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Book Details
Abstract
High throughput screening remains a key part of early stage drug and tool compound discovery, and methods and technologies have seen many fundamental improvements and innovations over the past 20 years. This comprehensive book provides a historical survey of the field up to the current state-of-the-art. In addition to the specific methods, this book also considers cultural and organizational questions that represent opportunities for future success.
Following thought-provoking foreword and introduction from Professor Stuart Schreiber and the editors, chapters from leading experts across academia and industry cover initial considerations for screening, methods appropriate for different goals in small molecule discovery, newer technologies that provide alternative approaches to traditional miniaturization procedures, and practical aspects such as cost and resourcing. Within the context of their historical development, authors explain common pitfalls and their solutions.
This book will serve as both a practical reference and a thoughtful guide to the philosophy underlying technological change in such a fast-moving area for postgraduates and researchers in academia and industry, particularly in the areas of chemical biology, pharmacology, structural biology and assay development.
Table of Contents
| Section Title | Page | Action | Price |
|---|---|---|---|
| Cover | Cover | ||
| Contents | xi | ||
| Foreword: Transforming Medicine by Innovating the Science of Therapeutics | v | ||
| Preface | vii | ||
| Chapter 1 HTS Methods: Assay Design and Optimisation | 1 | ||
| 1.1 Introduction | 1 | ||
| 1.2 HTS at AstraZeneca | 2 | ||
| 1.2.1 Criteria and Acceptance | 3 | ||
| 1.2.2 Robustness/Reliability | 4 | ||
| 1.2.3 Analysing Data to Define Robustness/Reliability | 8 | ||
| 1.2.4 As Simple to Run as Possible | 9 | ||
| 1.2.5 Assay Validation | 10 | ||
| 1.3 Summary | 14 | ||
| References | 14 | ||
| Chapter 2 Considerations Related to Small-molecule Screening Collections | 16 | ||
| 2.1 Introduction | 16 | ||
| 2.2 General Considerations Related to HTS Compound Collections | 17 | ||
| 2.2.1 Determination of Screening Objectives | 18 | ||
| 2.2.2 Size of HTS Compound Collections | 18 | ||
| 2.2.3 Chemical Diversity in Compound Collections | 19 | ||
| 2.2.4 Quality of Compounds in Screening Collections | 19 | ||
| 2.3 Sources of Compounds in Screening Collections | 20 | ||
| 2.3.1 Natural Products in Screening Collections | 20 | ||
| 2.3.2 Synthetic Drug-like Compounds | 23 | ||
| 2.3.3 Diverse Collections | 26 | ||
| 2.4 Performance of Compounds in Screening Collections | 29 | ||
| 2.4.1 Background | 29 | ||
| 2.4.2 Performance of Compounds from Different Sources | 29 | ||
| 2.4.3 Performance Diversity of Compound Collections | 31 | ||
| 2.4.4 Pan Assay Interference Compounds | 32 | ||
| 2.4.5 Dark Chemical Matter | 33 | ||
| 2.5 Conclusions and Discussion | 33 | ||
| References | 34 | ||
| Chapter 3 Combination Screening | 37 | ||
| 3.1 Introduction | 37 | ||
