Late stage assay provider Counterscreen for AddAB inhibitors: Cell-based colony-formation assay to identify inhibitors of recombination of E. coli transformed with p(addAB) and p(recA) (20 micromolar compound dose)
Name: Late stage assay provider Counterscreen for AddAB inhibitors: Cell-based colony-formation assay to identify inhibitors of recombination of E. coli transformed with p(addAB) and p(recA) (20 micromolar compound dose). ..more
BioActive Compound: 1
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: ECOLI-RECOMBINATION-ADDAB-RECA_INH_ABS_PETRI 2XFOLD MCSRUN 20uM
Name: Late stage assay provider Counterscreen for AddAB inhibitors: Cell-based colony-formation assay to identify inhibitors of recombination of E. coli transformed with p(addAB) and p(recA) (20 micromolar compound dose).
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.
assay provider, counterscreen, RecA, growth, 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, absorbance, 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 whether powder samples of compounds identified as AddAB inhibitor probe candidates can inhibit recombination in Hfr conjugational crosses. In this assay an E. coli recBCD deletion test strain transformed with H. pylori paddAB and precA is mated with an Hfr strain in the presence of compound, and the frequency of selected recombinants (His+ StrR) in the mixture is determined by differential plating. The viability of the donor and recipient strains are determined by plating on non-selective media. E. coli were exposed to test compounds for 1.5 hours. As designed, compounds that inhibit AddAB will reduce the frequency of recombinants. This assay is designed to determine if inhibitors of AddAB can enter living cells and inhibit the recombination-promoting function of AddAB. Probes should be active in this assay. Compounds were tested in duplicate experiments at single doses of 20 uM.
Recipient cells are grown for 1 hr in LB broth with or without compound. The viability of the donor and recipient strains are determined by plating on non-selective media. Donor (Hfr) and recipient (F-) cells are mixed in a ratio of 1:10, incubated for 30 min, vortexed to separate mating cells, and plated differentially to determine the frequency of reocmbinants (His+ StrR).
The relative viability of each culture was calculated as follows:
Relative_Viability = [colony forming units per ml of culture with compound] / [colony forming units per ml of culture with DMSO]
The fold reduction in recombinant frequency for each compound was calculated as follows:
Fold_Reduction = [Recombinant frequency of DMSO control] / [Recombinant frequency in presence of compound adjusted for relative viability of the culture]
Recombinant_Frequency = 100 * [His+ StrR recombinants per donor cell adjusted for the viability of the recipient]
PubChem Activity Outcome and Score:
Compounds that reduced recombinant frequency (viability) by 4-fold or greater compared to the DMSO control were considered active. Compounds that reduced recombinant frequency (viability) less than 4-fold were considered inactive.
Compounds were ranked by fold reduction, the largest fold reduction received a score of 100.
The PubChem Activity Score range for active compounds is 100-100, and for inactive compounds 22-16.
List of Reagents:
E. coli recBCD deletion transformed with AddAB and RecA plasmids; E. coli strain V1306 (HfrH)
This assay was run in 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.
Categorized Comment - additional comments and annotations
From BioAssay Depositor:
BAO: assay design: viability reporter: cell number
BAO: assay format: cell-based format
BAO: bioassay specification: assay biosafety level: bsl1
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: viability
BAO: detection technology: spectrophotometry: absorbance
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: Cell-based
** Test Concentration.
Data Table (Concise)