Chemogenomics: Methods and Protocols (Methods in Molecular Biology, 2706)

✍ Scribed by Daniel Merk (editor), Apirat Chaikuad (editor)

• Publisher: Humana
• Year: 2023
• Tongue: English
• Leaves: 240
• Edition: 1st ed. 2023
• Category: Library

✦ Synopsis

This volume presents both theoretical guidance and protocols on chemogenomics including chemogenomics library assembly, compound profiling, and phenotypic assays. The chapters in this book cover topics such as the assembly and use of Kinase Chemogenomics; data mining for chemogenomic compound candidates; protocols for protein family-focused assay systems to profile chemogenomic compounds; functional and target engagement assays in cellular settings for broad characterization; and a discussion on phenotypic assays where chemogenomic sets may be applied. Written in the highly successful Methods in Molecular Biology series format, chapters include 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, Chemogenomics: Methods and Protocols is a valuable resource for all researchers who are interested in learning more about this diverse and developing field.

✦ Table of Contents

Preface
Contents
Contributors
Chapter 1: An Introduction to Chemogenomics
1 Introduction: The Idea of Chemogenomics
2 What Makes a Chemogenomics Compound
3 Chemogenomics Libraries
4 How to Use Chemogenomics Compound Sets
5 Conclusion
References
Chapter 2: Developing a Kinase Chemogenomic Set: Facilitating Investigation into Kinase Biology by Linking Phenotypes to Targe...
1 Introduction
2 Developing the Kinase Chemogenomic Set
2.1 Previously Disclosed Kinase Sets
2.2 Kinase Activity Determination of KCGS
2.3 Size of Set
2.4 Chemotypes
2.5 Library Properties
2.6 Kinase Coverage
2.7 Plating
2.8 Distribution
3 How Researchers Have used PKIS and KCGS
4 Conclusion
References
Chapter 3: Compilation of Custom Compound/Bioactivity Datasets from Public Repositories
1 Introduction
2 Materials
2.1 Computational Framework - Software, Programming Language, and Tools
2.2 Databases
3 Methods
3.1 Data Preparation
3.1.1 HGNC File
3.1.2 IUPHAR/BPS
3.1.3 BindingDB
3.1.4 ChEMBL
3.2 Data Preparation for Merging
3.2.1 Standardize Gene Names and Flag Assay Types
3.2.2 Standardize Bioactivity Values
3.3 Merging
3.4 Activity Check
3.5 Structure Check
3.6 Dataset Curation
3.7 Application
3.7.1 Search for Potent Ligands of a Target of Interest
3.7.2 Extract Off-Targets and Find the Most Selective Compounds
3.7.3 Select Chemically Diverse Compounds
4 Notes
References
Chapter 4: Quality Control of Chemogenomic Library Using LC-MS
1 Introduction
2 Materials
3 Methods
3.1 Sample Plate Preparation
3.2 LC-MS QC for Chemogenomic Compound
3.2.1 The First-Pass Method
LC-MS Analytical Measurement for the First-Pass Method
LC-MS Data Evaluation for the First-Pass Method
3.2.2 The Second-Pass Method
LC-MS Analytical Measurement for the Second-Pass Method
4 Notes
References
Chapter 5: Annotation of the Effect of Chemogenomic Compounds on Cell Health Using High-Content Microscopy in Live-Cell Mode
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Preparation of Compounds
2.3 Cell Staining Dyes
2.4 Instruments for High-Content Imaging and Parameters
2.5 Analysis Software
3 Methods
3.1 Preparation of Cells for Live-Cell Imaging
3.2 Image Acquisition of Non-treated Cells
