High-Throughput Screening in Drug Discovery (Methods and Principles in Medicinal Chemistry)

362 Pages·2006·14.13 MB·English

by  Jörg Hüser (Editor)

High-ThroughputScreening
inDrugDiscovery
Editedby
JçrgH(cid:2)ser
Methods and Principles in Medicinal Chemistry
EditedbyR.Mannhold,H.Kubinyi,G.Folkers
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V
Foreword
Random screening of comprehensive compound collections constitutes a major
source of novel lead structures reflected by industry’s ongoing commitment to
investinextensivecompoundlibrariesandscreeningtechnologies.Duringthelast
decade, High-Throughput Screening (HTS)has evolved tobecome an innovative
multidisciplinary branch in biological chemistry combining aspects of medicinal
chemistry, biology, and laboratory automation. While basic medicinal chemistry
techniques and views are largely conserved throughout industry, HTS strategies
differ to a great extend. Each strategy can be justified by scientific rationale.
However, it is also the result of different scientific backgrounds, different ther-
apeuticareas,differenttechnicalexpertisewithinagroup,anddifferentwaysHTS
isintegratedwithintheoveralldiscoveryprocessinagivenorganization.Formost
strategies,thecloseinterrelationbetweenHTSandthemoleculartargetapproach
todrugdiscoveryrendersthevalidityofadiseaselinkforaselectedbiomolecular
targetanessentialprerequisiteforsuccess.Asaconsequence,acriticalassessment
of HTS has to incorporate also reflections on the discovery process from target
selectiontoappropriatescreeningcascades.Adifferentapproachemployingphe-
notypic readouts, e.g. cell proliferation, has a long tradition in screening, partic-
ularlyforchemotherapeuticprinciplesincancerandantiinfectivesresearch.Sim-
ilarly, chemical genetics makes use of small molecule perturbation of specific
cellularresponsestounraveltheunderlyinggeneandpathwaysfunction.Within
thelaterparadigm,High-ThroughputScreeningtechniqueshavegainedincreas-
ingrelevancealsoinacademicresearch.
Thecurrentbookpresentsacollectionofreview-stylepaperswrittenbyexpertsin
thefieldintendedtoprovideinsightsintoselectedaspectsoftheexperimentallead
discovery process in High-Throughput Screening. It is by no means claimed to
comprehensivelycovertheentirefield.Anumberofaspectshavebeendiscussedin
previous volumes within this series on “Methods and Principles in Medicinal
Chemistry”. It complements this book series by illustrating HTS as one of the
technologies of great relevance to the medicinal chemist and molecular pharma-
cologistworkinginpharmaceuticaloracademicresearch.
I am personally thankful to the Series Editors not only for providing the
opportunity to present High-Throughput Screening within a single dedicated
VI Foreword
volume, but also for their patience during the preparation of this volume. In
addition,thecontinuoussupportofmycolleagues,StefanMundt,NilsGriebenow
andPeterNell,isgratefullyacknowledged.
Wuppertal,July2006
JçrgH(cid:3)ser
VII
List of Contents
Preface XIV
ListofContributors XVI
PartI ConceptofScreening
1 ChemicalGenetics:UseofHigh-throughputScreeningtoIdentify
Small-moleculeModulatorsofProteinsInvolvedinCellularPathways
withtheAimofUncoveringProteinFunction
SaL.Chiang
1.1 Introduction 1
1.2 ClassicalandChemicalGenetics 1
1.2.1 ForwardandReverseScreens 3
1.3 IdentifyingBioactiveMolecules 4
1.4 TargetIdentification 5
1.4.1 Hypothesis-drivenTargetIdentification 5
1.4.2 Affinity-basedTargetIdentification 6
1.4.3 GenomicMethodsofTargetIdentification 7
