Assessing Sensitivity to Antibacterial Topoisomerase II Inhibitors

Sonia K. Morgan‐Linnell1, Hiroshi Hiasa2, Lynn Zechiedrich1, John L. Nitiss3

1 Baylor College of Medicine, Houston, Texas, 2 University of Minnesota Medical School‐Twin Cities, Minneapolis, Minnesota, 3 St. Jude Children's Research Hospital, Memphis, Tennessee
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 3.13
DOI:  10.1002/0471141755.ph0313s39
Online Posting Date:  December, 2007
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Abstract

Both prokaryotes and eukaryotes have two major classes of topoisomerases that make transient single‐ or double‐strand cuts in DNA. While these enzymes play critical roles in cellular processes, they are also important targets of therapeutic agents. This unit describes assays to use in characterizing topoisomerase II‐targeting agents in vitro and in bacterial cells. It provides protocols for characterizing the action of small molecules against bacterial type II topoisomerases in vitro and the in vivo effects of putative topoisomerase II‐targeting antibiotics, as well as for measuring trapped enzyme/DNA covalent complexes, the major cytotoxic lesion induced by fluoroquinolones. Curr. Protoc. Pharmacol. 39:3.13.1‐3.13.26. © 2007 by John Wiley & Sons, Inc.

Keywords: topoisomerase; gyrase; antibiotic; topoisomerase poison; fluoroquinolone; MIC; MPC; supercoil

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Determination of Inhibition of Bacterial Type II Topoisomerases: Assays for Inhibition of DNA Gyrase and Topoisomerase IV
  • Basic Protocol 2: In Vitro Inhibition of Supercoiling Activity of Gyrase
  • Basic Protocol 3: Determination of Inhibitor Effects on DNA Cleavage by Topoisomerase II Using a Plasmid Linearization Assay
  • Alternate Protocol 1: Determination of Topoisomerase Cleavage Using the K+/SDS Assay
  • Basic Protocol 4: Measurement of the Minimal Inhibitory Concentration (MIC) of Inhibitors of E. coli Gyrase and Topoisomerase IV Mutants
  • Basic Protocol 5: Measurement of the Mutant Prevention Concentration (MPC)
  • Basic Protocol 6: In Vivo Inhibition of Supercoiling Activity of Gyrase
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Determination of Inhibition of Bacterial Type II Topoisomerases: Assays for Inhibition of DNA Gyrase and Topoisomerase IV

  Materials
  • 10× topoisomerase IV reaction buffer (see recipe; also used for gyrase)
  • 20 mM ATP (diluted with H 2O from purchased stock solution; store in small aliquots up to 6 months at −20°C)
  • Kinetoplast DNA solution (TopoGen)
  • New chemical entity (NCE) to be tested
  • Positive control (e.g., ciprofloxacin; Sigma‐Aldrich), optional
  • Purified topoisomerase IV and/or DNA gyrase (TopoGen or Inspiralis; see Critical Parameters)
  • 250 mM EDTA
  • 5× loading dye (see recipe)
  • 0.8% agarose gel (see Voytas, ) prepared in 1× TAE (see recipe for 50× TAE buffer)
  • 10 mg/ml ethidium bromide stock solution: store at room temperature, protected from light
  • Camera suitable for gel documentation
  • Additional reagents and equipment for performing agarose gel electrophoresis, ethidium bromide staining, and gel photography (Voytas, )

Basic Protocol 2: In Vitro Inhibition of Supercoiling Activity of Gyrase

  Materials
  • 0.4 µg/ml DNA substrate: relaxed, covalently closed plasmid DNA (e.g., pBR322; New England Biolabs or see Critical Parameters)
  • Eukaryotic topoisomerase I (TopoGen)
  • 5× gyrase reaction buffer (see recipe)
  • New chemical entity (NCE) to be tested
  • Gyrase
  • 1 mM ciprofloxacin (Sigma‐Aldrich) or other topoisomerase II inhibitor as a positive control
  • Purified gyrase (TopoGen or Inspiralis; see Critical Parameters)
  • Gyrase dilution buffer (see recipe)
  • 28 mM ATP (diluted with H 2O from purchased stock solution; store up to 6 months in small aliquots at −20°C)
  • 24:1 chloroform/isoamyl alcohol
  • 5× loading dye (see recipe)
  • 0.8% agarose gel (Voytas, ) prepared with 1× TAE buffer (see recipe for 50× TAE buffer)
  • 10 mg/ml ethidium bromide stock solution: store at room temperature, protected from light
  • Camera suitable for gel documentation
  • Additional reagents and equipment for assessing DNA relaxation by eukaryotic topoisomerase I (unit 3.3), purifying and concentrating DNA ( appendix 3C), and performing agarose gel electrophoresis, ethidium bromide staining, and gel photography (Voytas, )

Basic Protocol 3: Determination of Inhibitor Effects on DNA Cleavage by Topoisomerase II Using a Plasmid Linearization Assay

