Preface .................................................. XIV
List of Contributors ..................................... XVI
Part I Concept of Screening
1 Chemical Genetics: Use of High-throughput Screening to
Identify Small-molecule Modulators of Proteins Involved
in Cellular Pathways with the Aim of Uncovering Protein
Function
So L. Chiang
1.1 Introduction ............................................... 1
1.2 Classical and Chemical Genetics
1.2.1 Forward and Reverse Screens ......................... 3
1.3 Identifying Bioactive Molecules ............................ 4
1.4 Target Identification ...................................... 5
1.4.1 Hypothesis-driven Target Identification ............. 5
1.4.2 Affinity-based Target Identification ................ 6
1.4.3 Genomic Methods of Target Identification ............ 7
1.4.4 Proteomic Methods ................................... 9
1.5 Discovery for Basic Research Versus Pharmacotherapy
Goals ..................................................... 10
1.6 Chemical Genetic Screens in the Academic Setting .......... 11
1.7 Conclusions ............................................... 12
2 High-throughput Screening for Targeted Lead Discovery
Jörg Hüser, Emanuel Lohrmann, Bernd Kalthof, Nils
Burkhardt, Ulf Brüggemeier, and Martin Bechern
2.1 Chemical Libraries for High-throughput Screening .......... 15
2.2 Properties of Lead Structures ............................. 17
2.3 Challenges to High-throughput Screening ................... 19
2.4 Assay Technologies for High-throughput Screening .......... 21
2.5 Laboratory Automation ..................................... 24
2.6 From Target Selection to Confirmed Hits - the HTS
Workflow and its Vocabulary ............................... 25
2.7 Separating Specific Modulators from Off-Target Effects .... 29
2.8 Data Analysis and Screening Results ....................... 32
2.9 Conclusions ............................................... 34
Part II Automation Technologies
3 Tools and Technologies that Facilitate Automated
Screening
John Comley
3.1 Introduction - the Necessity to Automate ................. 37
3.1.1 Compound Libraries ................................ 37
3.1.2 Targets and Data Points ........................... 38
3.1.3 Main Issues Facing HTS Groups Today ............... 38
3.1.4 Benefits of Miniaturization ....................... 39
3.1.5 Benefits of Automated HTS ......................... 39
3.1.6 Screening Strategies .............................. 40
3.1.7 Ultra HTS (UHTS) .................................. 40
3.2 Sample Carriers .......................................... 41
3.2.1 A Brief History of the Microplate ................. 41
3.2.2 Microplate Usage Today ............................ 41
3.2.3 Microplate Arrays ................................. 42
3.2.4 Non-microplate Alternatives ....................... 43
3.2.4.1 Labchips ................................. 43
3.2.4.2 LabCDs ................................... 43
3.2.4.3 LabBrick ................................. 44
3.2.4.4 Arrayed Compound Screening ............... 44
3.3 Liquid Handling Tools .................................... 45
3.3.1 Main Microplate Dispense Mechanisms ............... 45
3.3.1.1 Pin Tools ................................ 45
3.3.1.2 Air and Positive Displacement ............ 45
3.3.1.3 Peristaltic .............................. 46
3.3.1.4 Solenoid-syringe ......................... 47
3.3.1.5 Solenoid-pressure bottle ................. 47
3.3.1.6 Capillary Sipper ......................... 48
3.3.1.7 Piezoelectric ............................ 48
3.3.1.8 Acoustic Transducer ...................... 48
3.3.2 HTS Liquid Handling Applications and Dispensing
Technologies Used ................................. 49
3.3.2.1 Bulk Reagent and Cell Addition ........... 49
3.3.2.2 Compound Reformatting and Nanoliter
Dispensing ............................... 50
3.3.2.3 Cherry Picking and Serial Dilution ....... 51
3.3.2.4 Microplate Washing ....................... 52
3.4 Detection Technologies ................................... 53
3.4.1 Main Detection Modalities Used in HTS ............. 53