1.4.4 ProteomicMethods 9
1.5 DiscoveryforBasicResearchVersusPharmacotherapyGoals 10
1.6 ChemicalGeneticScreensintheAcademicSetting 11
1.7 Conclusions 12
2 High-throughputScreeningforTargetedLeadDiscovery
JçrgH(cid:2)ser,EmanuelLohrmann,BerndKalthof,NilsBurkhardt,
UlfBr(cid:2)ggemeier,andMartinBechem
2.1 ChemicalLibrariesforHigh-throughputScreening 15
2.2 PropertiesofLeadStructures 17
2.3 ChallengestoHigh-throughputScreening 19
2.4 AssayTechnologiesforHigh-throughputScreening 21
2.5 LaboratoryAutomation 24
2.6 FromTargetSelectiontoConfirmedHits–theHTSWorkflowandits
Vocabulary 25
VIII ListofContents
2.7 SeparatingSpecificModulatorsfromOff-TargetEffects 29
2.8 DataAnalysisandScreeningResults 32
2.9 Conclusions 34
PartII AutomationTechnologies
3 ToolsandTechnologiesthatFacilitateAutomatedScreening
JohnComley
3.1 Introduction–theNecessitytoAutomate 37
3.1.1 CompoundLibraries 37
3.1.2 TargetsandDataPoints 38
3.1.3 MainIssuesFacingHTSGroupsToday 38
3.1.4 BenefitsofMiniaturization 39
3.1.5 BenefitsofAutomatedHTS 39
3.1.6 ScreeningStrategies 40
3.1.7 UltraHTS(UHTS) 40
3.2 SampleCarriers 41
3.2.1 ABriefHistoryoftheMicroplate 41
3.2.2 MicroplateUsageToday 41
3.2.3 MicroplateArrays 42
3.2.4 Non-microplateAlternatives 43
3.2.4.1 Labchips 43
3.2.4.2 LabCDs 43
3.2.4.3 LabBrick 44
3.2.4.4 ArrayedCompoundScreening 44
3.3 LiquidHandlingTools 45
3.3.1 MainMicroplateDispenseMechanisms 45
3.3.1.1 PinTools 45
3.3.1.2 AirandPositiveDisplacement 45
3.3.1.3 Peristaltic 46
3.3.1.4 Solenoid-syringe 47
3.3.1.5 Solenoid-pressurebottle 47
3.3.1.6 CapillarySipper 48
3.3.1.7 Piezoelectric 48
3.3.1.8 AcousticTransducer 48
3.3.2 HTSLiquidHandlingApplicationsandDispensingTechnologies
Used 49
3.3.2.1 BulkReagentandCellAddition 49
3.3.2.2 CompoundReformattingandNanoliterDispensing 50
3.3.2.3 CherryPickingandSerialDilution 51
3.3.2.4 MicroplateWashing 52
3.4 DetectionTechnologies 53
3.4.1 MainDetectionModalitiesUsedinHTS 53
3.4.2 PlateReaders 54
3.4.3 PlateImagers 55
ListofContents IX
3.4.3.1 Macro-imaging 56
3.4.3.2 Micro-imaging 57
3.4.4 DispenseandReadDevices 60
3.4.5 OtherDetectionTechnologies 60
3.4.6 AutomationofDetectionTechnologies 61
3.4.7 PotentialSourcesofReadingError 61
3.5 LaboratoryRobotics 62
3.5.1 TraditionalWorkstations 64
3.5.2 RoboticSampleProcessors 64
3.5.3 PlateStorageDevices 64
3.5.4 PlateMovingDevices 65
3.5.5 FullyIntegratedRoboticSystems 65
3.5.6 TurnkeyWorkstations 66
3.5.7 AutomatedCellCultureSystems 66
3.5.8 CompoundManagementSystems 67
3.5.8.1 CurrentPracticeinCompoundManagement 67
3.5.8.2 Plate-basedversusTube-basedLiquidCompoundStorage 68
3.5.8.3 AssociatedAutomatedInstrumentation 70
3.5.8.4 SampleIntegrityandQCTesting 70
PartIII AssayTechnologies
4 FunctionalCell-basedAssaysforTargetedLeadDiscovery
inHigh-throughputScreening
JçrgH(cid:2)ser,BerndKalthof,andJochenStrayle
4.1 Introduction 75
4.2 ReporterGeneTechnologies 78
4.3 MembranePotentialIndicators 82
4.4 Ca2+Indicators 88
4.5 Conclusions 90
5 BiochemicalAssaysforHigh-throughputScreening
WilliamD.Mallender,MichaelBembenek,LawrenceR.Dick,MichaelKuranda,
PingLi,SaurabhMenon,EneidaPardoandTomParsons
5.1 GeneralConsiderationsforBiochemicalHigh-throughputScreening 93
5.2 ExpressionandPurificationofRecombinantEnzymes 95
5.2.1 DesignofExpressionConstructs 98
5.2.2 ExpressionAssessmentandOptimization 99
5.2.3 Purification 99
5.3 Peptidases 102
5.3.1 ApplicationofFluorogenicSubstratestoConfigurePeptidase
Screens 102
5.3.2 TheValueofContinuousAssays 107
5.4 Oxidoreductases 107
5.4.1 NAD(P)-dependentOxidoreductases 108