Absorbance-based bacterial cell-based high throughput dose response assay to identify inhibitors of RecBCD
Name: Absorbance-based bacterial cell-based high throughput dose response assay to identify inhibitors of RecBCD. ..more
Depositor Specified Assays
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: RECBCD_INH_ABS_1536_3XIC50 DRUN
Name: Absorbance-based bacterial cell-based high throughput dose response assay to identify inhibitors of RecBCD.
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).
1. Cromie, G. A. (2009) Phylogenetic ubiquity and shuffling of the bacterial RecBCD and AddAB recombination complexes, J Bacteriol 191, 5076-5084.
2. 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.
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.
Dose response DRUN, 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.
The purpose of this assay is to determine the efficacy of compounds in RecBCD 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 RecBCD recombination protein complex" (AID 651982), and were active in a set of experiments entitled, " Counterscreen for RecBCD inhibitors: absorbance-based high throughput cell-based assay to identify inhibitors of AddAB recombination protein complex." (AID 651984) and inactive in a set of experiments entitled, "Counterscreen for inhibitors of RecBCD: Absorbance-based cell-based high throughput assay to identify inhibitors of bacterial viability." (AID 651983). This bacterial cell-based 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 infect V66 E. coli (recBCD+), but V66 proliferate because of the RecBCD helicase and nuclease activity against the unprotected mutant phage. In this assay, V66 E. coli 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, thereby reducing V66 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.
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 ) )
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)
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.
Assay: Dictionary: Version: 0.1
Assay: CurveFit : Equation: =( ( [Maximal Response] * [Concentration]^[Hill Slope] ) / ( [Inflection Point Concentration]^[Hill Slope] + [Concentration]^[Hill Slope] ) ) + [Baseline Response]
Assay: CurveFit : Mask: Excluded Points
* Activity Concentration. ** Test Concentration.
Data Table (Concise)