3.4.2 Plate Readers ..................................... 54
3.4.3 Plate Imagers ..................................... 55
3.4.1 Macro-imaging ..................................... 56
3.4.3.2 Micro-imaging ............................ 57
3.4.4 Dispense and Read Devices ......................... 60
3.4.5 Other Detection Technologies ...................... 60
3.4.6 Automation of Detection Technologies .............. 61
3.4.7 Potential Sources of Reading Error ................ 61
3.5 Laboratory Robotics ...................................... 62
3.5.1 Traditional Workstations .......................... 64
3.5.2 Robotic Sample Processors ......................... 64
3.5.3 Plate Storage Devices ............................. 64
3.5.4 Plate Moving Devices .............................. 65
3.5.5 Fully Integrated Robotic Systems .................. 65
3.5.6 Turnkey Workstations .............................. 66
3.5.7 Automated Cell Culture Systems .................... 66
3.5.8 Compound Management Systems ....................... 67
3.5.8.1 Current Practice in Compound Management .. 67
3.5.8.2 Plate-based versus Tube-based Liquid
Compound Storage ......................... 68
3.5.8.3 Associated Automated Instrumentation ..... 70
3.5.8.4 Sample Integrity and QC Testing .......... 70
Part III Assay Technologies
4 Functional Cell-based Assays for Targeted Lead
Discovery in High-throughput Screening
Jörg Hüser, Bernd Kalthof and Jochen Strayle
4.1 Introduction ............................................ 75
4.2 Reporter Gene Technologies .............................. 78
4.3 Membrane Potential Indicators ........................... 82
4.4 Ca2+ Indicators ......................................... 88
4.5 Conclusions ............................................. 90
5 Biochemical Assays for High-throughput Screening
William D. Mattender, Michael Bembenek, Lawrence
R. Dick, Michael Kuranda, Ping Li, Saurabh Menon,
Eneida Pardo and Tom Parsons
5.1 General Considerations for Biochemical High-
throughput Screening .................................... 93
5.2 Expression and Purification of Recombinant Enzymes ...... 95
5.2.1 Design of Expression Constructs .................. 98
5.2.2 Expression Assessment and Optimization ........... 99
5.2.3 Purification ..................................... 99
5.3 Peptidases ............................................. 102
5.3.1 Application of Fluorogenic Substrates to
Configure Peptidase Screens ..................... 102
5.3.2 The Value of Continuous Assays .................. 107
5.4 Oxidoreductases ........................................ 107
5.4.1 NAD(P)-dependent Oxidoreductases ................ 108
5.4.2 Non-NAD(P) Cofactor-dependent Oxidoreductases ... 110
5.4.3 Oxidases or Oxygen-utilizing Oxidoreductases .... 111
5.4.4 General Considerations .......................... 113
5.5 Transferases, Synthetases and Lipid-modifying Enzymes .. 114
5.5.1 Streptavidin-Biotin Capture ..................... 114
5.5.2 Ionic Capture ................................... 117
5.5.3 Hydrophobic Capture ............................. 117
5.6 Kinases ................................................ 120
5.6.1 Streptavidin-Biotin Capture ..................... 120
5.6.2 Homogeneous Time-resolved Fluorescence (HTRF) ... 122
5.6.3 Pyruvate Kinase-Lactate Dehydrogenase Assay
System .......................................... 122
5.7 Pitfalls and Reasons for Assay Development Failures .... 125
6 Image-based High-content Screening - A View from
Basic Sciences
Peter Lipp and Lars Kaestner
6.1 Introduction ........................................... 129
6.2 HCS Systems Employing Confocal Optical Technologies .... 132
6.3 Single-point Scanning Technology ....................... 134
6.4 Line Scanning Technology ............................... 136
6.5 Multi-beam Technology .................................. 138
6.6 Structured Illumination ................................ 145
6.7 Summary and Perspectives ............................... 147
Part IV Data Analysis
7 Methods for Statistical Analysis, Quality Assurance
