Late stage assay provider counterscreen for AddAB inhibitors: Radioactivity-based biochemical dose response assay to identify inhibitors of the nuclease activity of purified RecBCD enzyme
Name: Late stage assay provider counterscreen for AddAB inhibitors: Radioactivity-based biochemical dose response assay to identify inhibitors of the nuclease activity of purified RecBCD enzyme. ..more
BioActive Compounds: 12
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-NUCLEASE_INH_RADIOACTIVITY_FILM_3XIC50 MCSRUN IC50
Name: Late stage assay provider counterscreen for AddAB inhibitors: Radioactivity-based biochemical dose response assay to identify inhibitors of the nuclease activity of purified RecBCD enzyme.
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.
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The purpose of this counterscreen assay is to determine whether powder samples of compounds identified as AddAB probe candidates can inhibit RecBCD enzymatic activities. Nuclease activities will be assayed to begin to elucidate the mechanism of inhibition. RecBCD purified from E. coli containing RecBCD will be assayed, depending upon availability. Nuclease activity is assayed as the formation of trichloroacetic acid-soluble radioactive material, detected with a scintillation counter, from uniformly labeled [3H] double-stranded (ds) DNA. As designed, compounds that inhibit RecBCD will reduce the nuclease activity (less acid-soluble material after standard incubation). Compounds are tested in triplicate using a dilution series starting at a nominal maximum test concentration of 100 uM.
Nuclease assays measured the formation of TCA-soluble radioactive material from phage T7 [3H] DNA (2 ug/mL; 6 uM nucleotides) substrate in a 20 min incubation at 37 C. RecBCD assays were in 50 mM Tris-HCl (pH 8.5), 10 mM MgCl2, polyvinylpyrrolidone (1 mg/mL), 1 mM DTT, and 25 uM ATP. 0.15 units of RecBCD enzyme (4.5 nM) was used in this assay.
Compounds were diluted in DMSO and added to enzyme in assay buffer on ice; final DMSO concentration was 5.0% (v/v) in each assay. DNA substrate was added, and after < 5 min the reactions were started by transferring the samples to 37 C. Reactions were stopped by addition of calf thymus DNA to 0.2 mg/mL and TCA to 5% (w/v). After 10 min on ice, the mixtures were centrifuged for 5 min at 16,100 x g, and the soluble radioactive material was determined in a scintillation counter.
The percent RecBCD activity following treatment with compounds was calculated as follows:
%_Enzyme_Activity = 100 * (1- ( ( Test_Compound - Background ) / ( Mean_DMSO_Control - Background ) ) )
Mean_DMSO_Control is defined as TCA-soluble radioactive product formed by RecBCD ds exonuclease in 20 min in the presence of 5.0 %(v/v) DMSO.
Background is defined as TCA-soluble radioactive product formed in the absence of RecBCD enzyme.
Test_Compound is defined as TCA-soluble radioactive product formed in the presence of compounds diluted in 5.0% (v/v) DMSO.
For each test compound, percent inhibition was plotted against compound concentration using Prism 5 software (GraphPad Software, Inc). The reported IC50 values were generated in GraphPad. In cases where the highest concentration tested (i.e. 100 uM) did not result in greater than 50% inhibition, the IC50 was determined manually as greater than 100 uM.
PubChem Activity Outcome and Score:
Compounds with an IC50 greater than 52 uM were considered inactive. Compounds with an IC50 equal to or less than 52 uM were considered active.
Activity score was then ranked by the potency of the compounds with fitted curves, with the most potent compounds assigned the highest activity scores.
The PubChem Activity Score range for active compounds is 100-32, and for inactive compounds 12-0.
List of Reagents:
Uniformly labeled phageT7 [3H] DNA; purified RecBCD enzyme.
This assay was performed by the assay provider's lab. This assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. 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 - additional comments and annotations
From BioAssay Depositor:
BAO: assay design: binding reporter: radioligand binding
BAO: assay format: biochemical format: protein format: single protein format
BAO: bioassay specification: assay biosafety level: bsl1
BAO: bioassay specification: assay footprint: vial
BAO: bioassay specification: assay measurement throughput quality: concentration response multiple replicates
BAO: bioassay specification: assay measurement type: endpoint assay
BAO: bioassay specification: assay readout content: assay readout method: regular screening
BAO: bioassay specification: assay readout content: content readout type: single readout
BAO: bioassay specification: assay stage: secondary: counter screening
BAO: bioassay specification: bioassay type: functional: enzyme activity
BAO: detection technology: radiometry: scintillation counting: scintillation proximity assay
BAO: meta target detail: binding reporter specification: interaction: protein-small molecule
BAO: meta target: biological process target: regulation of molecular function
BAO: meta target: molecular target: protein target: enzyme: generic hydrolase
BAO: version: 1.4b1090
Assay Format: Biochemical
* Activity Concentration.
Data Table (Concise)