Affinity and efficacy : the components of drug-receptor interactions = Components of drug-receptor interactions

The interaction of a drug with a receptor generates a code of information having components of affinity and efficacy. How this information is translated into a response depends on the unique cells, tissue, organ or system in which the receptor resides. This book describes how to analyze various responses to estimate the affinity and efficacy components of the initial drug-receptor interaction. More specifically, it describes how to measure the affinity and efficacy of drugs through the analysis of single receptor activity, the activation state of a population of receptors, and responses downstream from receptor activation. More light is thrown on ligand-gated ion channels and G protein-coupled receptors in this book.The topics discussed include radioligand binding, estimation of agonist affinity and efficacy, competitive antagonism, inverse agonism, allosteric agonists and modulators, agonist bias, modulation of pathway selectivity, and the estimation of ligand affinity for active and inactive receptor states. The natural history and structure of ligand-gated ion channels, G proteins, and G protein-coupled receptors are also discussed. Contents......Page 8 Acknowledgments......Page 10 Prologue: Drugs, Receptors and Therapeutic Messages......Page 12 Part I: Ligand-Gated Ion Channels......Page 46 Chapter 1: Ligand-Gated Ion Channels......Page 48 Nicotinic Acetylcholine Receptors......Page 49 Subunit composition and transmembrane topology of the nicotinic acetylcholine receptor from Torpedo......Page 52 The extracellular ligand binding domain......Page 56 The ion conduction pathway......Page 59 Ligand-induced channel opening......Page 62 Nicotinic receptor subtypes......Page 64 Electrical responses of nicotinic receptors......Page 65 GABAA Receptors......Page 68 The orthosteric ligand-binding site......Page 69 Allosteric binding sites......Page 70 GABAA receptor subtypes......Page 73 Allosteric modulation of electrical responses of GABAA receptors......Page 75 References......Page 77 Chapter 2: Affinity and Efficacy of Orthosteric Ligands at Ligand-Gated Ion Channels......Page 84 Population analysis......Page 85 Derivation of equilibrium equations......Page 87 Measurement of ligand-gated ion channel currents......Page 91 Estimation of observed affinity and efficacy......Page 93 Relationship between population parameters and microscopic constants......Page 99 Estimation of Kb and Ka......Page 102 Single Channel Analysis......Page 106 Measurement of single channel currents......Page 107 The population model applies to single receptor events......Page 108 Single channel behavior resembles a continuous Markov process......Page 111 Simulation of single channel events......Page 116 Distribution of channel open times......Page 121 Distribution of burst lengths......Page 128 Distribution of closed times......Page 130 Estimation of microscopic constants and the macroscopic constants, affinity and efficacy, from the distributions of single channel dwell times......Page 132 Estimation of observed affinity and efficacy from measurements of open probability......Page 135 Estimation of microscopic affinity constants from measurements open probability......Page 137 Population analysis......Page 139 Derivation of equilibrium equations for the cooperative model......Page 143 The meaning of observed affinity......Page 146 The meaning of observed efficacy......Page 148 Two agonist-binding sites are better than one......Page 150 Estimation of microscopic constants from the current response......Page 151 Estimation of observed affinity and mean efficacy from the population response......Page 157 Single Channel Analysis......Page 159 Simulation of single channel events for the cooperative model......Page 160 Estimation of microscopic constants from measurements of open probability......Page 163 Analysis of data from the literature......Page 168 Throwing Light on the Analysis of Agonist Action at G Protein Coupled Receptors......Page 169 References......Page 170 Chapter 3: Competitive Interactions between Orthostheric Ligands at Ligand-Gated Ion Channels......Page 172 Model for Competitive Inhibition......Page 174 Derivation of Equilibrium Equations for Competitive Inhibition......Page 175 Competition with a neutral antagonist......Page 176 Competition between an agonist and a less efficacious agonist......Page 179 The competitive effect of a