High Throughput Screening: Methods and Protocols (Methods in Molecular Biology, 1439)

✍ Scribed by William P. Janzen (editor)

• Publisher: Humana
• Year: 2016
• Tongue: English
• Leaves: 312
• Category: Library

✦ Synopsis

This third edition volume expands on the previous editions both by presenting more detailed protocols for the techniques described in the first and second editions of High Throughput Screening: Methods and Protocols and by covering important new procedures. The first chapter of this book provides an overview of important assay development techniques, while the rest of the chapters detail how to develop and execute screens at whatever throughput the user needs. Some chapter examples are: structure-based virtual screening, high throughput screening using mass spectrometry, identification of state-dependent blockers for voltage gated calcium channels, bioluminescence resonance energy transfer platform to monitor protein-protein interactions in live cells, high throughput flow cytometry, and application of imaging-based assays in microplate formats for high content screening. Written in the highly successful Methods in Molecular Biology series format, chaptersinclude introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Cutting edge and thorough, High Throughput Screening: Methods and Protocols, Third Edition, is a valuable resource for anyone who is interested in HTS research.

✦ Table of Contents

Preface
Contents
Contributors
Chapter 1: Design and Implementation of High-Throughput Screening Assays
1 Introduction to the HTS Process
2 Phenotypic Approaches to HTS
3 Choice of Therapeutic Target
4 Choice of Assay Method
4.1 Biochemical Assay Methods
4.2 Cell-Based Assay Methods
4.3 Matching Assay Method to Target Type
5 Assay Development and Validation
5.1 Critical Biochemical Parameters in HTS Assays
5.1.1 Enzymatic Assays
Substrate Concentration
Enzyme Concentration
Incubation Time and Degree of Substrate Depletion
Order of Reagent Addition
5.1.2 Binding Assays
Ligand Concentration
Receptor Concentration
Preincubation and Equilibrium
5.1.3 Cell-Based Assays
5.2 Assay Optimization
5.3 Statistical Evaluation of HTS Assay Quality
5.3.1 Signal-to-Background Ratio (S/B)
5.3.2 Coefficient of Variation (CV) of Signal and Background
5.3.3 Signal to Noise
5.3.4 Z Factor
5.4 Assay Validation
References
Chapter 2: Characterization of Inhibitor Binding Through Multiple Inhibitor Analysis: A Novel Local Fitting Method
1 Introduction
2 Materials
3 Methods
3.1 Assay Method
3.2 Data Analysis
4 Notes
References
Chapter 3: High-Throughput Screening Using Mass Spectrometry within Drug Discovery
1 Introduction
1.1 RapidFireTM System Overview
2 Buffer Preparation
2.1 Ligand Optimization Buffer
2.2 RapidFireTM-MS Running Buffers
3 Methods
3.1 Optimization of Ligand by Direct Infusion
3.2 Setup of RapidFireTM-MS
3.3 Experimental
3.4 HTS Screening Using RapidFireTM
3.5 Data Handling
4 Notes
References
Chapter 4: Structure-Based Virtual Screening of Commercially Available Compound Libraries
1 Introduction
2 Materials
2.1 Software
2.2 Databases
3 Methods
3.1 Workflow
3.2 Docking
3.3 Structural Protein-Ligand Interaction Fingerprints
3.4 SPLIF Reference Structures
3.5 Hit Analysis and Selection
3.6 Mer Microfluidic Capillary Electrophoresis Assay
3.7 Quality Control of Compound Samples
3.8 Post-screening Follow-Up
4 Notes
References
Chapter 5: AlphaScreen-Based Assays: Ultra-High-Throughput Screening for Small-Molecule Inhibitors of Challenging Enzymes and ...
1 Introduction
2 Materials
