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Cannabinoids and Their Receptors

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Contents

Contributors

Preface

References

Chapter One: Mouse Neuroblastoma CB1 Cannabinoid Receptor-Stimulated [35S]GTPɣS Binding: Total and Antibody-Targeted Gα P ...

1. Introduction

2. Membrane Preparations for Cultured Neuronal Cells

2.1. Equipment and Supplies

2.2. Buffers and Reagents

2.3. Neuroblastoma Cell Lines: Mouse N18TG2 Cell Culture Conditions

2.4. Procedure

3. Methods of Determining [35S]GTPɣS Binding to Total Gα in Membrane Preparations

3.1. Equipment and Supplies (PerkinElmer)

3.2. Buffers and Reagents

3.3. Procedures

4. Antibody-Targeted Scintillation Proximity Assay for [35S]GTPɣS Binding to Specific Gα Proteins in Membrane Preparations

4.1. Equipment and Supplies

4.2. Buffers and Reagents

4.3. Procedures

4.4. Antibody Verification by Western Blotting

5. Summary and Conclusion

Acknowledgments

References

Chapter Two: Protocols and Good Operating Practices in the Study of Cannabinoid Receptors

1. Introduction

2. Receptor Assays

2.1. Competitive Radioligand Binding

2.1.1. Equipment

2.1.2. Buffers and Reagents

2.1.3. Note

2.1.4. Procedure

2.1.4.1. Membrane Protein Preparation

2.1.4.2. Siliconization of Assay Tubes

2.1.4.3. Radioligand Binding and Detection

2.2. Functional Receptor Assay—[35S]GTPγS

2.2.1. Equipment

2.2.2. Buffers and Reagents

2.2.3. Notes

2.2.4. Procedure

2.2.4.1. Crude Whole Cell Protein Preparation

2.2.4.2. Siliconization of Assay Tubes

2.2.4.3. Binding Assay

2.2.5. Notes

2.3. Principle Receptor Signaling Assay—cAMP

2.3.1. Equipment

2.3.2. Buffers and Reagents

2.3.3. Procedure

2.3.4. Notes

2.4. Non-G Protein Signaling Assay—β-Arrestin

2.4.1. Equipment

2.4.2. Buffers and Reagents

2.4.3. Protocol

2.4.4. Notes

2.5. Reporter Assays of Downstream Signaling—Serum Response Element (SRE)

2.5.1. Equipment

2.5.2. Buffers and Reagents

2.5.3. Procedure

2.5.4. Notes

2.6. Methods for Compound Suspension

2.6.1. In Vitro Cellular Assays

2.6.2. In Vivo Assays

2.6.3. Notes

3. Summary and Conclusions

References

Chapter Three: Real-Time Measurement of Cannabinoid Receptor-Mediated cAMP Signaling

1. Introduction

1.1. Canonical Cannabinoid Receptors Are GPCRs

1.2. Importance of cAMP Signaling

1.3. cAMP Signaling of Cannabinoid Receptors

1.4. Temporal Control of GPCR Signaling

1.5. Types of cAMP Biosensor

2. Detecting Cytoplasmic cAMP Using CAMYEL

2.1. Protocol

2.1.1. Equipment

2.1.2. Cell Line Considerations

2.1.3. Reagents

2.1.4. Preparation of Cells—Seeding, Transfection, Reseeding of Adherent Cells

2.1.5. Performing the Assay, Including Strategies for Seeing Gi vs Gs Signaling

2.1.6. Cell Suspension Assay Variation

2.2. Data Analysis

3. Design and Validation of V8-CAMYEL

3.1. Validation of V8-CAMYEL

3.1.1. Stable Cell Lines

4. Summary

References

Chapter Four: Techniques for the Cellular and Subcellular Localization of Endocannabinoid Receptors and Enzymes in the Ma ...

1. Introduction

2. Use of Conventional Fluorescence Microscopy to Study the ECS

3. Localization-Based Microscopy

3.1. STORM to Study the ECS

4. Transmission Electron Microscopy

5. Confocal, TEM, and STORM Microscopy Applied to Study the ECS: A Technical Comparison

6. FRET

7. Conclusions

References

Further Reading

Chapter Five: Endocannabinoid Transport Proteins: Discovery of Tools to Study Sterol Carrier Protein-2

