Sequence: exonuclease V (RecBCD complex), beta subunit [Escherichia coli str. K-12 substr. MG1655] Protein Family: CRISPR/Cas system-associated protein Cas4 Gene:RECB     Related Protein 3D Structures

more...  

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: RECBCD_INH_ABS_1536_3X%INH CSRUN + phage

Name: Counterscreen for AddAB inhibitors: absorbance-based high throughput cell-based assay to identify inhibitors of RecBCD.

Description:

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.

References:

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.

Keywords:

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, exonuclease V, helicase, nuclease, RecBCD, RecBCD complex, recB, recC, recD, beta subunit, gamma chain, alpha chain, 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.

Assay Overview:

The purpose of this assay is to identify 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), also inhibit E. coli RecBCD, as an early indication of a compound that may have potential to elicit a broad-spectrum antibiotic like effect. This assay involves infecting E. coli with a T4 bacteriophage that carries three nonsense mutations in gene 2, whose wild-type protein product protects viral DNA from RecBCD-mediated degradation after infection. The mutant T4 phage is able to infect and block the growth of V67 E. coli (recB21, a recBCD null mutation), which lack RecBCD nuclease activity. The mutant phage also infect V66 E. coli (recBCD+), but V66 proliferate because of the RecBCD helicase and nuclease activity against the unprotected mutant phage. In this assay, the V66 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 RecBCD will allow the virus to replicate and block bacterial growth, leading to reduced well absorbance. Compounds are tested in triplicate at a nominal test concentration of 11.86 uM.

Protocol Summary:

Prior to the start of the assay, V66 and V67 bacterial cultures were grown at 37 C until it reached an OD600 of 0.05 or 2.5E7 cfu/mL. To start the assay, 3 uL of Assay Buffer (0.1% Glycerol + 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 V66 (recBCD+) or V67 (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 ) )

Where:

High_Control is defined as wells containing V66 + Ciprofloxacin + phage
Low_Control is defined as wells containing V66 + DMSO + phage.
Test_Compound is defined as wells containing V66 in the presence of test compound + phage.

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) three 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 100-3, and for inactive compounds 3-0.

List of Reagents:

V66 (recBCD+) and V67 (recB21) E. coli bacteria (supplied by Assay Provider)
T4 2 149 mutant bacteriophage (supplied by Assay Provider)
Ciprofloxacin (Sigma, part 17850)
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.

Grant Number: GM031693