and Management of Primary High-throughput Screening
Data
Hanspeter Cubler
7.1 Introduction ........................................... 151
7.1.1 Overview ........................................ 151
7.1.2 Problems during the Analysis of Primary HTS
Data ............................................ 151
7.2 Statistical Considerations in Assay Development ........ 155
7.3 Data Acquisition, Data Preprocessing, and HTS Data
Analysis Environment ................................... 158
7.4 Data Normalization ..................................... 160
7.5 Robust Statistics in HTS Data Analysis ................. 163
7.5.1 The General Problem ............................. 163
7.5.2 Threshold Setting - a Simple Model .............. 164
7.5.3 Threshold Setting - a Complex Model ............. 165
7.5.4 The Most Important Robust Estimation Methods
for Data Summaries .............................. 165
7.5.5 An Illustrative Example: Performance of
Location and Scale Estimators on Typical HTS
Data Activity Distributions ..................... 167
7.5.6 A Robust Outlier Detection Method ............... 168
7.5.7 Outlier-resistant Versions of Simple HTS Data
Quality Indicators .............................. 170
7.6 Measures of HTS Data Quality, Signaling of Possible
QC Problems, Visualizations ............................ 170
7.6.1 Trends and Change Points ........................ 171
7.6.2 Positional Effects - Summary Statistics and
Views ........................................... 175
7.6.3 Positional Effects - Heat Maps, Trellis Plots,
and Assay Maps .................................. 177
7.6.4 Distribution Densities - Histograms, Smoothed
Distributions ................................... 150
7.6.5 Numerical Diagnostics and Fully Automated QC
Assessment ...................................... 181
7.6.6 Possible Sources of Systematic Errors and
Trends .......................................... 182
7.7 Correction of Position-dependent Response Effects ...... 182
7.7.1 The General Problem ............................. 182
7.7.2 Plate Averaging (Multiple Plates) ............... 184
7.7.3 Median Polish Smoothing (Single Plates,
Multiple Plates) ................................ 185
7.7 A Change Point Detection (Multiple Plates, Plate
Sequences) ............................................. 186
7.7.5 Parametric (Polynomial) Surface Fitting
(Single Plates, Multiple Plates) ................ 187
7.7.6 Nonparametric Surface Fitting, Local
Regression, and Smoothing (Single Plates,
Multiple Plates) ................................ 189
7.7.7 Expansion into Orthogonal Basis Functions
(Single Plates) ................................. 190
7.7.8 Empirical Orthogonal Function (EOF) Analysis,
Singular Value Decomposition (SVD) (Multiple
Plates) ......................................... 191
7.7.9 Some Remarks on the Correction of Individual
Plates versus Complete Plate Sets ............... 194
7.7.10 Position-dependent Correction of Background
Response Surface ................................ 195
7.8 Hit Identification and Hit Scoring ..................... 199
7.9 Conclusion ............................................. 201
8 Chemoinformatic Tools for High-throughput Screening
Data Analysis
Peter C. Nell and Stefan M. Mundt
8.1 Introduction ........................................... 207
8.1.1 Definition of Chemoinformatics .................. 208
8.1.2 High-throughput Screening ....................... 208
8.1.2.1 Random Screening ....................... 209
8.1.2.2 Sequential Screening ................... 209
8.2 Workflow of High-throughput Screening and Use of
Chemoinformatics ....................................... 211
8.3 Chemoinformatic Methods Used in HTS Workflow ........... 214
8.3.1 Substructure Search/Similarity Search ........... 214
8.3.1.1 Structural Descriptors/Fingerprints .... 215
8.3.1.2 Measures of Similarity ................. 217
8.3.2 Clustering ...................................... 218
8.3.2.1 Hierarchical Clustering ................ 219
8.3.2.2 Nonhierarchical Clustering ............. 219
8.3.2.3 Partitioning Methods ................... 220
