Counterscreen for RECBCD inhibitors: absorbance-based high throughput cell-based assay to identify inhibitors of AddAB recombination protein complex
Name: Counterscreen for RecBCD inhibitors: absorbance-based high throughput cell-based assay to identify inhibitors of AddAB recombination protein complex. ..more
BioActive Compounds: 397
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: AddAB_INH_ABS_1536_3X%INH CSRUN for RecBCD
Name: Counterscreen for RecBCD inhibitors: absorbance-based high throughput cell-based assay to identify inhibitors of AddAB recombination protein complex.
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.
Counterscreen, 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, confirmatory, confirmation, triplicate, viability, cytotoxicity, secondary, 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 if 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), also inhibit AddAB, as an early indication of a compound that may have potential to elicit a broad-spectrum antibiotic like effect. Expression of the H. pylori addAB genes in Escherichia coli deleted for its recBCD genes allows assay of the H. pylori AddAB enzyme in easily cultured E. coli . A simple assay for intracellular AddAB nuclease activity is the blocking of growth of bacteriophage T4 gene 2 mutants: in wild-type phage the gene 2 protein binds to the ends of DNA and protects it from degradation by AddAB or RecBCD upon infection . Thus, T4 gene 2 mutants will grow in E. coli expressing H. pylori addAB only in the presence of an inhibitor of AddAB; a specific inhibitor of AddAB will block E. coli growth only in the presence of this phage. This assay employs E. coli that express the Helicobacter pylori addAB+ genes. These 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 T4 phage is able to infect and block the growth of V3069 E. coli, which lacks AddAB nuclease activity (AddAB-), but is not infective in V3065 E. coli, which contains a plasmid expressing the H. pylori addAB+ genes. 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 at a nominal test concentration of 11.86 uM.
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 2.5E7 cfu/mL. To start the assay, 3 uL of Assay Buffer (0.1% Glycerol + 100 ug/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 ) )
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
The average percent inhibition and standard deviation of each compound tested were calculated.
PubChem Activity Outcome and Score:
A mathematical algorithm was used to determine nominally inhibiting compounds in this counterscreen. Two values were calculated: (1) the average percent inhibition of all DMSO wells tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited an average % inhibition greater than the cutoff parameter 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 100-20, and for inactive compounds 19-0.
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)
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 not able to provide all compounds selected for testing in this assay.
** Test Concentration.
Data Table (Concise)