2.1 Materials for Detecting Substrate Depletion from AlphaScreen Assays
2.2 Materials for Detecting Protein-Protein Interactions from AlphaScreen Assays
2.3 Materials for Detection of Product Formation from Live Cells by AlphaScreen Assays
3 Methods
3.1 Methods for Detecting Substrate Depletion from AlphaScreen Assays
3.2 Methods for Detecting Protein-Protein Interactions from AlphaScreen Assays
3.3 Methods for Detection of Product Formation from Live Cells by AlphaScreen Assays
3.4 Identifying False-Positive Hit Compounds
4 Notes
References
Chapter 6: Instrument Quality Control
1 Introduction
2 Materials
2.1 Gravimetric Quality Control
2.2 Photometric Quality Control
3 Methods
3.1 Gravimetric Quality Control
3.2 Photometric Quality Control
3.2.1 Tartrazine Standard Curve
3.2.2 Photometric QC Setup
3.2.3 Photometric Quality Control of a 384 Multichannel Liquid Handler
Quality Control of Liquid Handler with Eight Independently Operating Tips
4 Notes
References
Chapter 7: Application of Fluorescence Polarization in HTS Assays
1 Introduction
1.1 Fluorescence Polarization Basic Theory
2 Methods
2.1 Direct FP Competition Assay
2.1.1 Design and Synthesis of an Appropriate Fluorescent Probe
2.1.2 Determination of Kd Between the Fluorescent Probe and the Target
2.1.3 Selection of Appropriate Screen Conditions for Direct FP Competition Assay
2.1.4 Validation of Direct FP Competition Assay
2.1.5 A Quality Control Parameter for a Direct FP Competition Screen
2.2 FP as a Detection Method in Enzymatic Assays
2.2.1 FP Method That Directly Measures the Product
2.2.2 FP Method That Measures the Product via Competition
2.3 Determination of Binding Mechanism from FP Competition Binding Assay
2.4 Limitations of Steady-State FP Measurements
3 Notes
4 Summary
References
Chapter 8: Time-Resolved Fluorescence Assays
1 Introduction
2 Materials
3 Methods
3.1 SUMO to SIM Containing Peptide Protein-Protein Interaction Screen
3.2 Cullin Neddylation Enzymatic Assay
4 Notes
References
Chapter 9: Protein Kinase Selectivity Profiling Using Microfluid Mobility Shift Assays
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 10: Screening for Inhibitors of Kinase Autophosphorylation
1 Introduction
2 Materials
3 Methods
3.1 Alk2 Autophosphorylation Assay
3.2 Alk2 ADP Formation Assay
4 Notes
References
Chapter 11: A Fluorescence-Based High-Throughput Screening Assay to Identify Growth Inhibitors of the Pathogenic Fungus Asperg...
1 Introduction
1.1 Assay Overview
2 Materials
2.1 Scientific Equipment
2.2 Reagents
3 Methods
3.1 Preparation of Aspergillus fumigatus Seed Stocks for Storage
3.2 Preparation of Conidial Working Stock for High-Throughput Screening
3.3 High-Throughput Screening Protocol in 1536w Format
4 Notes
References
Chapter 12: Mycobacterium tuberculosis High-Throughput Screening
1 Introduction
2 Materials (See Note 2)
2.1 Media/Broth and Agar Plates (See Note 3)
2.2 alamarBlue Assay
2.3 Mammalian Cell Cytotoxicity Assay
2.4 Equipment
3 Methods
3.1 Stock Mtb H37Rv Preparation
3.1.1 Subculture Growth
3.1.2 Preparation of Stock Mtb H37Rv
3.1.3 Stock Titration
3.2 Mtb Microtiter Plate Assay
3.2.1 Media Preparation
3.2.2 Compound Addition to Assay and Control Microtiter Plates
3.2.3 Control Plates
3.2.4 Mtb H37Rv Addition
3.2.5 alamarBlue End Point Addition
3.2.6 Alternate End Point-BacTiter Glo End Point Addition (See Note 14)
3.2.7 Dose-Response Assay
3.2.8 Preparation of Mammalian Cell Stocks
3.2.9 Mammalian Cell Cytotoxicity Assay (See Note 17)
3.3 Data Analysis
4 Notes
References