3.3 Image Acquisition of Treated Cells
3.4 Data Analysis Using CellPathfinder Software
3.5 Data Evaluation
4 Notes
References
Chapter 6: Characterization of Cellular Viability Using Label-Free Brightfield Live-Cell Imaging
1 Introduction
2 Materials
2.1 Cell Lines
2.2 Reagents and Perishables
2.3 Reference Compounds
2.4 Instruments and Related Software
3 Methods
3.1 Cell Seeding
3.2 Incucyte Image Acquisition Setup
3.3 Incucyte Plate Run
3.4 Incucyte Plate Analysis
3.5 Data Export and Analysis
3.6 Growth Rate Calculation
4 Notes
References
Chapter 7: Plate-Based Screening for DUB Inhibitors
1 Introduction
2 Materials
3 Methods
3.1 Pre-screening Setup
3.2 Single-Point Inhibitor Screen
3.3 Hit Verification and Follow-Up
4 Notes
References
Chapter 8: NanoBRET Live-Cell Kinase Selectivity Profiling Adapted for High-Throughput Screening
1 Introduction
2 Materials
2.1 Optimizing Cell Preparation and Transfection for HTS Kinase Selectivity Profiling
2.2 Optimization and Z′ Analysis for NanoBRET HTS Kinase Selectivity Assays
2.3 Validating Performance of Individual Kinase Assay in Measuring Compound Binding Affinity in Concentration-Response Experim...
2.4 Evaluating Percent Occupancy of Compounds in Single-Dose Selectivity Profiling
3 Methods
3.1 Key Considerations for Adapting NanoBRET Kinase Selectivity Assays to HTS
3.2 Cell Preparation and Transfection for HTS Kinase Selectivity Profiling
3.3 Optimization and Z′ Analysis for NanoBRET HTS Kinase Selectivity Assays
3.4 Validating Performance of Individual Kinase Assays in Measuring Compound Binding Affinity in Concentration-Response Experi...
3.5 Evaluating Percent Occupancy of Compounds in Single-Dose Selectivity Profiling
4 Notes
References
Chapter 9: A Fluorescence-Based Reporter Gene Assay to Characterize Nuclear Receptor Modulators
1 Introduction
2 Materials
2.1 Plasmids
2.2 Cell Culture
2.3 Test Compounds and References
2.4 Equipment
2.5 Software
3 Methods
3.1 General Considerations
3.2 Seeding Cells (0 h)
3.3 Transfection (24 h)
3.3.1 Preparations
3.3.2 Transfection Procedure
3.4 Test Compound Dilution and Incubation
3.4.1 Test Compound Dilution (27 h)
3.4.2 Incubation with Test Compounds (29 h)
3.5 Fluorescence Measurement (36-72 h)
3.6 Data Analysis
3.6.1 Data Analysis for Individual Assays
3.6.2 Data Analysis per Compound
4 Notes
References
Chapter 10: Measuring Protein-Protein Interactions in Cells using Nanoluciferase Bioluminescence Resonance Energy Transfer (Na...
1 Introduction
2 Materials
2.1 Constructs
2.2 Specific Reagents
2.3 Instrumentation
3 Methods
3.1 NanoBRET Optimization
3.1.1 Cell Plating
3.1.2 Cell Transfection
3.1.3 NanoBRET Measurement
3.2 Testing PPI Antagonists in NanoBRET
3.3 NanoBRET PPI Assays Using Isolated Domains and Short Peptide Sequences
3.4 NanoBRET PPI Assay Validation Using Genetic Mutants
4 Notes
References
Chapter 11: HiBiT Cellular Thermal Shift Assay (HiBiT CETSA)
1 Introduction
2 Materials
2.1 Cloning
2.2 Cell Culture
2.3 Compounds
2.4 HiBiT CETSA Assay
3 Methods
3.1 Generation of HiBiT Plasmids
3.1.1 Preparation of Restriction Enzyme-Digested HiBiT Acceptor Plasmids
3.1.2 Preparation of POI Insert and Cloning into a HiBiT Acceptor Plasmid
3.2 HiBiT CETSA Assay Setup
3.2.1 Transient Transfection of Cells with HiBiT Fusion Protein
