Counterscreen for AddAB inhibitors: absorbance-based bacterial cell-based high throughput confirmation assay for inhibitors of bacterial viability
Name: Counterscreen for AddAB inhibitors: absorbance-based bacterial cell-based high throughput confirmation assay for inhibitors of bacterial viability. ..more
BioActive Compounds: 306
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: ECOLIV3065VBLTY_INH_ABS_1536_3X%INH CSRUN
Name: Counterscreen for AddAB inhibitors: absorbance-based bacterial cell-based high throughput confirmation assay for inhibitors of bacterial viability.
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.
helicase, nuclease, exonuclease, AddAB, ADDAB, AddAB complex, RecBCD enzyme, beta subunit, gamma chain, alpha chain, Escherichia coli, E. coli, bacteria, phage, DNA, dsDNA, DNA damage, DNA repair, DNA binding, ATP-binding, homologous recombination, recombination, Chi, inhibition, inhibitor, optical density, OD, absorbance, HTS, high throughput screen, counterscreen, confirmatory, confirmation, triplicate, viability, cytotoxicity, secondary, 1536, 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 compounds identified as active in a set of previous experiments entitled, "Absorbance-based primary bacterial cell-based high throughput screening assay to identify inhibitors of AddAB recombination protein complex" (AID 435030), are nonselective inhibitors of the AddAB helicase-nuclease complex due to bacterial cytotoxicity. In this assay, V3065 bacteria are not infected with phage but, are incubated in the presence of test compounds alone, followed by measurement of well optical density as an indicator of bacterial growth. As designed, compounds that inhibit bacterial growth will reduce well absorbance. Compounds are tested in triplicate at a nominal test concentration of 11.86 uM.
Prior to the start of the assay, V3065 bacterial culture was grown at 37 C until it reached an OD600 of 0.05 or 2.5E7 cfu/mL. V3065 was diluted in assay buffer to achieve 1.25E7 cfu/mL. To start the assay, 3 uL of Assay Buffer (0.1% Glycerol + 100 ug/mL Ampicillin + Cation Mueller Hinton Broth) was dispensed into all wells. Next, 60 nL of test compound in DMSO, Ciprofloxacin (0.95 ug/ml final concentration) or DMSO alone (1.2% final concentration) were added to the appropriate wells.
Next, 2 uL of V3065 (addAB+) bacterial culture was dispensed into the appropriate wells. Plates were incubated for 18 hours at 37 C and absorbance (OD600) was read on a Envision microplate reader (PerkinElmer, Turku, Finland) using 10 flashes per well.
The percent inhibition for each compound was calculated as follows:
100 * ( ( Test_Compound - Median_Low_Control ) / ( Median_High_Control - Median_Low_Control ) )
High_Control is defined as wells containing V3065 + Ciprofloxacin.
Low_Control is defined as wells containing V3065 + DMSO.
Test_Compound is defined as wells containing V3065 in the presence of test compound.
The average percent inhibition and standard deviation of each compound tested were calculated. In addition, two values were calculated as well: (1) the average percent inhibition of the wells containing DMSO only, and (2) eight times their standard deviation. The sum of these two values was used as a hit cutoff parameter. Any compound that exhibited a percent inhibition greater than the calculated hit cutoff was declared active.
PubChem Activity Outcome and Score:
The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.
The PubChem Activity Score range for active compounds is 100-24, and for inactive compounds 23-0.
List of Reagents:
V3065 E.coli bacteria (supplied by Assay Provider)
Ciprofloxacin (Sigma, part 17850)
Ampicillin (Fisher, part BP1760-5)
Cation-Adjusted Mueller Hinton II Broth (BD, part 297963)
1536-well plates (Aurora, part 19326)
Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that modulate well fluorescence. 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 by the MLSMR. The MLSMR was not able to provide all compounds selected for testing in this assay.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)