| 3.2 Measures of Synergy | 38 | ||
| 3.2.1 Bliss Independence Model of Synergy | 39 | ||
| 3.2.2 Loewe Additivity | 40 | ||
| 3.2.3 Other Measures | 43 | ||
| 3.2.4 Reconciling Measures of Synergy | 46 | ||
| 3.3 Design of Combination Experiments | 47 | ||
| 3.4 Statistical Inference of Combination Effects | 49 | ||
| 3.4.1 The Error Distribution | 50 | ||
| 3.4.2 Bootstrap Intervals | 51 | ||
| 3.4.3 Intervals for Bliss Independence | 52 | ||
| 3.4.4 Intervals for Loewe Additivity Interaction Index | 53 | ||
| 3.5 Null Hypothesis Significance Testing | 54 | ||
| 3.5.1 Significance Testing of Bliss | 54 | ||
| 3.5.2 Significance Test of Loewe Additivity | 55 | ||
| 3.6 Concluding Remarks | 56 | ||
| References | 56 | ||
| Chapter 4 Modern Biophysical Methods for Screening and Drug Discovery | 58 | ||
| 4.1 Introduction | 58 | ||
| 4.2 Physicochemical Properties and High Concentration Screening | 59 | ||
| 4.2.1 Physicochemical Properties of Chemical Libraries | 59 | ||
| 4.2.2 High Concentration Screening | 63 | ||
| 4.3 Differential Scanning Fluorimetry | 64 | ||
| 4.4 Surface Plasmon Resonance | 65 | ||
| 4.5 Mass Spectrometry Techniques | 67 | ||
| 4.5.1 Affinity Selection MS | 68 | ||
| 4.5.2 Affinity Chromatography Methods | 69 | ||
| 4.5.3 Protein MS | 70 | ||
| 4.6 NMR Spectroscopy | 70 | ||
| 4.6.1 Protein NMR | 72 | ||
| 4.6.2 Ligand Observed NMR | 73 | ||
| 4.7 Calorimetric Methods | 75 | ||
| 4.7.1 Differential Scanning Calorimetry | 76 | ||
| 4.7.2 Isothermal Titration Calorimetry | 77 | ||
| 4.8 X-Ray Crystallography | 77 | ||
| 4.9 Newer Methods on the Horizon | 78 | ||
| 4.10 Summary and Recommendations | 79 | ||
| Acknowledgments | 80 | ||
| References | 80 | ||
| Chapter 5 Genetic Perturbation Methods, from the ‘Awesome Power' of Yeast Genetics to the CRISPR Revolution | 87 | ||
| 5.1 Introduction | 87 | ||
| 5.2 Genetic Methodologies | 88 | ||
| 5.2.1 Random Mutagenesis | 88 | ||
| 5.2.2 Targeted Genome-wide Deletions (Homozygous/Heterozygous) | 90 | ||
| 5.2.3 Random Genome-wide Deletions (Homozygous/Heterozygous) | 91 | ||
| 5.2.4 RNA Interference | 92 | ||
| 5.2.5 CRISPR | 95 | ||
| 5.2.6 Overexpression | 104 | ||
| 5.2.7 Synthetic Biology | 104 | ||
| 5.3 Concluding Remarks | 105 | ||
| Acknowledgments | 105 | ||
| References | 105 | ||
| Chapter 6 Understanding Luminescence Based Screens | 117 | ||
| 6.1 Why Luminescence? An Introduction to Bioluminescent and Chemiluminescent Assays | 117 | ||
| 6.1.1 Overview of Common Luminescent Enzymes Employed in Assays | 119 | ||
| 6.1.2 Chemiluminescence in HTS Assays | 122 | ||
| 6.2 Considerations and Applications of Bioluminescent Assays | 123 | ||
| 6.2.1 Prevalence of Luciferase Inhibitors in Compound Libraries | 123 | ||
| 6.2.2 Mechanisms of Luciferase Inhibition | 124 | ||
| 6.2.3 Ligand Based Stabilization of Luciferases: Impact on RGA Results | 129 | ||
| 6.2.4 Methods to Mitigate Luciferase Inhibitors in RGAs: Counter-Screens and Orthogonal Assay Formats | 131 | ||
| 6.2.5 Luciferases as Post-translational Sensors | 133 | ||
