Late stage assay provider Counterscreen for AddAB inhibitors: Cell-based colony formation assay to identify inhibitors of the recombination-promoting activity of RecBCD in V66 E. coli (100uM)
Name: Late stage assay provider Counterscreen for AddAB inhibitors: Cell-based colony formation assay to identify inhibitors of the recombination-promoting activity of RecBCD in V66 E. coli (100uM). ..more
BioActive Compounds: 7
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: HFR-RECOMBINATION_INH_ABS_PETRI MCSRUN 100uM
Name: Late stage assay provider Counterscreen for AddAB inhibitors: Cell-based colony formation assay to identify inhibitors of the recombination-promoting activity of RecBCD in V66 E. coli (100uM).
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, V66, phage, T4, T4 phage, gene 2, mutant, growth, RecBCD, chi cutting, 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, plate, 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 a test strain 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 donor and recipient are also plated on non-selective media to measure viability. As designed, compounds that inhibit RecBCD will reduce the frequency of recombinants. This assay is designed to determine if inhibitors of RecBCD can enter living cells and inhibit the recombination-promoting function of RecBCD. E. coli are exposed to test compounds for 1.5 hours. Probes should be active in this assay. Compounds are tested at a nominal concentration of 100 uM in duplicate experiments.
Recipient cells are grown for 1 hr in LB broth with or without compound. Donor and recipient cells are plated separately on non-selective media to measure viability. 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 recombinants (His+ StrR).
The relative viability of each culture was calculated as follows:
Relative_Viability = the number of colony forming units per ml of culture treated with compound normalized to the number of colony forming units per ml of culture treated with DMSO only.
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]
PubChem Activity Outcome and Score:
Compounds that reduced V66 E. coli recombinant frequency by less than or equal to 4-fold compared to the DMSO control were considered inactive. Compounds that reduced V66 E. coli recombinant frequency by greater than 4-fold compared to DMSO control were considered active.
Compounds were ranked by fold reduction, with the largest fold reduction receiving a score of 100.
The PubChem Activity Score range for active compounds is 100-0, and for inactive compounds 0-0.
List of Reagents:
E . coli strains V66 and V1306
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.
BAO: version: 1.4b1090
BAO: bioassay specification: assay stage: secondary: counter screening
BAO: bioassay specification: assay biosafety level: bsl1
BAO: assay format: cell-based format
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: meta target: molecular target: protein target: enzyme: generic hydrolase
BAO: meta target: biological process target: cell proliferation
BAO: meta target detail: binding reporter specification: interaction: protein-small molecule
BAO: assay design: viability reporter: cell number
BAO: detection technology: spectrophotometry: absorbance
BAO: bioassay specification: bioassay type: functional: viability
BAO: bioassay specification: assay measurement throughput quality: single concentration multiple replicates
** Test Concentration.
Data Table (Concise)