Integration of Label Free Detection Methods in GPCR

Drug Discovery 252

Oliver Nayler, Magdalena Birker-Robaczewska, and John Gatfield

11.1. Overview 252

11.2. Introduction 253

11.3. Label-Free Technologies—Past and Present 255

11.3.1. Automated Microscopes and Microbalances 256

11.3.2. Microphysiometry 257

11.3.3. Impedance/RWG 259

11.4. Discussion 270 Acknowledgments 272 References 272

12. Screening for Allosteric Modulators of G

Protein-Coupled Receptors 276

Christopher Langmead

12.1. Introduction 276

12.2. The Allosteric Ternary Complex Model,

Radioligand Binding, and Affinity 278

12.3. Beyond Affinity—Functional Assays, Efficacy, and Allosteric Agonism 281

12.4. Allosteric Modulator Titration Curves 286

12.5. The Impact of Functional Assay Format on

Allosteric Modulator Screening 289

12.6. Taking Advantage of Structural Understanding of Allosteric Binding Sites 293

12.7. Summary and Future Directions 294 References 295

13. Ultra-High-Throughput Screening Assays for GPCRs 300

Priya Kunapuli

13.1. Introduction 300

13.2. Assay Types for GPCRs in uHTS 303

13.2.1. Radioligand Displacement Assays 303

13.2.2. Functional Assays 305

13.3. Summary 317 Acknowledgments 319 References 319

14. New Techniques to Express and Crystallize

G Protein-Coupled Receptors 324

James C. Errey and Fiona H. Marshall

14.1. Introduction 324

14.2. Key Problems Limiting Production of 3D

GPCR Structures 327

14.3. History of GPCR Structures 329

14.3.1. Early Studies on Rhodopsin 329

14.3.2. Higher Resolution Structures of Bovine

Rhodopsin Using X-Ray Crystallography 333

14.3.3. Squid Rhodopsin 336

14.3.4. Activated Opsin and Binding to G Proteins 337

14.3.5. Rhodopsin as a Model for Other GPCRs 340

14.4. The Search for Other GPCR Structures 340

14.4.1. Expression of Recombinant Receptors 340

14.4.2. Factors Influencing GPCR Overexpression 349

14.4.3. Summary 350

14.5. Protein Purification and Solubilization 351

14.5.1. Choice of Detergents for Structural Studies 355

14.5.2. Crystallization Chaperones 356

14.6. In Cubo Crystallization 358

14.7. Engineering Receptor Stability 361

14.8. Structures of the P2AR 365

14.9. The Adenosine A2a Receptor 369

14.10. Conclusions and Future Developments 371 Acknowledgments 371 References 371

15. Structure and Modeling of GPCRs: Implications for

Drug Discovery 385

Kimberly A. Reynolds, Vsevolod Katritch, and Ruben Abagyan

15.1. Introduction 385

15.2. High-Resolution GPCR Modeling 389

15.2.1. From Electron Density to a Full Atom Model Suitable for Drug Discovery: Refinement of

Existing Crystal Structures 389

15.2.2. Ligand-Guided Modeling of Binding Pocket Conformation 392

15.2.3. Coupling LGM and TM Domain Motions to Capture Binding Site Conformational Changes Necessary for Agonist Recognition 397

15.2.4. VLS with High-Resolution Models: Antagonist/Agonist Selectivity 398

15.3. Constructing and Evaluating Homology Models of Other Receptor Types 402

15.3.1. A Note on De Novo Methods 402

15.3.2. Criteria for Homology Model Template Selection 403

15.3.3. Structure and Modeling of Loop Regions 409

15.3.4. GPCR Model Validation and Evaluation 411

15.3.5. Ligand Subtype Selectivity in GPCR Models 413

15.4. Modeling GPCR Functional Features—Analysis of Activation and Signaling 415

15.4.1. Activation-Related Conformational Changes in GPCRs 416

15.4.2. Macromolecular Complexes of GPCRs 417

15.5. Beyond Class A: Modeling of Other GPCR Families 418

15.5.1. Modeling Secretin (Class B) Family GPCRs 418

15.5.2. Glutamate/Class C 420

15.5.3. Orphan GPCRs 421

15.6. Summary and Conclusions 422 Acknowledgments 422 References 422

16. X-Ray Structure Developments for GPCR Drug Targets 434

Michael Sabio and Sidney W. Topiol

16.1. Overview 434

16.2. Introduction 434

16.3. Class A GPCRs 438

16.3.1. Sequence Homology 438

16.3.2. Stabilization of X-Ray Structures 438

16.3.3. The Overall Topology of the 7TM Region 440

16.3.4. The Binding Site 441

16.3.5. The ECL2 443

16.3.6. The Toggle Switch 443

16.3.7. The Ionic Lock 444

16.3.8. The ICL3 Region and Activation 445

16.3.9. Computational Chemistry Successes and Limitations 447

16.4. Class C GPCRs 449

16.4.1. Global Architecture 449

16.4.2. The VFT Domain 450

16.4.3. The C-rich Domain 451

16.4.4. Computational Studies 452

16.5. Conclusions 452 References 453

17. Pharmacological Chaperones: Potential for the Treatment of Hereditary Diseases Caused by Mutations in G Protein-Coupled Receptors 460

Kenneth J. Valenzano, Elfrida R. Benjamin,Patricia René, and Michel Bouvier

17.1. Overview 460

17.2. Introduction 461

17.3. NDI and the V2R 464

17.4. RP and the Rhodopsin Receptor 470

17.5. IHH and the Gonadotropin-Releasing

Hormone Receptor 476

17.6. Other Human Diseases Caused by Inactivating Mutations in GPCRs 479

17.6.1. Class A GPCRs 479

17.6.2. Class B GPCRs 482

17.6.3. Class C GPCRs 483

17.6.4. Family Frizzled/Smoothened GPCRs 484

17.7. Considerations for the Therapeutic Use of Pharmacological Chaperones 485

17.7.1. Pharmacogenetics 485

17.7.2. Dominant-Negative Effects 486

17.7.3. Function of Rescued GPCRs with

Missense Mutations 488

17.7.4. Biophysical Requirements of

Pharmacological Chaperones 488

17.7.5. Safety 490

17.8. Concluding Remarks 490 Acknowledgments 491 References 491

Index

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