1. Introduction

2. Materials and Methods

2.1. Materials

2.2. NBDS Displacement Assay

2.2.1. Binding Affinity of NBDS (Kd,NBDS) for SCP-2 Was Determined by Reverse and Forward Titrations

2.2.2. Displacement Assays Were Used to Determine Max % Displacement, EC50, and Ki

2.3. Computational Methods

3. Results and Discussion

3.1. Discovery of Inhibitor Probe Lead Compounds

3.2. SAR of Head Group-Substituted Fatty Acids

3.3. Hit-to-Lead Optimization

3.4. Hit Discovery by HTS In Silico

4. Conclusions

References

Further Reading

Chapter Six: Lipidomics: A Corrective Lens for Enzyme Myopia

1. Introduction

2. Molecule Specifist vs Generalist

3. Lipoamines (aka Fatty Acid Amides; Lipo Amino Acids; N-Acyl Amides; N-Acyl Amino Acids)

3.1. N-Acyl Ethanolamine, N-Acyl Glycine, and Novel N-Acyl Amino Acid Lipids in the Lipoamine Family

3.1.1. N-Acyl Ethanolamines

3.1.2. N-Acyl Glycines

3.1.3. Additional Lipoamines With Known Biological Significance

4. 2-Acyl Glycerols Beyond 2-AG

5. Prostaglandins Create Additional Complexity to the AA-Derived Signaling System

6. What Do Enzymes Tell Us About Lipoamines and What Do Lipoamines Tell Us about Enzymes?

6.1. The Many Faces of FAAH

7. Seeing Our Way Forward

References

Chapter Seven: Functional Analysis of Mitochondrial CB1 Cannabinoid Receptors (mtCB1) in the Brain

1. Introduction

2. Detection of mtCB1

2.1. Immunoelectron Microscopy

2.1.1. Transcardial Perfusion of Animals

2.1.2. Preembedding Silver-Intensified Immunogold Method for Electron Microscopy

2.1.3. Semiquantification of Immunogold Staining

2.2. Immunoprecipitation and Western Blot

2.2.1. Isolation of Brain Mitochondria

2.2.2. Immunoprecipitation

2.2.3. Western Blotting

3. Impact of mtCB1 on Mitochondrial Respiration

3.1. Oxygen Consumption of Isolated Mitochondria

3.2. Oxygen Consumption of Intact Cells

3.2.1. Transfection of Cells:

3.2.2. Cellular Respiration Using the Oroboros Oxygraph-2k

4. Impact of mtCB1 on Complex I Activity

5. Impact of mtCB1 on ATP Levels

6. Impact of mtCB1 on Mitochondrial Mobility in Neurons

6.1. Preparation of Primary Hippocampal Cultures

6.2. Transfection of Primary Neurons

6.3. Image Acquisition

6.4. Analysis of Mitochondrial Mobility

7. Conclusions

Acknowledgments

References

Chapter Eight: Modeling Neurodegenerative Disorders for Developing Cannabinoid-Based Neuroprotective Therapies

1. Introduction

2. The Biomedical Challenge of Neurodegenerative Disorders

3. Improving the Modeling of Neurodegenerative Disorders in Cells and Laboratory Animal Species

4. Cannabinoids as Neuroprotectant Agents

4.1. Cannabinoids Have a Broad-Spectrum Neuroprotective Profile

4.1.1. Effects Mediated by Endocannabinoid-Related Targets

4.1.2. Effects Mediated by Nonendocannabinoid Targets

4.2. Cannabinoid Targets Have a Key Cellular Location

4.3. Cannabinoids Mimic an Endogenous Protective Response

5. Concluding Remarks and Future Perspectives

Acknowledgments

References

Chapter Nine: Metabolic Profiling of CB1 Neutral Antagonists

1. Introduction

2. Background

2.1. Constitutive Activity of CB1R

3. Computational Modeling

4. Chemistry and Structure Proof and Stability Ki CB1 CB2

5. In Vivo Metabolic Effects

5.1. Methods

5.1.1. Animals

5.1.2. Experimental Protocol

5.1.3. Tissue Levels of PIMSR

5.1.4. Blood Chemistry

5.1.5. Glucose Tolerance (ipGTT) and Insulin Sensitivity Tests (ipIST)

5.1.6. Hepatic Triglyceride (TG) Content

5.1.7. Statistics

5.2. Results

5.2.1. PIMSR Is a Brain-Penetrant Neutral CB1R Antagonist

5.2.2. PIMSR Improves Metabolic Profile in Diet-Induced Obese Mice

6. PIMSR and Binge Alcoholic Hepatic Steatosis

6.1. Materials and Methods

6.1.1. Binge Alcohol-Induced Liver Steatosis

6.2. Results

6.2.1. Acute Alcohol-Induced Hepatic Steatosis and Prevention by PIMSR

7. Conclusions

Acknowledgments

References

Chapter Ten: Ligand-Assisted Protein Structure (LAPS): An Experimental Paradigm for Characterizing Cannabinoid-Receptor L ...

