to identify inhibitors of bacterial viability. ..more
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 Tested Compounds
 Tested Compounds
All(888)
 
 
Active(261)
 
 
Inactive(627)
 
 
 Tested Substances
 Tested Substances
All(892)
 
 
Active(263)
 
 
Inactive(629)
 
 
 Related BioAssays
 Related BioAssays
AID: 651983
Data Source: The Scripps Research Institute Molecular Screening Center (ECOLIV66VBLTY_INH_ABS_1536_3X%INH CSRUN for RECBCD)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2013-01-15

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 261
Related Experiments
AIDNameTypeComment
651602Absorbance-based primary bacterial cell-based high throughput screening assay to identify inhibitors of RecBCD (with phage)Screeningdepositor-specified cross reference: Primary screen (RECBCD inhibitors in singlicate)
651606Summary of the probe development efforts to identify inhibitors of RecBCDSummarydepositor-specified cross reference: Summary (RECBCD inhbitors)
652150Counterscreen for inhibitors of RecBCD: Absorbance-based cell-based high throughput dose response assay to identify inhibitors of bacterial viabilityConfirmatorydepositor-specified cross reference
652151Absorbance-based bacterial cell-based high throughput dose response assay to identify inhibitors of RecBCDConfirmatorydepositor-specified cross reference
651982Absorbance-based bacterial cell-based high throughput confirmation assay to identify inhibitors of RecBCDScreeningsame project related to Summary assay
651984Counterscreen for RECBCD inhibitors: absorbance-based high throughput cell-based assay to identify inhibitors of AddAB recombination protein complexScreeningsame 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: 1 R03 AI083736
Grant Proposal PI: Gerald R. Smith
External Assay ID: ECOLIV66VBLTY_INH_ABS_1536_3X%INH CSRUN for RECBCD

Name: Counterscreen for inhibitors of RecBCD: Absorbance-based cell-based high throughput assay to identify inhibitors of bacterial viability.

Description:

The RecBCD enzyme (Exonuclease V) of Escherichia coli is a helicase-nuclease that initiates the repair of double-stranded DNA breaks by homologous recombination. The RecBCD class of enzymes is widely distributed among bacteria (1) but is apparently not present in eukaryotes. The major pathway of double-strand DNA break repair in bacteria involves the coordinated action of nuclease and helicase activities provided by the three-subunit enzyme RecBCD, which is critical for DNA repair(2); recBCD null mutants have reduced cell viability, are hyper-sensitive to DNA damaging agents, and are deficient in homologous recombination involving linear DNA (3-5). Inhibitors of RecBCD would allow us to study the mechanism of this complex helicase-nuclease enzyme. Since this enzyme is crucial for bacterial survival during infection, we anticipate that some of these compounds will, in future work, lead to new, critically needed antibacterial drugs with few off-target effects for human use (6).

References:

1. Cromie, G. A. (2009) Phylogenetic ubiquity and shuffling of the bacterial RecBCD and AddAB recombination complexes, J Bacteriol 191, 5076-5084.
3. Howard-Flanders, P., and Theriot, L. (1966) Mutants of Escherichia coli K-12 defective in DNA repair and in genetic recombination, Genetics 53, 1137-1150.
4. Willetts, N. S., and Clark, A. J. (1969) Characteristics of some multiply recombination-deficient strains of Escherichia coli, J Bacteriol 100, 231-239.
5. Willetts, N. S., Clark, A. J., and Low, B. (1969) Genetic location of certain mutations conferring recombination deficiency in Escherichia coli, J Bacteriol 97, 244-249.
6. Amundsen, S. K., Spicer, T., Karabulut, A. C., Londono, L. M., Eberhardt, C., Fernandez Vega, V., Bannister, T. D., Hodder, P., and Smith, G. R. (2012) Small-Molecule Inhibitors of Bacterial AddAB and RecBCD Helicase-Nuclease DNA Repair Enzymes, ACS Chem Biol, 2012 May 18;7(5):879-91. Epub 2012 Mar 23.

Keywords:

exonuclease V, helicase, nuclease, RecBCD, RECBCD, RECBCDV66, RecBCD complex, recB, recC, recD, beta subunit, gamma chain, alpha chain, Escherichia coli, E. coli, bacteria, phage, T4, infection, DNA, dsDNA, DNA damage, DNA repair, DNA binding, ATP-binding, homologous recombination, recombination, Chi, inhibit, inhibition, inhibitor, optical density, OD, absorbance, HTS, high throughput screen, singlicate, confirmation, 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 whether compounds identified as active in a set of previous experiments entitled, ""Absorbance-based primary bacterial cell-based high throughput screening assay to identify inhibitors of RecBCD (with phage)" (AID 651602), are nonselective inhibitors of the RecBCD due to bacterial cytotoxicity. 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 at a nominal test concentration of 11.86 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.
PubChem Activity Outcome and Score:
The average percent inhibition and standard deviation of each compound tested were calculated. In addition, two values were calculated as well: (1) the average percent inhibition of the wells containing DMSO only, and (2) eight times their standard deviation. The sum of these two values was used as a hit cutoff parameter. Any compound that exhibited a percent inhibition greater than the calculated hit cutoff was declared active.
The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.
The PubChem Activity Score range for active compounds is 100-18, and for inactive compounds 18-0.
List of Reagents:
V66 (recBCD+) 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
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 absorbance. 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 unable to provide all compounds selected for testing in the assay.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Cell-based
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Average Inhibition at 11.86 uM (11.86μM**)Normalized percent inhibition of the counterscreen at a compound concentration of 11.86 micromolar.Float%
2Standard DeviationStandard deviation of the counterscreen assay derived from the normalized percent inhibition of the triplicate data for each compoundFloat
3Inhibition at 11.86 uM [1] (11.86μM**)Percent inhibition of the counterscreen at a compound concentration of 11.86 uM; replicate [1].Float%
4Inhibition at 11.86 uM [2] (11.86μM**)Percent inhibition of the counterscreen at a compound concentration of 11.86 uM; replicate [2].Float%
5Inhibition at 11.86 uM [3] (11.86μM**)Percent inhibition of the counterscreen at a compound concentration of 11.86 uM; replicate [3].Float%

** Test Concentration.
Additional Information
Grant Number: 1 R03 AI083736

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