| 6.2.6 Use of Luciferases in Biochemical Applications | 134 | ||
| 6.3 Considerations and Applications of Amplified Luminescent Proximity Homogenous Assays: AlphaScreen and AlphaLISA | 135 | ||
| 6.3.1 Example Protocols and Key Experiments | 135 | ||
| 6.3.2 Interferences with ALPHA Technology | 138 | ||
| 6.4 Conclusion | 138 | ||
| References | 139 | ||
| Chapter 7 High Throughput Screening Compatible Methods for Quantifying Protein Interactions in Living Cells | 143 | ||
| 7.1 Introduction | 143 | ||
| 7.2 Analysis of PPIs in Intact Cells | 145 | ||
| 7.2.1 Two-hybrid Systems | 145 | ||
| 7.2.2 Protein Fragment Complementation Technologies | 147 | ||
| 7.2.3 FRET for Analysis of PPIs | 150 | ||
| 7.3 Measuring Target Engagement in Cells | 153 | ||
| 7.3.1 Target Engagement by Protein Stability Analysis | 153 | ||
| 7.3.2 Real Time, Quantitative Analysis of Target Engagement via FRET | 155 | ||
| 7.4 Outlook | 159 | ||
| References | 159 | ||
| Chapter 8 Approaches to High Content Imaging and Multi-feature Analysis | 162 | ||
| 8.1 Introduction | 162 | ||
| 8.2 Imaging Hardware | 164 | ||
| 8.3 Image Analysis | 169 | ||
| 8.4 Quality Control of Image Acquisition and Well Level Data | 170 | ||
| 8.4.1 Quality Control of Image Acquisition | 170 | ||
| 8.4.2 Quality Control of Well Level Data | 171 | ||
| 8.5 Single Cell Analysis | 171 | ||
| 8.6 Analysis of Multiparametric Data | 173 | ||
| 8.6.1 Feature Selection and Dimensional Reduction | 174 | ||
| 8.6.2 Distance and Similarity | 174 | ||
| 8.7 Machine Learning: Supervised and Unsupervised Methods | 176 | ||
| 8.7.1 Supervised Learning (Classification) | 176 | ||
| 8.7.2 Unsupervised Learning (Clustering) | 178 | ||
| 8.8 Conclusion | 178 | ||
| References | 178 | ||
| Chapter 9 Pharmacological and Genetic Screening of Molecularly Characterized Cell Lines | 181 | ||
| 9.1 Introduction | 181 | ||
| 9.2 Cell Lines | 182 | ||
| 9.2.1 Applications in Cancer | 182 | ||
| 9.2.2 Mistaken Identities | 183 | ||
| 9.2.3 Authentication | 184 | ||
| 9.2.4 Molecular Characterizations | 184 | ||
| 9.3 In vitro Drug Screening towards Precision Medicine | 185 | ||
| 9.3.1 Drug Sensitivity Studies | 185 | ||
| 9.3.2 Molecular Predictors of Drug Response | 190 | ||
| 9.3.3 Datasets | 192 | ||
| 9.3.4 Drug Perturbation Studies | 196 | ||
| 9.3.5 Genetic Perturbation | 200 | ||
| 9.4 Conclusions and Outlook | 204 | ||
| References | 205 | ||
| Chapter 10 Multidimensional Profile Based Screening: Understanding Biology through Cellular Response Signatures | 214 | ||
| 10.1 Introduction | 214 | ||
| 10.2 Multidimensional Profiling through Multi-feature Measurement in a Single System | 216 | ||
| 10.2.1 LINCS Concept | 216 | ||
| 10.2.2 Gene Expression Profiling: CMap | 217 | ||
| 10.2.3 Proteomics Profiling and Other Measurements | 218 | ||
| 10.2.4 Cell Morphology Profiling: Cell Painting | 220 | ||
| 10.3 Multidimensional Profiling through Single Feature Measurement in Multiple Systems | 222 | ||
| 10.3.1 Expanding on the NCI-60: Building a Comprehensive Cell Viability Profile Matrix | 223 | ||
