Absorbance-based primary bacterial cell-based high throughput screening assay to identify inhibitors of AddAB recombination protein complex
Name: Absorbance-based primary bacterial cell-based high throughput screening assay to identify inhibitors of AddAB recombination protein complex. ..more
BioActive Compounds: 935
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: ADDAB_INH_ABS_1536_1X%INH PRUN +phage
Name: Absorbance-based primary bacterial cell-based high throughput screening assay to identify inhibitors of AddAB recombination protein complex.
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, 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, optical density, OD, absorbance, HTS, high throughput screen, primary, 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 identify compounds that act as inhibitors of bacterial AddAB activity. This bacterial cell-based assay employs E. coli that express the Helicobacter pylori AddAB+ genes. The bacteria are infected with a mutant T4 bacteriophage that carries three nonsense mutations in gene 2, whose protein product normally protects viral DNA from AddAB-mediated degradation after infection. The mutant phage infects and blocks the growth of V3069 E. coli, which lack AddAB nuclease activity (AddAB-). The mutant phage also infect V3065 E. coli, which contain a plasmid expressing the H. pylori addAB+ genes , but V3065 proliferate because of the AddAB directed nuclease and helicase activity against the unprotected mutant phage. In this assay, the V3065 E. coli cells are infected with mutant T4 phage in the presence of test compounds, followed by measurement of well optical density as an indicator of bacterial growth. As designed, compounds that inhibit AddAB will allow the virus to replicate and inhibit bacterial growth, leading to reduced well absorbance. Compounds are tested in singlicate at a nominal test concentration of 11.86 uM.
Prior to the start of the assay, V3065 and V3069 bacterial cultures were grown at 37 C until it reached an OD600 of 0.05 or 2.5e07 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. Then, 1 uL of V3065 (addAB+) or V3069 (phage control) bacterial cultures were dispensed into the appropriate wells and plates were incubated for 60 minutes at 37 C.
Next, 1 uL of mutant T4 2 149 bacteriophage was dispensed to the appropriate wells at a multiplicity of infection (MOI) of 0.02. Plates were centrifuged and after 18 hours of incubation at 37 C, 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 + phage.
Low_Control is defined as wells containing V3065 + DMSO + phage.
Test_Compound is defined as wells containing V3065 in the presence of test compound + phage
A mathematical algorithm was used to determine nominally inhibiting compounds in the primary screen. Two values were calculated: (1) the average percent inhibition of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition than the cutoff parameter 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 100-9, and for inactive compounds 9-0.
List of Reagents:
V3065 & V3069 E.coli bacteria (supplied by Assay Provider)
T4 2 149 mutant bacteriophage (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. In this case the results of each separate campaign were assigned "Active/Inactive" status based upon that campaign's specific compound activity cutoff value. 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.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)