New 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 ©2008HumanaPressInc. 999RiverviewDrive,Suite208 Totowa,NewJersey07512 www.humanapress.com Allrightsreserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic,mechanical,photocopying,microfilming,recording,orotherwisewithoutwrittenpermissionfromthePublisher. MethodsinMolecularMedicineTMisatrademarkofTheHumanaPressInc. Allpapers,comments,opinions,conclusions,orrecommendationsarethoseoftheauthor(s),anddonotnecessarilyreflect theviewsofthepublisher. Thispublicationisprintedonacid-freepaper.(cid:2)(cid:3) ANSIZ39.48-1984(AmericanStandardsInstitute)PermanenceofPaperforPrintedLibraryMaterials. ProductionEditor:ChristinaM.Thomas Coverdesignby:W.ScottChampney Coverillustration:Thecovershowsthechemicalstructuresofanumberofantibioticscurrentlyusedagainstsomeofthe targetsdescribedinthisbook. PhotocopyAuthorizationPolicy: Authorizationtophotocopyitemsforinternalorpersonaluse,ortheinternalorpersonaluseofspecificclients,isgranted byHumanaPressInc.,providedthatthebasefeeofUS$30percopyispaiddirectlytotheCopyrightClearanceCenterat 222RosewoodDrive,Danvers,MA01923.Forthoseorganizationsthathavebeengrantedaphotocopylicensefromthe CCC,aseparatesystemofpaymenthasbeenarrangedandisacceptabletoHumanaPressInc.Thefeecodeforusersof theTransactionalReportingServiceis:[978-1-58829-915-4/08$30]. PrintedintheUnitedStatesofAmerica.10987654321 LibraryofCongressCataloginginPublicationData:2007933473 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
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