| 10.3.2 Current Logistical Constraints in Screening: Large Numbers of Cell Lines or Large Numbers of Compounds? | 223 | ||
| 10.3.3 PRISM: Multiplexed Cancer Cell Line HTS | 224 | ||
| 10.4 Analysis Approaches for using Cell Based Profiles to Identify Mechanism of Action | 225 | ||
| 10.4.1 NCI-60 COMPARE | 227 | ||
| 10.4.2 CMap: Computational Aspects | 227 | ||
| 10.5 Comprehensive Cell Line Profiling Requires Comprehensive Reference Annotations | 231 | ||
| 10.5.1 Importance of Compound Annotations | 231 | ||
| 10.5.2 Importance of Cell Line Annotations and Quality Control | 232 | ||
| 10.6 Summary and Needs for the Future | 234 | ||
| 10.6.1 Increasing the Number of Cell Lines for Screening and Call for Common Quality Control | 234 | ||
| 10.6.2 Increasing the Perturbagen Dimension to include Genetic Perturbagens | 235 | ||
| 10.7 Conclusion | 235 | ||
| Acknowledgments | 236 | ||
| References | 236 | ||
| Chapter 11 3D Cell Culture and Dish Based Organogenesis: Optimizing In vitro Cellular Physiology | 239 | ||
| 11.1 Introduction | 239 | ||
| 11.2 3D Culture Gels, Scaffolds and Bioprinting | 240 | ||
| 11.2.1 Hydrogels | 240 | ||
| 11.2.2 Scaffolds | 241 | ||
| 11.2.3 3D Bioprinting | 243 | ||
| 11.3 3D Culture and Drug Discovery | 244 | ||
| 11.4 Organs-on-Chips | 246 | ||
| 11.5 3D Cell Culture Limitations | 248 | ||
| 11.6 Conclusion | 249 | ||
| References | 249 | ||
| Chapter 12 Small-molecule-mediated Targeted Protein Degradation for Drug Discovery | 252 | ||
| 12.1 Introduction | 252 | ||
| 12.2 E3-ligase-targeting Heterobifunctional Degraders | 253 | ||
| 12.2.1 The State of the Art | 253 | ||
| 12.2.2 CRBN-recruiting Heterobifunctional Degraders | 254 | ||
| 12.2.3 VHL-recruiting Heterobifunctional Degraders | 257 | ||
| 12.2.4 The Generality of the Bifunctional Degrader Approach | 259 | ||
| 12.2.5 Towards the Mechanism of Action of Bifunctional Degraders | 260 | ||
| 12.2.6 Towards a Therapeutic | 261 | ||
| 12.2.7 Scope and Limitations | 262 | ||
| 12.3 Alternative Approaches to Small-molecule-mediated Targeted Protein Degradation | 266 | ||
| 12.3.1 Hydrophobic Tag | 267 | ||
| 12.3.2 Chaperone Mediated Autophagy | 267 | ||
| 12.3.3 Molecular Glue Degraders | 268 | ||
| 12.4 Identifying Suitable Targets for Small-molecule-mediated Protein Degradation | 269 | ||
| 12.5 Conclusion and Outlook | 269 | ||
| References | 270 | ||
| Chapter 13 Phenotypic Screens with Model Organisms | 275 | ||
| 13.1 Introduction to Large-scale Screens | 275 | ||
| 13.2 What Animal Models to Choose for a Screen? | 276 | ||
| 13.3 Problems and Questions Addressed with Phenotypic Screens | 282 | ||
| 13.3.1 Genetic Screens | 282 | ||
| 13.3.2 Small Bioactive Molecule Screens | 283 | ||
| 13.3.3 Genome Editing Assisted Targeted Screens | 284 | ||
| 13.3.4 Summary | 285 | ||
| 13.4 The Output of Large Scale Screens | 286 | ||
| 13.4.1 Features of Different Screen Models | 286 | ||
| 13.4.2 Behavioral Readout: Motion Index versus Complex Behavior | 286 | ||
| 13.4.3 Physiological Readout: Electrophysiology and Imaging | 288 | ||
