
High Throughput Screening: Methods, Techniques and Applications
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Content
- Intro
- Contents
- Preface
- Acronyms
- Chapter 1
- Assay Development for High Throughput Screening
- Abstract
- Introduction
- Commonalities between Developing Biochemical and Cell-Based Assays
- Considerations for Developing Cell-Based Assays for HTS
- Literature Review
- Relevance of Cell Type Used for the HTS
- Practicality and Safety of the Biological Material
- Culturing Cells Free of Contamination and Cross-Contamination
- Surface Treatments and Seeding Cells for an Assay
- Cell Confluency and Contact Inhibition
- Cell Passage Number
- Edge Effects
- Assay Variabilities Due to Change in Incubators
- Cell Tolerance to DMSO or Other Reagent Vehicles
- Miniaturization of the Cell-Based Assay into 1536 Well Format
- Automation for Cell-Based Assays
- Cell-Based Assay Development for HTS with Alternative Platforms
- Considerations for Developing Biochemical Assays for HTS
- Conclusion
- References
- Chapter 2
- Application of Fluorescence Technologies to High Throughput Screening Assays
- Abstract
- Introduction
- Fluorescence Intensity
- Fluorescence Interference by Library Compounds
- Fluorescence Polarization and Anisotropy
- Considerations for Assay Development and Implementation
- Flagging Assay Artifacts and Interferences for FP
- Applications of Fluorescence Polarization and Anisotropy to HTS
- Förster Resonance Energy Transfer
- Considerations for Assay Development
- Applications of FRET to HTS
- Time-Resolved Förster Resonance Energy Transfer
- Conclusion
- References
- Chapter 3
- Luminescence Technologies for High Throughput Compound Screening
- Abstract
- Luminescence-Based Assays
- Luciferase-Based Luminescent Systems
- Luciferase-Based Assay Strategies in HTS Campaigns
- Luciferase Reporter Gene Assays (RGA)
- Multi-Colour-Luciferase Reporter Assays:
- Protein-Protein Interaction (PPI) Assays
- Split Luciferase Complementation Assay (SLCA)
- Bioluminescence Resonance Energy Transfer (BRET)
- Sequential BRET-FRET (SRET)
- Luciferase Assays to Measure ATP
- Cell-Based Viability Assay
- Kinase/ATPase Assays
- ATP-Related Functional Assays
- Caged Bioluminogenic Probes
- Protease Assays
- Histone Deacetylases
- Metabolic Enzymes
- NAD(P)H-Based Assays
- Calcium Sensors
- Recent Advancements in Bioluminescence-Based Technologies with the Potential for HTS
- Enhanced Nano-Lantern (eNL)
- Chemiluminescence and Assay Strategies Used in HTS Campaigns
- Alpha Technology (Amplified Luminescence Proximity Homogenous Assay)
- Scintillation Proximity Assay (SPA)
- Electrochemiluminescence
- General Consideration in HTS Assay Design
- Single versus Dual Reporter Strategies in HTS
- Assay Interference
- Instrumentation and Materials
- Conclusion
- Acknowledgments
- References
- Chapter 4
- Acoustic Mist Ionization Mass Spectrometry (AMI-MS) in High Throughput Screening
- Abstract
- Introduction
- Methods
- Instrument Setup
- AMI-MS Settings
- Assay Development
- Plate Type
- Initial Scoping
- Data Processing
- Data Analysis
- Screening
- Automation of AMI-MS Assays
- Triage Assays
- Future Perspectives in AMI-MS
- References
- Chapter 5
- High Throughput Flow Cytometry: Principles, Practice and Prospects
- Abstract
- Introduction
- HTFC Technology
- Basic Description of Flow Cytometers
- Cytometer Basics
- Making a Cytometer into a High Throughput Instrument
- Examples of HTFC Applications
- Unique Features of HTFC that Can Be Exploited for Assays
- Examples of the Capabilities of HTFC from Our Research
- Cell-Based Assays for Immunologically Active Compounds
- Our Use in Multiplexed Assays Based on Genetically-Encoded CFP-YFP FRET Sensors
- Alternatives to Our Use of HTFC
- Some Uses of HTFC by Others
- The Future of HTFC
- Conclusion
- References
- Chapter 6
- Perspectives on High Content Imaging as an HTS Technology: Now and in the Future
- Abstract
- Introduction
- Benefits of High Content Imaging
- Workflow for HTS Imaging Screens
- Quality Control, Data Management, Processing and Analysis
- Image Analysis
- Quantification of Image and Assay Quality
- Maximizing the Speed of Image Acquisition and Analysis
- To Store or Not to Store?
