Late stage assay provider Counterscreen for AddAB inhibitors: Cell-based colony-formation dose response assay to identify inhibitors of the viability of V66 E. coli in Hfr recombination assays
Name: Late stage assay provider Counterscreen for AddAB inhibitors: Cell-based colony-formation dose response assay to identify inhibitors of the viability of V66 E. coli in Hfr recombination assays. ..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: HFR-RECOMBINATION_INH_ABS_PETRI_IC50 MDCSRUN
Name: Late stage assay provider Counterscreen for AddAB inhibitors: Cell-based colony-formation dose response assay to identify inhibitors of the viability of V66 E. coli in Hfr recombination assays.
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.
V66, phage, viability, counterscreen,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, dose response, triplicate, 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 reduce the viability of the recipients in Hfr conjugational crosses. In this assay a test strain is grown in the presence of compound for 1 hr, then 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 are determined by plating on non-selective media. E. coli were exposed to test compounds for 1.5 hours. As designed, compounds that reduce strain V66 viability could reduce the apparent frequency of recombinants. This assay is designed to determine if compounds can enter living cells and reduce viability. Compounds were tested using a 5-point dilution series of doses ranging from 0.03 to 100 uM.
Recipient cells are grown for 1 hr in LB broth with or without compound. Donor and recipient cells are plated on non-selective media to determine 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 percent inhibition for each compound was calculated as follows:
%_Inhibition = 100 * ( 1- ( [colony forming units per ml of V66 in presence of compound] / [colony forming units per ml of V66 in DMSO control] ) )
This value is used to adjust the frequency of His+ StrR recombinants determined in assays of Hfr recombination.
PubChem Activity Outcome and Score:
Compounds that reduced V66 E. coli viability to less than 80% of the DMSO control (set as 100%) at all concentrations were considered active. Compounds that reduced V66 E. coli viability to 80% or greater at all concentrations were considered inactive.
Compounds were ranked by percent viability, with the lowest percent receiving a score of 100.
The PubChem Activity Score range for active compounds is 100-100, and for inactive compounds 12-1.
List of Reagents:
E. coli strain V66 and 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: 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: filter 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: Cell-based
Data Table (Concise)