| 13.5 Criteria, Sensitivity, Performance and Visualization of the Screen | 290 | ||
| 13.5.1 Defining a ‘‘Hit\": Variance and Dimensionality | 290 | ||
| 13.5.2 Assessing, Visualizing and Ranking Performance | 291 | ||
| 13.5.3 Current Limitations and the Future | 294 | ||
| Acknowledgments | 294 | ||
| References | 294 | ||
| Chapter 14 Encoded Compound Libraries to Accelerate Small-molecule Therapeutic Discovery | 303 | ||
| 14.1 Introduction | 303 | ||
| 14.2 Combinatorial Chemistry | 304 | ||
| 14.3 Encoding of Compound Libraries | 307 | ||
| 14.3.1 First-generation Encoding | 308 | ||
| 14.3.2 Second-generation Encoding | 310 | ||
| 14.3.3 DNA Encoded Small-molecule Libraries | 311 | ||
| 14.3.4 Associated Synthesis and Encoding | 312 | ||
| 14.3.5 Disassociated Synthesis and Encoding | 313 | ||
| 14.4 Screening of DNA Encoded Compound Libraries | 315 | ||
| 14.5 Data Processing and Analysis | 318 | ||
| 14.6 Conclusion | 320 | ||
| References | 321 | ||
| Chapter 15 Research Data Management | 324 | ||
| 15.1 Introduction | 324 | ||
| 15.2 Evolution of Research Data Management | 326 | ||
| 15.3 Principles for Data Management in the 21st Century | 328 | ||
| 15.4 Assembling Data from the Data Ecosystem | 331 | ||
| 15.5 Annotating, Curating and Describing Data Derived from the Data Ecosystem | 332 | ||
| 15.6 Predictive Analytics | 335 | ||
| 15.7 Towards Understanding: Moving from Prediction to Action | 338 | ||
| 15.8 Lessons Learned | 340 | ||
| 15.9 Conclusion | 340 | ||
| References | 340 | ||
| Chapter 16 Small-molecule Bioactivity Databases | 344 | ||
| 16.1 Introduction | 344 | ||
| 16.2 Public Bioactivity Databases | 345 | ||
| 16.2.1 BindingDB | 347 | ||
| 16.2.2 PubChem | 347 | ||
| 16.2.3 ChEMBL | 351 | ||
| 16.2.4 GtoPdb | 354 | ||
| 16.2.5 Public Data in the CDD Vault | 354 | ||
| 16.3 Data Quality | 360 | ||
| 16.4 Conclusions | 364 | ||
| Acknowledgments | 365 | ||
| References | 365 | ||
| Chapter 17 ‘‘So You Want to Run a High-throughput Screen: Do You Know How Much That Costs?\"; Costs of High Throughput Screens and How to Fund Them | 372 | ||
| 17.1 Introduction: Planning for High Throughput Screening | 372 | ||
| 17.2 Costs Associated with HTS | 373 | ||
| 17.2.1 Do not Forget the Overheads | 373 | ||
| 17.2.2 Example of a Budget for a High Throughput Screen | 374 | ||
| 17.2.3 Beyond the HTS: Secondary and Tertiary Assays | 377 | ||
| 17.2.4 Improving Cost Efficiency of HTS Execution | 377 | ||
| 17.3 Organizational Models | 379 | ||
| 17.3.1 Academic Core Facilities | 379 | ||
| 17.3.2 Pharmaceutical Screening Facilities | 382 | ||
| 17.3.3 Contract Research Organizations | 383 | ||
| 17.3.4 Balancing Innovation and Economies of Scale | 384 | ||
| 17.4 Funding Opportunities | 384 | ||
| 17.4.1 Government Grants for Screening | 385 | ||
| 17.4.2 Philanthropic Societies | 385 | ||
| 17.4.3 Industrial Collaborations | 386 | ||
| 17.4.4 Crowdsourcing | 387 | ||
| 17.5 Future Outlook for HTS Funding | 387 | ||
| Acknowledgments | 388 | ||
| References | 388 | ||
| Subject Index | 390 |