highly efficacious agonist on the current response of a less efficacious agonist......Page 184 Single Channel Analysis......Page 185 Derivation of the equation for the analysis of competitive interactions at equilibrium......Page 186 Summary......Page 187 References......Page 188 Chapter 4: Analysis of Allosteric Interactions at Ligand-Gated Ion Channels......Page 190 Conformational Changes and Allosterism......Page 192 Benefits of Allosteric Modulation......Page 194 Why do Ligand-Gated Ion Channels have Allosteric Sites?......Page 195 Receptor activation models......Page 198 Ligand-binding model for cooperative interactions......Page 202 Receptor activation function for the case one allosteric site......Page 205 Open channel block......Page 207 Receptor binding functions for the case of one allosteric site......Page 208 Receptor activation function for the case of two allosteric sites......Page 212 Receptor binding functions for the case of two allosteric sites......Page 214 Reciprocal modulation of observed binding affinity......Page 216 Relationship between microscopic constants and observed cooperativities......Page 220 Estimation of observed affinity and cooperativity constants......Page 222 Simulation and Analysis of Allosteric Modulation of the Agonist-Induced Current Response......Page 224 Analysis of allosterism for the case of one allosteric site......Page 225 Analysis of open channel block......Page 228 Analysis of allosterism for the case of two allosteric sites......Page 229 Single Channel Analysis......Page 232 Simulation of Single Channel Events......Page 233 Analysis of the Effect of Allosteric Modulation on Open Probability......Page 237 Analysis of Allosteric Modulation of the GABAA Receptor......Page 238 Probe-Dependence of Allosteric Interactions......Page 245 Observed Affinity and Efficacy......Page 247 Derivation of Equations for the Allosteric Modulation of Observed Affinity and Mean Efficacy......Page 248 Estimation of mean efficacy and observed affinity for the case of one allosteric site......Page 249 Estimation of mean efficacy and observed affinity for the case of two allosteric sites......Page 251 Analysis of allosteric modulation of observed affinity and mean efficacy......Page 253 Relation to Endogenous Signaling and Protein-Protein Interactions......Page 257 References......Page 258 Part II: G Protein-Coupled Receptors......Page 262 Light Detection by Rhodopsin......Page 264 G Protein-Coupled Receptors......Page 270 Rhodopsin Family......Page 272 Secretin Receptor Family......Page 275 Adhesion Receptor Family......Page 277 Frizzled/Taste2 Family......Page 278 Glutamate Receptor Family......Page 279 Heterotrimeric G Proteins......Page 282 Structure of G protein subunits......Page 283 Receptor-catalyzed guanine nucleotide exchange......Page 284 G protein inactivation and hydrolysis of GTP......Page 287 Types of G protein......Page 288 Specificity of the Receptor-G Protein Interaction......Page 290 The Receptosome......Page 295 β1-Adrenoceptor signaling in the heart......Page 298 M2 muscarinic receptor signaling in the heart......Page 299 M3 muscarinic receptor mediated phosphoinositide hydrolysis in smooth muscle......Page 300 What is the Measure of Receptor Activation at G Protein Coupled Receptors?......Page 302 References......Page 304 Chapter 6: Affinity and Efficacy of the Agonist-Receptor-G-Protein Interaction......Page 312 The Receptor-G Protein Activation Cycle......Page 313 Analysis of the Receptor-G Protein-Effector Interaction as a Series of Linked Allosteric Steps......Page 317 Intuitive Explanation of the Receptor-G Protein Cycle......Page 329 Measuring the G Protein Cycle with Labeled Guanine Nucleotides......Page 331 Definition of Observed Affinity and Intrinsic Efficacy at GPCRs at the Population Level of Analysis......Page 332 Modeling the Agonist-Receptor-G Protein Interaction......Page 333 Derivation of Equations for the Quaternary Complex Model......Page 338 Behavior of the Quaternary Complex Model, Population Analysis......Page 345 How Well Does the Model Predict the Behavior of G Protein-Coupled Receptors?......Page 354 Conformational Analysis of the Ternary Complex Model with Guanine Nucleotide......Page 361 Conformational Changes of the G Protein......Page 379 A Simple Function for Agonist Activation of GPCRs......Page 381 References......Page 382 Chapter 7: Estimating Affinity and Efficacy by Reverse-Engineering and Response-Clamp Analysis......Page 388 Reverse Engineering