Table Of ContentNew Antibiotic Targets
M E T H O D S I N M O L E C U L A R M E D I C I N ETM
John M. Walker, SERIES EDITOR
142. NewAntibioticTargets,editedbyW.Scott 120. BreastCancerResearchProtocols,editedby
Champney,2008 SusanA.BrooksandAdrianHarris,2006
141. ClinicalBioinformatics,editedbyRonald 119. HumanPapillomaviruses:MethodsandProtocols,
J.A.Trent,2008 editedbyClareDavyandJohnDoorbar,2005
140. TissueEngineering,editedbyHansjörgHauser 118. AntifungalAgents:MethodsandProtocols, edited
andMartinFussenegger,2008 byErikaJ.ErnstandP.DavidRogers,2005
139. VascularBiologyProtocols,editedbyNair 117. FibrosisResearch:MethodsandProtocols,edited
SreejayanandJunRen,2007 byJohnVarga,DavidA.Brenner,andSemH.
138. AllergyMethodsandProtocols,editedby Phan,2005
MeinirG.JonesandPennyLympany,2007 116. InteferonMethodsandProtocols,editedby
137. MicrotubuleProtocols,editedbyJunZhou,2007 DanielJ.J.Carr,2005
136. ArthritisResearch:MethodsandProtocols, 115. Lymphoma:MethodsandProtocols,editedby
Vol.2,editedbyAndrewP.Cope,2007 TimothyIllidgeandPeterW.M.Johnson,2005
135. ArthritisResearch:MethodsandProtocols, 114. MicroarraysinClinicalDiagnostics,editedby
Vol.1,editedbyAndrewP.Cope,2007 ThomasO.JoosandPaoloFortina,2005
134. BoneMarrowandStemCellTransplantation, 113. MultipleMyeloma:MethodsandProtocols,edited
editedbyMeralBeksac,2007 byRossD.BrownandP.JoyHo,2005
133. CancerRadiotherapy,editedbyRobertA. 112. MolecularCardiology:MethodsandProtocols,
HuddartandVedangMurthy,2007 editedbyZhongjieSun,2005
132. SingleCellDiagnostics:MethodsandProtocols, 111. Chemosensitivity:Volume2,InVivoModels,
editedbyAlanThornhill,2007 Imaging,andMolecularRegulators,editedby
131. AdenovirusMethodsandProtocols,Second RosalynD.Blumethal,2005
Edition,Vol.2:AdProteins,RNA,Lifecycle,Host 110. Chemosensitivity:Volume1,InVitroAssays,
Interactions,andPhylogenetics,editedbyWilliam editedbyRosalynD.Blumethal,2005
S.M.WoldandAnnE.Tollefson,2007 109. AdoptiveImmunotherapy:Methodsand
130. AdenovirusMethodsandProtocols,Second Protocols,editedbyBurkhardLudewigand
Edition,Vol.1:Adenoviruses,AdVectors, MatthiasW.Hoffman,2005
Quantitation,andAnimalModels,editedby 108. Hypertension:MethodsandProtocols,editedby
WilliamS.M.WoldandAnnE.Tollefson,2007 JérômeP.FennellandAndrew H.Baker,2005
129. CardiovascularDisease:MethodsandProtocols, 107. HumanCellCultureProtocols,SecondEdition,
Volume2:MolecularMedicine,editedbyQingK. editedbyJoannaPicot,2005
Wang,2006 106. AntisenseTherapeutics,SecondEdition,editedby
128. CardiovascularDisease:MethodsandProtocols, M.IanPhillips,2005
Volume1:Genetics,editedbyQingK.Wang,2006 105. DevelopmentalHematopoiesis:Methodsand
127. DNAVaccines:MethodsandProtocols, Protocols,editedbyMargaretH.Baron,2005
SecondEdition,editedbyMarkW.Saltzman,Hong 104. StrokeGenomics:MethodsandReviews,editedby
Shen,andJanetL.Brandsma,2006 SimonJ.ReadandDavidVirley,2004
126. CongenitalHeartDisease:MolecularDiagnostics, 103. PancreaticCancer:MethodsandProtocols,edited
editedbyMaryKearns-Jonker,2006 byGloriaH.Su,2004
125. MyeloidLeukemia:MethodsandProtocols,edited 102. Autoimmunity:MethodsandProtocols,editedby
byHarryIland,MarkHertzberg,andPaula AndrasPerl,2004
Marlton,2006 101. CartilageandOsteoarthritis:Volume2,Structure
124. MagneticResonanceImaging:Methodsand andInVivoAnalysis,editedbyFrédéricDe
BiologicApplications,editedbyPottumarthi Ceuninck,MassimoSabatini,andPhilippe
V.Prasad,2006 Pastoureau,2004
123. MarijuanaandCannabinoidResearch: Methods 100. CartilageandOsteoarthritis:Volume1,Cellular
andProtocols,editedbyEmmanuelS.Onaivi,2006 andMolecularTools,editedbyMassimoSabatini,
122. PlacentaResearchMethodsandProtocols: PhilippePastoureau,andFrédéricDeCeuninck,
Volume2,editedbyMichaelJ.Soaresand 2004
JoanS.Hunt,2006 99. PainResearch:MethodsandProtocols,editedby
121. PlacentaResearchMethodsandProtocols: DavidZ.Luo,2004
Volume1,editedbyMichaelJ.SoaresandJoanS. 98. TumorNecrosisFactor:MethodsandProtocols,
Hunt,2006 editedbyAngeloCortiandPietroGhezzi,2004
M E T H O D S I N M O L E C U L A R M E D I C I N ETM