1. Introduction

2. Ligand-Assisted Protein Structure

3. Application of LAPS to Endocannabinoid-System GPCRs

3.1. Contextual and Strategic Precedents

3.2. Proof-of-Principal Studies With a Classical Cannabinoid Probe, AM841

3.3. Applied LAPS Methodology for Characterizing Cannabinoid-Receptor-Binding Motifs

3.3.1. hCB2R Biarylpyrazole Antagonist/Inverse Agonist Binding Motif

3.3.2. hCB2R Classical Cannabinoid Agonist Binding Motif

4. Conclusions

Acknowledgments

References

Chapter Eleven: New Methods for the Synthesis of Cannabidiol Derivatives

1. Introduction

2. Synthesis of CBD Derivatives

2.1. Synthesis of CBD

2.2. Synthesis of Selected CBD Derivatives

2.3. Synthesis of N-Heterocyclic Derivatives of CBD

3. Experimental Details for Schemes 13 and 14

3.1. Materials

3.2. Methods

4. Conclusions

Acknowledgments

References

Chapter Twelve: Approaches to Assess Biased Signaling at the CB1R Receptor

1. Introduction

2. Quantifying CB1R Ligand Bias

3. Assays Used to Examine CB1R Ligand Bias

3.1. G Protein Coupling

3.2. β-Arrestin Recruitment

3.3. Inhibition of cAMP Accumulation

3.4. Changes in Ca2+

3.5. Receptor Internalization

3.6. Protein Phosphorylation and Changes in Gene Expression

4. Important Considerations and Caveats

4.1. Cell Type and Receptor Density

4.2. Proximal vs Distal Effects to the Receptor

4.3. Ligand Kinetics

4.4. Applying Measurements of Bias to Allosteric Ligands

4.5. Crystal Structures of CB1R

5. Conclusions

Acknowledgment

References

Chapter Thirteen: Design and Synthesis of Cannabinoid 1 Receptor (CB1R) Allosteric Modulators: Drug Discovery Applications

1. Introduction

1.1. GPCR Allosteric Modulation

1.2. Cannabinoid 1 Receptor

2. CB1R Allosteric Modulators

2.1. CB1R Negative Allosteric Modulators

2.1.1. Indole-2-Carboxamides Scaffold

2.1.2. Diaryl Urea Scaffold

2.1.3. Miscellaneous CB1 NAMs

2.2. CB1R Positive Allosteric Modulators

2.2.1. 2-Phenylindole-Based CB1R PAMs

2.2.2. Miscellaneous CB1R PAMs

3. Designer Probes for Characterizing CB1R Allosteric Site(s)

4. Summary and Future Directions

Acknowledgment

References

Chapter Fourteen: Assessing Allosteric Modulation of CB1 at the Receptor and Cellular Levels

1. Introduction

1.1. Cannabinoid Receptor System

1.2. Allosteric Modulation of the CB1 Receptor

2. Methods of Allosteric Ligand Binding Analysis

2.1. Impact of CB1 Allosteric Ligands on Orthosteric Ligand Affinity

2.2. Equilibrium Binding Assays With Allosteric Modulators

2.2.1. Equipment and Reagents for Equilibrium Binding

2.2.2. Protocol for Equilibrium Binding

2.3. Kinetic Binding Assays With Allosteric Modulators

3. G-Protein Coupling

4. Cellular Internalization of CB1

4.1. Protocol for Cellular Internalization of CB1

5. Analysis of Kinase Phosphorylation

5.1. ERK1/2 Phosphorylation

5.2. Phosphorylation of Other Kinases

5.2.1. Reagents for Kinase Phosphorylation

5.2.2. Protocol for Kinase Phosphorylation

6. Perspectives

Acknowledgments

References

Chapter Fifteen: Purification of Functional CB1 and Analysis by Site-Directed Fluorescence Labeling Methods

1. Introduction

2. Expression and Purification of Functional CB1

2.1. Overview and Description of shCB1

2.2. Alterations of shCB1 to Increase Receptor Expression and Detergent Solubility

2.3. Transient Transfection of COS1 Cells

2.3.1. Equipment

2.3.2. Buffers and Reagents

2.3.3. Procedure

2.3.4. Notes

2.4. Purification of CB1 Mutants

2.4.1. Equipment

2.4.2. Buffers and Reagents

2.4.3. Procedure

2.4.4. Notes

2.5. Radioligand Binding Measurements for Purified, Solubilized CB1

2.5.1. Equipment

2.5.2. Buffers and Reagents

2.5.3. Procedures

2.5.4. Notes

2.6. G-Protein Activation Assays Using Purified, Solubilized CB1

2.6.1. Equipment

2.6.2. Buffers and Reagents

2.6.3. Procedure

2.6.4. Notes

3. Site-Directed Fluorescence Labeling Studies of CB1

3.1. Construction of CB1 Mutants Containing Single Reactive Cysteines

3.1.1. Equipment

3.1.2. Buffers and Reagents

3.1.3. Procedure

3.1.4. Notes

3.2. Fluorescence Labeling of CB1

3.2.1. Equipment

3.2.2. Buffers and Reagents

3.2.3. Procedure

3.2.4. Notes

3.3. Monitoring Ligand Binding Induced Changes in CB1 by Fluorescence

3.3.1. Equipment

3.3.2. Buffers and Reagents

3.3.3. Procedure

3.3.4. Notes

4. Summary and Conclusion

References

Chapter Sixteen: Mass Spectrometry Analysis of Human CB2 Cannabinoid Receptor and Its Associated Proteins