and Receptor Theory......Page 389 Properties......Page 393 What can you get from a single concentration-response curve?......Page 397 Estimating the affinity of a partial agonist......Page 399 Estimating affinity and relative efficacy......Page 402 Null Methods......Page 405 Estimating affinity and efficacy......Page 406 Estimating the affinity of a partial agonist by analysis of its concentration-response curve and that of a full agonist......Page 410 Philosophical significance of the null method approach......Page 413 Estimation of Agonist RAi Values......Page 414 Estimating RAi values from a series of agonist concentration-response curves......Page 415 Relationship of the RAi value to the EC50 and Emax values of agonist......Page 418 Estimating the relative affinities and efficacies of partial agonists is easy when the slope factor is equivalent to one......Page 420 The relationship of RAi value to the initial slope of the agonist concentration-response curve......Page 422 Agonist Activity in Different Assay Systems......Page 424 The reverse engineering approach is appropriate for a variety of assay procedures......Page 425 Signaling pathways amplify the response and increase the potency of the agonist......Page 431 Effect of the sensitivity of the assay system on agonist activity......Page 436 Is the inactivity of a partial agonist caused by the insensitivity of the system?......Page 440 Influence of cytosolic GTP on agonist responses......Page 442 Measuring the Selectivity of the Agonist for the Active and Inactive States of the Receptor......Page 445 Efficacy and selectivity for the active state......Page 446 RAi and the microscopic affinity constant of the active state......Page 449 RIi and the microscopic affinity constant of the inactive state......Page 453 Proof of the relationships among microscopic affinity constants and RAi and RIi......Page 459 Toward a Facile Estimation of the Microscopic Affinity Constants of Agonists......Page 462 Appendix......Page 463 References......Page 469 Chapter 8: Analysis of Agonism and Inverse Agonism in Signaling Pathways that Exhibit Constitutive Activity......Page 470 Constitutive Receptor Activity......Page 471 Modifying Receptor Theory to Account for Constitutive Receptor Activity......Page 476 Operational Model for Constitutive Receptor Activity......Page 479 Estimation of the Observed Affinity Constants of Partial and Inverse Agonists......Page 482 Estimation of the Observed Affinity Constants Orthosteric Ligands by Means of Partial Receptor Inactivation......Page 490 Estimation of the Observed Intrinsic Efficacies of Orthosteric Ligands......Page 495 Estimation of the Microscopic Affinity Constants of the Active and Inactive States of the Receptor......Page 498 Estimating Kb values from functional data......Page 501 Estimating Ka values from functional data......Page 504 Estimation of Kb, Kobs and relative efficacy when the transducer slope factor is equivalent to one (m = 1)......Page 509 Constitutive Activity and the Threshold for Generating a Response......Page 514 Constitutive Activity and Drug Discovery......Page 518 Appendix......Page 520 References......Page 526 Chapter 9: Analysis of Ligand Bias in Receptor Signaling through Different G Protein Pathways......Page 530 Simple model for two active receptor states......Page 537 Model of a receptor with two active states that exhibit selectivity for different G proteins......Page 541 Estimating Kobs and Kb by the method of partial receptor inactivation......Page 545 Analysis of a series of orthosteric ligand concentration-response curves for ligand bias......Page 548 Estimation Ka and RIi values when there is more than one active state......Page 553 Estimation of Kb, RAi , Kobs and Relative Efficacy When the Hill Slope is One......Page 555 Why Multiple Active States......Page 556 Appendix......Page 557 References......Page 560 Chapter 10: Competitive Interactions Between Orthosteric Ligands in Functional Assays on G Protein-Coupled Receptors......Page 562 Competitive Inhibition of the Stimulus......Page 567 Estimation of antagonist affinity in functional assays lacking constitutive activity......Page 568 Null method......Page 569 Operational model......Page 571 Testing the hypothesis of competitive inhibition......Page 575 Interpretation of Deviations from Competitive Inhibition......Page 585 Analysis of Irreversible and Slowly Dissociating Orthosteric Ligands......Page 587 Operational model for competitive interactions at