  Materials
  • 10× topoisomerase IV reaction buffer (when using topoisomerase IV; see recipe) or 5× DNA gyrase reaction buffer (when using DNA gyrase; see recipe)
  • Supercoiled plasmid DNA (e.g., 0.28 µg pBR322 or 0.18 µg pUC18; see Critical Parameters)
  • New chemical entity (NCE) stock solution
  • 1 mM positive control (e.g., ciprofloxacin from Sigma‐Aldrich)
  • 20 mM ATP (diluted with H 2O from purchased stock solution; store up to 6 months in small aliquots at −20°C), optional
  • 4 to 20 U/µl purified topoisomerase II (DNA topoisomerase IV or DNA gyrase; TopoGen or Inspiralis; see Critical Parameters)
  • 10% (w/v) SDS
  • 250 mM EDTA
  • 4 mg/ml stock solution of proteinase K: dilute to 0.8 mg/ml before use
  • 0.8% agarose gel (Voytas, ) prepared with 1× TAE (see recipe for 50× TAE)
  • Restriction enzyme appropriate for cutting plasmid once
  • 10 mg/ml ethidium bromide stock solution: store at room temperature, protected from light
  • Camera suitable for gel documentation
  • Additional reagents and equipment for performing agarose gel electrophoresis, ethidium bromide staining, and gel photography (Voytas, )

Alternate Protocol 1: Determination of Topoisomerase Cleavage Using the K+/SDS Assay

  Materials
  • DNA substrate: plasmid DNA (e.g., pUC18 or pBR322; New England Biolabs)
  • EcoRI restriction endonuclease (New England Biolabs)
  • 5× loading dye (see recipe)
  • 0.8% agarose gel (see Voytas, ) prepared in 1× TAE (see recipe for 50× TAE)
  • 10 mg/ml ethidium bromide stock solution: store at room temperature, protected from light
  • TE buffer, pH 8.0 ( appendix 2A)
  • 5 mM dCTP, dGTP, and dTTP
  • 10× Klenow buffer (provided with enzyme; New England Biolabs)
  • 10 mCi/ml [α‐32P]dATP (800 Ci/mmol; Amersham)
  • 5 U/µl Klenow fragment of DNA polymerase I (New England Biolabs)
  • Sephadex G‐25 spin columns (Pharmacia Biotech)
  • 10× topoisomerase IV reaction buffer (see recipe) or 5× gyrase reaction buffer (see recipe)
  • New chemical entity (NCE) solutions: several concentrations diluted in H 2O
  • 20 to 40 U/µl purified topoisomerase IV or gyrase (Inspiralis; see Critical Parameters)
  • Topoisomerase stop solution (see recipe), 37°C (for topoisomerase) or 25°C (for gyrase)
  • 325 mM KCl
  • Topoisomerase wash solution (see recipe)
  • Scintillation fluid (Fisher Scientific)
  • 65°C heating block
  • Additional reagents and equipment for performing agarose gel electrophoresis (Voytas, ) and purifying DNA by phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation ( appendix 3C)

Basic Protocol 4: Measurement of the Minimal Inhibitory Concentration (MIC) of Inhibitors of E. coli Gyrase and Topoisomerase IV Mutants

  Materials
  • Mueller‐Hinton broth medium (Becton Dickinson)
  • E. coli isogenic wild‐type and mutant strains: e.g., C600 derivatives LZ23, gyrase gyrAS83L and LZ21, gyrAS83LparCS80L (available from E. Lynn Zechiedrich, Baylor College of Medicine)
  • New chemical entity (NCE) solution to be tested
  • 32 mg/ml ciprofloxacin (Sigma‐Aldrich; reference standard antibiotic) solution
  • Mueller‐Hinton agar plates (Becton Dickinson)
  • Sterile 13 × 100–mm culture tubes
  • 250‐ml Erlenmeyer flask

Basic Protocol 5: Measurement of the Mutant Prevention Concentration (MPC)

  Materials
  • Mueller‐Hinton broth (Becton Dickinson): prepare according to the manufacturer's directions
  • Mueller‐Hinton powdered medium (Becton Dickinson)
  • New chemical entity (NCE) stock solution
  • E. coli K12 wild‐type strain: e.g., C600; American Type Culture Collection (ATCC) #23724
  • E. coli National Committee for Clinical Laboratory Standards (CLSI) quality control strain: ATCC #25922
  • Sterile 13 × 100–mm culture tubes
  • 250‐ml Erlenmeyer flasks

Basic Protocol 6: In Vivo Inhibition of Supercoiling Activity of Gyrase

  Materials
  • Bacterial strain encoding resistant topoisomerase IV (e.g., parCL80; available from E. Lynn Zechiedrich, Baylor College of Medicine)
  • Plasmid DNA (e.g., pUC18, pBR322, or any plasmid that carries a selectable marker such as ampicillin resistance)
  • Mueller‐Hinton broth media (Becton Dickinson) with and without antibiotic (appropriate for selection of the plasmid used)
  • New chemical entity (NCE)
  • 5× loading dye (see recipe)
  • 0.8% agarose gel (Voytas, ) prepared with 1× TAE buffer (see recipe for 50× TAE)
  • 10 mg/ml ethidium bromide stock solution: store at room temperature, protected from light
  • Sterile 26 × 250–mm test tubes
  • Camera for gel documentation
  • Additional reagents and equipment for transforming bacteria using CaCl 2 or electroporation (Seidman et al., ), purifying DNA by phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation ( appendix 3C), and performing agarose gel electrophoresis, ethidium bromide staining, and gel photography (Voytas, )
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Figures

Videos

Literature Cited

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Key References
   Anderson et al., 1998. See above.
  A fine detailed description concerning mechanistic questions of inhibiting bacterial topoisomerases.
   Hooper, 2001. See above.
  The importance of fluoroquinolones has led to a large literature on these agents. These two references are a fine starting point.
   Drlica and Malik, 2003. See above.
  This review covers basic biochemical and in vivo mechanisms concerning how fluoroquinolones target bacteria.
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