3.2.2 Live-Cell Format Nano-Glo Cellular Thermal Shift Assay Protocol
3.2.3 Permeabilized Cell Format Nano-Glo Cellular Thermal Shift Assay Protocol
3.2.4 Data Processing and Analysis of HiBiT CETSA Data
3.3 Considerations for Experimental Design
3.3.1 Comparison of N-Versus C-Terminally Tagged HiBiT Protein
3.3.2 Isothermal Compound Screening
3.3.3 Dose-Response Compound Screening: Total Temperature Gradient v/s Isothermal Screening
3.4 Other Applications of HiBiT CETSA
4 Notes
References
Chapter 12: Detection of Cellular Target Engagement for Small-Molecule Modulators of Striatal-Enriched Protein Tyrosine Phosph...
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Assay Components
2.3 Instrumentation
3 Methods
3.1 Cell Culture and Transient Transfection with Target Engagement Plasmid
3.2 Cell Detachment and Assay Plate Preparation
3.3 Thermal Pulse and Assay Quantification
4 Notes
References
Chapter 13: Target Deconvolution by Limited Proteolysis Coupled to Mass Spectrometry
1 Introduction
2 Materials
2.1 Lysis
2.2 Limited Proteolysis Step
2.3 Tryptic Digest
2.4 Peptide Desalting
2.5 Peptide Resuspension
2.6 MS Measurement
3 Methods
3.1 Lysis
3.2 Limited Proteolysis Step
3.3 Tryptic Digest
3.4 Peptide Desalting
3.5 Peptide Resuspension
3.6 MS Measurement
3.7 Data Evaluation
4 Notes
References
Chapter 14: Global Assessment of Drug Target Engagement and Selectivity of Covalent Cysteine-Reactive Inhibitors Using Alkyne-...
1 Introduction
2 Materials
2.1 Preparation of Buffers for Click Reaction and Cell Lysis
2.2 In-Gel Fluorescence
2.3 Affinity Enrichment, Digestion, and Desalting
3 Methods
3.1 Reactive Probe Treatment and Cell Lysis
3.2 Click Reaction
3.3 In-Gel Fluorescence Detection
3.4 Affinity Enrichment
3.5 MS Sample Preparation
3.6 Desalting
3.7 MS Instrument Settings and Data Analysis
4 Notes
References
Chapter 15: 3D Spheroid Invasion Assay for High-Throughput Screening of Small-Molecule Libraries
1 Introduction
2 Materials
2.1 Cells
2.2 Cell Culture Reagents
2.3 Cell Culture Accessories
2.4 Imaging and Analysis
3 Methods (Fig. 1)
3.1 Pre-setup Day: - 2
3.2 Setup Day: - 1
3.2.1 Preparation of Cells
3.2.2 Seeding of Cells
3.2.3 Preparation for the Next Day
3.3 Setup Day: 0
3.3.1 Addition of the Cell Matrix
3.3.2 Drug Treatments
3.4 Imaging Days: 0, 3, and 5 and Analysis
3.4.1 Spheroid Imaging
3.4.2 Image Analysis
Image Masking Using ILASTIK
Cell Segmentation and Image Analysis Using ImageJ FIJI
3.4.3 Data Visualization and Statistics
4 Notes
References
Chapter 16: Live-Cell High-Throughput Screen for Monitoring Autophagy Flux
1 Introduction
2 Materials
2.1 Reporter Cell Line
2.2 Preparation of Compound Plates
2.3 Cell Seeding and Screen
3 Methods
3.1 Cell Seeding (Day 1)
3.2 Preparation of Compound Plates (Days 1 and 2)
3.3 Screening (Day 2)
3.4 Data Analysis and Basic Quality Control
4 Notes
References
Chapter 17: Phenotypic Chemical Screening in CD4+ T Cells to Identify Epigenetic Inhibitors
1 Introduction
2 Materials
2.1 Isolating Mononuclear Cells
2.2 Isolating Naïve CD4+ T Lymphocytes
2.3 Culture and Differentiation Conditions
2.4 Chemical Library
2.5 ELISAs
2.6 Cell Viability
3 Methods
3.1 Isolating Mononuclear Cells
3.2 Isolating Naïve CD4+ T Lymphocytes
3.3 Culture and Differentiation Conditions
3.4 Chemical Library
3.5 ELISAs
3.6 Cell Viability
4 Notes
References
Index