constitutively active receptors......Page 590 Analysis of simulated competitive inhibition data......Page 592 Can the standard null equation be used to analyze data with inverse and partial agonists?......Page 594 Theory of resultant analysis......Page 596 Estimation of the affinity constant p-FHHSiD for muscarinic receptors in smooth muscle......Page 601 Using Antagonists to Identify Receptor Function......Page 603 Examples of using antagonists to identify receptor function......Page 605 Simulation of antagonism of responses mediated by two receptors......Page 607 Clinical Uses of Antagonists......Page 612 Appendix......Page 613 References......Page 617 Chapter 11: Analysis of Allosterism in Functional Assays on G Protein-coupled Receptors......Page 620 General population model......Page 624 Operational model for allosterism......Page 628 Simple two-state model......Page 630 Complex two-state model......Page 633 Simulation and Analysis of Allosteric Interactions......Page 642 Changes in the sensitivity of the signaling pathway affect the behavior of allosterism but not the estimates of K2 and γ......Page 643 Estimating the cooperativity constant α using the method of partial receptor inactivation......Page 648 Analyzing the effect of an allosteric agonist......Page 651 Analyzing allosterism at constitutively active receptors......Page 653 Multiple active states of the receptor give rise to biased allosteric effects......Page 655 Allosteric modulation of pathway selectivity......Page 659 Summary of the determinants of allosteric modulation in affinity and efficacy......Page 660 Relationship between allosteric modulation and occupancy of the allosteric site......Page 663 Analysis of steric inhibitory effects on receptor function......Page 667 Analysis of Data from the Literature......Page 671 Positive allosteric modulation of the metabotropic glutamate receptor 4......Page 672 Negative allosteric modulation of the M2 muscarinic receptor......Page 674 What Does the Allosteric Modulation of GPCRs Tell us About Receptor States?......Page 676 Analyzing Allosterism Using a Combination of Functional and Binding Methods......Page 682 References......Page 683 Part III: Radioligand Binding......Page 686 Chapter 12: Analysis of Drug-receptor Interactions Using Radioligand Binding Assays on G Protein-coupled Receptors......Page 688 Nature of reversible binding interactions......Page 692 Estimation of the equilibrium binding parameters of the radioligand......Page 700 Competitive Inhibition of Radioligand Binding......Page 708 Estimating the affinity constants of nonlabeled competitors......Page 709 How is Occupancy by the Inhibitor Related to Displacement of Radioligand Binding......Page 715 Testing the hypothesis of competitive inhibition......Page 716 Allosteric Modification of Radioligand Binding......Page 721 Estimating the affinity constants of allosteric ligands......Page 723 How is Occupancy by the Modulator Related to Displacement of Radioligand Binding......Page 730 Testing the hypothesis of allosterism......Page 732 How is occupancy by the modulator related to its effect on the saturation curve of the radioligand?......Page 736 Measuring the interaction between the modulator and a nonlabeled orthosteric ligand......Page 737 References......Page 741 Index......Page 742 "The interaction of a drug with a receptor encodes a vector of information having components of affinity and efficacy. How this information is translated into a response depends on the unique cells, tissue, organ or system in which the receptor resides. This book describes how to analyze various responses to estimate the affinity and efficacy components of the initial drug-receptor interaction. More specifically, it describes how to measure the affinity and efficacy of drugs through the analysis of single receptor activity, the activation state of a population of receptors, and responses downstream from receptor activation. More light is shed on ligand-gated ion channels and G protein-coupled receptors in this book. The topics discussed include radio ligand binding, estimation of agonist affinity and efficacy, competitive antagonism, inverse agonism, allosteric agonists and modulators, ligand-directed signaling, modulation of pathway selectivity, and the analysis of the loss of function in tissues from receptor knockout mice. The natural history and structure of ligand-gated ion channels, G proteins, and G protein-coupled receptors are also discussed."--Page 4 of cover