1. Introduction

2. Mass Spectrometry Analysis of Human CB2 Cannabinoid Receptor

2.1. Membrane Preparation and Solubilization

2.2. Purification by Ni Chromatography

2.3. In-Gel Digestion

2.4. MALDI-TOF-MS

2.5. ESI-MS/MS

3. Functional Proteomic Analysis of CB2-Associated Proteins

3.1. Membrane Preparation and Solubilization

3.2. Immunoprecipitation

3.3. In-Gel Digestion

3.4. MALDI-TOF-MS

4. Chemical Cross-linking and Mass Spectrometry Analysis of CB2-G Protein Complex

4.1. Chemical Cross-linking and Solubilization

4.2. Purification by Affinity Chromatography

4.3. In-Gel Digestion

4.4. ESI-MS/MS

5. Conclusion

Acknowledgment

References

Chapter Seventeen: Expression and NMR Structural Studies of Isotopically Labeled Cannabinoid Receptor Type II

1. Introduction

2. Preparation of Stable Isotope-Labeled Cannabinoid Receptor—An Overview of the Method

3. Materials

4. Expression of CB2 in a Fermenter

4.1. Composition of the Mineral Salt Medium

4.2. Adaptation of Cells to MSM

4.3. Preparation of the Fermenter

4.4. Fermentation in MSM

5. Purification of Recombinant CB2 Receptor

6. Reconstitution of CB2 Into Lipid Bilayers

7. Acquisition of NMR Spectra

7.1. NMR Sample Preparation

7.2. MAS NMR Experiments

8. Protocol Notes

9. Conclusions

Acknowledgments

References

Chapter Eighteen: Methods for the Development of In Silico GPCR Models

1. Introduction

2. Sequence Alignment

2.1. Ballesteros-Weinstein Numbering System

2.2. Downloading GPCR Sequences

2.3. Cautions About Alignment Programs

2.4. Species Differences

2.5. GPR3 Sequence Alignment

3. Choosing the Appropriate Template for Model Construction

4. Model Development

4.1. Mutating the Crystal Structure Template

4.2. Appreciating the Effect of Sequence Deviations

4.2.1. Calculating a New TMH Conformation With Conformational Memories

4.2.2. Calculating a New TMH7 Conformation in GPR3

4.3. Loop Modeling

4.4. Energy Minimization

4.5. Active vs Inactive States

4.5.1. G-Protein-Dependent Signaling

4.5.2. G-Protein-Independent β-Arrestin Signaling

5. Docking Studies

5.1. GPR3 Docking Studies

6. Model Refinement

7. Summary

References

Further Reading

Chapter Nineteen: Molecular Dynamics Methodologies for Probing Cannabinoid Ligand/Receptor Interaction

1. Introduction

2. General Methods

2.1. Construction of the Simulation Cell

2.1.1. Determine the Simulation Cell Size

2.1.2. Generate the Unsolvated Membrane Cell

2.1.3. Placement of All-Atom Phospholipids

2.1.4. Relaxation of the Phospholipid Acyl Tails

2.2. Ion Addition and Solvation With Water

2.3. Full Minimization and Warming

2.4. Details of MD Simulations

3. Methods for Each System

3.1. 2-AG Embedded in a POPC Model Membrane

3.1.1. Details of the Construction of the 2-AG/POPC Simulation Cell

3.1.2. Initial Minimization

3.1.3. Details of MD Simulations

3.1.4. Calculation of the Mass/Number Density Distributions

3.2. 2-AG Binds to CB2

3.3. MD of ORG27569 in Water and a POPC Model Bilayer

3.3.1. ORG27569 Initiated in the Aqueous Phase

3.3.2. ORG27569 Initiated at the Phospholipid Bilayer Interface

3.4. ORG27569 Binds to and Activates CB1

3.4.1. Equilibration of the CB1 Receptor and Construction of the ORG27569/CB1 Simulation Cell

3.4.2. Initial Minimization and Details of MD

3.4.3. Addition of ORG27569 to the Simulation

4. Results and Discussion

4.1. 2-AG in a POPC Bilayer

4.2. 2-AG Spontaneously Binds to CB2 via a Lipid Pathway

4.3. ORG27569 in POPC Bilayer

4.4. ORG27569 Binds and Activates the CB1 β-Arrestin-1 Signaling Path

5. Conclusions

References

Author Index

Subject Index

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