Chapter 13: Identification of State-Dependent Blockers for Voltage-Gated Calcium Channels Using a FLIPR-Based Assay
1 Introduction
2 Materials
2.1 Cell Line and Culture Medium
2.2 Reagents and Buffers
3 Methods
4 Notes
References
Chapter 14: A Luciferase Reporter Gene System for High-Throughput Screening of gamma-Globin Gene Activators
1 Introduction
2 Materials
3 Methods
3.1 Dual-Glo Luciferase Activity Measurement
3.1.1 Compound Plate Preparation and Cell Treatment
3.1.2 Dual-Glo Luciferase Activity Detection
3.2 Firefly Luciferase Reporter Assay Development for HTS
3.2.1 Cell Density Titration in 384-Well Plates
3.2.2 Serum Titration in 384-Well Plates
3.2.3 DMSO Titration in 1536-Well Plates
3.2.4 Comparison of Freshly Cultured Cells with Cryopreserved Cells in 1536-Well Plates
3.3 HTS Campaign and Outcomes
3.3.1 Compound Stamping
3.3.2 Cell Plating and Treatment
3.3.3 Firefly Luciferase Detection
3.4 Confirmation of HTS Hits with Triage Assays
3.4.1 Cell Lysis System with Steady-Glo
3.4.2 Non-Lysis Substrate System Activity Measurement
3.4.3 Full Dose Curve Confirmation
4 Notes
References
Chapter 15: A High-Throughput Flow Cytometry Assay for Identification of Inhibitors of 3,5-Cyclic Adenosine Monophosphate Ef
1 Introduction
2 Materials
2.1 Components for Fluorescent Cyclic AMP Loading
2.2 cAMP Efflux Assay Components
2.3 High-Throughput Screening Components
3 Methods
3.1 Loading F-cAMP into Cells
3.2 Optimization of F-cAMP Efflux Positive Control and Determining Cell F-cAMP Efflux Ability
3.3 High-Throughput F-cAMP Efflux Assay
4 Notes
References
Chapter 16: High-Throughput Cell Toxicity Assays
1 Introduction
2 Materials
2.1 Cell Culture Components for All Three Toxicity Assays
2.2 Assay Ready Plate Generation
2.3 CellTiter-Blue Components
2.4 CellTox Green Components
2.5 Intellicyt MultiCyt 4-Plex Apoptosis Kit Components
3 Methods
3.1 Routine Cell Culture Method
3.2 Cell Culture Preparation and Cell Plating
3.3 Preparation of Assay Ready Screening Plates (ARPs)
3.4 Cytotoxicity Assays
3.5 CellTiter-Blue Method
3.6 Compound Fluorescence Quench Assay for CellTiter-Blue
3.7 CellTox Green Method
3.8 Intellicyt MultiCyt 4-plex Apoptosis Assay Method
3.9 Example Data
4 Notes
References
Chapter 17: BRET: NanoLuc-Based Bioluminescence Resonance Energy Transfer Platform to Monitor Protein-Protein Interactions in ...
1 Introduction
2 Materials
2.1 Plasmids for NLuc-tagged TEAD2 and Venus-tagged YAP1 Construction(see Note 1)
2.2 Other Reagents
3 Methods
3.1 Plasmid Construction
3.2 Plating Cells
3.3 Transient Transfection
3.4 BRETn Measurement
3.5 Data Analysis
4 Notes
References
Chapter 18: Application of Imaging-Based Assays in Microplate Formats for High-Content Screening
1 Introduction
1.1 General Considerations in HCS Assay Design and Execution
1.1.1 Start from Robust Biology in an Appropriate Model System
1.1.2 Controls Define the Assay but Won´t Always Predict the Behavior of Bioactive Agents in a Screen
1.1.3 Always Be on the Lookout for False Positives Related to Toxicity
1.1.4 Imaging Hardware: Balancing Throughput, Signal, and Resolution
1.1.5 Managing High-Content Data: Invest in a Lot of Storage!
1.1.6 Analyzing High-Content Data: Making a Mole-Hill from a Mountain?
1.2 The Anatomy of a HCS Assay: Segmentation Strategy in the Parkin Assay Example
2 Materials
2.1 Cell Line Generation
2.2 384-Well HCS Assay
2.3 Screening Hardware and Imaging
2.4 Image Processing and Data Analysis
3 Methods
3.1 Cell Line Generation
3.2 384-Well Plate HCS Assay
3.3 Screening Hardware and Imaging
3.4 Image Processing and Data Analysis
4 Notes
References
Index