Late 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
Name: Late 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. ..more
BioActive Compounds: 3
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: GM031693
Grant Proposal PI: Gerald R. Smith
External Assay ID: ADDAB_INH_ABS_1536_3XIC50 MDRUN + phage
Name: Late 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.
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.
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.
late stage, late stage AID, chemistry, purchased, synthesis, synthesized, powders, helicase, nuclease, exonuclease, ATP-dependent nuclease, AddAB, ADDAB, AddAB complex, RecBCD enzyme, beta subunit, gamma chain, alpha chain, Escherichia coli, E. coli, bacteria, Helicobacter pylori, phage, T4, DNA, dsDNA, DNA damage, DNA repair, DNA binding, ATP-binding, homologous recombination, recombination, Chi, inhibition, inhibitor, optical density, OD, absorbance, turbidity, HTS, high throughput screen, dose response, triplicate, 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 AddAB inhibition dose response curves for powder samples of compounds identified as possible AddAB probe candidates. This bacterial cell-based assay employs E. coli that express the Helicobacter pylori AddAB+ genes. The 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 phage infects and blocks the growth of V3069 E. coli, which lack AddAB nuclease activity (AddAB-). The mutant phage also infect V3065 E. coli, which contain a plasmid expressing the H. pylori addAB+ genes , but V3065 proliferate because of the AddAB directed nuclease and helicase activity against the unprotected mutant phage. 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 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, V3065 and V3069 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 + 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
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 active compounds is 100-92, and for inactive compounds 87-0.
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)
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.
Assay: Dictionary: Version: 0.1
Assay: CurveFit : Equation: ( ( [Maximal Response] * [Concentration]^[Hill Slope] ) / ( [Inflection Point Concentration]^[Hill Slope] + [Concentration]^[Hill Slope] ) ) + [Baseline Response]
* Activity Concentration. ** Test Concentration.
Data Table (Concise)