New Antibiotic Targets
Edited by
W. Scott Champney
Department of Biochemistry and Molecular Biology,
East Tennessee State University,
Johnson, Tennessee
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Preface
A crisis is developing in the treatment of infectious diseases. Resistance of
microorganismstocurrentlyprescribedantibioticsisincreasingatasteadyrate,
and fewer and fewer antimicrobial agents are available to treat these infec-
tions.Organismsdisplayingamultipledrugresistancephenotypearebecoming
common in both nosocomial and community-acquired infections. In some
instances, there remains only a single antibiotic therapy suitable for treating
the infection.
This situation has stimulated several avenues of research in an effort
to identify new drugs and new drug targets. Advances in combinatorial
chemistry and high-throughput drug screening have provided a large number
of compounds to be tested as new antimicrobial agents. The examination of
microbialgenomesequenceshasprovidedlistsofpotentialnewtargetsforsuch
agents. This book is an attempt to bring these two areas together in the form
of specific techniques that can be used to examine new drug targets and the
effectiveness of new antibiotics.
The major areas of vulnerability in microbial cells include cell wall and
membrane synthesis and the processes of DNA replication, RNA transcription,
and protein synthesis. Many of the currently used antibiotics have a specific
inhibitory effect on one of these activities. Both new antimicrobial agents
and modifications of existing drugs are being tested for improved antibac-
terial activity against these targets. In addition, a number of novel cellular
targets are being examined as potential drug sites. These include the process
of fatty acid biosynthesis, the activity of certain aminoacyl tRNA synthetases,
the function of peptide deformylase, and the process of ribosomal subunit
formation. Inhibitors of efflux pumps that lower cellular drug levels are also
currently being sought. Additional potential drug targets include the inacti-
vation of enzymes that modify antibiotics and the inhibition of enzymes that
alter cellular antibiotic targets leading to resistance.
Chapter 1 is a description of biocomputational methods for examining
microbial genome sequences for novel inhibitory targets. Chapters 2 and 3
describemethodologiesforexamininginhibitorsofDNAreplicationthataffect
topoisomerases (Chapter 2) and DNA polymerase III (Chapter 3). New assays
for inhibitors of the bacterial RNA polymerase are discussed in Chapter 4.
v
vi Preface
Proteinbiosynthesishasbeenamajortargetformanydifferentantimicrobial
agents. Methods to target the aminoacyl tRNA synthetases are presented in
Chapter 5. Chapters 6 and 7 present methods to examine inhibitors of bacterial
ribosome biogenesis, a newly described target for antibiotics. Chapters 8
and 9 describe specific assays for inhibitors of ribosomal functions in trans-
lation including initiation (Chapter 8) and peptidyltransferase (Chapter 9). An
additional promising new target is peptide deformylase; assays for inhibitors
of its function are given in Chapter 10.
Thebacterialcellwallandmembraneremainattractivetargetsforantibiotics.
Chapter 11 discusses assays for penicillin-binding proteins of the cell wall,
and Chapter 12 presents methods to assay inhibitors of lipopolysaccharide
biosynthesis, an exciting new target. Membrane structure and function are the
topics of Chapters 13 and 14, including measures of cationic antimicrobial
peptide function (Chapter 13) and assays for changes in permeability using
flow cytometry (Chapter 14). Chapter 15 describes several assays for detecting
inhibitors of the ubiquitous efflux pumps in bacterial membranes.
Two novel targets are described in Chapters 16 and 17. Inhibition of fatty
acid synthesis (Chapter 16) is a promising new target, as are compounds
that affect the important two-component signal transduction pathways in cells
(Chapter 17).