- Emerging Technologies and Future Perspectives
- Improving the Physiological Relevance of Cell Models
- Miniaturization
- Target Agnostic Morphological Profiling to Characterize Chemical or Genetic Perturbations
- Using the Cell Painting Assay to Generate Morphological Profiles
- Label Free Imaging to Quantify Cellular Responses
- Artificial Intelligence and Machine Learning for Phenotypic Hit Identification
- Reflections and Summary
- Acknowledgments
- References
- Chapter 7
- Biophysical Hit Evaluation
- Abstract
- Introduction
- Surface Plasmon Resonance
- Overview
- Utility of SPR
- Challenges and Limitations of SPR
- Future Outlook for SPR
- Nuclear magnetic Resonance
- Overview
- Utility of NMR
- Challenges and Limitations of NMR
- Future Outlook for NMR
- Mass Spectrometry
- Overview
- Utility of MS
- Challenges and Limitations of MS
- Future Outlook for MS
- Thermal Shift Assay
- Overview
- Utility of FTSA
- Challenges and Limitations of FTSA
- Future Outlook for FTSA
- Microscale Thermophoresis
- Overview
- Utility of MST
- Challenges and Limitations of MST
- Future Outlook for MST
- Isothermal Titration Calorimetry
- Overview
- Utility of ITC
- Challenges and Limitations of ITC
- Future Outlook for ITC
- Conclusion
- Acknowledgement
- References
- Chapter 8
- CETSA in High Throughput Screening
- Abstract
- Introduction
- Application of High Throughput CETSA: Target Engagement
- Melt Curves and Tm Shift: Detection of Small Molecule Target Engagement
- Relative Potencies from Isothermal Dose Response Curves: The ITDRF
- Methods
- Antibody Detection CETSA with AlphaLISA Readout
- Reporter Assay CETSA: Beta-Galactosidase Enzyme Fragment Complementation
- Melt Curve Experiments in 384 Well Plates
- Permeabilized Cell Protocol for 384 Well Plates
- High Throughput 384 Well Isothermal Dose Response Fingerprint (ITDRFCETSA) Experiments
- High Throughput 1536 Well Single Concentration Compound Testing Protocol
- Reporter Assay CETSA: NanoLuciferase Enzyme Fragment Complementation
- Caveats and Artefacts
- Detection Method-Based Artefacts
- Challenges When Tool Molecules are Not Available
- Melt Curve Not Observed
- Potency and Response Amplitude in CETSA
- False Negatives: Molecules That Bind but Do Not Produce Thermal Stabilization
- Sensitivity to Stoichiometry of Protein Target and Detection Reagents
- High Throughput CETSA: Other Uses and Applications
- Rationalizing Differences in Cellular and Biochemical Potencies
- Intracellular Conversion to Active Kinetics
- Specificity Assays
- Hit Identification and High Throughput Screening
- Conclusion
- Future Considerations
- Acknowledgments
- References
- Chapter 9
- Droplet-Based Microfluidics for High Throughput Screening
- Abstract
- Introduction
- Fabrication of Microfluidic Devices
- Droplet Manipulation via Droplet-Based Microfluidics
- Droplet Generation
- Droplet Content Modification
- Droplet Detection
- Droplet Sorting
- Applications of Droplet-Based Microfluidics
- Directed Evolution of Enzymes
- Small Molecule Screening
- Antibody Screening and Drug Discovery
- Single Cell RNA Sequencing
- Outlook
- Instrumentation and Surfactants
- New Analytical Detection Techniques and Biosensors
- Conclusion
- References
- Chapter 10
- Encoded Library Technologies as Screening Platforms for Drug Discovery
- Abstract
- Introduction
- DNA-Encoded Libraries
- Peptide Discovery Platform
- Encoded Library Technologies at Novartis
- Future Directions of Encoded Library Technologies
- References
- Chapter 11
- Data Analysis in High Throughput Screening
- Abstract
- Introduction
- The Screening Cascade
- Systems in High Throughput Screening
- Raw Data Types in HTS
- Zero-Dimensional Readouts - Endpoint Measurements
- One Dimensional Readout - Time Series or Spectral
- Two-Dimensional Readouts - Images
- Plate Maps and Assay Annotations
- Unified Processing Workflows for HTS Data
- Data Reduction to Well-Based Results
- Data Standardization
- Data Normalization
- Using Normalization for the Visual Review of Data
- Two-Point Normalization
- One-Point Normalization
- Robust Z-Score Normalization
- Standard Curve Normalization
- Normalization Using Control Plates
- Pattern Correction
- Median Correction
- B-Score and R-Score
- Genedata Screener Pattern Correction
- Data Reduction to Compound/Analyte Results
- Result Compilation, Interpretation and Decision-making
- Concentration-Response Analysis
- Dose-Response Curve Fitting
- Logistic Model - Hill Equation
- Important Additional Results from a Dose-Response Curve Fit
- Robust Fitting and Outlier Handling
- Lowest-Effective Concentration
- Biphasic Model
- Curve Diagnostic and Classification
- Curve Diagnostic
- Solubility Issues
- Replicate Issues
- Common Problems in Dose-Response Curve Fitting
- Curve Classification
- Quality Assurance and Validity of Results
- Plate and Assay Statistics
- Typical QC Metrics
- Robust Z-Factor and Robust Z'-Factor
- Assuring Screening Process Quality
- Assuring Quality and Validity of Screening Results
- High Throughput Imaging and Phenotypic Screening
- Conclusion
- Acknowledgments
- References
- Chapter 12
- The Application of Iterative Screening to Hit Identification
- Abstract
- Introduction
- Examples from the Literature
- Active Learning
- Integrating Bioactivity Data
- Confidence Predictors
- Iterative Screening Using Gain-Cost Functions
- Starting from Weakly Active Compounds
- Challenges and Future Developments
- Conclusion and Outlook
- Acknowledgments
- References
- About the Editors
- Index
- Blank Page
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