Late stage assay provider counterscreen for AddAB inhibitors: Radioactivity-based biochemical assay to identify inhibitors of the helicase/Chi cutting activity of purified RecBCD
Name: Late stage assay provider counterscreen for AddAB inhibitors: Radioactivity-based biochemical assay to identify inhibitors of the helicase/Chi cutting activity of purified RecBCD. ..more
BioActive Compounds: 16
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-CHI-CUTTING_INH_RADIOACTIVITY_FILM_3X%INH MCSRUN 50uM
Name: Late stage assay provider counterscreen for AddAB inhibitors: Radioactivity-based biochemical assay to identify inhibitors of the helicase/Chi cutting activity of purified RecBCD.
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 assay is to determine whether powder samples of compounds identified as AddAB inhibitor probe candidates can inhibit the ability of E. coli RecBCD enzyme to cut DNA at Chi sequences during DNA unwinding. In this assay linear, 5'end-labeled duplex DNA with Chi is incubated with RecBCD in the absence and presence of compound, and the products are analyzed by gel electrophoresis and autoradiography. A labeled fragment ending at or near Chi, but absent in reactions with DNA lacking Chi, reflects Chi-specific cutting. Controls include native and boiled DNA without RecBCD. As designed an inhibitor of RecBCD will decrease the amount of the Chi-specific fragment. If the compound inhibits DNA unwinding, less full length single-stranded DNA, with mobility equal to that of boiled DNA, will be seen. This assay is designed to determine if compounds inhibit either the unwinding of DNA or specific cutting at Chi hotspots of recombination. Compounds were tested at a nominal concentration of 50 uM.
Helicase assays measured the formation of ss DNA from 5'[32P] pBR322 Chi+F (or Chi_o control) DNA (0.1 nM molecules) linearized by digestion with HindIII enzyme.RecBCD unwinding assays were in 25 mM Tris-acetate (pH 7.5), 2.0 mM Mg(OAc)2, 5 mM ATP. RecBCD assays used 0.15 nM enzyme and without SSB.
Compounds were added to the reaction mixture containing all the reagents except ATP; final DMSO concentration was 2.5% (v/v) for each assay. (All other percentages in this paragraph are w/v.) Reactions were started by addition of ATP and were at 37 C for 1 min . Reactions were stopped by addition of 1/3 vol of stop buffer (2.5% SDS, 100 mM EDTA, 0.125% bromophenol blue, 0.125% xylene cyanol FF, and 10% Ficoll), and the products were subjected to electrophoresis in a 1.25% agarose gel at 5 V/cm for 2.5 h in TAE buffer (40 mM Tris base, 20 mM acetic acid, 1 mM EDTA). Gels were dried under vacuum, and the products were detected by autoradiography or analyzed with a Typhoon Trio PhosphorImager (GE Lifesciences) and ImageQuant TL software (Amersham). With RecBCD, this assay also detects cutting of DNA at the Chi site Chi+F, which produces a 1.46 kb fragment.
The fold reduction in RecBCD activity for each compound was determined according to the degree of reduction of Chi cutting as observed on the X-Ray film of the dried gels.
Fold_Reduction = ( Test_Compound_Chi_Band_Intensity )/( DMSO_Control_Chi_Band_Intensity)
DMSO_Control_Chi_Band_Intensity is defined as the intensity of the Chi band formed with the presence of 5% DMSO control.
Test_Compound_Chi_Band_Intensity is defined as the intensity of the Chi band formed in the presence of test compound dissolved in 5% final concentration of DMSO in the assay mixture.
For each test compound, inhibition of RecBCD Chi cutting was measured as the reduction of Chi band intensity in the presence of test compound compared to DMSO control and was plotted against compound concentration or manually estimated. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Prism 5 (GraphPad Software Inc) or or . 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 or manual estimation of the reaction product band intensities on X-ray images of the dried gels. In cases where the highest concentration tested (i.e. 500 uM) did not result in greater than 50% inhibition, the IC50 was determined manually as greater than 500 uM.
PubChem Activity Outcome and Score:
Any compound giving Chi cutting activity value less than or equal to a 2-fold reduction of Chi-Band intensity compared to DMSO control was considered inactive. Any compound giving Chi cutting activity value greater than a 2-fold reduction of Chi-Band intensity was considered active.
Active compounds were assigned a score of 100; and inactive compounds were assigned a score of 0.
The PubChem Activity Score range for active compounds is 100-100, and for inactive compounds 0-0.
List of Reagents:
E. coli RecBCD enzyme; 5' [32P] linear DNA with Chi (5' GCTGGTGG 3')
This assay was run 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: single concentration 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
** Test Concentration.
Data Table (Concise)