The remaining chapters describe assays for measuring target modification
by ribosomal methyltransferases (Chapter 18), a discussion of inhibitors of
(cid:2)-lactamase activity (Chapter 19), and assays for aminoglycoside-modifying
enzymes (Chapter 20).
The chapters in the book describe in detail specific methods that can be
used to identify and assay these targets. As additional new compounds are
identifiedandnewtargetsindifferentmicroorganismsarefound,thesemethods
will aid in testing the antibacterial potential of these products. It is anticipated
that this book will be primarily useful to laboratory investigators in academic,
pharmaceutical, and medical institutions.
W. Scott Champney
Contents
Preface............................................................... v
Contributors........................................................... ix
1 Biocomputational Strategies for Microbial Drug Target Identification
Kishore R. Sakharkar, Meena K. Sakharkar,
and Vincent T. K. Chow........................................ 1
2 Methods to Assay Inhibitors of DNA Gyrase and Topoisomerase
IV Activities
L. Mark Fisher and Xiao-Su Pan................................... 11
3 A Method to Assay Inhibitors of DNA Polymerase IIIC Activity
Michelle M. Butler and George E. Wright......................... 25
4 Methods to Identify and Characterize Inhibitors of Bacterial RNA
Polymerase
A. Simon Lynch and Qun Du...................................... 37
5 Methods to Assay Inhibitors of tRNA Synthetase Activity
Dieter Beyer, Hein Peter Kroll, and Heike Brötz-Oesterhelt....... 53
6 Three Methods to Assay Inhibitors of Ribosomal Subunit Assembly
W. Scott Champney............................................... 63
7 Inhibition of Chaperone-Dependent Bacterial Ribosome Biogenesis
Abdalla Al Refaii and Jean-Hervé Alix............................. 75
8 Assays for the Identification of Inhibitors Targeting Specific
Translational Steps
Letizia Brandi, John Dresios, and Claudio O. Gualerzi............. 87
9 SPARK: A New Peptidyl Transferase Activity Assay
Alexander S. Mankin and Norbert Polacek........................ 107
10 High-Throughput Screening of Peptide Deformylase Inhibitors
Kiet T. Nguyen and Dehua Pei.................................... 117
11 A Method to Assay Penicillin-Binding Proteins
Michael J. Pucci and Thomas J. Dougherty........................ 131
12 A Method to Assay Inhibitors of Lipopolysaccharide Synthesis
Marcy Hernick and Carol A. Fierke............................... 143
vii
viii Contents
13 Methods for Assessing the Structure and Function of Cationic
Antimicrobial Peptides
Michelle Pate and Jack Blazyk.................................... 155
14 Flow Cytometry of Bacterial Membrane Potential and Permeability
Howard M. Shapiro...............................................175
15 Bacterial Efflux Pump Inhibitors
Barbara J. Kamicker, Michael T. Sweeney, Frank Kaczmarek,
Fadia Dib-Hajj, Wenchi Shang, Kim Crimin, Joan Duignan,
and Thomas D. Gootz..........................................187
16 Mycobacterium tuberculosis (cid:2)-Ketoacyl Acyl Carrier Protein
Synthase III (mtFabH) Assay: Principles and Method
Sarbjot Sachdeva and Kevin A. Reynolds.......................... 205
17 Screening for Compounds That Affect the Interaction Between
Bacterial Two-Component Single Transduction Response
Regulator Protein and Cognate Promoter DNA
Matthew G. Erickson, Andrew T. Ulijasz, and Bernard Weisblum.. 215
18 The Activity of rRNA Resistance Methyltransferases Assessed
by MALDI Mass Spectrometry
Stephen Douthwaite, Rikke Lind Jensen, and Finn Kirpekar........ 223
19 Assays for (cid:2)-Lactamase Activity and Inhibition
Thammaiah Viswanatha, Laura Marrone, Valerie Goodfellow,
and Gary I. Dmitrienko........................................ 239
20 Studies of Enzymes That Cause Resistance to Aminoglycoside
Antibiotics
Engin H. Serpersu, Can Özen, and Edward Wright................ 261
Index................................................................. 273
Contributors
Jean-Hervé Alix • Institute de Biologie Physico-Chimique, UPR 9073 du CNRS,
University Paris 7 – Denis Diderot, Paris, France
Abdulla Al Refaii • Institute de Biologie Physico-Chimique, UPR 9073 du CNRS,