8.3.2.4 Principal Components Analysis .......... 221
8.3.3 Maximum Common Substructure - Distill ........... 222
8.3.4 Mode of Action - Profiling ...................... 223
8.3.5 Artificial Neural Networks (ANNs) ............... 224
8.3.6 Decision Trees/Recursive Partitioning ........... 226
8.3.7 Reduced Graph-based Methods ..................... 226
8.3.7.1 Reduced Graph Theory Analysis:
Baytree ................................ 227
8.3.8 Fragment-based Methods - Structural Units
Analysis ........................................ 230
8.4 Chemoinformatic Methods in the Design of a Screening
Library ................................................ 231
8.4.1 Drag and Lead Likeness .......................... 231
8.4.2 ADME Parameters ................................. 233
8.4.2.1 Absorption ............................. 233
8.4.2.2 Distribution ........................... 234
8.4.2.3 Metabolism ............................. 234
8.4.2.4 Excretion .............................. 235
8.4.2.5 Toxicity ............................... 235
8.4.3 Diversity ....................................... 236
8.5 Integrated Software Packages ........................... 238
8.5.1 Commercially Available Packages ................. 239
8.5.1.1 Accelrys: DIVA® ........................ 239
8.5.1.2 BioSolveIT: HTSview .................... 239
8.5.1.3 SciTegic: Pipeline PilotTM ............. 240
8.5.1.4 Bioreason: ClassPharmerTM Suite ......... 241
8.5.1.5 Spotfire Lead Discovery ................ 242
8.5.1.6 LeadScope .............................. 243
8.5.1.7 OmniViz ................................ 244
8.5.1.8 SARNavigator ........................... 245
8.5.2 In-house Packages ............................... 246
8.6 Conclusions ............................................ 247
9 Combinatorial Chemistry and High-throughput Screening
Roger A. Smith and Nils Criebenow
9.1 Introduction ........................................... 259
9.2 Categories of Compound Libraries for High-throughput
Screening .............................................. 260
9.3 Synthesis Techniques and Library Formats ............... 261
9.3.1 Solid-phase Synthesis ........................... 262
9.3.1.1 Parallel Solid-phase Synthesis
Techniques and Tools ................... 265
9.3.1.2 Pool/Split Techniques with Encoding .... 265
9.3.2 Solution-phase Synthesis ........................ 269
9.3.2.1 Polymer-supported Reagents and
Scavengers ............................. 269
9.3.2.2 Extraction Techniques for
Purification ........................... 271
9.3.2.3 Purification by Chromatography ......... 273
9.3.3 Library Formats ................................. 273
9.3.3.1 One-bead One-compound Libraries ........ 274
9.3.3.2 Pre-encoded Libraries .................. 275
9.3.3.3 Spatially Addressable Libraries ........ 276
9.4 Library Design and Profiling Approaches ................ 277
9.5 Impact of Combinatorial Libraries on Drug Discovery
9.5.1 Lead Identification ............................. 278
9.5.2 Lead Optimization ............................... 283
9.5.3 Clinical Drug Candidates ........................ 286
9.6 Conclusion ............................................. 290
10 High-throughput Screening and Data Analysis
Jeremy S. Caldwell and Jeff Janes
10.1 Introduction ........................................... 297
10.2 Analysis of Cellular Screening Data
10.2.1 Quality Control and Analysis .................... 298
10.2.2 Enrichment for Hits ............................. 301
10.2.3 Meta-data Analysis .............................. 302
10.3 Massively Parallel Cellular Screens
10.3.1 Data Analysis of Multidimensional Datasets ...... 307
10.3.2 Multidimensional Cellular Profiling for MOA
prediction ......................................
10.3.3 Cellular Profiling in Lead Exploration .......... 314
10.4 Systematic Serendipity ................................. 317
10.5 Conclusion ............................................. 320
Appendix ............................................... 323
Index .................................................. 333
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