University Paris 7 – Denis Diderot, Paris, France
Dieter Beyer • Bayer HealthCare AG, Wuppertal, Germany
JackBlazyk • DepartmentofBiomedicalSciences,OhioUniversityCollegeofOsteo-
pathic Medicine, Athens, OH
Letizia Brandi • Department of Biology MCA, University of Camerino, Camerino
(MC), Italy
Heike Brötz-Oesterhelt • AiCuris GmbH & Co. KG, Wuppertal, Germany
Michelle M. Butler • Microbiotix Inc., Worcester, MA
W. Scott Champney • Department of Biochemistry and Molecular Biology, East
Tennessee State University, Johnson City, TN
Vincent T. K. Chow • Human Genome Laboratory, Department of Microbiology,
Yong Loo Lin School of Medicine, National University of Singapore, Singapore
Kim Crimin • Pfizer Global R & D, MS, Groton, CT
Fadia Dib-Hajj • Pfizer Global R & D, MS, Eastern Point Road, Groton, CT
GaryI.Dmitrienko • DepartmentofChemistry,UniversityofWaterloo,W.Waterloo,
Ontario, Canada
JohnDresios • DepartmentofNeurobiology,ScrippsResearchInstitute,LaJolla,CA
Thomas J. Dougherty • Pfizer Global Research and Development, Pfizer, Inc.,
Groton, CT
StephenDouthwaite • DepartmentofBiochemistryandMolecularBiology,University
of Southern Denmark, Campusvej, Denmark
Qun Du • Cumbre Pharmaceuticals Inc., Dallas, TX
Joan Duignan • Pfizer Global R & D, MS, Groton, CT
MatthewG.Erickson • DepartmentofPharmacology,UniversityofWisconsinMedical
School, Madison, WI
Carol A. Fierke • Department of Chemistry, University of Michigan, Ann Arbor, MI
L. Mark Fisher • Director, Molecular and Metabolic Signalling Centre, Division
of Basic Medical Sciences, St George’s, University of London, Cranmer Terrace,
London, UK
ValerieGoodfellow • DepartmentofChemistry,UniversityofWaterloo,W.,Waterloo,
Ontario, Canada
Thomas D. Gootz • Pfizer Global R & D, MS, Groton, CT
ix
x Contributors
ClaudioO.Gualerzi • DepartmentofBiologyMCA,UniversityofCamerino,Camerino
(MC), Italy
Marcy Hernick • Department of Biochemistry, Virginia Tech, Blacksburg, VA
Rikke Lind Jensen • Department of Biochemistry and Molecular Biology, University
of Southern Denmark, Campusvej, Denmark
Barbara J. Kamicker • Pfizer Global R & D, MS, Groton, CT
Frank Kaczmarek • Pfizer Global R & D, MS, Groton, CT
Finn Kirpekar • Department of Biochemistry and Molecular Biology, University of
Southern Denmark, Campusvej, Denmark
Hein Peter Kroll • Bayer HealthCare AG, Wuppertal, Germany
A. Simon Lynch • Cumbre Pharmaceuticals Inc., Dallas, TX
Alexander S. Mankin • Center for Pharmaceutical Biotechnology, University of
Illinois, Chicago, IL
Laura Marrone • Department of Chemistry, University of Waterloo, W., Waterloo,
Ontario, Canada
Kiet T. Nguyen • Wadsworth Center, New York State Department of Health, CMS,
Albany, NY
Can Özen • Graduate School of Genome Science and Technology, University of
Tennessee, Knoxville, TN
Michelle Pate • Department of Biomedical Sciences, Ohio University College of
Osteopathic Medicine, Athens, OH
Xiao-Su Pan • Department of Basic Medical Sciences, St George’s, University of
London, Cranmer Terrace, London, UK
Dehua Pei • Department of Chemistry and Ohio State Biochemistry Program, The
Ohio State University, Columbus, OH
NorbertPolacek • InnsbruckBiocenter,DivisionofGenomicsandRNomics,Innsbruck
Medical University, Innsbruck, Austria
Michael J. Pucci • Achillion Pharmaceuticals, New Haven, CT
Kevin A. Reynolds • Portland State University, Portland, OR
Sarbjot Sachdeva • Portland State University, Portland, OR
Kishore R. Sakharkar • Human Genome Laboratory, Department of Microbiology,
Yong Loo Lin School of Medicine, National University of Singapore, Singapore
Meena K. Sakharkar • Nanyang Centre for Supercomputing and Visualization,
Nanyang Technological University, Singapore
Engin H. Serpersu • Department of Biochemistry, Cellular and Molecular Biology,
University of Tennessee, Knoxville, TN
Howard M. Shapiro • The Center for Microbial Cytometry and Howard M. Shapiro,
M.D., P.C., West Newton, MA
Wenchi Shang • Pfizer Global R & D, MS, Eastern Point Road, Groton, CT
Michael T. Sweeney • Pfizer Global R & D, MS, Eastern Point Road Groton, CT
Andrew T. Ulijasz • Department of Pharmacology, University of Wisconsin Medical
School, Madison, WI
